WO1996021469A1 - Multi-armed, monofunctional, and hydrolytically stable derivatives of poly(ethylene glycol) and related polymers for modification of surfaces and molecules - Google Patents
Multi-armed, monofunctional, and hydrolytically stable derivatives of poly(ethylene glycol) and related polymers for modification of surfaces and molecules Download PDFInfo
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- WO1996021469A1 WO1996021469A1 PCT/US1996/000474 US9600474W WO9621469A1 WO 1996021469 A1 WO1996021469 A1 WO 1996021469A1 US 9600474 W US9600474 W US 9600474W WO 9621469 A1 WO9621469 A1 WO 9621469A1
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
- C08G65/32—Polymers modified by chemical after-treatment
- C08G65/329—Polymers modified by chemical after-treatment with organic compounds
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G65/34—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from hydroxy compounds or their metallic derivatives
- C08G65/48—Polymers modified by chemical after-treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
- Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
- Y10S530/815—Carrier is a synthetic polymer
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
- Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
- Y10S530/815—Carrier is a synthetic polymer
- Y10S530/816—Attached to the carrier via a bridging agent
Definitions
- This invention relates to monofunctional derivatives of poly(ethylene glycol) and related polymers and to methods for their synthesis and
- enzymes that exhibit specific biocatalytic activity sometimes are less useful than they otherwise might be because of problems of low stability and solubility in organic solvents.
- many proteins are cleared from circulation too rapidly. Some proteins have less water solubility than is optimal for a therapeutic agent that circulates through the bloodstream. Some proteins give rise to immunological problems when used as therapeutic agents. Immunological problems have been reported. from
- polypeptides as drugs or biocatalysts
- link suitable hydrophilic or amphiphilic polymer derivatives to the polypeptide to create a polymer cloud surrounding the polypeptide. If the polymer derivative is soluble and stable in organic solvents, then enzyme conjugates with the polymer may acquire that solubility and stability. Biocatalysis can be extended to organic media with enzyme and polymer combinations that are soluble and stable in organic solvents.
- the polymer cloud can help to protect the compound from chemical attack, to limit adverse side effects of the compound when injected into the body, and to increase the size of the compound, potentially to render useful compounds that have some medicinal benefit, but otherwise are not useful or are even harmful to an organism.
- the polymer cloud surrounding a protein can reduce the rate of renal excretion and immunological complications and can increase resistance of the protein to proteolytic breakdown into simpler, inactive substances.
- linking moieties are toxic. Some linkages reduce the activity of the protein or enzyme, thereby rendering the protein or enzyme less effective.
- the structure of the protein or enzyme dictates the location of reactive sites that form the loci for linkage with polymers.
- Proteins are built of various sequences of alpha-amino acids, which have the general structure
- the alpha amino moiety (H 2 N-) of one amino acid joins to the carboxyl moiety (-COOH) of an adjacent amino acid to form amide linkages, which can be represented as where n can be hundreds or thousands.
- the terminal amino acid of a protein molecule contains a free alpha amino moiety that is reactive and to which a polymer can be attached.
- the fragment represented by R can contain reactive sites for protein biological activity and for attachment of polymer.
- lysine which is an amino acid forming part of the backbone of most proteins
- a reactive amino (-NH 2 ) moiety is present in the epsilon position as well as in the alpha position.
- the epsilon -NH 2 is free for reaction under conditions of basic pH.
- Much of the art has been directed to developing polymer derivatives having active moieties for attachment to the epsilon -NH 2 moiety of the lysine fraction of a protein. These polymer derivatives all have in common that the lysine amino acid fraction of the protein typically is modified by polymer attachment, which can be a drawback where lysine is important to protein activity.
- PEG poly(ethylene glycol), which is commonly referred to simply as "PEG,” has been the nonpeptidic polymer most used so far for attachment to proteins.
- the PEG molecule typically is linear and can be
- the PEG molecule is difunctional, and is sometimes referred to as "PEG diol.”
- the terminal portions of the PEG molecule are relatively nonreactive hydroxyl moieties, -OH, that can be activated, or converted to functional moieties, for attachment of the PEG to other compounds at reactive sites on the compound.
- terminal moieties of PEG diol have been functionalized as active carbonate ester for selective reaction with amino moieties by
- succinimidyl ester moiety can be represented structurally as
- Difunctional PEG functionalized as the succinimidyl carbonate, has a structure that can be represented as
- Difunctional succinimidyl carbonate PEG has been reacted with free lysine monomer to make high molecular weight polymers.
- Free lysine monomer which is also known as alpha, epsilon diaminocaproic acid, has a structure with reactive alpha and epsilon amino moieties that can be represented as
- These high molecular weight polymers from difunctional PEG and free lysine monomer have multiple, pendant reactive carboxyl groups extending as branches from the polymer backbone that can be represented structurally as
- the pendant carboxyl groups typically have been used to couple nonprotein pharmaceutical agents to the polymer. Protein pharmaceutical agents would tend to be cross linked by the multifunctional polymer with loss of protein activity.
- Multiarmed PEGs having a reactive terminal moiety on each branch have been prepared by the
- Nonreactive end moiety so that the PEG molecule is monofunctional.
- Monofunctional PEGs are usually preferred for protein modification to avoid cross linking and loss of activity.
- One hydroxyl moiety on the terminus of the PEG diol molecule typically is substituted with a nonreactive methyl end moiety, CH 3 -.
- the opposite terminus typically is converted to a reactive end moiety that can be activated for
- molecule such as a protein.
- PEG molecules having a methyl end moiety are sometimes referred to as monomethoxy-poly (ethylene glycol) and are sometimes referred to simply as "mPEG.”
- mPEG polymer derivatives can be represented
- n typically equals from about 45 to 115 and -Z is a functional moiety that is active for selective attachment to a reactive site on a molecule or surface or is a reactive moiety that can be converted to a functional moiety.
- mPEG polymers are linear polymers of molecular weight in the range of from about 1,000 to 5,000. Higher molecular weights have also been
- these mPEGs typically are not of high purity and have not normally been useful in PEG and protein chemistry.
- these high molecular weight mPEGs typically contain significant percentages of PEG diol.
- Proteins and other molecules typically have a limited number and distinct type of reactive sites available for coupling, such as the epsilon -NH 2 moiety of the lysine fraction of a protein. Some of these reactive sites may be responsible for a protein's biological activity. A PEG derivative that attached to a sufficient number of such sites to impart the desired characteristics can adversely affect the activity of the protein, which offsets many of the advantages otherwise to be gained.
- PEG derivatives have been developed that have a single functional moiety located along the polymer backbone for attachment to another molecule or surface, rather than at the terminus of the polymer. Although these compounds can be considered linear, they are often referred to as "branched” and are distinguished from conventional, linear PEG derivatives since these molecules typically comprise a pair of mPEG- molecules that have been joined by their reactive end moieties to another moiety, which can be represented structurally as -T-, and that includes a reactive moiety, -Z, extending from the polymer backbone. These compounds have a general structure that can be represented as
- mPEG-disubstituted chlorotriazine molecule reacts with water, thus substantially precluding purification of the branched mPEG structure by commonly used
- a branched mPEG with a single activation site based on coupling of mPEG to a substituted benzene ring is disclosed in European Patent Application Publication No. 473 084 A2.
- this structure contains a benzene ring that could have toxic effects if the structure is destroyed in a living organism.
- Another branched mPEG with a single activation site has been prepared through a complex synthesis in which an active succinate moiety is attached to the mPEG through a weak ester linkage that is susceptible to hydrolysis.
- An mPEG-OH is reacted with succinic anhydride to make the succinate.
- the reactive succinate is then activated as the
- succinimide The synthesis, starting with the active succinimide, includes the following steps, represented structurally below.
- the mPEG activated as the succinimide, mPEG succinimidyl succinate, is reacted in the first step as shown above with norleucine.
- the symbol -R in the synthesis represents the n-butyl moiety of norleucine.
- the mPEG and norleucine conjugate (A) is activated as the succinimide in the second step by reaction with N- hydroxy succinimide.
- the mPEG and norleucine conjugate activated as the succinimide (B) is coupled to the alpha and epsilon amino moieties of lysine to create an mPEG
- the mPEG disubstituted lysine is activated as the succinimide.
- ester linkage formed from the reaction of the mPEG-OH and succinic anhydride molecules is a weak linkage that is hydrolytically unstable. In vivo application is therefore limited. Also, purification of the branched mPEG is precluded by commonly used chromatographic techniques in water, which normally would destroy the molecule.
- the molecule also has relatively large molecular fragments between the carboxyl group
- linkers or “spacer arms,” and have the potential to act as antigenic sites promoting the formation of antibodies upon injection and initiating an undesirable immunological response in a living organism.
- the invention provides a branched or "multi-armed" amphiphilic polymer derivative that is
- monofunctional, hydrolytically stable can be prepared in a simple, one-step reaction, and possesses no aromatic moieties in the linker fragments forming the linkages with the polymer moieties.
- the derivative can be prepared without any toxic linkage or potentially toxic fragments. Relatively pure polymer molecules of high molecular weight can be created.
- the polymer can be purified by chromotography in water.
- a multi-step method can be used if it is desired to have polymer arms that differ in molecular weight.
- the polymer arms are capped with relatively nonreactive end groups.
- the derivative can include a single reactive site that is located along the polymer backbone rather than on the terminal portions of the polymer moieties.
- reactive site can be activated for selective reactions.
- the multi-armed polymer derivative of the invention having a single reactive site can be used for, among other things, protein modification with a high retention of protein activity. Protein and enzyme activity can be preserved and in some cases is enhanced.
- the single reactive site can be converted to a functional group for highly selective coupling to proteins, enzymes, and surfaces. A larger, more dense polymer cloud can be created surrounding a biomolecule with fewer attachment points to the biomolecule as compared to conventional polymer derivatives having terminal functional groups. Hydrolytically weak ester linkages can be avoided. Potentially harmful or toxic products of hydrolysis can be avoided. Large linker fragments can be avoided so as to avoid an antigenic response in living organisms. Cross linking is
- the molecules of the invention can be any organic radicals.
- the molecules of the invention can be any organic radicals.
- Poly a and poly b represent nonpeptidic and substantially nonreactive water soluble polymeric arms that may be the same or different.
- C represents carbon.
- P and Q represent linkage fragments that may be the same or different and that join polymer arms poly a and poly b , respectively, to C by hydrolytically stable linkages in the absence of aromatic rings in the linkage fragments.
- R is a moiety selected from the group consisting of H, substantially nonreactive, usually alkyl, moieties, and linkage fragments attached by a hydrolytically stable linkage in the absence of aromatic rings to a nonpeptidic and substantially nonreactive water soluble polymeric arm.
- the moiety -Z comprises a moiety selected from the group consisting of moieties having a single site reactive toward nucleophilic moieties, sites that can be converted to sites reactive toward nucleophilic moieties, and the reaction product of a nucleophilic moiety and moieties having a single site reactive toward nucleophilic moieties.
- the moiety -P-CR(-Q-)-Z is the reaction product of a linker moiety and the reactive site of monofunctional, nonpeptidic polymer
- nonpeptidic polymers and can be selected from polymers that have a single reactive moiety that can be
- the linker has the general structure X-CR-(Y)-Z, in which X and Y represent fragments that contain reactive sites for coupling to the polymer reactive site W to form linkage fragments P and Q, respectively.
- At least one of the polymer arms is a poly (ethylene glycol) moiety capped with an essentially nonreactive end group, such as a monomethoxy-poly (ethylene glycol) moiety
- mPEG- which is capped with a methyl end group, CH 3 -.
- the other branch can also be an mPEG moiety of the same or different molecular weight, another
- poly (ethylene glycol) moiety that is capped with an essentially nonreactive end group other than methyl, or a different nonpeptidic polymer moiety that is capped with a nonreactive end group such as a capped
- poly a and poly b are each monomethoxy-poly (ethylene glycol) ("mPEG") of the same or different molecular weight.
- the mPEG- disubstituted derivative has the general structure mPEG a -P-CH(-Q-mPEG b )-Z.
- the moieties mPEG a - and mPEG b - have the structure CH 3 -(CH 2 CH 2 O) n CH 2 CH 2 - and n may be the same or different for mPEG a and mPEG b .
- Molecules having values of n of from 1 to about 1,150 are contemplated.
- the linker fragments P and Q contain
- linker fragments typically are alkyl fragments
- linker fragments P and Q can include reactive sites for joining additional monofunctional nonpeptidic polymers to the multi-armed structure.
- the moiety -R can be a hydrogen atom, H, a nonreactive fragment, or, depending on the degree of substitution desired, R can include reactive sites for joining additional monofunctional nonpeptidic polymers to the multi-armed structure.
- the moiety -Z can include a reactive moiety for which the activated nonpeptidic polymers are not selective and that can be subsequently activated for attachment of the derivative to enzymes, other
- the moiety -Z can include a linkage fragment -R z .
- the R z fragment can include reactive sites for joining additional
- the -Z moiety includes terminal functional moieties for providing linkages to reactive sites on proteins, enzymes, nucleotides, lipids, liposomes, and other materials.
- the moiety -Z is intended to have a broad interpretation and to include the reactive moiety of monofunctional polymer
- the invention includes biologically active conjugates comprising a biomolecule, which is a biologically active molecule, such as a protein or enzyme, linked through an activated moiety to the branched polymer derivative of the invention.
- the invention includes biomaterials comprising a solid such as a surface or particle linked through an activated moiety to the polymer derivatives of the invention.
- the polymer moiety is an mPEG moiety and the polymer derivative is a two-armed mPEG derivative based upon hydrolytically stable coupling of mPEG to lysine.
- the mPEG moieties are represented structurally as CH 3 O-(CH 2 CH 2 O) n CH 2 CH 2 - wherein n may be the same or different for poly a - and poly b - and can be from 1 to about 1,150 to provide molecular weights of from about 100 to 100,000.
- the -R moiety is hydrogen.
- the -Z moiety is a reactive carboxyl moiety.
- the molecule is represented structurally as follows:
- hydrolytically stable mPEG-disubstituted lysine which can also be called alpha, epsilon-mPEG lysine, provides a site for interacting with ion exchange chromatography media and thus provides a mechanism for purifying the product.
- These purifiable, high molecular weight, monofunctional compounds have many uses.
- mPEG-disubstituted lysine, activated as succinimidyl ester reacts with amino groups in enzymes under mild aqueous conditions that are compatible with the
- the mPEG-disubstituted lysine of the invention activated as the succinimidyl ester, is represented as follows:
- the invention includes methods of
- the methods comprise reacting an active suitable polymer having the structure poly-W with a linker moiety having the structure X-CR-(Y)Z to form poly a -P-CR(-Q-poly b )-Z.
- the poly moiety in the structure poly-W can be either poly a or poly b and is a polymer having a single reactive moiety W.
- the W moiety is an active moiety that is linked to the polymer moiety directly or through a hydrolytically stable linkage.
- the moieties X and Y in the structure X-CR-(Y)Z are reactive with W to form the linkage fragments Q and P, respectively.
- R includes reactive sites similar to those of X and Y, then R can also be modified with a poly-W, in which the poly can be the same as or different from poly a or poly b .
- the moiety Z normally does not include a site that is reactive with W.
- X, Y, R, and Z can each include one or more such reactive sites for preparing monofunctional polymer derivatives having more than two branches.
- the method of the invention typically can be accomplished in one or two steps.
- the method can include additional steps for preparing the compound poly-W and for converting a reactive Z moiety to a functional group for highly selective reactions.
- the active Z moiety includes a reactive moiety that is not reactive with W and can be activated subsequent to formation of poly a -P-CR(-Q-poly b )-Z for highly selective coupling to selected reactive moieties of enzymes and other proteins or surfaces or any molecule having a reactive nucleophilic moiety for which it is desired to modify the characteristics of the molecule.
- the invention provides a multi-armed mPEG derivative for which preparation is simple and straightforward.
- Figures 1(a), 1(b), and 1(c) illustrate the time course of digestion of ribonuclease ( ⁇ ),
- Figures 2(a) and 2(b) illustrate stability toward heat (Figure 2(a)) and pH (Figure 2(b)) of ribonuclease ( ⁇ ), linear mPEG-modified ribonuclease (O), and ribonuclease modified with a multi-armed mPEG of the invention ( ⁇ ).
- Figure 2 (a) is based on data taken after a 15 minute incubation period at the indicated temperatures.
- Figure 2(b) is based on data taken over a 20 hour period at different pH values.
- Figures 3 (a) and 3 (b) illustrate the time course of digestion for catalase ( ⁇ ), linear mPEG-modified catalase (O), and catalase modified with a multi-armed mPEG of the invention ( ⁇ ) as assessed by enzyme activity upon incubation with pronase ( Figure 3(a)) and trypsin ( Figure 3 (b)).
- Figure 4 illustrates the stability of catalase ( ⁇ ), linear mPEG-modified catalase (D) , and catalase modified with a multi-armed mPEG of the invention (O) for 20 hours incubation at the indicated pH values.
- Figure 5 illustrates the time course of digestion of asparaginase ( ⁇ ), linear mPEG-modified asparaginase (O), and asparaginase modified with a multi-armed mPEG of the invention ( ⁇ ) as assessed by enzyme activity assay upon trypsin incubation.
- Figure 6 illustrates the time course of autolysis of trypsin ( ⁇ ), linear mPEG-modified trypsin ( ⁇ ), and trypsin modified with a multi-armed mPEG of the invention (A) evaluated as residual activity towards TAME (alpha N-p-tosyl-arginine methyl ester).
- the first procedure is a two step procedure, meaning that the lysine is substituted with each of the two mPEG moieties in separate reaction steps.
- Monomethoxy-poly (ethylene glycol) arms of different lengths or of the same length can be substituted onto the lysine molecule, if
- the second procedure is a one step procedure in which the lysine molecule is substituted with each of the two mPEG moieties in a single reaction step.
- the one step procedure is suitable for preparing mPEG-disubstituted lysine having mPEG moieties of the same length.
- moiety “moiety,” “active moiety,” “reactive site,” “radical,” and similar terms are somewhat synonymous in the chemical arts and are used in the art and herein to refer to distinct, definable portions or units of a molecule or fragment of a molecule.
- Reactive site “functional group,” and “active moiety” refer to units that perform some function or have a chemical activity and are reactive with other molecules or portions of molecules. In this sense a protein or a protein residue can be considered as a molecule and as a functional moiety when coupled to a polymer.
- polymer such as mPEG-COOH has a reactive site, the carboxyl moiety, -COOH, that can be converted to a functional group for selective reactions and attachment to proteins and linker moieties.
- the converted polymer is said to be activated and to have an active moiety, while the -COOH group is relatively nonreactive in comparison to an active moiety.
- nonreactive is used herein
- nonreactive should be understood to exclude carboxyl and hydroxyl moieties, which, although relatively nonreactive, can be converted to functional groups that are of selective reactivity.
- biologically active means a substance, such as a protein, lipid, or nucleotide that has some activity or function in a living organism or in a substance taken from a living organism.
- an enzyme can catalyze chemical reactions.
- biomaterial is somewhat imprecise, and is used herein to refer to a solid material or particle or surface that is compatible with living organisms or tissue or fluids. For example, surfaces that contact blood, whether in vitro or in vivo, can be made
- an activated mPEG is prepared for coupling to free lysine monomer and then the lysine monomer is disubstituted with the activated mPEG in two steps.
- the first step occurs in aqueous buffer.
- the second step occurs in dry
- the active moiety of the mPEG for coupling to the lysine monomer can be selected from a number of activating moieties having leaving moieties that are reactive with the amino moieties of lysine monomer.
- the two step procedure can be represented structurally as follows:
- Step 1 Preparation of mPEG-monosubstituted lysine. Modification of a single lysine amino group was accomplished with mPEG-p-nitrophenylcarbonate in aqueous solution where both lysine and mPEG-p- nitrophenylcarbonate are soluble.
- the mPEG-p- nitrophenylcarbonate has only limited stability in aqueous solution. However, lysine is not soluble in organic solvents in which the activated mPEG is stable. Consequently, only one lysine amino group is modified by this procedure. NMR confirms that the epsilon amino group is modified.
- Step 2 Preparation of mPEG-Disubstituted Lysine.
- the mPEG-monosubstituted lysine product from step 1 above is soluble in organic solvents and so modification of the second lysine amino moiety can be achieved by reaction in dry methylene chloride.
- Activated mPEG, mPEG-p-nitrophenylcarbonate is soluble and stable in organic solvents and can be used to modify the second lysine amino moiety.
- Triethylamine (“TEA") was added to 4.5 grams of mPEG-monosubstituted lysine, which is about 0.86 millimoles.
- the mixture of TEA and mPEG-monosubstituted lysine was dissolved in 10 milliliters of anhydrous methylene chloride to reach a pH of 8.0.
- the mPEG-disubstituted lysine was also separated from unmodified mPEG-OH and purified by an alternative method. Ion exchange chromatography was performed on a QAE Sephadex A50 column (Pharmacia) that measured 5 centimeters by 80 centimeters. An 8.3 mM borate buffer of pH 8.9 was used. This alternative procedure permitted fractionation of a greater amount of material per run than the other method above
- the purified mPEG-disubstituted lysine was also characterized by 1 H-NMR on a 200 MHz Bruker instrument in dimethyl sulfoxide, d6, at a 5% weight to volume concentration. The data confirmed the expected molecular weight of 10,000 for the polymer.
- the chemical shifts and assignments of the protons in the mPEG-disubstituted lysine are as follows: 1.2-1.4 ppm (multiplet, 6H, methylenes 3,4,5 of lysine); 1.6 ppm (multiplet, 2H, methylene 6 of lysine); 3.14 ppm (s, 3H, terminal mPEG methoxy); 3.49 ppm (s, mPEG backbone methylene); 4.05 ppm (t, 2H, -CH 2 , -OCO-); 7.18 ppm (t, 1H, -NH- lysine); and 7.49 ppm (d,l H, -NH- lysine).
- the two step procedure described above allows polymers of different types and different lengths to be linked with a single reactive site between them.
- the polymer can be designed to provide a polymer cloud of custom shape for a particular application.
- mPEG disubstituted lysine is prepared from lysine and an activated mPEG in a single step as represented structurally below:
- the mPEG disubstituted lysine of the one step procedure does not differ structurally from the mPEG disubstituted lysine of the two step procedure. It should be recognized that the identical compound, having the same molecular weight, can be prepared by either method.
- Succinimidylcarbonate mPEG of molecular weight about 20,000 was added in an amount of 10.8 grams, which is 5.4 ⁇ 10 -4 moles, to 40 milliliters of lysine HCl solution.
- the lysine HCL solution was in a borate buffer of pH 8.0.
- the concentration was 0.826
- the solution was diluted with 300 milliliters of deionized water.
- the pH of the solution was adjusted to 3.0 by the addition of oxalic acid.
- mPEG disubstituted lysine of molecular weight 20,000 was eluted with 10 mM NaCl.
- the pH of the eluate was adjusted to 3.0 with oxalic acid and then mPEG
- the one step procedure is simple in application and is useful for producing high molecular weight dimers that have polymers of the same type and length linked with a single reactive site between them.
- Succinimidylcarbonate mPEG was prepared by dissolving 30 grams of mPEG-OH of molecular weight 20,000, which is about 1.5 millimoles, in 120
- Succinimidylcarbonate mPEG of molecular weight about 20,000 was precipitated in ethyl ether and dried in vacuum for a minimum of 8 hours. The yield was 90%.
- Succinimidylcarbonate-mPEG is available commercially from Shearwater Polymers in Huntsville, Alabama. The mPEG disubstituted lysine of the
- -P-CR(-Q-)-Z is the reaction product of a precursor linker moiety having two reactive amino groups and active monofunctional precursors of poly a and poly b that have been joined to the linker moiety at the reactive amino sites.
- Linker fragments Q and P contain carbamate linkages formed by joining the amino
- linker fragments are selected from -O-C(O)NH(CH 2 ) 4 - and -O-C(O)NH- and are different in the exemplified polymer derivative.
- P and Q could both be -O-C(O)NH(CH 2 ) 4 - or
- R is hydrogen, H.
- the moiety represented by Z is the carboxyl group, -COOH.
- the moieties P, R, Q, and Z are all joined to a central carbon atom.
- the nonpeptidic polymer arms, poly a and poly b are mPEG moieties mPEG a and mPEG b , respectively, and are the same on each of the linker fragments Q and P for the examples above.
- the mPEG moieties have a structure represented as CH 3 O-(CH 2 CH 2 O) n CH 2 CH 2 -.
- n is about 454 to provide a molecular weight for each mPEG moiety of 20,000 and a dimer molecular weight of
- n is about 114 to provide a molecular weight for each mPEG moiety of 5,000 and a dimer molecular weight of 10,000.
- Lysine disubstituted with mPEG and having as dimer molecular weights of 10,000 and 40,000 and procedures for preparation of mPEG-disubstituted lysine have been shown. However, it should be recognized that mPEG disubstituted lysine and other multi-armed
- compounds of the invention can be made in a variety of molecular weights, including ultra high molecular weights. High molecular weight monofunctional PEGs are otherwise difficult to obtain.
- Polymerization cf ethylene oxide to yield mPEGs usually produces molecular weights of up to about 20,000 to 25,000 g/mol. Accordingly, two-armed mPEG disubstituted lysines of molecular weight of about 40,000 to 50,000 can be made according to the
- disubstituted PEGs can be made if the chain length of the linear mPEGs is increased, up to about 100,000. Higher molecular weights can also be obtained by adding additional monofunctional nonpeptidic polymer arms to additional reactive sites on a linker moiety, within practical limits of steric hindrance. However, no unreacted active sites on the linker should remain that could interfere with the monofunctionality of the multi -armed derivative. Lower molecular weight
- disubstituted mPEGs can also be made, if desired, down to a molecular weight of about 100 to 200.
- linker fragments P and Q are available, although not necessarily with equivalent results, depending on the precursor linker moiety and the functional moiety with which the activated mPEG or other nonpeptidic
- linker fragments will contain the reaction products of
- linker moieties that have reactive amino and/or thiol moieties and suitably activated
- nonpeptidic, monofunctional, water soluble polymers are nonpeptidic, monofunctional, water soluble polymers.
- activated mPEGs are available that form a wide variety of
- Linkages can be selected from the group consisting of amide, amine, ether, carbamate, which are also called urethane linkages, urea, thiourea,
- Hydrolytic stability of the linkages means that the linkages between the polymer arms and the linker moiety are stable in water and that the linkages do not react with water at useful pHs for an extended period of time of at least several days, and
- One or both of the reactive amino moieties, -NH 2 , of lysine or another linker moiety can be replaced with thiol moieties, -SH.
- the linker moiety has a reactive thiol moiety instead of an amino moiety
- the linkages can be selected from the group consisting of thioester, thiocarbonate, thiocarbamate, dithiocarbamate, thioether linkages, and others.
- mPEG or other monofunctional polymer reactants can be prepared with a reactive amino moiety and then linked to a suitable linker moiety having reactive groups such as those shown above on the mPEG molecule to form hydrolytically stable linkages as discussed above.
- a suitable linker moiety having reactive groups such as those shown above on the mPEG molecule to form hydrolytically stable linkages as discussed above.
- the amine linkage could be formed as follows:
- hydrolytically stable linkages in the absence of aromatic moieties include trifluoroethylsulfonate, isocyanate, isosthiocyanate, active esters, active carbonates, various aldehydes, various sulfones, including chloroethylsulfone and vinylsulfone,
- Active esters include N-hydroxylsuccinimidyl ester.
- Active carbonates include N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate, and trichlorophenylcarbonate. These electrophilic moieties are examples of those that are suitable as Ws in the structure poly-W and as Xs and Ys in the linker structure X-CR(-Y)-Z.
- linkages can be amino, thiol, and hydroxyl. Hydroxyl moieties form hydrolytically stable linkages with isocyanate electrophilic moieties. Also, it should be recognized that the linker can be substituted with different nucleophilic or electrophilic moieties or both electrophilic and nucleophilic moieties depending on the active moieties on the monofunctional polymers with which the linker moiety is to be substituted.
- Linker moieties other than lysine are
- moieties include those having more than one reactive site for attachment of various monofunctional polymers.
- Linker moieties can be synthesized to include multiple reactive sites such as amino, thiol, or hydroxyl groups for joining multiple suitably activated mPEGs or other nonpeptidic polymers to the molecule by hydrolytically stable linkages, if it is desired to design a molecule having multiple nonpeptidic polymer branches extending from one or more of the linker arm fragments.
- the linker moieties should also include a reactive site, such as a carboxyl or alcohol moiety, represented as -Z in the general structure above, for which the activated polymers are not selective and that can be subsequently activated for selective reactions for joining to enzymes, other proteins, surfaces, and the like.
- one suitable linker moiety is a diamino alcohol having the structure
- the diamino alcohol can be disubstituted with activated mPEG or other suitable activated polymers similar to disubstitution of lysine and then the hydroxyl moiety can be activated as follows:
- diamino alcohols and alcohols having more than two amino or other reactive groups for polymer attachment are useful.
- a suitably activated mPEG or other monofunctional, nonpeptidic, water soluble polymer can be attached to the amino groups on such a diamino alcohol similar to the method by which the same polymers are attached to lysine as shown above.
- the amino groups can be replaced with thiol or other active groups as discussed above.
- only one hydroxyl group, which is relatively nonreactive, should be present in the -Z moiety, and can be activated subsequent to polymer substitution.
- the moiety -Z can include a reactive moiety or functional group, which normally is a carboxyl moiety, hydroxyl moiety, or activated carboxyl or hydroxyl moiety.
- the carboxyl and hydroxyl moieties are somewhat nonreactive as compared to the thiol, amino, and other moieties discussed above.
- carboxyl and hydroxyl moieties typically remain intact when the polymer arms are attached to the linker moiety and can be subsequently activated.
- the carboxyl and hydroxyl moieties also provide a mechanism for
- the carboxyl and hydroxyl moieties provide a site for interacting with ion exchange chromatography media.
- the moiety -Z may also include a linkage fragment, represented as R z in the moiety, which can be substituted or unsubstituted, branched or linear, and joins the reactive moiety to the central carbon.
- R z a linkage fragment
- R z a linkage fragment
- the -Z moiety has the structure -R z -COOH if the R z fragment is present.
- the structure is -R z -OH.
- R z is CH 2 . It should be understood that the carboxyl and hydroxyl moieties normally will extend from the R z terminus, but need not necessarily do so.
- R z can also include the reaction product of one or more reactive moieties including reactive amino, thiol, or other moieties, and a suitably activated mPEG arm or related nonpeptidic polymer arm.
- R z can have the structure (-L-poly c )-COOH or (-L-poly c )-OH in which -L- is the reaction product of a portion of the linker moiety and a suitably activated nonpeptidic polymer, poly c -W, which is selected from the same group as poly a -W and poly b -W but can be the same or different from poly a -W and poly b -W.
- -Z have a broad definition.
- the moiety -Z is intended to represent not only the reactive site of the multisubstituted
- polymeric derivative that subsequently can be converted to an active form and its attachment to the central carbon, but the activated reactive site and also the conjugation of the precursor activated site with another molecule, whether that molecule be an enzyme, other protein or polypeptide, a phospholipid, a
- preformed liposome or on a surface to which the polymer derivative is attached.
- Z encompasses the currently known activating moieties in PEG chemistry and their conjugates. It should also be recognized that, although the linker fragments
- Q and P and R z should not contain aromatic rings or hydrolytically weak linkages such as ester linkages, such rings and such hydrolytically weak linkages may be present in the active site moiety of -Z or in a molecule joined to such active site. It may be desirable in some instances to provide a linkage between, for example, a protein or enzyme and a
- multisubstituted polymer derivative that has limited stability in water.
- Some amino acids contain aromatic moieties, and it is intended that the structure Z include conjugates of multisubstituted monofunctional polymer derivatives with such molecules or portions of molecules. Activated Zs and Zs including attached proteins and other moieties are discussed below.
- R is H.
- R can be designed to have another substantially nonreactive moiety, such as a nonreactive methyl or other alkyl group, or can be the reaction product of one or more reactive moieties including reactive amino, thiol, or other moieties, and a suitably activated mPEG arm or related nonpeptidic polymer arm.
- R can have the structure -M-poly d , in which -M- is the reaction product of a portion of the linker moiety and a suitably activated nonpeptidic polymer, poly d -W, which is selected from the same group as poly a -W and poly b -W but can be the same or different from poly a -W
- multi-armed structures can be made having one or more mPEGs or other nonpeptidic polymer arms extending from each portion P, Q, R, and R z , all of which portions extend from a central carbon atom, C, which multi-armed structures have a single reactive site for subsequent activation included in the structure represented by Z.
- linker fragments P and Q are located at least one active site for which the monofunctional, nonpeptidic polymers are selective. These active sites include amino moieties, thiol moieties, and other moieties as described above.
- linker fragment length of from 1 to 10 carbon atoms or the equivalent has been determined to be useful to avoid a length that could provide an antigenic site. Also, for all the linker fragments P, Q, R, and R 2 , there should be an absence of aromatic moieties in the structure and the linkages should be hydrolytically stable.
- Poly(ethylene glycol) is useful in the practice of the invention for the nonpeptidic polymer arms attached to the linker fragments.
- PEG is used in biological applications because it has properties that are highly desirable and is generally approved for biological or biotechnical applications. PEG typically is clear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, does not hydrolyze or deteriorate, and is nontoxic.
- Poly(ethylene glycol) is considered to be
- PEG is capable of coexistence with living tissues or organisms without causing harm. More specifically, PEG is not
- the activated PEGs of the invention should be substantially non-toxic and should not tend substantially to produce an immune response or cause clotting or other
- PEG is used in the art and herein to describe any of several condensation polymers of ethylene glycol having the general formula represented by the structure
- PEG is also known as polyoxyethylene, polyethylene oxide, polyglycol, and polyether glycol. PEG can be prepared as copolymers of ethylene oxide and many other monomers.
- poly(vinyl alcohol) (“PVA”); other poly (alkylene oxides) such as poly(propylene glycol) (“PPG”) and the like; and poly(oxyethylated polyols) such as poly (oxyethylated glycerol), poly(oxyethylated sorbitol), and
- poly(oxyethylated glucose), and the like can be homopolymers or random or block copolymers and terpolymers based on the monomers of the above
- suitable additional polymers include poly(oxazoline),
- PAcM poly(acryloylmorpholine)
- PVP poly(vinylpyrrolidone)
- disubstituted compound can be purified, activated, and used in various reactions for modification of molecules and surfaces similarly to the mPEG-disubstituted lysine described above.
- PAcM can be used similarly to mPEG or PVP to create multi-armed structures and ultra-high molecular weight polymers.
- An example of a PAcM-disubstituted lysine having a single carboxyl moiety available for activation is shown below.
- the disubstituted compound can be purified, activated, and used in various
- multi- armed monofunctional polymers of the invention can be used for attachment to a linker moiety to create a highly branched monofunctional structure, within the practical limits of steric hindrance.
- DCC N,N-dicyclohexylcarbodiimide
- multisubstituted polymer derivatives for attachment to surfaces and molecules.
- Any of the activating groups of the known derivatives of PEG can be applied to the multisubstituted structure.
- the mPEG-disubstituted lysine of the invention was
- succinimidyl ester which can be attached to protein amino groups.
- moieties available for activation of carboxilic acid polymer moieties for attachment to various surfaces and molecules include trifluoroethylsulfonate, isocyanate, isosthiocyanate, active esters, active carbonates, various aldehydes, various sulfones, including chloroethylsulfone and vinylsulfone, maleimide, iodoacetamide, and
- Active esters include N-hydroxylsuccinimidyl ester.
- Active carbonates include N-hydroxylsuccinimidyl carbonate,
- a highly useful, new activating group that can be used for highly selective coupling with thiol moieties instead of amino moieties on molecules and surfaces is the vinyl sulfone moiety described in co pending U.S. Patent Application No. 08/151,481, which was filed on November 12, 1993, the contents of which are incorporated herein by reference.
- Various sulfone moieties can be used to activate a multi-armed
- linker fragments represented by Q and P should not contain aromatic rings or hydrolytically weak linkages such as ester linkages, such rings and such
- hydrolytically weak linkages may be present in the moiety represented by -Z. It may be desirable in some instances to provide a linkage between, for example, a protein or enzyme and a multisubstituted polymer derivative that has limited stability in water. Some amino acids contain aromatic moieties, and it is intended that the structure -Z include conjugates of multisubstituted monofunctional polymer derivatives with such molecules or portions of molecules.
- Enzymes were modified with activated, two- armed, mPEG-disubstituted lysine of the invention of molecular weight about 10,000 that had been prepared according to the two step procedure and activated as the succinimidyl ester as discussed above.
- enzymes were also modified with activated, conventional, linear mPEG of molecular weight 5,000, which was mPEG with a norleucine amino acid spacer arm activated as the succinimide.
- conventional, linear mPEG derivatives with which enzymes are modified are referred to as "linear mPEG.”
- the activated, two- armed, mPEG-disubstituted lysine of the invention is referred to as "two-armed mPEG.”
- Different procedures were used for enzyme modification depending upon the type of enzyme and the polymer used so that a similar extent of amino group modification or attachment for each enzyme could be obtained. Generally, higher molar ratios of the two-armed mPEG were used.
- the enzymes were dissolved in a 0.2 M borate buffer of pH 8.5 to dissolve proteins.
- the polymers were added in small portions for about 10 minutes and stirred for over 1 hour.
- the amount of polymer used for modification was calculated on the basis of available amino groups in the enzyme.
- Ribonuclease in a concentration of 1.5 milligrams per milliliter of buffer was modified at room temperature.
- Linear and two-armed mPEGs as described were added at a molar ratio of polymer to protein amino groups of 2.5:1 and 5:1, respectively.
- Ribonuclease has a molecular weight of 13,700 D and 11 available amino groups.
- Catalase has a molecular weight of 250,000 D with 112 available amino groups.
- Trypsin has a molecular weight of 23,000 D with 16 available amino groups.
- Erwinia Caratimora asparaginase has a molecular weight of 141,000 D and 92 free amino groups.
- Catalase in a concentration of 2.5 milligrams per milliliter of buffer was modified at room
- Trypsin in a concentration of 4 milligrams per milliliter of buffer was modified at 0°C.
- Linear and two-armed mPEGs as described were added at a molar ratio of polymer to protein amino groups of 2.5:1.
- Asparaginase in a concentration of 6 milligrams per milliliter of buffer was modified with linear mPEG at room temperature.
- Linear mPEG as described was added at a molar ratio of polymer to protein amino groups of 3:1.
- Asparaginase in a concentration of 6 milligrams per milliliter of buffer was modified with linear mPEG at room temperature.
- Linear mPEG as described was added at a molar ratio of polymer to protein amino groups of 3:1.
- Asparaginase in a concentration of 6 milligrams per milliliter of buffer was modified with linear mPEG at room temperature.
- Linear mPEG as described was added at a molar ratio of polymer to protein amino groups of 3:1.
- Asparaginase in a concentration of 6 milligrams per milliliter of buffer was modified with linear mPEG at room temperature.
- Linear mPEG as described was added at a molar ratio of polymer to protein amino groups of 3:1.
- concentration of 6 milligrams per milliliter of buffer was modified with two-armed mPEG at 37°C.
- Two-armed mPEG of the invention as described was added at a molar ratio of polymer to protein amino groups of 3.3:1.
- the polymer and enzyme conjugates were purified by ultrafiltration and concentrated in an
- Protein concentration for the native forms of ribonuclease, catalase, and trypsin was evaluated spectrophotometrically using molar extinction
- concentration of native asparaginase was evaluated by biuret assay.
- Biuret assay was also used to evaluate concentrations of the protein modified forms.
- the extent of protein modification was evaluated by one of three methods. The first is a colorimetric method described in Habeeb, A. F. S. A. (1966) Determination of free amino groups in protein by trmitrobenzensulphonic acid. Anal. Biochem. 14, 328-336. The second method is amino acid analysis after acid hydrolysis. This method was accomplished by two procedures: 1) the post-column procedure of Benson, J. V., Gordon, M. J., and Patterson, J. A. (1967)
- Trypsin modification was at the level of 50% and 57% of amino groups with linear mPEG and with two-armed mPEG, respectively.
- Asparaginase with 53% and 40% modified protein amino groups was obtained by coupling with linear mPEG and two-armed mPEG,
- Enzymatic activity was increased, relative to the free enzyme, to 110% for the linear mPEG conjugate and to 133% for the two-armed mPEG conjugate.
- Enzymatic activity of native and modified enzyme was evaluated by the following methods.
- ribonuclease the method was used of Crook, E. M., Mathias, A. P., and Rabin, B.R. (1960)
- conjugates were assayed according to the method of Zwilling, R., and Neurath, H. (1981) Invertrebate protease. Methods Enzymol . 80, 633-664. Four enzymes were used: ribonuclease, catalase, trypsin, and asparaginase. From each enzyme solution, aliquots were taken at various time intervals and enzyme activity was assayed spectrophotometrically.
- proteolytic digestion was performed in 0.05 M phosphate buffer of pH 7.0.
- the free enzyme, linear mPEG and protein conjugate, and two-armed mPEG-protein conjugates were exposed to the known proteolytic enzymes trypsin, pronase, elastase or subtilisin under conditions as follows.
- trypsin autolysis rate i.e., the rate at which trypsin digests trypsin
- Protein can provoke an immune response when injected into the bloodstream. Reduction of protein
- Anti-SOD antibodies were obtained from rabbit and purified by affinity chromatography.
- the antigens (SOD, linear mPEG-SOD, and two-armed mPEG-SOD) were labelled with tritiated succinimidyl propionate to facilitate tracing. Reaction of antigen and antibody were evaluated by radioactive counting. In a 500 ⁇ L sample, the antigen (in the range of 0-3 ⁇ g) was incubated with 2.5 ⁇ g of antibody. The results show the practical disappearance of antibody recognition for two-armed mPEG-SOD, while an appreciable
- antibody-antigen complex was formed for linear mPEG-SOD and native SOD.
- Proteins and enzymes can be usefully modified by attachment to the polymer derivatives of the invention. Proteins and enzymes can be derived from animal sources, humans, microorganisms, and plants and can be produced by genetic engineering or
- cytokines such as various interferons (e.g. interferon- ⁇ , interferon- ⁇ , interferon- ⁇ ), interleukin-2 and interleukin-3), hormones such as insulin, growth hormone-releasing factor (GRF), calcitonin, calcitonin gene related peptide (CGRP), atrial natriuretic peptide (ANP), vasopressin, corticortropin-releasing factor (CRF), vasoactive intestinal peptide (VIP), secretin, ⁇ -melanocyte-stimulating hormone ( ⁇ -MSH),
- interferons e.g. interferon- ⁇ , interferon- ⁇ , interferon- ⁇
- hormones such as insulin, growth hormone-releasing factor (GRF), calcitonin, calcitonin gene related peptide (CGRP), atrial natriuretic peptide (ANP), vasopressin, corticortropin-releasing factor (CR
- ACTH adrenocorticotropic hormone
- CCK cholecystokinin
- PTH parathyroid hormone
- somatostatin somatostatin, endothelin, substance P, dynorphin, oxytocin and growth hormone-releasing peptide, tumor necrosis factor binding protein, growth factors such as growth hormone (GH), insulin-like growth factor (IGF-I, IGF-II), ⁇ -nerve growth factor ( ⁇ -NGF), basic
- fibroblast growth factor transforming growth factor
- G-CSF granulocyte colony-stimulating factor
- GM-CSF granulocyte macrophage colony-stimulating factor
- PDGF platelet-derived growth factor
- EGF epidermal growth factor
- enzymes such as tissue plasminogen activator (t-PA), elastase, superoxide dismutase (SOD), bilirubin
- the two-armed polymer derivative ofe the invention has a variety of related applications. Small molecules attached to two-armed activated mPEG derivatives of the invention can be expected to show enhanced solubility in either aqueous or organic solvents. Lipids and liposomes attached to the
- derivative of the invention can be expected to show long blood circulation lifetimes.
- Other particles than lipids and surfaces having the derivative of the invention attached can be expected to show nonfouling characteristics and to be useful as biomaterials having increased blood compatibility and avoidance of protein adsorption.
- Polymer-ligand conjugates can be prepared that are useful in two phase affinity partitioning.
- the polymers of the invention could be attached to various forms of drugs to produce prodrugs. Small drugs having the multisubstituted derivative attached can be expected to show altered solubility, clearance time, targeting, and other properties.
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Abstract
Multi-armed, monofunctional, and hydrolytically stable polymers are described having the structure (I) wherein Z is a moiety that can be activated for attachment to biologically active molecules such as proteins and wherein P and Q represent linkage fragments that join polymer arms polya and polyb, respectively, to central carbon atom, C, by hydrolytically stable linkages in the absence of aromatic rings in the linkage fragments. R typically is hydrogen or methyl, but can be a linkage fragment that includes another polymer arm. A specific example is an mPEG disubstituted lysine having the structure (II) where mPEGa and mPEGb have the structure CH3O-(CH2CH2O)nCH2CH2- wherein n may be the same or different for polya- and polyb- and can be from 1 to about 1,150 to provide molecular weights of from about 100 to 100,000.
Description
MULTI-ARMED, MONOFUNCTIONAL, AND HYDROLYTICALLY STABLE
DERIVATIVES OF
POLY(ETHYLENE GLYCOL) AND
RELATED POLYMERS FOR
MODIFICATION OF SURFACES AND MOLECULES
This application is related to and claims the benefit of the filing date of USSN 08/371,065, which was filed on January 10, 1995 and is entitled MULTI- ARMED, MONOFUNCTIONAL, AND HYDROLYTICALLY STABLE
DERIVATIVES OF POLY (ETHYLENE GLYCOL) AND RELATED
POLYMERS FOR MODIFICATION OF SURFACES AND MOLECULES.
FIELD OF THE INVENTION
This invention relates to monofunctional derivatives of poly(ethylene glycol) and related polymers and to methods for their synthesis and
activation for use in modifying the characteristics of surfaces and molecules.
BACKGROUND OF THE INVENTION
Improved chemical and genetic methods have made many enzymes, proteins, and other peptides and polypeptides available for use as drugs or biocatalysts having specific catalytic activity. However,
limitations exist to use of these compounds.
For example, enzymes that exhibit specific biocatalytic activity sometimes are less useful than they otherwise might be because of problems of low stability and solubility in organic solvents. During in vivo use, many proteins are cleared from circulation too rapidly. Some proteins have less water solubility
than is optimal for a therapeutic agent that circulates through the bloodstream. Some proteins give rise to immunological problems when used as therapeutic agents. Immunological problems have been reported. from
manufactured proteins even where the compound
apparently has the same basic structure as the
homologous natural product. Numerous impediments to the successful use of enzymes and proteins as drugs and biocatalysts have been encountered.
One approach to the problems that have arisen in the use of polypeptides as drugs or biocatalysts has been to link suitable hydrophilic or amphiphilic polymer derivatives to the polypeptide to create a polymer cloud surrounding the polypeptide. If the polymer derivative is soluble and stable in organic solvents, then enzyme conjugates with the polymer may acquire that solubility and stability. Biocatalysis can be extended to organic media with enzyme and polymer combinations that are soluble and stable in organic solvents.
For in vivo use, the polymer cloud can help to protect the compound from chemical attack, to limit adverse side effects of the compound when injected into the body, and to increase the size of the compound, potentially to render useful compounds that have some medicinal benefit, but otherwise are not useful or are even harmful to an organism. For example, the polymer cloud surrounding a protein can reduce the rate of renal excretion and immunological complications and can increase resistance of the protein to proteolytic breakdown into simpler, inactive substances.
However, despite the benefits of modifying polypeptides with polymer derivatives, additional problems have arisen. These problems typically arise in the linkage of the polymer to the polypeptide. The linkage may be difficult to form. Bifunctional or multifunctional polymer derivatives tend to cross link
proteins, which can result in a loss of solubility in water, making a polymer-modified protein unsuitable for circulating through the blood stream of a living organism. Other polymer derivatives form
hydrolytically unstable linkages that are quickly destroyed on injection into the blood stream. Some linking moieties are toxic. Some linkages reduce the activity of the protein or enzyme, thereby rendering the protein or enzyme less effective.
The structure of the protein or enzyme dictates the location of reactive sites that form the loci for linkage with polymers. Proteins are built of various sequences of alpha-amino acids, which have the general structure
The alpha amino moiety (H2N-) of one amino acid joins to the carboxyl moiety (-COOH) of an adjacent amino acid to form amide linkages, which can be represented as
where n can be hundreds or thousands. The terminal amino acid of a protein molecule contains a free alpha amino moiety that is reactive and to which a polymer can be attached. The fragment represented by R can contain reactive sites for protein biological activity and for attachment of polymer.
For example, in lysine, which is an amino acid forming part of the backbone of most proteins, a reactive amino (-NH2) moiety is present in the epsilon position as well as in the alpha position. The epsilon
-NH2 is free for reaction under conditions of basic pH. Much of the art has been directed to developing polymer derivatives having active moieties for attachment to the epsilon -NH2 moiety of the lysine fraction of a protein. These polymer derivatives all have in common that the lysine amino acid fraction of the protein typically is modified by polymer attachment, which can be a drawback where lysine is important to protein activity.
Poly (ethylene glycol), which is commonly referred to simply as "PEG," has been the nonpeptidic polymer most used so far for attachment to proteins. The PEG molecule typically is linear and can be
or, more simply, as HO-PEG-OH. As shown, the PEG molecule is difunctional, and is sometimes referred to as "PEG diol." The terminal portions of the PEG molecule are relatively nonreactive hydroxyl moieties, -OH, that can be activated, or converted to functional moieties, for attachment of the PEG to other compounds at reactive sites on the compound.
For example, the terminal moieties of PEG diol have been functionalized as active carbonate ester for selective reaction with amino moieties by
substitution of the relatively nonreactive hydroxyl moieties, -OH, with succinimidyl active ester moieties from N-hydroxy succinimide. The succinimidyl ester moiety can be represented structurally as
Difunctional PEG, functionalized as the succinimidyl carbonate, has a structure that can be represented as
Difunctional succinimidyl carbonate PEG has been reacted with free lysine monomer to make high molecular weight polymers. Free lysine monomer, which is also known as alpha, epsilon diaminocaproic acid, has a structure with reactive alpha and epsilon amino moieties that can be represented as
These high molecular weight polymers from difunctional PEG and free lysine monomer have multiple, pendant reactive carboxyl groups extending as branches from the polymer backbone that can be represented structurally as
The pendant carboxyl groups typically have been used to couple nonprotein pharmaceutical agents to the polymer. Protein pharmaceutical agents would tend to be cross linked by the multifunctional polymer with loss of protein activity.
Multiarmed PEGs having a reactive terminal moiety on each branch have been prepared by the
polymerization of ethylene oxide onto multiple hydroxyl groups of polyols including glycerol. Coupling of this type of multi-functional, branched PEG to a protein normally produces a cross-linked product with
considerable loss of protein activity.
It is desirable for many applications to cap the PEG molecule on one end with an essentially
nonreactive end moiety so that the PEG molecule is monofunctional. Monofunctional PEGs are usually preferred for protein modification to avoid cross linking and loss of activity. One hydroxyl moiety on the terminus of the PEG diol molecule typically is substituted with a nonreactive methyl end moiety, CH3-. The opposite terminus typically is converted to a reactive end moiety that can be activated for
attachment at a reactive site on a surface or a
molecule such as a protein.
PEG molecules having a methyl end moiety are sometimes referred to as monomethoxy-poly (ethylene glycol) and are sometimes referred to simply as "mPEG." The mPEG polymer derivatives can be represented
where n typically equals from about 45 to 115 and -Z is a functional moiety that is active for selective attachment to a reactive site on a molecule or surface or is a reactive moiety that can be converted to a functional moiety.
Typically, mPEG polymers are linear polymers of molecular weight in the range of from about 1,000 to 5,000. Higher molecular weights have also been
examined, up to a molecular weight of about 25,000, but these mPEGs typically are not of high purity and have not normally been useful in PEG and protein chemistry. In particular, these high molecular weight mPEGs typically contain significant percentages of PEG diol.
Proteins and other molecules typically have a limited number and distinct type of reactive sites available for coupling, such as the epsilon -NH2 moiety of the lysine fraction of a protein. Some of these reactive sites may be responsible for a protein's biological activity. A PEG derivative that attached to a sufficient number of such sites to impart the desired characteristics can adversely affect the activity of the protein, which offsets many of the advantages otherwise to be gained.
Attempts have been made to increase the polymer cloud volume surrounding a protein molecule without further deactivating the protein. Some PEG derivatives have been developed that have a single functional moiety located along the polymer backbone for attachment to another molecule or surface, rather than at the terminus of the polymer. Although these compounds can be considered linear, they are often referred to as "branched" and are distinguished from conventional, linear PEG derivatives since these
molecules typically comprise a pair of mPEG- molecules that have been joined by their reactive end moieties to another moiety, which can be represented structurally as -T-, and that includes a reactive moiety, -Z, extending from the polymer backbone. These compounds have a general structure that can be represented as
These monofunctional mPEG polymer derivatives show a branched structure when linked to another compound. One such branched form of mPEG with a single active binding site , - Z , has been prepared by
substitution of two of the chloride atoms of
trichloro-s-triazine with mPEG to make mPEG-disubstituted chlorotriazine. The third chloride is used to bind to protein. An mPEG disubstituted
chlorotriazine and its synthesis are disclosed in Wada, H., Imamura, l., Sako, M., Katagiri, S., Tarui, S., Nishimura, H., and Inada, Y. (1990) Antitumor enzymes: polyethylene glycol-modified asparaginase. Ann. N. Y. Acad . Sci . 613, 95-108. Synthesis of mPEG
disubstituted chlorotriazine is represented
structurally below.
However, mPEG-disubstituted chlorotriazine and the procedure used to prepare it present severe limitations because coupling to protein is highly nonselective. Several types of amino acids other than
lysine are attacked and many proteins are inactivated. The intermediate is toxic. Also, the
mPEG-disubstituted chlorotriazine molecule reacts with water, thus substantially precluding purification of the branched mPEG structure by commonly used
chromatographic techniques in water.
A branched mPEG with a single activation site based on coupling of mPEG to a substituted benzene ring is disclosed in European Patent Application Publication No. 473 084 A2. However, this structure contains a benzene ring that could have toxic effects if the structure is destroyed in a living organism.
Another branched mPEG with a single activation site has been prepared through a complex synthesis in which an active succinate moiety is attached to the mPEG through a weak ester linkage that is susceptible to hydrolysis. An mPEG-OH is reacted with succinic anhydride to make the succinate. The reactive succinate is then activated as the
succinimide. The synthesis, starting with the active succinimide, includes the following steps, represented structurally below.
The mPEG activated as the succinimide, mPEG succinimidyl succinate, is reacted in the first step as shown above with norleucine. The symbol -R in the synthesis represents the n-butyl moiety of norleucine. The mPEG and norleucine conjugate (A) is activated as the succinimide in the second step by reaction with N- hydroxy succinimide. As represented in the third step, the mPEG and norleucine conjugate activated as the succinimide (B) is coupled to the alpha and epsilon amino moieties of lysine to create an mPEG
disubstituted lysine (C) having a reactive carboxyl moiety. In the fourth step, the mPEG disubstituted lysine is activated as the succinimide.
The ester linkage formed from the reaction of the mPEG-OH and succinic anhydride molecules is a weak linkage that is hydrolytically unstable. In vivo
application is therefore limited. Also, purification of the branched mPEG is precluded by commonly used chromatographic techniques in water, which normally would destroy the molecule.
The molecule also has relatively large molecular fragments between the carboxyl group
activated as the succinimide and the mPEG moieties due to the number of steps in the synthesis and to the number of compounds used to create the fragments.
These molecular fragments are sometimes referred to as "linkers" or "spacer arms," and have the potential to act as antigenic sites promoting the formation of antibodies upon injection and initiating an undesirable immunological response in a living organism.
SUMMARY OF THE INVENTION
The invention provides a branched or "multi-armed" amphiphilic polymer derivative that is
monofunctional, hydrolytically stable, can be prepared in a simple, one-step reaction, and possesses no aromatic moieties in the linker fragments forming the linkages with the polymer moieties. The derivative can be prepared without any toxic linkage or potentially toxic fragments. Relatively pure polymer molecules of high molecular weight can be created. The polymer can be purified by chromotography in water. A multi-step method can be used if it is desired to have polymer arms that differ in molecular weight. The polymer arms are capped with relatively nonreactive end groups. The derivative can include a single reactive site that is located along the polymer backbone rather than on the terminal portions of the polymer moieties. The
reactive site can be activated for selective reactions.
The multi-armed polymer derivative of the invention having a single reactive site can be used for, among other things, protein modification with a high retention of protein activity. Protein and enzyme activity can be preserved and in some cases is
enhanced. The single reactive site can be converted to a functional group for highly selective coupling to proteins, enzymes, and surfaces. A larger, more dense polymer cloud can be created surrounding a biomolecule with fewer attachment points to the biomolecule as compared to conventional polymer derivatives having terminal functional groups. Hydrolytically weak ester linkages can be avoided. Potentially harmful or toxic products of hydrolysis can be avoided. Large linker fragments can be avoided so as to avoid an antigenic response in living organisms. Cross linking is
avoided.
The molecules of the invention can be
represented structurally as polya-P-CR(-Q-polyb)-Z or:
Polya and polyb represent nonpeptidic and substantially nonreactive water soluble polymeric arms that may be the same or different. C represents carbon. P and Q represent linkage fragments that may be the same or different and that join polymer arms polya and polyb, respectively, to C by hydrolytically stable linkages in the absence of aromatic rings in the linkage fragments. R is a moiety selected from the group consisting of H, substantially nonreactive, usually alkyl, moieties, and linkage fragments attached by a hydrolytically stable linkage in the absence of aromatic rings to a nonpeptidic and substantially nonreactive water soluble polymeric arm. The moiety -Z comprises a moiety selected from the group consisting of moieties having a single site reactive toward nucleophilic moieties, sites that can be converted to sites reactive toward nucleophilic moieties, and the reaction product of a nucleophilic moiety and moieties
having a single site reactive toward nucleophilic moieties.
Typically, the moiety -P-CR(-Q-)-Z is the reaction product of a linker moiety and the reactive site of monofunctional, nonpeptidic polymer
derivatives, polya-W and polyb-W, in which W is the reactive site. Polymer arms polya and polyb are
nonpeptidic polymers and can be selected from polymers that have a single reactive moiety that can be
activated for hydrolytically stable coupling to a suitable linker moiety. The linker has the general structure X-CR-(Y)-Z, in which X and Y represent fragments that contain reactive sites for coupling to the polymer reactive site W to form linkage fragments P and Q, respectively.
In one embodiment, at least one of the polymer arms is a poly (ethylene glycol) moiety capped with an essentially nonreactive end group, such as a monomethoxy-poly (ethylene glycol) moiety
("mPEG-"), which is capped with a methyl end group, CH3-. The other branch can also be an mPEG moiety of the same or different molecular weight, another
poly (ethylene glycol) moiety that is capped with an essentially nonreactive end group other than methyl, or a different nonpeptidic polymer moiety that is capped with a nonreactive end group such as a capped
poly (alkylene oxide), a poly (oxyethylated polyol), a poly (olefinic alcohol), or others.
For example, in one embodiment polya and polyb are each monomethoxy-poly (ethylene glycol) ("mPEG") of the same or different molecular weight. The mPEG- disubstituted derivative has the general structure mPEGa-P-CH(-Q-mPEGb)-Z. The moieties mPEGa- and mPEGb- have the structure CH3-(CH2CH2O)nCH2CH2- and n may be the same or different for mPEGa and mPEGb. Molecules having values of n of from 1 to about 1,150 are contemplated.
The linker fragments P and Q contain
hydrolytically stable linkages that may be the same or different depending upon the functional moiety on the mPEG molecules and the molecular structure of the linker moiety used to join the mPEG moieties in the method for synthesizing the multi-armed structure. The linker fragments typically are alkyl fragments
containing amino or thiol residues forming a linkage with the residue of the functional moiety of the polymer. Depending on the degree of substitution desired, linker fragments P and Q can include reactive sites for joining additional monofunctional nonpeptidic polymers to the multi-armed structure.
The moiety -R can be a hydrogen atom, H, a nonreactive fragment, or, depending on the degree of substitution desired, R can include reactive sites for joining additional monofunctional nonpeptidic polymers to the multi-armed structure.
The moiety -Z can include a reactive moiety for which the activated nonpeptidic polymers are not selective and that can be subsequently activated for attachment of the derivative to enzymes, other
proteins, nucleotides, lipids, liposomes, other
molecules, solids, particles, or surfaces. The moiety -Z can include a linkage fragment -Rz. Depending on the degree of substitution desired, the Rz fragment can include reactive sites for joining additional
monofunctional nonpeptidic polymers to the multi-armed structure.
Typically, the -Z moiety includes terminal functional moieties for providing linkages to reactive sites on proteins, enzymes, nucleotides, lipids, liposomes, and other materials. The moiety -Z is intended to have a broad interpretation and to include the reactive moiety of monofunctional polymer
derivatives of the invention, activated derivatives, and conjugates of the derivatives with polypeptides and
other substances. The invention includes biologically active conjugates comprising a biomolecule, which is a biologically active molecule, such as a protein or enzyme, linked through an activated moiety to the branched polymer derivative of the invention. The invention includes biomaterials comprising a solid such as a surface or particle linked through an activated moiety to the polymer derivatives of the invention.
In one embodiment, the polymer moiety is an mPEG moiety and the polymer derivative is a two-armed mPEG derivative based upon hydrolytically stable coupling of mPEG to lysine. The mPEG moieties are represented structurally as CH3O-(CH2CH2O)nCH2CH2- wherein n may be the same or different for polya- and polyb- and can be from 1 to about 1,150 to provide molecular weights of from about 100 to 100,000. The -R moiety is hydrogen. The -Z moiety is a reactive carboxyl moiety. The molecule is represented structurally as follows:
The reactive carboxyl moiety of
hydrolytically stable mPEG-disubstituted lysine, which can also be called alpha, epsilon-mPEG lysine, provides a site for interacting with ion exchange chromatography media and thus provides a mechanism for purifying the product. These purifiable, high molecular weight, monofunctional compounds have many uses. For example, mPEG-disubstituted lysine, activated as succinimidyl ester, reacts with amino groups in enzymes under mild aqueous conditions that are compatible with the
stability of most enzymes. The mPEG-disubstituted
lysine of the invention, activated as the succinimidyl ester, is represented as follows:
The invention includes methods of
synthesizing the polymers of the invention. The methods comprise reacting an active suitable polymer having the structure poly-W with a linker moiety having the structure X-CR-(Y)Z to form polya-P-CR(-Q-polyb)-Z. The poly moiety in the structure poly-W can be either polya or polyb and is a polymer having a single reactive moiety W. The W moiety is an active moiety that is linked to the polymer moiety directly or through a hydrolytically stable linkage. The moieties X and Y in the structure X-CR-(Y)Z are reactive with W to form the linkage fragments Q and P, respectively. If the moiety R includes reactive sites similar to those of X and Y, then R can also be modified with a poly-W, in which the poly can be the same as or different from polya or polyb. The moiety Z normally does not include a site that is reactive with W. However, X, Y, R, and Z can each include one or more such reactive sites for preparing monofunctional polymer derivatives having more than two branches.
The method of the invention typically can be accomplished in one or two steps. The method can include additional steps for preparing the compound poly-W and for converting a reactive Z moiety to a functional group for highly selective reactions.
The active Z moiety includes a reactive moiety that is not reactive with W and can be activated
subsequent to formation of polya-P-CR(-Q-polyb)-Z for highly selective coupling to selected reactive moieties of enzymes and other proteins or surfaces or any molecule having a reactive nucleophilic moiety for which it is desired to modify the characteristics of the molecule.
In additional embodiments, the invention provides a multi-armed mPEG derivative for which preparation is simple and straightforward.
Intermediates are water stable and thus can be
carefully purified by standard aqueous chromatographic techniques. Chlorotriazine activated groups are avoided and more highly selective functional groups are used for enhanced selectivity of attachment and much less loss of activity upon coupling of the mPEG
derivatives of the invention to proteins, enzymes, and other peptides. Large spacer arms between the coupled polymer and protein are avoided to avoid introducing possible antigenic sites. Toxic groups, including triazine, are avoided. The polymer backbone contains no hydrolytically weak ester linkages that could break down during in vivo applications. Monofunctional polymers of double the molecular weight as compared to the individual mPEG moieties can be provided, with mPEG dimer structures having molecular weights of up to at least about 50,000, thus avoiding the common problem of difunctional impurities present in conventional, linear mPEGs.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1(a), 1(b), and 1(c) illustrate the time course of digestion of ribonuclease (●),
conventional, linear mPEG-modified ribonuclease (O), and ribonuclease modified with a multi-armed mPEG of the invention (■) as assessed by enzyme activity upon incubation with pronase (Figure 1(a)), elastase (Figure 1(b)), and subtilisin (Figure 1(c)).
Figures 2(a) and 2(b) illustrate stability toward heat (Figure 2(a)) and pH (Figure 2(b)) of ribonuclease (●), linear mPEG-modified ribonuclease (O), and ribonuclease modified with a multi-armed mPEG of the invention (□). Figure 2 (a) is based on data taken after a 15 minute incubation period at the indicated temperatures. Figure 2(b) is based on data taken over a 20 hour period at different pH values.
Figures 3 (a) and 3 (b) illustrate the time course of digestion for catalase (●), linear mPEG-modified catalase (O), and catalase modified with a multi-armed mPEG of the invention (■) as assessed by enzyme activity upon incubation with pronase (Figure 3(a)) and trypsin (Figure 3 (b)).
Figure 4 illustrates the stability of catalase (●), linear mPEG-modified catalase (D) , and catalase modified with a multi-armed mPEG of the invention (O) for 20 hours incubation at the indicated pH values.
Figure 5 illustrates the time course of digestion of asparaginase (●), linear mPEG-modified asparaginase (O), and asparaginase modified with a multi-armed mPEG of the invention (■) as assessed by enzyme activity assay upon trypsin incubation.
Figure 6 illustrates the time course of autolysis of trypsin (●), linear mPEG-modified trypsin (■), and trypsin modified with a multi-armed mPEG of the invention (A) evaluated as residual activity towards TAME (alpha N-p-tosyl-arginine methyl ester). DETAILED DESCRIPTION
I. Prepara tion of a Hydrolytically Stable mPEG-Disubsti tuted Lysine .
Two procedures are described for the
preparation of a hydrolytically stable, two-armed, mPEG-disubstituted lysine. The first procedure is a two step procedure, meaning that the lysine is
substituted with each of the two mPEG moieties in separate reaction steps. Monomethoxy-poly (ethylene glycol) arms of different lengths or of the same length can be substituted onto the lysine molecule, if
desired, using the two step procedure. The second procedure is a one step procedure in which the lysine molecule is substituted with each of the two mPEG moieties in a single reaction step. The one step procedure is suitable for preparing mPEG-disubstituted lysine having mPEG moieties of the same length.
Unlike prior multisubstituted structures, no aromatic ring is present in the linkage joining the nonpeptidic polymer arms produced by either the one or two step methods described below that could result in toxicity if the molecule breaks down in vivo. No hydrolytically weak ester linkages are present in the linkage. Lengthy linkage chains that could promote an antigenic response are avoided.
The terms "group, " "functional group,"
"moiety," "active moiety," "reactive site," "radical," and similar terms are somewhat synonymous in the chemical arts and are used in the art and herein to refer to distinct, definable portions or units of a molecule or fragment of a molecule. "Reactive site," "functional group," and "active moiety" refer to units that perform some function or have a chemical activity and are reactive with other molecules or portions of molecules. In this sense a protein or a protein residue can be considered as a molecule and as a functional moiety when coupled to a polymer. A
polymer, such as mPEG-COOH has a reactive site, the carboxyl moiety, -COOH, that can be converted to a functional group for selective reactions and attachment to proteins and linker moieties. The converted polymer is said to be activated and to have an active moiety, while the -COOH group is relatively nonreactive in comparison to an active moiety.
The term "nonreactive" is used herein
primarily to refer to a moiety that does not readily react chemically with other moieties, such as the methyl alkyl moiety. However, the term "nonreactive" should be understood to exclude carboxyl and hydroxyl moieties, which, although relatively nonreactive, can be converted to functional groups that are of selective reactivity.
The term "biologically active" means a substance, such as a protein, lipid, or nucleotide that has some activity or function in a living organism or in a substance taken from a living organism. For example, an enzyme can catalyze chemical reactions. The term "biomaterial" is somewhat imprecise, and is used herein to refer to a solid material or particle or surface that is compatible with living organisms or tissue or fluids. For example, surfaces that contact blood, whether in vitro or in vivo, can be made
nonfouling by attachment of the polymer derivatives of the invention so that proteins do not become attached to the surface.
A. Two Step Procedure
For the two step procedure, an activated mPEG is prepared for coupling to free lysine monomer and then the lysine monomer is disubstituted with the activated mPEG in two steps. The first step occurs in aqueous buffer. The second step occurs in dry
methylene chloride. The active moiety of the mPEG for coupling to the lysine monomer can be selected from a number of activating moieties having leaving moieties that are reactive with the amino moieties of lysine monomer. A commercially available activated mPEG, mPEG-p-nitrophenylcarbonate, the preparation of which is discussed below, was used to exemplify the two step procedure.
Step 1. Preparation of mPEG-monosubstituted lysine. Modification of a single lysine amino group was accomplished with mPEG-p-nitrophenylcarbonate in aqueous solution where both lysine and mPEG-p- nitrophenylcarbonate are soluble. The mPEG-p- nitrophenylcarbonate has only limited stability in aqueous solution. However, lysine is not soluble in organic solvents in which the activated mPEG is stable. Consequently, only one lysine amino group is modified by this procedure. NMR confirms that the epsilon amino group is modified. Nevertheless, the procedure allows ready chloroform extraction of mPEG-monosubstituted lysine from unreacted lysine and other water soluble by-products, and so the procedure provides a desirable monosubstituted product for disubstitution.
To prepare the mPEG-monosubstituted lysine, 353 milligrams of lysine, which is about 2.5
millimoles, was dissolved in 20 milliliters of water at a pH of about 8.0 to 8.3. Five grams of mPEG-p- nitrophenylcarbonate of molecular weight 5,000, which is about 1 millimole, was added in portions over 3 hours. The pH was maintained at 8.3 with 0.2 N NaOH. The reaction mixture was stirred overnight at room temperature. Thereafter, the reaction mixture was cooled to 0°C and brought to a pH of about 3 with 2 N
HCl. Impurities were extracted with diethyl ether. The mPEG monosubstituted lysine, having the mPEG substituted at the epsilon amino group of lysine as confirmed by NMR analysis, was extracted three times with chloroform. The solution was dried. After concentration, the solution was added drop by drop to diethyl ether to form a precipitate. The precipitate was collected and then crystallized from absolute ethanol. The percentage of modified amino groups was 53%, calculated by colorimetric analysis.
Step 2. Preparation of mPEG-Disubstituted Lysine. The mPEG-monosubstituted lysine product from step 1 above is soluble in organic solvents and so modification of the second lysine amino moiety can be achieved by reaction in dry methylene chloride.
Activated mPEG, mPEG-p-nitrophenylcarbonate, is soluble and stable in organic solvents and can be used to modify the second lysine amino moiety.
Triethylamine ("TEA") was added to 4.5 grams of mPEG-monosubstituted lysine, which is about 0.86 millimoles. The mixture of TEA and mPEG-monosubstituted lysine was dissolved in 10 milliliters of anhydrous methylene chloride to reach a pH of 8.0. Four and nine tenths grams of mPEG-p-nitrophenycarbonate of molecular weight 5,000, which is 1.056 millimoles, was added over 3 hours to the
solution. If it is desirable to make an mPEG
disubstituted compound having mPEG arms of different lengths, then a different molecular weight mPEG could have been used. The pH was maintained at 8.0 with TEA. The reaction mixture was refluxed for 72 hours, brought to room temperature, concentrated, filtered,
precipitated with diethyl ether and then crystallized in a minimum amount of hot ethanol. The excess of activated mPEG, mPEG-p-nitrophenycarbonate, was
deactivated by hydrolysis in an alkaline aqueous medium by stirring overnight at room temperature. The
solution was cooled to 0°C and brought to a pH of about 3 with 2 N HCl.
p-Nitrophenol was removed by extraction with diethyl ether. Monomethyl-poly (ethylene glycol)-disubstituted lysine and remaining traces of mPEG were extracted from the mixture three times with chloroform, dried, concentrated, precipitated with diethyl ether and crystallized from ethanol. No unreacted lysine amino groups remained in the polymer mixture as
assessed by colorimetric analysis.
Purification of mPEG-disubstituted lysine and removal of mPEG were accomplished by gel filtration chromatography using a Bio Gel P100 (Bio-Rad) column. The column measured 5 centimeters by 50 centimeters. The eluent was water. Fractions of 10 milliliters were collected. Up to 200 milligrams of material could be purified for each run. The fractions corresponding to mPEG-disubstituted lysine were revealed by iodine reaction. These fractions were pooled, concentrated, and then dissolved in ethanol and concentrated. The mPEG-disubstituted lysine product was dissolved in methylene chloride, precipitated with diethyl ether, and crystallized from ethanol.
The mPEG-disubstituted lysine was also separated from unmodified mPEG-OH and purified by an alternative method. Ion exchange chromatography was performed on a QAE Sephadex A50 column (Pharmacia) that measured 5 centimeters by 80 centimeters. An 8.3 mM borate buffer of pH 8.9 was used. This alternative procedure permitted fractionation of a greater amount of material per run than the other method above
described (up to four grams for each run).
For both methods of purification, purified mPEG-disubstituted lysine of molecular weight 10,000, titrated with NaOH, showed that 100% of the carboxyl groups were free carboxyl groups. These results
indicate that the reaction was complete and the product pure.
The purified mPEG-disubstituted lysine was also characterized by 1H-NMR on a 200 MHz Bruker instrument in dimethyl sulfoxide, d6, at a 5% weight to volume concentration. The data confirmed the expected molecular weight of 10,000 for the polymer. The chemical shifts and assignments of the protons in the mPEG-disubstituted lysine are as follows: 1.2-1.4 ppm (multiplet, 6H, methylenes 3,4,5 of lysine); 1.6 ppm (multiplet, 2H, methylene 6 of lysine); 3.14 ppm (s, 3H, terminal mPEG methoxy); 3.49 ppm (s, mPEG backbone methylene); 4.05 ppm (t, 2H, -CH2, -OCO-); 7.18 ppm (t, 1H, -NH- lysine); and 7.49 ppm (d,l H, -NH- lysine).
The above signals are consistent with the reported structure since two different carbamate NH protons are present. The first carbamate NH proton (at 7.18 ppm) shows a triplet for coupling with the
adjacent methylene group. The second carbamate NH proton (at 7.49 ppm) shows a doublet because of
coupling with the α-CH of lysine. The intensity of these signals relative to the mPEG methylene peak is consistent with the 1:1 ratio between the two amide groups and the expected molecular weight of 10,000 for the polymer.
The two step procedure described above allows polymers of different types and different lengths to be linked with a single reactive site between them. The polymer can be designed to provide a polymer cloud of custom shape for a particular application.
The commercially available activated mPEG, mPEG-p-nitrophenylcarbonate, is available from
Shearwater Polymers, Inc. in Huntsville, Alabama. This compound was prepared by the following procedure, which can be represented structurally as follows:
Five grams of mPEG-OH of molecular weight 5,000, or 1 millimole, were dissolved in 120
milliliters of toluene and dried azeotropically for 3 hours. The solution was cooled to room temperature and concentrated under vacuum. Reactants added to the concentrated solution under stirring at 0°C were 20 milliliters of anhydrous methylene chloride and 0.4 g of p-nitrophenylchloroformate, which is 2 millimoles. The pH of the reaction mixture was maintained at 8 by adding 0.28 milliliters of triethylamine ("TEA"), which is 2 millimoles. The reaction mixture was allowed to stand overnight at room temperature. Thereafter, the reaction mixture was concentrated under vacuum to about 10 milliliters, filtered, and dropped into 100
milliliters of stirred diethyl ether. A precipitate was collected from the diethyl ether by filtration and crystallized twice from ethyl acetate. Activation of mPEG was determined to be 98%. Activation was
calculated spectrophotometrically on the basis of the absorption at 400 nm in alkaline media after 15 minutes of released 4-nitrophenol (e of p-nitrophenol at 400 nm equals 17,000).
B. One Step Procedure
In the one step procedure, mPEG disubstituted lysine is prepared from lysine and an activated mPEG in a single step as represented structurally below:
Except for molecular weight attributable to a longer PEG backbone in the activated mPEG used in the steps below, the mPEG disubstituted lysine of the one step procedure does not differ structurally from the mPEG disubstituted lysine of the two step procedure. It should be recognized that the identical compound, having the same molecular weight, can be prepared by either method.
Preparation of mPEG disubstituted lysine by the one step procedure proceeded as follows:
Succinimidylcarbonate mPEG of molecular weight about 20,000 was added in an amount of 10.8 grams, which is 5.4 × 10-4 moles, to 40 milliliters of lysine HCl solution. The lysine HCL solution was in a borate buffer of pH 8.0. The concentration was 0.826
milligrams succinimidylcarbonate mPEG per milliliter of lysine HCL solution, which is 1.76 × 10-4 moles. Twenty milliliters of the same buffer was added. The solution pH was maintained at 8.0 with aqueous NaOH solution for the following 8 hours. The reaction mixture was stirred at room temperature for 24 hours.
Thereafter, the solution was diluted with 300 milliliters of deionized water. The pH of the solution was adjusted to 3.0 by the addition of oxalic acid.
The solution was then extracted three times with dichloromethane. The combined dichloromethane extracts were dried with anhydrous sodium sulphate and filtered. The filtrate was concentrated to about 30 milliliters. The product, an impure mPEG disubstituted lysine, was
precipitated with about 200 milliliters of cold ethyl ether. The yield was 90%.
Nine grams of the above impure mPEG-disubstituted lysine reaction product was dissolved in 4 liters of distilled water and then loaded onto a column of DEAE Sepharose FF, which is 500 milliliters of gel equilibrated with 1500 milliliters of boric acid in a 0.5% sodium hydroxide buffer at a pH of 7.0. The loaded system was then washed with water. Impurities of succinimidylcarbonate mPEG and mPEG-monosubstituted lysine, both of molecular weight about 20,000, were washed off the column. However, the desired mPEG disubstituted lysine of molecular weight 20,000 was eluted with 10 mM NaCl. The pH of the eluate was adjusted to 3.0 with oxalic acid and then mPEG
disubstituted lysine was extracted with
dichloromethane, dried with sodium sulphate,
concentrated, and precipitated with ethyl ether. Five and one tenth grams of purified mPEG disubstituted lysine were obtained. The molecular weight was
determined to be 38,000 by gel filtration
chromatography and 36,700 by potentiometric titration.
The one step procedure is simple in application and is useful for producing high molecular weight dimers that have polymers of the same type and length linked with a single reactive site between them.
Additional steps are represented below for preparing succinimidylcarbonate mPEG for disubstitution of lysine.
Succinimidylcarbonate mPEG was prepared by dissolving 30 grams of mPEG-OH of molecular weight 20,000, which is about 1.5 millimoles, in 120
milliliters of toluene. The solution was dried
azeotropically for 3 hours. The dried solution was cooled to room temperature. Added to the cooled and dried solution were 20 milliliters of anhydrous
dichloromethane and 2.33 milliliters of a 20% solution of phosgene in toluene. The solution was stirred continuously for a minimum of 16 hours under a hood due to the highly toxic fumes.
After distillation of excess phosgene and solvent, the remaining syrup, which contained mPEG chlorocarbonate, was dissolved in 100 milliliters of anhydrous dichloromethane, as represented above . To this solution was added 3 millimoles of triethylamine and 3 millimoles of N-hydroxysuccinimide. The reaction mixture remained standing at room temperature for 24 hours. Thereafter, the solution was filtered through a silica gel bed of pore size 60 Angstroms that had been wetted with dichloromethane. The filtrate was
concentrated to 70 milliliters. Succinimidylcarbonate mPEG of molecular weight about 20,000 was precipitated in ethyl ether and dried in vacuum for a minimum of 8 hours. The yield was 90%. Succinimidylcarbonate-mPEG is available commercially from Shearwater Polymers in Huntsville, Alabama.
The mPEG disubstituted lysine of the
invention can be represented structurally more
generally as polya-P-CR(-Q-polyb)-Z or:
For the mPEG disubstituted lysines described above, -P-CR(-Q-)-Z is the reaction product of a precursor linker moiety having two reactive amino groups and active monofunctional precursors of polya and polyb that have been joined to the linker moiety at the reactive amino sites. Linker fragments Q and P contain carbamate linkages formed by joining the amino
containing portions of the lysine molecule with the functional group with which the mPEG was substituted. The linker fragments are selected from -O-C(O)NH(CH2)4- and -O-C(O)NH- and are different in the exemplified polymer derivative. However, it should be recognized that P and Q could both be -O-C(O)NH(CH2)4- or
-O-C(O)NH- or some other linkage fragment, as discussed below. The moiety represented by R is hydrogen, H.
The moiety represented by Z is the carboxyl group, -COOH. The moieties P, R, Q, and Z are all joined to a central carbon atom.
The nonpeptidic polymer arms, polya and polyb, are mPEG moieties mPEGa and mPEGb, respectively, and are the same on each of the linker fragments Q and P for the examples above. The mPEG moieties have a structure represented as CH3O-(CH2CH2O)nCH2CH2-. For the mPEG disubstituted lysine made by the one step method, n is about 454 to provide a molecular weight for each mPEG moiety of 20,000 and a dimer molecular weight of
40,000. For the mPEG disubstituted lysine made by the two step method, n is about 114 to provide a molecular
weight for each mPEG moiety of 5,000 and a dimer molecular weight of 10,000.
Lysine disubstituted with mPEG and having as dimer molecular weights of 10,000 and 40,000 and procedures for preparation of mPEG-disubstituted lysine have been shown. However, it should be recognized that mPEG disubstituted lysine and other multi-armed
compounds of the invention can be made in a variety of molecular weights, including ultra high molecular weights. High molecular weight monofunctional PEGs are otherwise difficult to obtain.
Polymerization cf ethylene oxide to yield mPEGs usually produces molecular weights of up to about 20,000 to 25,000 g/mol. Accordingly, two-armed mPEG disubstituted lysines of molecular weight of about 40,000 to 50,000 can be made according to the
invention. Higher molecular weight lysine
disubstituted PEGs can be made if the chain length of the linear mPEGs is increased, up to about 100,000. Higher molecular weights can also be obtained by adding additional monofunctional nonpeptidic polymer arms to additional reactive sites on a linker moiety, within practical limits of steric hindrance. However, no unreacted active sites on the linker should remain that could interfere with the monofunctionality of the multi -armed derivative. Lower molecular weight
disubstituted mPEGs can also be made, if desired, down to a molecular weight of about 100 to 200.
It should be recognized that a wide variety of linker fragments P and Q are available, although not necessarily with equivalent results, depending on the precursor linker moiety and the functional moiety with which the activated mPEG or other nonpeptidic
monofunctional polymer is substituted and from which the linker fragments result. Typically, the linker fragments will contain the reaction products of
portions of linker moieties that have reactive amino
and/or thiol moieties and suitably activated
nonpeptidic, monofunctional, water soluble polymers.
For example, a wide variety of activated mPEGs are available that form a wide variety of
hydrolytically stable linkages with reactive amino moieties. Linkages can be selected from the group consisting of amide, amine, ether, carbamate, which are also called urethane linkages, urea, thiourea,
thiocarbamate, thiocarbonate, thioether, thioester, dithiocarbonate linkages, and others. However, hydrolytically weak ester linkages and potentially toxic aromatic moieties are to be avoided.
Hydrolytic stability of the linkages means that the linkages between the polymer arms and the linker moiety are stable in water and that the linkages do not react with water at useful pHs for an extended period of time of at least several days, and
potentially indefinitely. Most proteins could be expected to lose their activity at a caustic pH of 11 or higher, so the derivatives should be stable at a pH of less than about 11.
Examples of the above linkages and their formation from activated mPEG and lysine are
represented structurally below.
One or both of the reactive amino moieties, -NH2, of lysine or another linker moiety can be replaced with thiol moieties, -SH. Where the linker moiety has a reactive thiol moiety instead of an amino moiety, then the linkages can be selected from the group consisting of thioester, thiocarbonate, thiocarbamate, dithiocarbamate, thioether linkages, and others. The above linkages and their formation from activated mPEG and lysine in which both amino moieties have been replaced with thiol moieties are represented
It should be apparent that the mPEG or other monofunctional polymer reactants can be prepared with a reactive amino moiety and then linked to a suitable linker moiety having reactive groups such as those shown above on the mPEG molecule to form hydrolytically stable linkages as discussed above. For example, the amine linkage could be formed as follows:
Examples of various active electrophilic moieties useful for activating polymers or linking moieties for biological and biotechnical applications in which the active moiety is reacted to form
hydrolytically stable linkages in the absence of aromatic moieties include trifluoroethylsulfonate, isocyanate, isosthiocyanate, active esters, active carbonates, various aldehydes, various sulfones, including chloroethylsulfone and vinylsulfone,
maleimide, iodoacetamide, and iminoesters. Active esters include N-hydroxylsuccinimidyl ester. Active carbonates include N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate, and trichlorophenylcarbonate. These electrophilic moieties are examples of those that are suitable as Ws in the structure poly-W and as Xs and Ys in the linker structure X-CR(-Y)-Z.
Nucleophilic moieties for forming the
linkages can be amino, thiol, and hydroxyl. Hydroxyl moieties form hydrolytically stable linkages with isocyanate electrophilic moieties. Also, it should be recognized that the linker can be substituted with different nucleophilic or electrophilic moieties or both electrophilic and nucleophilic moieties depending on the active moieties on the monofunctional polymers with which the linker moiety is to be substituted.
Linker moieties other than lysine are
available for activation and for disubstitution or
multisubstitution with mPEG and related polymers for creating multi-armed structures in the absence of aromatic moieties in the structure and that are
hydrolytically stable. Examples of such linker
moieties include those having more than one reactive site for attachment of various monofunctional polymers.
Linker moieties can be synthesized to include multiple reactive sites such as amino, thiol, or hydroxyl groups for joining multiple suitably activated mPEGs or other nonpeptidic polymers to the molecule by hydrolytically stable linkages, if it is desired to design a molecule having multiple nonpeptidic polymer branches extending from one or more of the linker arm fragments. The linker moieties should also include a reactive site, such as a carboxyl or alcohol moiety, represented as -Z in the general structure above, for which the activated polymers are not selective and that can be subsequently activated for selective reactions for joining to enzymes, other proteins, surfaces, and the like.
The diamino alcohol can be disubstituted with activated mPEG or other suitable activated polymers similar to disubstitution of lysine and then the hydroxyl moiety can be activated as follows:
Other diamino alcohols and alcohols having more than two amino or other reactive groups for polymer attachment are useful. A suitably activated mPEG or other monofunctional, nonpeptidic, water soluble polymer can be attached to the amino groups on such a diamino alcohol similar to the method by which the same polymers are attached to lysine as shown above. Similarly, the amino groups can be replaced with thiol or other active groups as discussed above. However, only one hydroxyl group, which is relatively nonreactive, should be present in the -Z moiety, and can be activated subsequent to polymer substitution.
The moiety -Z can include a reactive moiety or functional group, which normally is a carboxyl moiety, hydroxyl moiety, or activated carboxyl or hydroxyl moiety. The carboxyl and hydroxyl moieties are somewhat nonreactive as compared to the thiol, amino, and other moieties discussed above. The
carboxyl and hydroxyl moieties typically remain intact when the polymer arms are attached to the linker moiety and can be subsequently activated. The carboxyl and hydroxyl moieties also provide a mechanism for
purification of the multisubstituted linker moiety. The carboxyl and hydroxyl moieties provide a site for interacting with ion exchange chromatography media.
The moiety -Z may also include a linkage fragment, represented as Rz in the moiety, which can be
substituted or unsubstituted, branched or linear, and joins the reactive moiety to the central carbon. Where a reactive group of the -Z moiety is carboxyl, for activation after substitution with nonpeptidic
polymers, then the -Z moiety has the structure -Rz-COOH if the Rz fragment is present. For hydroxyl, the structure is -Rz-OH. For example, in the diamino alcohol structure discussed above, Rz is CH2. It should be understood that the carboxyl and hydroxyl moieties normally will extend from the Rz terminus, but need not necessarily do so.
Rz can also include the reaction product of one or more reactive moieties including reactive amino, thiol, or other moieties, and a suitably activated mPEG arm or related nonpeptidic polymer arm. In the latter event, Rz can have the structure (-L-polyc)-COOH or (-L-polyc)-OH in which -L- is the reaction product of a portion of the linker moiety and a suitably activated nonpeptidic polymer, polyc-W, which is selected from the same group as polya-W and polyb-W but can be the same or different from polya-W and polyb-W.
It is intended that -Z have a broad definition. The moiety -Z is intended to represent not only the reactive site of the multisubstituted
polymeric derivative that subsequently can be converted to an active form and its attachment to the central carbon, but the activated reactive site and also the conjugation of the precursor activated site with another molecule, whether that molecule be an enzyme, other protein or polypeptide, a phospholipid, a
preformed liposome, or on a surface to which the polymer derivative is attached.
The skilled artisan should recognize that Z encompasses the currently known activating moieties in PEG chemistry and their conjugates. It should also be recognized that, although the linker fragments
represented by Q and P and Rz should not contain
aromatic rings or hydrolytically weak linkages such as ester linkages, such rings and such hydrolytically weak linkages may be present in the active site moiety of -Z or in a molecule joined to such active site. It may be desirable in some instances to provide a linkage between, for example, a protein or enzyme and a
multisubstituted polymer derivative that has limited stability in water. Some amino acids contain aromatic moieties, and it is intended that the structure Z include conjugates of multisubstituted monofunctional polymer derivatives with such molecules or portions of molecules. Activated Zs and Zs including attached proteins and other moieties are discussed below.
When lysine, the diamino alcohol shown above, or many other compounds are linkers, then the central carbon has a nonreactive hydrogen, H, attached thereto. In the general structure polya-P-CR(-Q-polyb)-Z, R is H. It should be recognized that the moiety R can be designed to have another substantially nonreactive moiety, such as a nonreactive methyl or other alkyl group, or can be the reaction product of one or more reactive moieties including reactive amino, thiol, or other moieties, and a suitably activated mPEG arm or related nonpeptidic polymer arm. In the latter event, R can have the structure -M-polyd, in which -M- is the reaction product of a portion of the linker moiety and a suitably activated nonpeptidic polymer, polyd-W, which is selected from the same group as polya-W and polyb-W but can be the same or different from polya-W
and polyb-W.
For example, multi-armed structures can be made having one or more mPEGs or other nonpeptidic polymer arms extending from each portion P, Q, R, and Rz, all of which portions extend from a central carbon atom, C, which multi-armed structures have a single reactive site for subsequent activation included in the structure represented by Z. Upon at least the linker
fragments P and Q are located at least one active site for which the monofunctional, nonpeptidic polymers are selective. These active sites include amino moieties, thiol moieties, and other moieties as described above.
The nonpeptidic polymer arms tend to mask antigenic properties of the linker fragment, if any. A linker fragment length of from 1 to 10 carbon atoms or the equivalent has been determined to be useful to avoid a length that could provide an antigenic site. Also, for all the linker fragments P, Q, R, and R2, there should be an absence of aromatic moieties in the structure and the linkages should be hydrolytically stable.
Poly(ethylene glycol) is useful in the practice of the invention for the nonpeptidic polymer arms attached to the linker fragments. PEG is used in biological applications because it has properties that are highly desirable and is generally approved for biological or biotechnical applications. PEG typically is clear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, does not hydrolyze or deteriorate, and is nontoxic.
Poly(ethylene glycol) is considered to be
biocompatible, which is to say that PEG is capable of coexistence with living tissues or organisms without causing harm. More specifically, PEG is not
immunogenic, which is to say that PEG does not tend to produce an immune response in the body. When attached to a moiety having some desirable function in the body, the PEG tends to mask the moiety and can reduce or eliminate any immune response so that an organism can tolerate the presence of the moiety. Accordingly, the activated PEGs of the invention should be substantially non-toxic and should not tend substantially to produce an immune response or cause clotting or other
undesirable effects.
The term "PEG" is used in the art and herein to describe any of several condensation polymers of ethylene glycol having the general formula represented by the structure
or, more simply, as HO-PEG-OH. PEG is also known as polyoxyethylene, polyethylene oxide, polyglycol, and polyether glycol. PEG can be prepared as copolymers of ethylene oxide and many other monomers.
Other water soluble polymers than PEG are suitable for similar modification to create multi-armed structures that can be activated for selective
reactions. These other polymers include poly(vinyl alcohol) ("PVA"); other poly (alkylene oxides) such as poly(propylene glycol) ("PPG") and the like; and poly(oxyethylated polyols) such as poly (oxyethylated glycerol), poly(oxyethylated sorbitol), and
poly(oxyethylated glucose), and the like. The polymers can be homopolymers or random or block copolymers and terpolymers based on the monomers of the above
polymers, straight chain or branched, or substituted or unsubstituted similar to mPEG and other capped,
monofunctional PEGs having a single active site
available for attachment to a linker.
Specific examples of suitable additional polymers include poly(oxazoline),
poly(acryloylmorpholine) ("PAcM"), and
poly(vinylpyrrolidone) ( "PVP"). PVP and poly(oxazoline) are well known polymers in the art and theihr
preparation and use in the syntheses described above for mPEG should be readily apparent to the skilled artisan.
An example of the synthesis of a PVP disubstituted lysine having a single carboxyl moiety
available for activation is shown below. The
disubstituted compound can be purified, activated, and used in various reactions for modification of molecules and surfaces similarly to the mPEG-disubstituted lysine described above.
Poly(acryloylmorpholine) "(PAcM)"
functionalized at one end is a new polymer, the
structure, preparation, and characteristics of which are described in Italian Patent Application No. MI 92 A 0002616, which was published May 17, 1994 and is entitled, in English, "Polymers Of N-Acryloylmorpholine Functionalized At One End And Conjugates With Bioactive Materials And Surfaces." Dimer polymers of molecular weight up to at least about 80,000 can be prepared using this polymer. The contents of the Italian patent application are incorporated herein by reference.
PAcM can be used similarly to mPEG or PVP to create multi-armed structures and ultra-high molecular weight polymers. An example of a PAcM-disubstituted lysine having a single carboxyl moiety available for activation is shown below. The disubstituted compound can be purified, activated, and used in various
reactions for modification of molecules and surfaces
similarly to the mPEG- and PVP-disubstituted lysines described above.
It should also be recognized that the multi- armed monofunctional polymers of the invention can be used for attachment to a linker moiety to create a highly branched monofunctional structure, within the practical limits of steric hindrance.
II. Activation of mPEG-Disubstituted Lysine and Modification of Protein Amino Groups.
Schemes are represented below for activating the mPEG-disubstituted lysine product made by either the one step or two step procedures and for linking the activated mPEG-disubstituted lysine through a stable carbamate linkage to protein amino groups to prepare
polymer and protein conjugates. Various other
multisubstituted polymer derivatives as discussed above can be activated similarly.
A. Activation of mPEG Disubstituted Lysine. Purified mPEG-disubstituted lysine produced in accordance with the two step procedure discussed above was activated with N-hydroxysuccinimide to produce mPEG-disubstituted lysine activated as the succinimidyl ester. The reaction is represented structurally below:
Six and two tenths grams of mPEG- disubstituted lysine of molecular weight 10,000, which is about 0.6 millimoles, was dissolved in 10
milliliters of anhydrous methylene chloride and cooled to 0°C. N-hydroxysuccinimide and
N,N-dicyclohexylcarbodiimide ("DCC") were added under stirring in the amounts, respectively, of 0.138
milligrams, which is about 1.2 millimoles, and 0.48 milligrams, which is about 1.2 millimoles. The
reaction mixture was stirred overnight at room
temperature. Precipitated dicyclohexylurea was removed by filtration and the solution was concentrated and precipitated with diethyl ether. The product, mPEG disubstituted lysine activated as the succinimidyal ester, was crystallized from ethyl acetate. The yield of esterification, calculated on the basis of
hydroxysuccinimide absorption at 260 nm (produced by hydrolysis), was over 97% (∊ of hydroxysuccinimide at 260 nm = 8,000 m-1cm-1). The NMR spectrum was identical
to that of the unactivated carboxylic acid except for the new succinimide singlet at 2.80 ppm (2Hs)
The procedure previously described for the activation of the mPEG-disubstituted lysine of
molecular weight 10,000 was also followed for the activation of the higher molecular weight polymer of molecular weight approximately 40,000 that was produced in accordance with the one step procedure discussed above. The yield was over 95% of high molecular weight mPEG-disubstituted lysine activated as the
succinimidyal ester.
It should be recognized that a number of activating groups can be used to activate the
multisubstituted polymer derivatives for attachment to surfaces and molecules. Any of the activating groups of the known derivatives of PEG can be applied to the multisubstituted structure. For example, the mPEG-disubstituted lysine of the invention was
functionalized by activation as the succinimidyl ester, which can be attached to protein amino groups.
However, there are a wide variety of functional
moieties available for activation of carboxilic acid polymer moieties for attachment to various surfaces and molecules. Examples of active moieties used for biological and biotechnical applications include trifluoroethylsulfonate, isocyanate, isosthiocyanate, active esters, active carbonates, various aldehydes, various sulfones, including chloroethylsulfone and vinylsulfone, maleimide, iodoacetamide, and
iminoesters. Active esters include N-hydroxylsuccinimidyl ester. Active carbonates include N-hydroxylsuccinimidyl carbonate,
p-nitrophenylcarbonate, and trichlorophenylcarbonate.
A highly useful, new activating group that can be used for highly selective coupling with thiol moieties instead of amino moieties on molecules and surfaces is the vinyl sulfone moiety described in co
pending U.S. Patent Application No. 08/151,481, which was filed on November 12, 1993, the contents of which are incorporated herein by reference. Various sulfone moieties can be used to activate a multi-armed
structure in accordance with the invention for thiol selective coupling.
Various examples of activation of -Z reactive moieties to created -Z activated moieties are presented as follows:
It should also be recognized that, although the linker fragments represented by Q and P should not contain aromatic rings or hydrolytically weak linkages such as ester linkages, such rings and such
hydrolytically weak linkages may be present in the moiety represented by -Z. It may be desirable in some instances to provide a linkage between, for example, a protein or enzyme and a multisubstituted polymer derivative that has limited stability in water. Some amino acids contain aromatic moieties, and it is intended that the structure -Z include conjugates of multisubstituted monofunctional polymer derivatives with such molecules or portions of molecules.
B. Enzyme Modification
Enzymes were modified with activated, two- armed, mPEG-disubstituted lysine of the invention of molecular weight about 10,000 that had been prepared according to the two step procedure and activated as the succinimidyl ester as discussed above. The
reaction is represented structurally below:
For comparison, enzymes were also modified with activated, conventional, linear mPEG of molecular weight 5,000, which was mPEG with a norleucine amino acid spacer arm activated as the succinimide. In the discussion of enzyme modification below, conventional, linear mPEG derivatives with which enzymes are modified are referred to as "linear mPEG." The activated, two- armed, mPEG-disubstituted lysine of the invention is referred to as "two-armed mPEG." Different procedures were used for enzyme modification depending upon the
type of enzyme and the polymer used so that a similar extent of amino group modification or attachment for each enzyme could be obtained. Generally, higher molar ratios of the two-armed mPEG were used. However, in all examples the enzymes were dissolved in a 0.2 M borate buffer of pH 8.5 to dissolve proteins. The polymers were added in small portions for about 10 minutes and stirred for over 1 hour. The amount of polymer used for modification was calculated on the basis of available amino groups in the enzyme.
Ribonuclease in a concentration of 1.5 milligrams per milliliter of buffer was modified at room temperature. Linear and two-armed mPEGs as described were added at a molar ratio of polymer to protein amino groups of 2.5:1 and 5:1, respectively. Ribonuclease has a molecular weight of 13,700 D and 11 available amino groups. Catalase has a molecular weight of 250,000 D with 112 available amino groups. Trypsin has a molecular weight of 23,000 D with 16 available amino groups. Erwinia Caratimora asparaginase has a molecular weight of 141,000 D and 92 free amino groups.
Catalase in a concentration of 2.5 milligrams per milliliter of buffer was modified at room
temperature. Linear and two-armed mPEGs as described were added at a molar ratio of polymer to protein amino groups of 5:1 and 10:1, respectively.
Trypsin in a concentration of 4 milligrams per milliliter of buffer was modified at 0°C. Linear and two-armed mPEGs as described were added at a molar ratio of polymer to protein amino groups of 2.5:1.
Asparaginase in a concentration of 6 milligrams per milliliter of buffer was modified with linear mPEG at room temperature. Linear mPEG as described was added at a molar ratio of polymer to protein amino groups of 3:1. Asparaginase in a
concentration of 6 milligrams per milliliter of buffer
was modified with two-armed mPEG at 37°C. Two-armed mPEG of the invention as described was added at a molar ratio of polymer to protein amino groups of 3.3:1.
The polymer and enzyme conjugates were purified by ultrafiltration and concentrated in an
Amicon system with a PM 10 membrane (cut off 10,000) to eliminate N-hydroxysuccinimide and reduce polymer concentration. The conjugates were further purified from the excess of unreacted polymer by gel filtration chromatography on a Pharmacia Superose 12 column, operated by an FPLC instrument, using 10 mM phosphate buffer of pH 7.2, 0.15 M in NaCl, as eluent.
Protein concentration for the native forms of ribonuclease, catalase, and trypsin was evaluated spectrophotometrically using molar extinction
coefficients of 945×103 M-1 cm-1, 1.67×105 M-1 cm-1 and 3.7×104 M-1 cm-1 at 280 nm, respectively. The
concentration of native asparaginase was evaluated by biuret assay. Biuret assay was also used to evaluate concentrations of the protein modified forms.
The extent of protein modification was evaluated by one of three methods. The first is a colorimetric method described in Habeeb, A. F. S. A. (1966) Determination of free amino groups in protein by trmitrobenzensulphonic acid. Anal. Biochem. 14, 328-336. The second method is amino acid analysis after acid hydrolysis. This method was accomplished by two procedures: 1) the post-column procedure of Benson, J. V., Gordon, M. J., and Patterson, J. A. (1967)
Accelerated chromatographic analysis of amino acid in physiological fluids containing vitamin and asparagine. Anal. Biol. Chem. 18, 288-333, and 2) pre-column derivatization by phenylisothiocyanate (PITC) according to Bidlingmeyer, B. A., Cohen, S. A., and Tarvin, T. L. (1984) Rapid analysis of amino acids using pre-column derivatization. J. Chromatography 336, 93-104.
The amount of bound linear mPEG was evaluated from norleucine content with respect to other protein amino acids. The amount of two-armed, mPEG-disubstituted lysine was determined from the increase in lysine content. One additional lysine is present in the hydrolysate for each bound polymer.
III. Analysis of Polymer and Enzyme Conjugates
Five different model enzymes, ribonuclease, catalase, asparaginase, trypsin and uricase, were modified with linear, conventional mPEG of molecular weight 5000 having a norleucine amino acid spacer arm activated as succinimidl ester and with a two-armed, mPEG-disubstituted lysine of the invention prepared from the same linear, conventional mPEG as described above in connection with the two step procedure. The molecular weight of the two-armed mPEG disubstituted lysine of the invention was approximately 10,000.
A. Comparison of Enzyme Activity. The catalytic properties of the modified enzymes were determined and compared and the results are presented in Table 1 below. To facilitate comparison, each enzyme was modified with the two polymers to a similar extent by a careful choice of polymer to enzyme ratios and reaction temperature.
Ribonuclease with 50% and 55% of the amino groups modified with linear mPEG and two-armed mPEG, respectively, presented 86% and 94% residual activity with respect to the native enzyme. Catalase was modified with linear mPEG and with two-armed mPEG to obtain 43% and 38% modification of protein amino groups, respectively. Enzyme activity was not
significantly changed after modification. Trypsin modification was at the level of 50% and 57% of amino groups with linear mPEG and with two-armed mPEG, respectively. Esterolytic activity for enzyme modified with linear mPEG and two-armed mPEG, assayed on the small substrate TAME, was increased by the modification
to 120% and 125%, respectively. Asparaginase with 53% and 40% modified protein amino groups was obtained by coupling with linear mPEG and two-armed mPEG,
respectively. Enzymatic activity was increased, relative to the free enzyme, to 110% for the linear mPEG conjugate and to 133% for the two-armed mPEG conjugate.
While not wishing to be bound by theory, it is possible that in the case of trypsin and
asparaginase, that modification produces a more active form of the enzyme. The Km values of the modified and unmodified forms are similar.
For the enzyme uricase a particularly
dramatic result was obtained. Modification of uricase with linear mPEG resulted in total loss of activity.
While not wishing to be bound by theory, it is believed that the linear mPEG attached to an amino acid such as lysine that is critical for activity. In direct contrast, modification of 40% of the lysines of uricase with two-armed mPEG gave a conjugate retaining 70% activity.
It is apparent that modification of enzymes with two-armed mPEG gives conjugates of equal or greater activity than those produced by conventional linear mPEG modification with monosubstituted
structures, despite the fact that two-armed mPEG modification attaches twice as much polymer to the enzyme .
Coupling two-armed mPEG to asparaginase with chlorotriazine activation as described in the
background of the invention gave major loss of
activity. Presumably the greater activity of enzymes modified with a two-armed mPEG of the invention results because the bulky two-armed mPEG structure is less likely than monosubstituted linear mPEG structures to penetrate into active sites of the proteins.
-
Enzymatic activity of native and modified enzyme was evaluated by the following methods. For ribonuclease, the method was used of Crook, E. M., Mathias, A. P., and Rabin, B.R. (1960)
Spectrophotometric assay of bovine pancreatic
ribonuclease by the use of cytidine 2':3' phosphate. Biochem. J. 74, 234-238. Catalase activity was
determined by the method of Beers, R. F. and Sizer, I W. (1952) A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol .
Chem. 195,133-140. The esterolytic activity of trypsin and its derivatives was determined by the method of Laskowski, M. (1955) Trypsinogen and trypsin. Methods Enzymol . 2, 26-36. Native and modified asparaginase were assayed according to a method reported by Cooney, D. A., Capizzi, R. L. and Handschumacher, R. E. (1970) Evaluation of L-asparagine metabolism in animals and man. Cancer Res . 30, 929-935. In this method, 1 . 1 ml containing 120 μg of α-ketoglutaric acid, 20 Ul of glutamic-oxalacetic transaminase, 30 Ul of malate dehydrogenase, 100 μg of NADH, 0.5 μg of asparaginase and 10 μmoles of asparagine were incubated in 0.122 M Tris buffer, pH 8.35, while the NADH absorbance
decrease at 340 nm was followed.
B. Proteolytic Digestion of Free Enzyme and
Conjugates. The rates at which proteolytic enzymes digest and destroy proteins was determined and compared for free enzyme, enzyme modified by attachment of linear activated mPEG, and enzyme modified by
attachment of an activated two-armed mPEG of the invention. The proteolytic activities of the
conjugates were assayed according to the method of Zwilling, R., and Neurath, H. (1981) Invertrebate protease. Methods Enzymol . 80, 633-664. Four enzymes were used: ribonuclease, catalase, trypsin, and asparaginase. From each enzyme solution, aliquots were
taken at various time intervals and enzyme activity was assayed spectrophotometrically.
Proteolytic digestion was performed in 0.05 M phosphate buffer of pH 7.0. The free enzyme, linear mPEG and protein conjugate, and two-armed mPEG-protein conjugates were exposed to the known proteolytic enzymes trypsin, pronase, elastase or subtilisin under conditions as follows.
For native ribonuclease and its linear and two-armed mPEG conjugates, 0.57 mg protein was digested at room temperature with 2.85 mg of pronase, or 5.7 mg of elastase, or with 0.57 mg of subtilisin in a total volume of 1 ml. Ribonuclease with 50% and 55% of the amino groups modified with linear mPEG and two-armed mPEG, respectively, was studied for stability to proteolytic digestion by pronase (Figure 1(a)),
elastase (Figure 1(b)) and subtilisin (Figure 1(c)). Polymer modification greatly increases the stability to digestion by all three proteolytic enzymes, but the protection offered by two-armed mPEG is much more effective as compared to linear mPEG.
For native and linear and two-armed mPEG-modified catalase, 0.58 mg of protein were
digested at room temperature with 0.58 mg of trypsin or 3 .48 mg of pronase in a total volume of 1 ml . Catalase was modified with linear mPEG and two-armed mPEG to obtain 43% and 38% modification of protein amino groups, respectively. Proteolytic stability was much greater for the two-armed mPEG derivative than for the monosubstituted mPEG derivative, particularly toward pronase (Figure 3 (a)) and trypsin (Figure 3 (b)), where no digestion took place.
Autolysis of trypsin and its linear and two- armed mPEG derivatives at 37°C was evaluated by
esterolytic activity of protein solutions at 25 mg/ml of TAME. Trypsin modification was at the level of 50% and 57% of amino groups with linear mPEG and two-armed
mPEG, respectively. Modification with linear mPEG and two-armed mPEG reduced proteolytic activity of trypsin towards casein, a high molecular weight substrate:
activity relative to the native enzyme was found, after 20 minutes incubation, to be 64% for the linear mPEG and protein conjugate and only 35% for the two-armed mPEG conjugate. In agreement with these results, the trypsin autolysis rate (i.e., the rate at which trypsin digests trypsin), evaluated by enzyme esterolytic activity, was totally prevented in two-armed
mPEG-trypsin but only reduced in the linear mPEG-trypsin conjugate. To prevent autolysis with linear mPEG, modification of 78% of the available protein amino groups was required.
For native and linear mPEG- and two-armed mPEG-modified asparaginase, 2.5 μg were digested at 37°C with 0.75 mg of trypsin in a total volume of 1 ml. Asparaginase with 53% and 40% modified protein amino groups was obtained by coupling with linear mPEG and two-armed mPEG, respectively. Modification with two-armed mPEG had an impressive influence on stability towards proteolytic enzyme. Increased protection was achieved at a lower extent of modification with respect to the derivative obtained with the two-armed polymer (Figure 5).
These data clearly show that two-armed mPEG coupling is much more effective than conventional linear mPEG coupling in providing a protein with protection against proteolysis. While not wishing to be bound by theory, it is believed that the two-armed mPEG, having two polymer chains bound to the same site, presents increased hindrance to approaching
macromolecules in comparison to linear mPEG.
C. Reduction of Protein Antigenicitv.
Protein can provoke an immune response when injected into the bloodstream. Reduction of protein
immunogenicity by modification with linear and
two-armed mPEG was determined and compared for the enzyme superoxidedismutase ("SOD").
Anti-SOD antibodies were obtained from rabbit and purified by affinity chromatography. The antigens (SOD, linear mPEG-SOD, and two-armed mPEG-SOD) were labelled with tritiated succinimidyl propionate to facilitate tracing. Reaction of antigen and antibody were evaluated by radioactive counting. In a 500 μL sample, the antigen (in the range of 0-3 μg) was incubated with 2.5 μg of antibody. The results show the practical disappearance of antibody recognition for two-armed mPEG-SOD, while an appreciable
antibody-antigen complex was formed for linear mPEG-SOD and native SOD.
D. Blood Clearance Times. Increased blood circulation half lives are of enormous pharmaceutical importance. The degree to which mPEG conjugation of proteins reduces kidney clearance of proteins from the blood was determined and compared for free protein, protein modified by attachment of conventional, linear activated mPEG, and protein modified by attachment of the activated two-armed mPEG of the invention. Two proteins were used. These experiments were conducted by assaying blood of mice for the presence of the protein.
For linear mPEG-uricase and two-armed
mPEG-uricase, with 40% modification of lysine groups, the half life for blood clearance was 200 and 350 minutes, respectively. For unmodified uricase the result was 50 minutes.
For asparaginase, with 53% modification with mPEG and 40% modification with two armed mPEG, the half lives for blood clearance were 1300 and 2600 minutes, respectively. For unmodified asparaginase the result was 27 minutes.
E. Thermal Stability of Free and Conjugated Enzymes. Thermal stability of native ribonuclease,
catalase and asparaginase and their linear mPEG and two-armed mPEG conjugates was evaluated in 0.5 M phosphate buffer pH 7.0 at 1 mg/ml, 9 μg/ml and 0.2 mg/ml respectively. The samples were incubated at the specified temperatures for 15 min., 10 min., and 15 min, respectively, cooled to room temperature and assayed spectrophotometrically for activity.
Increased thermostability was found for the modified forms of ribonuclease, as shown in Figure 2, at pH 7.0, after 15 min. incubation at different temperatures, but no significant difference between the two polymers was observed. Data for catalase, not reported here, showed that modification did not
influence catalase thermostability. A limited increase in thermal stability of linear and two-armed mPEG-modified asparaginase was also noted, but is not reported.
F. pH Stability of the Free and Conjugated Enzymes. Unmodified and polymer-modified enzymes were incubated for 20 hrs in the following buffers: sodium acetate 0.05 M at a pH of from 4.0 to 6.0, sodium phosphate 0.05 M at pH 7.0 and sodium borate 0.05 M at a pH of from 8.0 to 11. The enzyme concentrations were 1 mg/ml, 9 μg/ml, 5 μg/ml for ribonuclease, catalase, and asparaginase respectively. The stability to incubation at various pH was evaluated on the basis of enzyme activity.
As shown in Figure 2b, a decrease in pH stability at acid and alkline pH values was found for the linear and two-armed mPEG-modified ribonuclease forms as compared to the native enzyme. As shown in Figure 4, stability of the linear mPEG and two-armed mPEG conjugates with catalase was improved for
incubation at low pH as compared to native catalase. However, the two-armed mPEG and linear mPEG conjugates showed equivalent pH stability. A limited increase in pH stability at acid and alkaline pH values was noted
for linear and two-armed mPEG-modified asparaginase as compared to the native enzyme.
It should be recognized that there are thousands of proteins and enzymes that can be usefully modified by attachment to the polymer derivatives of the invention. Proteins and enzymes can be derived from animal sources, humans, microorganisms, and plants and can be produced by genetic engineering or
synthesis. Representatives include: cytokines such as various interferons (e.g. interferon-α, interferon-β, interferon-γ), interleukin-2 and interleukin-3), hormones such as insulin, growth hormone-releasing factor (GRF), calcitonin, calcitonin gene related peptide (CGRP), atrial natriuretic peptide (ANP), vasopressin, corticortropin-releasing factor (CRF), vasoactive intestinal peptide (VIP), secretin, α-melanocyte-stimulating hormone (α-MSH),
adrenocorticotropic hormone (ACTH), cholecystokinin (CCK), glucagon, parathyroid hormone (PTH),
somatostatin, endothelin, substance P, dynorphin, oxytocin and growth hormone-releasing peptide, tumor necrosis factor binding protein, growth factors such as growth hormone (GH), insulin-like growth factor (IGF-I, IGF-II), β-nerve growth factor (β-NGF), basic
fibroblast growth factor (bFGF), transforming growth factor, erythropoietin, granulocyte colony-stimulating factor (G-CSF) , granulocyte macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), enzymes such as tissue plasminogen activator (t-PA), elastase, superoxide dismutase (SOD), bilirubin
oxydase, catalase, uricase and asparaginase, other proteins such as ubiquitin, islet activating protein (IAP), serum thymic factor (STF), peptide-T and trypsin inhibitor, and derivatives thereof. In addition to protein modification, the two-armed polymer derivative ofe the invention has a variety of related applications.
Small molecules attached to two-armed activated mPEG derivatives of the invention can be expected to show enhanced solubility in either aqueous or organic solvents. Lipids and liposomes attached to the
derivative of the invention can be expected to show long blood circulation lifetimes. Other particles than lipids and surfaces having the derivative of the invention attached can be expected to show nonfouling characteristics and to be useful as biomaterials having increased blood compatibility and avoidance of protein adsorption. Polymer-ligand conjugates can be prepared that are useful in two phase affinity partitioning. The polymers of the invention could be attached to various forms of drugs to produce prodrugs. Small drugs having the multisubstituted derivative attached can be expected to show altered solubility, clearance time, targeting, and other properties.
The invention claimed herein has been
described with respect to particular exemplified embodiments. However, the foregoing description is not intended to limit the invention to the exemplified embodiments, and the skilled artisan should recognize that variations can be made within the scope and spirit of the invention as described in the foregoing
specification. The invention includes all
alternatives, modifications, and equivalents that may be included within the true spirit and scope of the invention as defined by the appended claims.
Claims
1. A polymeric derivative represented by the structure wherein polya and polyb are nonpeptidic and substantially nonreactive water soluble polymeric arms that may be the same or different, wherein C is carbon, wherein P and Q comprise linkage fragments that may be the same or different and join polymeric arms polya and polyb, respectively, to C by hydrolytically stable linkages in the absence of aromatic rings in said linkage fragments, wherein R is a moiety selected from the group consisting of H, substantially nonreactive moieties, and linkage fragments having attached thereto by a hydrolytically stable linkage in the absence of aromatic rings one or more nonpeptidic and substantially nonreactive water soluble polymeric arms, and wherein Z comprises a moiety selected from the group consisting of moieties having a single site reactive toward nucleophilic moieties, sites that can be converted to sites reactive toward nucleophilic moieties, and the reaction product of a nucleophilic moiety and moieties having a single site reactive toward nucleophilic moieties.
2. The polymeric derivative of Claim 1 wherein said hydrolytically stable linkages are selected from the group consisting of amide, amine, ether, carbamate, thiourea, urea, thiocarbamate, thiocarbonate, thioether, thioester, and dithiocarbamate linkages.
3. The polymeric derivative of Claim 1 wherein said nucleophilic moieties are selected from the group consisting of amino, thiol, and hydroxyl moieties.
4. The polymeric derivative of Claim 1 wherein said nucleophilic moiety is a biologically active molecule.
5. The polymeric derivative of Claim 4 wherein said biologically active molecule is selected from the group consisting of polypeptides,
polynucleotides, and lipids.
6. The polymeric derivative of Claim 1 wherein said nucleophilic moiety is a solid surface or a particle.
7. The polymeric derivative of Claim 6 wherein said solid particle is a liposome.
8. The polymeric derivative of Claim 1 wherein Z is selected from the group consisting of carboxyl, hydroxyl, activated carboxyl, activated hydroxyl, and conjugates of activated carboxyl or hydroxyl sites and molecules having at least one reactive nucleophilic moiety.
9. The polymeric derivative of Claim 1 wherein Z comprises a moiety selected from the group consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, and iminoesters.
10. The polymeric derivative of Claim 9 wherein said active ester is N-hydroxylsuccinimidyl ester and said active carbonates are selected from the group consisting of N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate, and trichlorophenylcarbonate.
11. The polymeric derivative of Claim 1 wherein said nonpeptidic polymeric arms are selected from the group consisting of poly(alkylene oxides), poly(oxyethylated polyols), and poly(oxyethylated glucose).
12. The polymeric derivative of Claim 1 wherein said nonpeptidic polymeric arms are selected from the group consisting of poly(ethylene glycol), poly(vinyl alcohol), poly(propylene glycol),
poly(oxyethylated glycerol), poly(oxyethylated
sorbitol), poly(oxyethylated glucose), poly(oxazoline), poly(acryloylmorpholine), and poly(vinylpyrrolidone).
13. The polymeric derivative of Claim 1 wherein said nonpeptidic polymeric arms are linear mPEGs of molecular weight of from about 50 to 50,000.
14. The polymeric derivative of Claim 1 wherein said linkage fragments P and Q comprise
hydrolytically stable linkages in the absence of aromatic rings to one or more nonpeptidic and water soluble polymeric arms.
15. The polymeric derivative of Claim 1 wherein R comprises a linkage fragment attached by a hydrolytically stable linkage in the absence of
aromatic rings to a nonpeptidic and substantially nonreactive water soluble polymeric arm.
16. The polymeric derivative of Claim 15 wherein R is represented by the general structure
-M-polyd, wherein polyd is said polymeric arm and M is said linkage fragment.
17. The polymeric derivative of Claim 1 wherein Z further comprises a linkage fragment attached by a hydrolytically stable linkage in the absence of aromatic rings to a nonpeptidic and substantially nonreactive water soluble polymeric arm.
18. A polymeric derivative represented by the structure
wherein polya and polyb may be the same or different and are selected from the group consisting of linear poly(ethylene glycol), poly(vinyl alcohol),
poly(propylene glycol), poly(oxyethylated glycerol), poly(oxyethylated sorbitol), poly(oxyethylated
glucose), poly(oxazoline), poly(acryloylmorpholine), and poly(vinylpyrrolidone); wherein C is carbon;
wherein P and Q comprise linkage fragments that may be the same or different and join polymeric arms polya and polyb, respectively, to C by hydrolytically stable linkages selected from the group consisting of amide, amine, ether, carbamate, thiourea, urea, thiocarbamate, thiocarbonate, thioether, thioester, and
dithiocarbamate linkages; wherein R is a moiety
selected from the group consisting of H, substantially nonreactive moieties, and linkage fragments having attached thereto by a hydrolytically stable linkage in the absence of aromatic rings one or more nonpeptidic and substantially nonreactive water soluble polymeric arms; and wherein Z comprises a moiety selected from the group consisting of carboxyl, hydroxyl,
trifluoroethylsulfonate, isocyanate, isothiocyanate, N-hydroxylsuccinimidyl ester, N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate,
trichlorophenylcarbonate, aldehyde, vinylsulfone, maleimide, iodoacetamide, and iminoesters.
19. A multi-armed monofunctional polymeric derivative that is the reaction product of at least one monofunctional nonpeptidic polymer derivative and a linker moiety having two or more active sites that form linkages with said monofunctional nonpeptidic polymer derivatives in the absence of aromatic moieties, wherein said linkages between said linker moiety and said monofunctional nonpeptidic polymer derivatives are hydrolytically stable.
20. The multi-armed monofunctional polymeric derivative of Claim 19 wherein said linker moiety is selected from the group consisting of monohydroxy alcohols and monocarboxylic acids.
21. The multi-armed monofunctional polymer derivative of Claim 19 wherein said active sites on said linker moiety are nucleophilic moieties.
22. The multi-armed monofunctional polymer derivative of Claim 21 wherein said nucleophilic moieties are selected from the group consisting of amino, thiol, and hydroxyl moieties.
23. The multi-armed monofunctional polymer derivative of Claim 19 wherein said active sites on said linker moiety are electrophilic moieties.
24. The multi-armed monofunctional polymer derivative of Claim 23 wherein said electrophilic moieties are selected from the group consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde,
vinylsulfone, maleimide, iodoacetamide, and
iminoesters.
25. The multi-armed monofunctional polymeric derivative of Claim 24 wherein said active esters are N-hydroxylsuccinimidyl ester and said active carbonates are selected from the group consisting of N-hydroxylsuccinimidyl carbonates,
p-nitrophenylcarbonates, and trichlorophenylcarbonates.
26. The multi-armed monofunctional polymeric derivative of Claim 19 wherein said hydrolytically stable linkages in the absence of aromatic rings are selected from the group consisting of amide, amine, ether, carbamate, thiourea, urea, thiocarbamate, thiocarbonate, thioether, thioester, and
dithiocarbamate linkages.
27. The multi-armed monofunctional polymeric derivative of Claim 19 wherein said monofunctionality is selected from the group consisting of carboxyl, hydroxyl, activated carboxyl, activated hydroxyl, and conjugates of activated carboxyl or hydroxyl sites and molecules having at least one reactive nucleophilic moiety.
28. The multi-armed monofunctional polymeric derivative of Claim 19 wherein said monofunctionality is selected from the group consisting of
trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, and
iminoesters.
29. The multi-armed monofunctional polymeric derivative of Claim 28 wherein said active ester is N-hydroxylsuccinimide and said active carbonates are selected from the group consisting of N-hydroxylsuccinimide carbonates,
p-nitrophenylcarbonates, and trichlorophenylcarbonates.
30. The multi-armed monofunctional polymeric derivative of Claim 19 wherein said nonpeptidic
polymeric derivative is selected from the group
consisting of poly(alkylene oxides), poly(oxyethylated polyols), and poly(oxyethylated glucose).
31. The multi-armed monofunctional polymeric derivative of Claim 19 wherein said nonpeptidic polymer derivative is selected from the group consisting of activated poly(ethylene glycol), poly(vinyl alcohol), poly(propylene glycol), poly(oxyethylated glycerol), poly(oxyethylated sorbitol), poly(oxyethylated
glucose), poly(oxazoline), poly(acryloylmorpholine), and poly(vinylpyrrolidone).
32. The multi-armed monofunctional polymeric derivative of Claim 19 wherein said nonpeptidic polymer derivative is a linear mPEG of molecular weight of from about 50 to 50,000 and the multi-armed monofunctional polymeric derivative has two arms of said linear mPEG.
33. A material comprising a solid surface or particle having attached thereto compounds of the structure claimed in Claim 19.
34. The material of Claim 33 wherein said solid surface or particle is a liposome.
35. A biologically active structure
comprising a biologically active molecule having attached thereto one or more compounds of the structure claimed in Claim 19.
36. The biologically active structure of
Claim 35 wherein said biologically active molecule is selected from the group consisting of polypeptides, polynucleotides, and lipids.
37. The biologically active structure of Claim 36 wherein said polypeptide is selected from the group consisting of asparaginase, catalase,
ribonuclease, subtilisine, trypsin, and uricase.
38. A two-armed polymeric derivative having a structure selected from the group consisting of:
wherein polya and polyb may be the same or different and comprise moieties selected from the group consisting of poly(ethylene glycol), poly(vinyl alcohol),
poly(propylene glycol), poly(oxyethylated glycerol), poly(oxyethylated sorbitol), poly(oxyethylated
glucose), poly(oxazoline), poly(acryloylmorpholine), and poly(vinylpyrrolidone) moieties; and wherein Z comprises a moiety selected from the group consisting of moieties having a single site reactive toward nucleophilic moieties, sites that can be converted to sites reactive toward nucleophilic moieties, and the reaction product of a nucleophilic moiety and moieties having a single site reactive toward nucleophilic moieties.
39. The two-armed polymeric derivative of
Claim 38 wherein said reactive site is selected from the group consisting of carboxyl, activated carboxyl, hydroxyl, activated hydroxyl, and conjugates of
activated carboxyl or hydroxyl sites and molecules having at least one reactive nucleophilic moiety.
40. The polymeric derivative of Claim 38 wherein Z comprises a moiety selected from the group consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, and iminoesters.
41. The polymeric derivative of Claim 40 wherein said active ester is N-hydroxylsuccinimidyl ester and said active carbonates are selected from the group consisting of N-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate, and trichlorophenylcarbonate.
42. A molecule having the structure
CH3-(CH2CH2O)nCH2CH2-, wherein n equals from 1 to about 1,150, and wherein n may be the same or different for mPEGa and mPEGb.
43. The molecule of Claim 42 wherein n equals from 1 to about 570.
44. A method of synthesizing a multi-armed, water soluble, monofunctional polymeric molecule comprising reacting one or more nonpeptidic
monofunctional polymers of the structure poly-W, wherein W is an active moiety providing the
monofunctionality for the polymer, with a linker moiety having two or more active sites with which W is
reactive, and forming hydrolytically stable linkages in the absence of aromatic rings between the
monofunctional polymer and the linker moiety at the linker moiety active sites, the linker moiety having a reactive site for which said active moiety -W is not reactive to provide the monofunctionality for the multi-armed molecule.
45. The method of Claim 44 wherein the method further comprises activating the reactive site after the multi-armed polymeric compound is formed with an electrophilic moiety.
46. The method of Claim 45 wherein the electrophilic moiety is reactive with nucleophilic moieties selected from the group consisting of amino, thiol, and hydroxyl moieties.
47. The method of Claim 44 wherein the active moiety W is an electrophilic moiety selected from the group consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde, vinylsulfone, maleimide,
iodoacetamide, and iminoesters.
48. The method of Claim 47 wherein the active ester is N-hydroxylsuccinimidyl ester and the active carbonates are selected from the group
consisting of N-hydroxylsuccinimidyl carbonate,
p-nitrophenylcarbonate, and trichlorophenylcarbonate.
49. The method of Claim 44 wherein the active moiety W is a nucleophilic moiety selected from the group consisting of amino, thiol, and hydroxyl moieties.
50. The method of Claim 44 wherein the active sites on the linker moiety are nucleophilic moieties selected from the group consisting of amino, thiol, and hydroxyl moieties.
51. The method of Claim 44 wherein the active sites on the linker moiety are electrophilic moieties selected from the group consisting of
trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde,
vinylsulfone, maleimide, iodoacetamide, and
iminoesters.
52. The method of Claim 51 wherein the active ester is N-hydroxylsuccinimidyl ester and the active carbonates are selected from the group
consisting of N-hydroxylsuccinimidyl carbonate,
p-nitrophenylcarbonate, and trichlorophenylcarbonate.
53. The method of Claim 44 wherein the hydrolytically stable linkages are selected from the group consisting of amide, amine, ether, carbamate, thiourea, urea, thiocarbamate, thiocarbonate,
thioether, thioester, and dithiocarbamate linkages.
54. A method for preparing a polymeric derivative represented by the structure
a) reacting nonpeptidic, water soluble, monofunctional polymers of the structure polya-W and polyb-W with a linker moiety having at least two active sites for which W is selective, a reactive site Z for which W is not selective, and a moiety R which is substantially nonreactive, wherein W is an active electrophilic moiety selected from the group consisting of trifluoroethylsulfonate, isocyanate, isothiocyanate, active esters, active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, and
iminoesters, and may be the same or different on polya and polyb, wherein polya and polyb are polymer moieties selected from the group consisting of poly (ethylene glycol), poly(vinyl alcohol), poly(propylene glycol), poly(oxyethylated glycerol), poly(oxyethylated
sorbitol), poly(oxyethylated glucose), poly(oxazoline), poly(acryloylmorpholine), and poly(vinylpyrrolidone) and may be the same or different, and wherein the active sites of the linker moiety are nucleophilic sites selected from the group consisting of amino, thiol, and hydroxyl; and
b) forming hydrolytically stable linkages P and Q, which may be the same or different, in the absence of aromatic rings between the polymer and the linker moiety that are selected from the group
consisting of amide, amine, ether, carbamate,
thiourea, urea, thiocarbamate, thiocarbonate,
thioether, thioester, and dithiocarbamate linkages.
55. The method of Claim 54 wherein the linker moiety is substituted with polymer at each active site in one step.
56. The method of Claim 55 wherein the linker moiety is substituted with polymer at each active site in more than one step.
57. The multi-armed polymeric derivative of Claim 54 wherein said linker moiety is selected from the group consisting of monohydroxy alcohols and monocarboxilic acids having two or more active moieties selected from the group consisting of thiol, amino, and hydroxyl moieties.
58. The multi-armed polymeric derivative of Claim 1 wherein Z is selected from the group consisting of carboxyl, hydroxyl, activated carboxyl, activated hydroxyl, and conjugates of precursor activated
carboxyl or hydroxyl sites and molecules having sites for which said precursor activated sites are active.
59. A method for forming monofunctional monomethoxy-poly(ethylene glycol) disubstituted lysene comprising the following step:
61. The method of Claim 59 further
comprising the steps of
62. The method of Claim 61 wherein steps a) and b) take place in methylene chloride.
63. The method of Claim 59 further
comprising the steps of activating the carboxyl moiety and reacting the activated carboxyl moiety with an active moiety to join the disubstituted lysine to the active moiety.
64. A method for forming a monofunctional monomethoxy-poly(ethylene glycol) disubstituted compound comprising the following steps:
65. The method of Claim 64 further
comprising the steps of activating the carboxyl moiety and reacting the activated carboxyl moiety with an active moiety to join the disubstituted lysine to the active moiety.
66. The method of Claim 64 wherein step a) takes place in aqueous buffer.
67. The method of Claim 64 wherein step b) takes place in methylene chloride.
Priority Applications (1)
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AU47555/96A AU4755596A (en) | 1995-01-10 | 1996-01-11 | Multi-armed, monofunctional, and hydrolytically stable derivatives of poly(ethylene glycol) and related polymers for modification of surfaces and molecules |
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US37106595A | 1995-01-10 | 1995-01-10 | |
US08/371,065 | 1995-01-10 | ||
US08/443,383 US5932462A (en) | 1995-01-10 | 1995-05-17 | Multiarmed, monofunctional, polymer for coupling to molecules and surfaces |
US08/443,383 | 1995-05-17 |
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Cited By (181)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5859228A (en) * | 1995-05-04 | 1999-01-12 | Nexstar Pharmaceuticals, Inc. | Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes |
WO1999006071A1 (en) * | 1997-07-30 | 1999-02-11 | The Procter & Gamble Company | Modified polypeptides with high activity and reduced allergenicity |
WO1999022770A1 (en) * | 1997-11-05 | 1999-05-14 | Shearwater Polymers, Inc. | Delivery of poly(ethylene glycol)-conjugated molecules from degradable hydrogels |
WO1999027897A1 (en) * | 1997-12-03 | 1999-06-10 | Applied Research Systems Ars Holding N.V. | Site-specific preparation of polyethylene glycol-grf conjugates |
WO1999045964A1 (en) * | 1998-03-12 | 1999-09-16 | Shearwater Polymers, Incorporated | Poly(ethylene glycol) derivatives with proximal reactive groups |
WO1999055376A1 (en) * | 1998-04-28 | 1999-11-04 | Applied Research Systems Ars Holding N.V. | Peg-lhrh analog conjugates |
US6011020A (en) * | 1990-06-11 | 2000-01-04 | Nexstar Pharmaceuticals, Inc. | Nucleic acid ligand complexes |
US6025325A (en) * | 1995-05-05 | 2000-02-15 | Hoffman-La Roche Inc. | Pegylated obese (ob) protein compositions |
US6025324A (en) * | 1996-05-15 | 2000-02-15 | Hoffmann-La Roche Inc. | Pegylated obese (ob) protein compositions |
US6051698A (en) * | 1997-06-06 | 2000-04-18 | Janjic; Nebojsa | Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes |
EP1040151A1 (en) * | 1997-12-12 | 2000-10-04 | MacroMed, Inc. | Heterofunctionalized star-shaped poly(ethylene glycols) for protein modification |
US6147204A (en) * | 1990-06-11 | 2000-11-14 | Nexstar Pharmaceuticals, Inc. | Nucleic acid ligand complexes |
US6168778B1 (en) | 1990-06-11 | 2001-01-02 | Nexstar Pharmaceuticals, Inc. | Vascular endothelial growth factor (VEGF) Nucleic Acid Ligand Complexes |
US6229002B1 (en) | 1995-06-07 | 2001-05-08 | Nexstar Pharmaceuticlas, Inc. | Platelet derived growth factor (PDGF) nucleic acid ligand complexes |
EP1107813A1 (en) * | 1998-08-26 | 2001-06-20 | Neomend, Inc. | Compositions, systems, and methods for creating in situ, chemically cross-linked, mechanical barriers or covering structures |
WO2001048052A1 (en) | 1999-12-24 | 2001-07-05 | Kyowa Hakko Kogyo Co., Ltd. | Branched polyalkylene glycols |
US6303119B1 (en) | 1999-09-22 | 2001-10-16 | The Procter & Gamble Company | Personal care compositions containing subtilisin enzymes bound to water insoluble substrates |
WO2001093914A2 (en) * | 2000-06-08 | 2001-12-13 | La Jolla Pharmaceutical Company | Multivalent platform molecules comprising high molecular weight polyethylene oxide |
US6410017B1 (en) | 1998-09-22 | 2002-06-25 | The Procter & Gamble Company | Personal care compositions containing active proteins tethered to a water insoluble substrate |
US6426335B1 (en) | 1997-10-17 | 2002-07-30 | Gilead Sciences, Inc. | Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes |
US6465188B1 (en) | 1990-06-11 | 2002-10-15 | Gilead Sciences, Inc. | Nucleic acid ligand complexes |
WO2003032990A2 (en) * | 2001-10-18 | 2003-04-24 | Nektar Therapeutics Al, Corporation | Polymer conjugates of opioid antagonists |
US6583251B1 (en) | 1997-09-08 | 2003-06-24 | Emory University | Modular cytomimetic biomaterials, transport studies, preparation and utilization thereof |
US6638500B1 (en) | 1998-04-28 | 2003-10-28 | Applied Research Systems Ars Holding N.V. | Polyol-IFN-βconjugates modified at Cys-17 and composition containing same |
WO2003093346A1 (en) * | 2002-05-06 | 2003-11-13 | Universita' Degli Studi Di Trieste | Multifunctional polyethylene glycol derivatives: preparation and use |
WO2004000366A1 (en) | 2002-06-21 | 2003-12-31 | Novo Nordisk Health Care Ag | Pegylated factor vii glycoforms |
EP1434589A2 (en) * | 2001-10-09 | 2004-07-07 | Nektar Therapeutics Al, Corporation | Thioester-terminated water soluble polymers and method of modifying the n-terminus of a polypeptide therewith |
WO2004061094A1 (en) | 2002-12-30 | 2004-07-22 | Gryphon Therapeutics, Inc. | Water-soluble thioester and selenoester compounds and methods for making and using the same |
WO2005035727A2 (en) | 2003-10-09 | 2005-04-21 | Ambrx, Inc. | Polymer derivatives |
US6899889B1 (en) | 1998-11-06 | 2005-05-31 | Neomend, Inc. | Biocompatible material composition adaptable to diverse therapeutic indications |
US6914121B2 (en) | 1998-04-28 | 2005-07-05 | Applied Research Systems Ars Holding N.V. | PEG-LHRH analog conjugates |
US6936298B2 (en) | 2000-04-13 | 2005-08-30 | Emory University | Antithrombogenic membrane mimetic compositions and methods |
EP1588717A1 (en) * | 1998-04-28 | 2005-10-26 | Applied Research Systems ARS Holding N.V. | PEG-LHRH analog conjugates |
WO2005108463A2 (en) * | 2004-05-03 | 2005-11-17 | Nektar Therapeutics Al, Corporation | Branched polyethylen glycol derivates comprising an acetal or ketal branching point |
WO2006009901A2 (en) | 2004-06-18 | 2006-01-26 | Ambrx, Inc. | Novel antigen-binding polypeptides and their uses |
WO2006069246A2 (en) | 2004-12-22 | 2006-06-29 | Ambrx, Inc. | Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides |
EP1704878A2 (en) | 1995-12-18 | 2006-09-27 | AngioDevice International GmbH | Crosslinked polymer compositions and methods for their use |
WO2006134173A2 (en) | 2005-06-17 | 2006-12-21 | Novo Nordisk Health Care Ag | Selective reduction and derivatization of engineered proteins comprising at least one non-native cysteine |
US7201897B2 (en) | 1996-05-31 | 2007-04-10 | Hoffmann-La Roche Inc. | Interferon conjugates |
EP1776132A2 (en) * | 2004-05-26 | 2007-04-25 | Nobex Corporation | Natriuretic compounds, conjugates, and uses thereof |
US7244830B2 (en) | 2001-01-12 | 2007-07-17 | Emory University | Glycopolymers and free radical polymerization methods |
WO2007062610A3 (en) * | 2005-11-30 | 2007-09-20 | Ct Ingenieria Genetica Biotech | Four branched dendrimer-peg for conjugation to proteins and peptides |
EP1891231A2 (en) * | 2005-05-25 | 2008-02-27 | Neose Technologies, Inc. | Glycopegylated factor ix |
WO2008030558A2 (en) | 2006-09-08 | 2008-03-13 | Ambrx, Inc. | Modified human plasma polypeptide or fc scaffolds and their uses |
WO2008060780A2 (en) | 2006-10-04 | 2008-05-22 | Novo Nordisk A/S | Glycerol linked pegylated sugars and glycopeptides |
EP1982732A2 (en) | 2000-02-11 | 2008-10-22 | Maxygen Holdings Ltd. | Factor VII or VIIA-like molecules |
WO2009023826A1 (en) | 2007-08-16 | 2009-02-19 | Pharmaessentia Corp. | Protein-polymer conjugates |
EP2042196A2 (en) | 2001-10-10 | 2009-04-01 | Neose Technologies, Inc. | Remodelling and glycoconjugation of Granulocyte Colony Stimulating Factor (G-CSF) |
EP2055189A1 (en) | 2003-04-09 | 2009-05-06 | Neose Technologies, Inc. | Glycopegylation methods and proteins/peptides produced by the methods |
WO2009067636A2 (en) | 2007-11-20 | 2009-05-28 | Ambrx, Inc. | Modified insulin polypeptides and their uses |
EP2080771A2 (en) | 2001-02-27 | 2009-07-22 | Maxygen Aps | New interferon beta-like molecules |
JP2009541333A (en) * | 2006-06-23 | 2009-11-26 | クインテセンス バイオサイエンシーズ インコーポレーティッド | Modified ribonuclease |
US7632492B2 (en) | 2006-05-02 | 2009-12-15 | Allozyne, Inc. | Modified human interferon-β polypeptides |
EP2133098A1 (en) | 2000-01-10 | 2009-12-16 | Maxygen Holdings Ltd | G-CSF conjugates |
WO2010011735A2 (en) | 2008-07-23 | 2010-01-28 | Ambrx, Inc. | Modified bovine g-csf polypeptides and their uses |
US7655747B2 (en) * | 1996-09-26 | 2010-02-02 | Nektar Therapeutics | Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution |
US7662773B2 (en) | 2002-11-26 | 2010-02-16 | Biocon Limited | Natriuretic compounds, conjugates, and uses thereof |
US7696163B2 (en) | 2001-10-10 | 2010-04-13 | Novo Nordisk A/S | Erythropoietin: remodeling and glycoconjugation of erythropoietin |
US7713544B2 (en) | 2000-07-28 | 2010-05-11 | Emory University | Biological component comprising artificial membrane |
US7736872B2 (en) | 2004-12-22 | 2010-06-15 | Ambrx, Inc. | Compositions of aminoacyl-TRNA synthetase and uses thereof |
EP2213733A2 (en) | 2006-05-24 | 2010-08-04 | Novo Nordisk Health Care AG | Factor IX analogues having prolonged in vivo half life |
US7786133B2 (en) | 2003-12-16 | 2010-08-31 | Nektar Therapeutics | Chemically modified small molecules |
US7795210B2 (en) | 2001-10-10 | 2010-09-14 | Novo Nordisk A/S | Protein remodeling methods and proteins/peptides produced by the methods |
US7803777B2 (en) | 2003-03-14 | 2010-09-28 | Biogenerix Ag | Branched water-soluble polymers and their conjugates |
US7816320B2 (en) | 2004-12-22 | 2010-10-19 | Ambrx, Inc. | Formulations of human growth hormone comprising a non-naturally encoded amino acid at position 35 |
US7824672B2 (en) | 2004-03-26 | 2010-11-02 | Emory University | Method for coating living cells |
US7829659B2 (en) | 2006-05-02 | 2010-11-09 | Allozyne, Inc. | Methods of modifying polypeptides comprising non-natural amino acids |
US7833978B2 (en) | 2004-02-20 | 2010-11-16 | Emory University | Thrombomodulin derivatives and conjugates |
US7842661B2 (en) | 2003-11-24 | 2010-11-30 | Novo Nordisk A/S | Glycopegylated erythropoietin formulations |
WO2010144629A1 (en) | 2009-06-09 | 2010-12-16 | Prolong Pharmaceuticals, LLC | Hemoglobin compositions |
EP2263684A1 (en) | 2003-10-10 | 2010-12-22 | Novo Nordisk A/S | IL-21 derivatives |
EP2279756A2 (en) | 2005-04-05 | 2011-02-02 | Instituto di Ricerche di Biologia Molecolare p Angeletti S.P.A. | Method for shielding functional sites or epitopes on proteins |
EP2284191A2 (en) | 2004-12-22 | 2011-02-16 | Ambrx, Inc. | Process for the preparation of hGH |
EP2298354A2 (en) | 2001-10-10 | 2011-03-23 | BioGeneriX AG | Remodelling and glycoconjugation of interferon-beta |
US7932364B2 (en) | 2003-05-09 | 2011-04-26 | Novo Nordisk A/S | Compositions and methods for the preparation of human growth hormone glycosylation mutants |
US7947473B2 (en) | 2004-12-22 | 2011-05-24 | Ambrx, Inc. | Methods for expression and purification of pegylated recombinant human growth hormone containing a non-naturally encoded keto amino acid |
EP2327724A2 (en) | 2004-02-02 | 2011-06-01 | Ambrx, Inc. | Modified human growth hormone polypeptides and their uses |
US7956032B2 (en) | 2003-12-03 | 2011-06-07 | Novo Nordisk A/S | Glycopegylated granulocyte colony stimulating factor |
US8008252B2 (en) | 2001-10-10 | 2011-08-30 | Novo Nordisk A/S | Factor VII: remodeling and glycoconjugation of Factor VII |
US8012931B2 (en) | 2007-03-30 | 2011-09-06 | Ambrx, Inc. | Modified FGF-21 polypeptides and their uses |
WO2011107591A1 (en) | 2010-03-05 | 2011-09-09 | Rigshospitalet | Chimeric inhibitor molecules of complement activation |
EP2386571A2 (en) | 2005-04-08 | 2011-11-16 | BioGeneriX AG | Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants |
WO2011143274A1 (en) | 2010-05-10 | 2011-11-17 | Perseid Therapeutics | Polypeptide inhibitors of vla4 |
US8067431B2 (en) | 2003-12-16 | 2011-11-29 | Nektar Therapeutics | Chemically modified small molecules |
US8071737B2 (en) | 1995-05-04 | 2011-12-06 | Glead Sciences, Inc. | Nucleic acid ligand complexes |
US8093356B2 (en) | 2005-06-03 | 2012-01-10 | Ambrx, Inc. | Pegylated human interferon polypeptides |
US8114630B2 (en) | 2007-05-02 | 2012-02-14 | Ambrx, Inc. | Modified interferon beta polypeptides and their uses |
WO2012024452A2 (en) | 2010-08-17 | 2012-02-23 | Ambrx, Inc. | Modified relaxin polypeptides and their uses |
US8207112B2 (en) | 2007-08-29 | 2012-06-26 | Biogenerix Ag | Liquid formulation of G-CSF conjugate |
US8268967B2 (en) | 2004-09-10 | 2012-09-18 | Novo Nordisk A/S | Glycopegylated interferon α |
US8278418B2 (en) | 2008-09-26 | 2012-10-02 | Ambrx, Inc. | Modified animal erythropoietin polypeptides and their uses |
EP2514757A2 (en) | 2005-01-10 | 2012-10-24 | BioGeneriX AG | Glycopegylated granulocyte colony stimulating factor |
WO2013006706A1 (en) | 2011-07-05 | 2013-01-10 | Bioasis Technologies Inc. | P97-antibody conjugates and methods of use |
WO2013004607A1 (en) | 2011-07-01 | 2013-01-10 | Bayer Intellectual Property Gmbh | Relaxin fusion polypeptides and uses thereof |
EP2548967A2 (en) | 2006-09-21 | 2013-01-23 | The Regents of The University of California | Aldehyde tags, uses thereof in site-specific protein modification |
US8377466B2 (en) | 1995-12-18 | 2013-02-19 | Angiotech Pharmaceuticals (Us), Inc. | Adhesive tissue repair patch |
US8383144B2 (en) | 1998-11-06 | 2013-02-26 | Neomend, Inc. | Tissue adhering compositions |
US8420792B2 (en) | 2006-09-08 | 2013-04-16 | Ambrx, Inc. | Suppressor tRNA transcription in vertebrate cells |
EP2586456A1 (en) | 2004-10-29 | 2013-05-01 | BioGeneriX AG | Remodeling and glycopegylation of fibroblast growth factor (FGF) |
EP2633866A2 (en) | 2003-10-17 | 2013-09-04 | Novo Nordisk A/S | Combination therapy |
WO2013185115A1 (en) | 2012-06-08 | 2013-12-12 | Sutro Biopharma, Inc. | Antibodies comprising site-specific non-natural amino acid residues, methods of their preparation and methods of their use |
US8633157B2 (en) | 2003-11-24 | 2014-01-21 | Novo Nordisk A/S | Glycopegylated erythropoietin |
WO2014022515A1 (en) | 2012-07-31 | 2014-02-06 | Bioasis Technologies, Inc. | Dephosphorylated lysosomal storage disease proteins and methods of use thereof |
WO2014036492A1 (en) | 2012-08-31 | 2014-03-06 | Sutro Biopharma, Inc. | Modified amino acids comprising an azido group |
US8716240B2 (en) | 2001-10-10 | 2014-05-06 | Novo Nordisk A/S | Erythropoietin: remodeling and glycoconjugation of erythropoietin |
US8716239B2 (en) | 2001-10-10 | 2014-05-06 | Novo Nordisk A/S | Granulocyte colony stimulating factor: remodeling and glycoconjugation G-CSF |
US8791070B2 (en) | 2003-04-09 | 2014-07-29 | Novo Nordisk A/S | Glycopegylated factor IX |
US8791066B2 (en) | 2004-07-13 | 2014-07-29 | Novo Nordisk A/S | Branched PEG remodeling and glycosylation of glucagon-like peptide-1 [GLP-1] |
WO2014118382A1 (en) | 2013-02-04 | 2014-08-07 | W. L. Gore & Associates, Inc. | Coating for substrate |
US8841439B2 (en) | 2005-11-03 | 2014-09-23 | Novo Nordisk A/S | Nucleotide sugar purification using membranes |
US8846624B2 (en) | 2006-09-11 | 2014-09-30 | Emory University | Modified protein polymers |
WO2014160438A1 (en) | 2013-03-13 | 2014-10-02 | Bioasis Technologies Inc. | Fragments of p97 and uses thereof |
US8853376B2 (en) | 2002-11-21 | 2014-10-07 | Archemix Llc | Stabilized aptamers to platelet derived growth factor and their use as oncology therapeutics |
EP2805964A1 (en) | 2009-12-21 | 2014-11-26 | Ambrx, Inc. | Modified bovine somatotropin polypeptides and their uses |
EP2805965A1 (en) | 2009-12-21 | 2014-11-26 | Ambrx, Inc. | Modified porcine somatotropin polypeptides and their uses |
US8911967B2 (en) | 2005-08-19 | 2014-12-16 | Novo Nordisk A/S | One pot desialylation and glycopegylation of therapeutic peptides |
US8916360B2 (en) | 2003-11-24 | 2014-12-23 | Novo Nordisk A/S | Glycopegylated erythropoietin |
WO2015006555A2 (en) | 2013-07-10 | 2015-01-15 | Sutro Biopharma, Inc. | Antibodies comprising multiple site-specific non-natural amino acid residues, methods of their preparation and methods of their use |
US8969532B2 (en) | 2006-10-03 | 2015-03-03 | Novo Nordisk A/S | Methods for the purification of polypeptide conjugates comprising polyalkylene oxide using hydrophobic interaction chromatography |
WO2015031673A2 (en) | 2013-08-28 | 2015-03-05 | Bioasis Technologies Inc. | Cns-targeted conjugates having modified fc regions and methods of use thereof |
US9006407B2 (en) | 2008-10-01 | 2015-04-14 | Quintessence Biosciences, Inc. | Therapeutic ribonucleases |
US9005625B2 (en) | 2003-07-25 | 2015-04-14 | Novo Nordisk A/S | Antibody toxin conjugates |
WO2015054658A1 (en) | 2013-10-11 | 2015-04-16 | Sutro Biopharma, Inc. | Modified amino acids comprising tetrazine functional groups, methods of preparation, and methods of their use |
WO2015081282A1 (en) | 2013-11-27 | 2015-06-04 | Redwood Bioscience, Inc. | Hydrazinyl-pyrrolo compounds and methods for producing a conjugate |
US9050304B2 (en) | 2007-04-03 | 2015-06-09 | Ratiopharm Gmbh | Methods of treatment using glycopegylated G-CSF |
US9121025B2 (en) | 2008-09-26 | 2015-09-01 | Ambrx, Inc. | Non-natural amino acid replication-dependent microorganisms and vaccines |
US9133495B2 (en) | 2006-09-08 | 2015-09-15 | Ambrx, Inc. | Hybrid suppressor tRNA for vertebrate cells |
US9150848B2 (en) | 2008-02-27 | 2015-10-06 | Novo Nordisk A/S | Conjugated factor VIII molecules |
US9187532B2 (en) | 2006-07-21 | 2015-11-17 | Novo Nordisk A/S | Glycosylation of peptides via O-linked glycosylation sequences |
US9192656B2 (en) | 2006-07-17 | 2015-11-24 | Quintessence Biosciences, Inc. | Methods and compositions for the treatment of cancer |
US9272075B2 (en) | 2013-02-04 | 2016-03-01 | W.L. Gore & Associates, Inc. | Coating for substrate |
US9310374B2 (en) | 2012-11-16 | 2016-04-12 | Redwood Bioscience, Inc. | Hydrazinyl-indole compounds and methods for producing a conjugate |
US9393319B2 (en) | 2004-04-13 | 2016-07-19 | Quintessence Biosciences, Inc. | Non-natural ribonuclease conjugates as cytotoxic agents |
US9434778B2 (en) | 2014-10-24 | 2016-09-06 | Bristol-Myers Squibb Company | Modified FGF-21 polypeptides comprising an internal deletion and uses thereof |
US9488660B2 (en) | 2005-11-16 | 2016-11-08 | Ambrx, Inc. | Methods and compositions comprising non-natural amino acids |
US9493499B2 (en) | 2007-06-12 | 2016-11-15 | Novo Nordisk A/S | Process for the production of purified cytidinemonophosphate-sialic acid-polyalkylene oxide (CMP-SA-PEG) as modified nucleotide sugars via anion exchange chromatography |
EP3103880A1 (en) | 2008-02-08 | 2016-12-14 | Ambrx, Inc. | Modified leptin polypeptides and their uses |
US9567386B2 (en) | 2010-08-17 | 2017-02-14 | Ambrx, Inc. | Therapeutic uses of modified relaxin polypeptides |
US9579390B2 (en) | 2012-11-12 | 2017-02-28 | Redwood Bioscience, Inc. | Compounds and methods for producing a conjugate |
EP3135690A1 (en) | 2012-06-26 | 2017-03-01 | Sutro Biopharma, Inc. | Modified fc proteins comprising site-specific non-natural amino acid residues, conjugates of the same, methods of their preparation and methods of their use |
US9605078B2 (en) | 2012-11-16 | 2017-03-28 | The Regents Of The University Of California | Pictet-Spengler ligation for protein chemical modification |
WO2017132617A1 (en) | 2016-01-27 | 2017-08-03 | Sutro Biopharma, Inc. | Anti-cd74 antibody conjugates, compositions comprising anti-cd74 antibody conjugates and methods of using anti-cd74 antibody conjugates |
WO2017199033A1 (en) | 2016-05-18 | 2017-11-23 | NZP UK Limited | Intermediates for the synthesis of bile acid derivatives, in particular of obeticholic acid |
US9920106B2 (en) | 2003-12-18 | 2018-03-20 | Novo Nordisk A/S | GLP-1 compounds |
US10131688B2 (en) | 2014-11-19 | 2018-11-20 | NZP UK Limited | 5.beta.-6-alkyl-7-hydroxy-3-one steroids as intermediates for the production of steroidal FXR modulators |
WO2019023316A1 (en) | 2017-07-26 | 2019-01-31 | Sutro Biopharma, Inc. | Methods of using anti-cd74 antibodies and antibody conjugates in treatment of t-cell lymphoma |
WO2019055909A1 (en) | 2017-09-18 | 2019-03-21 | Sutro Biopharma, Inc. | Anti-folate receptor alpha antibody conjugates and their uses |
US10266578B2 (en) | 2017-02-08 | 2019-04-23 | Bristol-Myers Squibb Company | Modified relaxin polypeptides comprising a pharmacokinetic enhancer and uses thereof |
US10301350B2 (en) | 2014-11-19 | 2019-05-28 | NZP UK Limited | 6-alkyl-7-hydroxy-4-en-3-one steroids as intermediates for the production of steroidal FXR modulators |
WO2019133399A1 (en) | 2017-12-26 | 2019-07-04 | Becton, Dickinson And Company | Deep ultraviolet-excitable water-solvated polymeric dyes |
US10407482B2 (en) | 2006-05-02 | 2019-09-10 | Allozyne, Inc. | Amino acid substituted molecules |
WO2019191482A1 (en) | 2018-03-30 | 2019-10-03 | Becton, Dickinson And Company | Water-soluble polymeric dyes having pendant chromophores |
US10538550B2 (en) | 2014-11-19 | 2020-01-21 | NZP UK Limited | 6.alpha.-alkyl-3,7-dione steroids as intermediates for the production of steroidal FXR modulators |
WO2020023300A1 (en) | 2018-07-22 | 2020-01-30 | Bioasis Technologies, Inc. | Treatment of lymmphatic metastases |
WO2020056066A1 (en) | 2018-09-11 | 2020-03-19 | Ambrx, Inc. | Interleukin-2 polypeptide conjugates and their uses |
US10597423B2 (en) | 2014-11-19 | 2020-03-24 | NZP UK Limited | 6.alpha.-alkyl-6,7-dione steroids as intermediates for the production of steroidal FXR modulators |
WO2020060944A1 (en) | 2018-09-17 | 2020-03-26 | Sutro Biopharma, Inc. | Combination therapies with anti-folate receptor antibody conjugates |
WO2020061670A1 (en) * | 2018-09-25 | 2020-04-02 | Universidade Federal Do Rio De Janeiro | Liposomal formulation, pharmaceutical composition, use of a liposomal formulation, method for treating cancer, and process for preparing a liposomal formulation |
WO2020082057A1 (en) | 2018-10-19 | 2020-04-23 | Ambrx, Inc. | Interleukin-10 polypeptide conjugates, dimers thereof, and their uses |
WO2020168017A1 (en) | 2019-02-12 | 2020-08-20 | Ambrx, Inc. | Compositions containing, methods and uses of antibody-tlr agonist conjugates |
US10766921B2 (en) | 2016-05-18 | 2020-09-08 | NZP UK Limited | Process and intermediates for the 6,7-alpha-epoxidation of steroid 4,6-dienes |
WO2020227105A1 (en) | 2019-05-03 | 2020-11-12 | Sutro Biopharma, Inc. | Anti-bcma antibody conjugates |
WO2020252043A1 (en) | 2019-06-10 | 2020-12-17 | Sutro Biopharma, Inc. | 5H-PYRROLO[3,2-d]PYRIMIDINE-2,4-DIAMINO COMPOUNDS AND ANTIBODY CONJUGATES THEREOF |
WO2020257235A1 (en) | 2019-06-17 | 2020-12-24 | Sutro Biopharma, Inc. | 1-(4-(aminomethyl)benzyl)-2-butyl-2h-pyrazolo[3,4-c]quinolin-4-amine derivatives and related compounds as toll-like receptor (tlr) 7/8 agonists, as well as antibody drug conjugates thereof for use in cancer therapy and diagnosis |
WO2021178597A1 (en) | 2020-03-03 | 2021-09-10 | Sutro Biopharma, Inc. | Antibodies comprising site-specific glutamine tags, methods of their preparation and methods of their use |
WO2021183832A1 (en) | 2020-03-11 | 2021-09-16 | Ambrx, Inc. | Interleukin-2 polypeptide conjugates and methods of use thereof |
WO2021236526A1 (en) | 2020-05-18 | 2021-11-25 | Bioasis Technologies, Inc. | Compositions and methods for treating lewy body dementia |
WO2021255524A1 (en) | 2020-06-17 | 2021-12-23 | Bioasis Technologies, Inc. | Compositions and methods for treating frontotemporal dementia |
WO2022040596A1 (en) | 2020-08-20 | 2022-02-24 | Ambrx, Inc. | Antibody-tlr agonist conjugates, methods and uses thereof |
US11273202B2 (en) | 2010-09-23 | 2022-03-15 | Elanco Us Inc. | Formulations for bovine granulocyte colony stimulating factor and variants thereof |
WO2022103983A2 (en) | 2020-11-11 | 2022-05-19 | Sutro Biopharma, Inc. | Fluorenylmethyloxycarbonyl and fluorenylmethylaminocarbonyl compounds, protein conjugates thereof, and methods for their use |
WO2022212899A1 (en) | 2021-04-03 | 2022-10-06 | Ambrx, Inc. | Anti-her2 antibody-drug conjugates and uses thereof |
EP4155349A1 (en) | 2021-09-24 | 2023-03-29 | Becton, Dickinson and Company | Water-soluble yellow green absorbing dyes |
WO2024006542A1 (en) | 2022-06-30 | 2024-01-04 | Sutro Biopharma, Inc. | Anti-ror1 antibodies and antibody conjugates, compositions comprising anti-ror1 antibodies or antibody conjugates, and methods of making and using anti-ror1 antibodies and antibody conjugates |
WO2024007016A2 (en) | 2022-07-01 | 2024-01-04 | Beckman Coulter, Inc. | Novel fluorescent dyes and polymers from dihydrophenanthrene derivatives |
WO2024006272A1 (en) | 2022-06-27 | 2024-01-04 | Sutro Biopharma, Inc. | β-GLUCURONIDE LINKER-PAYLOADS, PROTEIN CONJUGATES THEREOF, AND METHODS THEREOF |
WO2024015229A1 (en) | 2022-07-15 | 2024-01-18 | Sutro Biopharma, Inc. | Protease/enzyme cleavable linker-payloads and protein conjugates |
EP4083108A4 (en) * | 2019-12-27 | 2024-02-14 | Nof Corp | Refinement method for polyethylene glycol compound |
WO2024044327A1 (en) | 2022-08-26 | 2024-02-29 | Beckman Coulter, Inc. | Dhnt monomers and polymer dyes with modified photophysical properties |
WO2024044780A1 (en) | 2022-08-26 | 2024-02-29 | Sutro Biopharma, Inc. | Interleukin-18 variants and uses thereof |
US11931420B2 (en) | 2021-04-30 | 2024-03-19 | Celgene Corporation | Combination therapies using an anti-BCMA antibody drug conjugate (ADC) in combination with a gamma secretase inhibitor (GSI) |
Families Citing this family (504)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6395888B1 (en) * | 1996-02-01 | 2002-05-28 | Gilead Sciences, Inc. | High affinity nucleic acid ligands of complement system proteins |
US6191105B1 (en) | 1993-05-10 | 2001-02-20 | Protein Delivery, Inc. | Hydrophilic and lipophilic balanced microemulsion formulations of free-form and/or conjugation-stabilized therapeutic agents such as insulin |
KR100361933B1 (en) * | 1993-09-08 | 2003-02-14 | 라 졸라 파마슈티칼 컴파니 | Chemically defined nonpolymeric bonds form the platform molecule and its conjugate |
US5919455A (en) | 1993-10-27 | 1999-07-06 | Enzon, Inc. | Non-antigenic branched polymer conjugates |
US6057287A (en) | 1994-01-11 | 2000-05-02 | Dyax Corp. | Kallikrein-binding "Kunitz domain" proteins and analogues thereof |
US5545553A (en) * | 1994-09-26 | 1996-08-13 | The Rockefeller University | Glycosyltransferases for biosynthesis of oligosaccharides, and genes encoding them |
US5932462A (en) * | 1995-01-10 | 1999-08-03 | Shearwater Polymers, Inc. | Multiarmed, monofunctional, polymer for coupling to molecules and surfaces |
US8003705B2 (en) * | 1996-09-23 | 2011-08-23 | Incept Llc | Biocompatible hydrogels made with small molecule precursors |
WO1998012274A1 (en) | 1996-09-23 | 1998-03-26 | Chandrashekar Pathak | Methods and devices for preparing protein concentrates |
US6056973A (en) * | 1996-10-11 | 2000-05-02 | Sequus Pharmaceuticals, Inc. | Therapeutic liposome composition and method of preparation |
US6743248B2 (en) | 1996-12-18 | 2004-06-01 | Neomend, Inc. | Pretreatment method for enhancing tissue adhesion |
US20030191496A1 (en) * | 1997-03-12 | 2003-10-09 | Neomend, Inc. | Vascular sealing device with microwave antenna |
US6371975B2 (en) | 1998-11-06 | 2002-04-16 | Neomend, Inc. | Compositions, systems, and methods for creating in situ, chemically cross-linked, mechanical barriers |
US20040176801A1 (en) * | 1997-03-12 | 2004-09-09 | Neomend, Inc. | Pretreatment method for enhancing tissue adhesion |
JP2002515932A (en) * | 1997-04-18 | 2002-05-28 | カリフォルニア インスティチュート オブ テクノロジー | Multifunctional polymeric tissue coating |
EP1002064B1 (en) * | 1997-06-25 | 2007-10-10 | Novozymes A/S | A modified polypeptide |
CN1276730A (en) * | 1997-09-18 | 2000-12-13 | 霍夫曼-拉罗奇有限公司 | Use of IFN-alpha and amantadine for treatment of chronic hepatitis C |
US6258782B1 (en) | 1998-05-20 | 2001-07-10 | Trimeris, Inc. | Hybrid polypeptides with enhanced pharmacokinetic properties |
US6656906B1 (en) * | 1998-05-20 | 2003-12-02 | Trimeris, Inc. | Hybrid polypeptides with enhanced pharmacokinetic properties |
IL139786A0 (en) * | 1998-06-08 | 2002-02-10 | Hoffmann La Roche | Use of peg-ifn-alpha and ribavirin for the treatment of chronic hepatitis c |
ES2245114T3 (en) | 1998-08-06 | 2005-12-16 | Mountain View Pharmaceuticals, Inc. | CONJUGATES OF PEG-OXIDASA DE URATO AND ITS USE. |
US6783965B1 (en) * | 2000-02-10 | 2004-08-31 | Mountain View Pharmaceuticals, Inc. | Aggregate-free urate oxidase for preparation of non-immunogenic polymer conjugates |
US6632457B1 (en) | 1998-08-14 | 2003-10-14 | Incept Llc | Composite hydrogel drug delivery systems |
US6703381B1 (en) | 1998-08-14 | 2004-03-09 | Nobex Corporation | Methods for delivery therapeutic compounds across the blood-brain barrier |
US6994686B2 (en) | 1998-08-26 | 2006-02-07 | Neomend, Inc. | Systems for applying cross-linked mechanical barriers |
US6458147B1 (en) | 1998-11-06 | 2002-10-01 | Neomend, Inc. | Compositions, systems, and methods for arresting or controlling bleeding or fluid leakage in body tissue |
US7279001B2 (en) * | 1998-11-06 | 2007-10-09 | Neomend, Inc. | Systems, methods, and compositions for achieving closure of vascular puncture sites |
US6949114B2 (en) | 1998-11-06 | 2005-09-27 | Neomend, Inc. | Systems, methods, and compositions for achieving closure of vascular puncture sites |
WO2000033764A1 (en) | 1998-12-04 | 2000-06-15 | Pathak Chandrashekhar P | Biocompatible crosslinked polymers |
US6458953B1 (en) * | 1998-12-09 | 2002-10-01 | La Jolla Pharmaceutical Company | Valency platform molecules comprising carbamate linkages |
US6958212B1 (en) * | 1999-02-01 | 2005-10-25 | Eidgenossische Technische Hochschule Zurich | Conjugate addition reactions for the controlled delivery of pharmaceutically active compounds |
CA2359318C (en) | 1999-02-01 | 2009-06-30 | Donald Elbert | Biomaterials formed by nucleophilic addition reaction to conjugated unsaturated groups |
DE60041255D1 (en) * | 1999-04-28 | 2009-02-12 | Eidgenoess Tech Hochschule | POLYIONIC COATINGS FOR ANALYTICAL AND SENSOR DEVICES |
WO2000075105A1 (en) * | 1999-06-08 | 2000-12-14 | La Jolla Pharmaceutical Company | Valency platform molecules comprising aminooxy groups |
US6309633B1 (en) | 1999-06-19 | 2001-10-30 | Nobex Corporation | Amphiphilic drug-oligomer conjugates with hydroyzable lipophile components and methods for making and using the same |
JO2291B1 (en) | 1999-07-02 | 2005-09-12 | اف . هوفمان لاروش ايه جي | Erythopintin derivatives |
CZ299516B6 (en) * | 1999-07-02 | 2008-08-20 | F. Hoffmann-La Roche Ag | Erythropoietin glycoprotein conjugate, process for its preparation and use and pharmaceutical composition containing thereof |
US6541508B2 (en) * | 1999-09-13 | 2003-04-01 | Nobex Corporation | Taxane prodrugs |
US6380405B1 (en) | 1999-09-13 | 2002-04-30 | Nobex Corporation | Taxane prodrugs |
US6713454B1 (en) * | 1999-09-13 | 2004-03-30 | Nobex Corporation | Prodrugs of etoposide and etoposide analogs |
KR100619612B1 (en) * | 1999-10-04 | 2006-09-01 | 넥타르 테라퓨틱스 에이엘, 코포레이션 | Polymer stabilized neuropeptides |
US6348558B1 (en) | 1999-12-10 | 2002-02-19 | Shearwater Corporation | Hydrolytically degradable polymers and hydrogels made therefrom |
US7074878B1 (en) * | 1999-12-10 | 2006-07-11 | Harris J Milton | Hydrolytically degradable polymers and hydrogels made therefrom |
US6638906B1 (en) | 1999-12-13 | 2003-10-28 | Nobex Corporation | Amphiphilic polymers and polypeptide conjugates comprising same |
WO2001045796A2 (en) | 1999-12-22 | 2001-06-28 | Shearwater Corporation | Method for the preparation of 1-benzotriazolyl carbonate esters of poly(ethylene glycol) |
AU2001257577A1 (en) | 2000-02-28 | 2001-09-03 | Shearwater Corporation | Water-soluble polymer conjugates of artelinic acid |
US6756037B2 (en) | 2000-03-31 | 2004-06-29 | Enzon, Inc. | Polymer conjugates of biologically active agents and extension moieties for facilitating conjugation of biologically active agents to polymeric terminal groups |
US6777387B2 (en) | 2000-03-31 | 2004-08-17 | Enzon Pharmaceuticals, Inc. | Terminally-branched polymeric linkers containing extension moieties and polymeric conjugates containing the same |
US7291673B2 (en) * | 2000-06-02 | 2007-11-06 | Eidgenossiche Technische Hochschule Zurich | Conjugate addition reactions for the controlled delivery of pharmaceutically active compounds |
KR20030032977A (en) * | 2000-07-12 | 2003-04-26 | 그리폰 테라퓨틱스, 인코포레이티드 | Chemokine receptor modulators, production and use |
US7118737B2 (en) | 2000-09-08 | 2006-10-10 | Amylin Pharmaceuticals, Inc. | Polymer-modified synthetic proteins |
RU2003109746A (en) * | 2000-09-08 | 2005-01-27 | Грифон Терапьютикс, Инк. (Us) | SYNTHETIC PROTEINS STIMULATING ERYTHROPOESIS |
US7052686B2 (en) * | 2000-09-29 | 2006-05-30 | Schering Corporation | Pegylated interleukin-10 |
JP4070605B2 (en) * | 2000-10-19 | 2008-04-02 | アイトゲノッシスシェ・テヒニッシュ・ホーホシューレ・ツューリヒ | Block copolymers for multifunctional self-assembled systems |
US7053150B2 (en) * | 2000-12-18 | 2006-05-30 | Nektar Therapeutics Al, Corporation | Segmented polymers and their conjugates |
TW593427B (en) * | 2000-12-18 | 2004-06-21 | Nektar Therapeutics Al Corp | Synthesis of high molecular weight non-peptidic polymer derivatives |
US7265186B2 (en) * | 2001-01-19 | 2007-09-04 | Nektar Therapeutics Al, Corporation | Multi-arm block copolymers as drug delivery vehicles |
TWI246524B (en) | 2001-01-19 | 2006-01-01 | Shearwater Corp | Multi-arm block copolymers as drug delivery vehicles |
CA2436623C (en) * | 2001-01-30 | 2011-08-02 | Kyowa Hakko Kogyo Co., Ltd. | Branched polyalkylene glycols |
US7060675B2 (en) * | 2001-02-15 | 2006-06-13 | Nobex Corporation | Methods of treating diabetes mellitus |
MXPA03008498A (en) * | 2001-03-20 | 2005-06-30 | Univ Zuerich | Two-phase processing of thermosensitive polymers for use as biomaterials. |
US6828305B2 (en) | 2001-06-04 | 2004-12-07 | Nobex Corporation | Mixtures of growth hormone drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same |
US6828297B2 (en) | 2001-06-04 | 2004-12-07 | Nobex Corporation | Mixtures of insulin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same |
US6713452B2 (en) | 2001-06-04 | 2004-03-30 | Nobex Corporation | Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same |
US7713932B2 (en) | 2001-06-04 | 2010-05-11 | Biocon Limited | Calcitonin drug-oligomer conjugates, and uses thereof |
US20040077835A1 (en) * | 2001-07-12 | 2004-04-22 | Robin Offord | Chemokine receptor modulators, production and use |
WO2003018665A1 (en) | 2001-08-22 | 2003-03-06 | Bioartificial Gel Technologies Inc. | Process for the preparation of activated polyethylene glycols |
KR100761652B1 (en) * | 2001-08-25 | 2007-10-04 | 동아제약주식회사 | multi-branched polymer used in conjugating protein or peptide, and resulting conjugator |
US7312192B2 (en) * | 2001-09-07 | 2007-12-25 | Biocon Limited | Insulin polypeptide-oligomer conjugates, proinsulin polypeptide-oligomer conjugates and methods of synthesizing same |
US6913903B2 (en) * | 2001-09-07 | 2005-07-05 | Nobex Corporation | Methods of synthesizing insulin polypeptide-oligomer conjugates, and proinsulin polypeptide-oligomer conjugates and methods of synthesizing same |
US6770625B2 (en) | 2001-09-07 | 2004-08-03 | Nobex Corporation | Pharmaceutical compositions of calcitonin drug-oligomer conjugates and methods of treating diseases therewith |
US7030082B2 (en) * | 2001-09-07 | 2006-04-18 | Nobex Corporation | Pharmaceutical compositions of drug-oligomer conjugates and methods of treating disease therewith |
US7166571B2 (en) * | 2001-09-07 | 2007-01-23 | Biocon Limited | Insulin polypeptide-oligomer conjugates, proinsulin polypeptide-oligomer conjugates and methods of synthesizing same |
US7179617B2 (en) * | 2001-10-10 | 2007-02-20 | Neose Technologies, Inc. | Factor IX: remolding and glycoconjugation of Factor IX |
US7157277B2 (en) | 2001-11-28 | 2007-01-02 | Neose Technologies, Inc. | Factor VIII remodeling and glycoconjugation of Factor VIII |
US7026440B2 (en) | 2001-11-07 | 2006-04-11 | Nektar Therapeutics Al, Corporation | Branched polymers and their conjugates |
AU2002359364A1 (en) * | 2001-11-09 | 2003-05-26 | Enzon, Inc. | Polymeric thiol-linked prodrugs employing benzyl elimination systems |
US20030171285A1 (en) * | 2001-11-20 | 2003-09-11 | Finn Rory F. | Chemically-modified human growth hormone conjugates |
US7473680B2 (en) | 2001-11-28 | 2009-01-06 | Neose Technologies, Inc. | Remodeling and glycoconjugation of peptides |
EP1468036B1 (en) | 2002-01-14 | 2008-10-08 | The General Hospital Corporation | Biodegradable polyketal polymers and methods for their formation and use |
US7144978B2 (en) * | 2002-01-15 | 2006-12-05 | Pan Asia Bio Co., Ltd. | Multidrop tree branching functional polyethylene glycol, methods of preparing and using same |
CN1176137C (en) * | 2002-01-15 | 2004-11-17 | 泛亚生物技术有限公司 | Multi-arm fork type functional polyethylene glycol preparation method and its application in medicine |
WO2003062290A1 (en) | 2002-01-16 | 2003-07-31 | Biocompatibles Uk Limited | Polymer conjugates |
EA009783B1 (en) | 2002-01-18 | 2008-04-28 | Байоджен Айдек Ма Инк. | Polyalkylene glycolhaving a moiety for conjugation of a biologically active compound |
US20030229333A1 (en) * | 2002-02-22 | 2003-12-11 | Control Delivery Systems, Inc. | Methods for treating otic disorders |
EP1489167A4 (en) * | 2002-03-01 | 2006-06-07 | Nat Inst Of Advanced Ind Scien | Immobilized cells and liposomes and method of immobilizing the same |
US20030179692A1 (en) * | 2002-03-19 | 2003-09-25 | Yoshitaka Ohotomo | Storage medium |
WO2003078461A1 (en) * | 2002-03-20 | 2003-09-25 | Biopolymed Inc. | Preparation of g-csf stoichiometrically conjugated with biocompatible polymers at cystein residue |
US8282912B2 (en) * | 2002-03-22 | 2012-10-09 | Kuros Biosurgery, AG | Compositions for tissue augmentation |
ATE518885T1 (en) * | 2002-05-28 | 2011-08-15 | Ucb Pharma Sa | PEG POSITION ISOMER OF AN ANTIBODY TO TNFALPHA (CDP870) |
JP2005534647A (en) | 2002-06-07 | 2005-11-17 | ダイアックス、コープ | Prevention and reduction of blood loss |
US7153829B2 (en) | 2002-06-07 | 2006-12-26 | Dyax Corp. | Kallikrein-inhibitor therapies |
DE60336555D1 (en) | 2002-06-21 | 2011-05-12 | Novo Nordisk Healthcare Ag | PEGYLATED GLYCO FORMS OF FACTOR VII |
WO2004002469A1 (en) * | 2002-06-29 | 2004-01-08 | Aquanova German Solubilisate Technologies (Agt) Gmbh | Isoflavone concentrate and method for production thereof |
US8227411B2 (en) * | 2002-08-20 | 2012-07-24 | BioSurface Engineering Technologies, Incle | FGF growth factor analogs |
US7598224B2 (en) | 2002-08-20 | 2009-10-06 | Biosurface Engineering Technologies, Inc. | Dual chain synthetic heparin-binding growth factor analogs |
US7166574B2 (en) | 2002-08-20 | 2007-01-23 | Biosurface Engineering Technologies, Inc. | Synthetic heparin-binding growth factor analogs |
AU2003265361A1 (en) * | 2002-08-28 | 2004-03-19 | Pharmacia Corporation | Stable ph optimized formulation of a modified antibody |
WO2004019860A2 (en) * | 2002-08-28 | 2004-03-11 | Pharmacia Corporation | Formulations of modified antibodies and methods of making the same |
AU2003270118A1 (en) * | 2002-08-30 | 2004-03-19 | F. Hoffmann-La Roche Ag | Scatter factor/hepatocyte growth factor antagonist nk4 for the treatment of glioma |
CA2498062C (en) * | 2002-09-27 | 2010-05-25 | F. Hoffmann-La Roche Ag | Conjugates of insulin-like growth factor binding protein-4 and poly(ethylene glycol) |
US20040062748A1 (en) * | 2002-09-30 | 2004-04-01 | Mountain View Pharmaceuticals, Inc. | Polymer conjugates with decreased antigenicity, methods of preparation and uses thereof |
US8129330B2 (en) * | 2002-09-30 | 2012-03-06 | Mountain View Pharmaceuticals, Inc. | Polymer conjugates with decreased antigenicity, methods of preparation and uses thereof |
TWI281864B (en) * | 2002-11-20 | 2007-06-01 | Pharmacia Corp | N-terminally monopegylated human growth hormone conjugates and process for their preparation |
TWI364295B (en) * | 2002-12-26 | 2012-05-21 | Mountain View Pharmaceuticals | Polymer conjugates of cytokines, chemokines, growth factors, polypeptide hormones and antagonists thereof with preserved receptor-binding activity |
PL219741B1 (en) * | 2002-12-26 | 2015-07-31 | Mountain View Pharmaceuticals | Polymer conjugates of interferon-beta with enhanced biological potency |
CA2509260C (en) | 2002-12-31 | 2012-10-02 | Nektar Therapeutics Al, Corporation | Maleamic acid polymer derivatives and their bioconjugates |
KR101090784B1 (en) * | 2002-12-31 | 2011-12-08 | 넥타르 테라퓨틱스 | Polymeric reagents comprising a ketone or a related functional group |
US7432331B2 (en) | 2002-12-31 | 2008-10-07 | Nektar Therapeutics Al, Corporation | Hydrolytically stable maleimide-terminated polymers |
US7432330B2 (en) * | 2002-12-31 | 2008-10-07 | Nektar Therapeutics Al, Corporation | Hydrolytically stable maleimide-terminated polymers |
DE602004029646D1 (en) | 2003-01-06 | 2010-12-02 | Nektar Therapeutics San Carlos | Thiolselektive wasserlösliche polymerderivate |
US7553930B2 (en) * | 2003-01-06 | 2009-06-30 | Xencor, Inc. | BAFF variants and methods thereof |
US20050221443A1 (en) * | 2003-01-06 | 2005-10-06 | Xencor, Inc. | Tumor necrosis factor super family agonists |
US20050130892A1 (en) * | 2003-03-07 | 2005-06-16 | Xencor, Inc. | BAFF variants and methods thereof |
US20060014248A1 (en) * | 2003-01-06 | 2006-01-19 | Xencor, Inc. | TNF super family members with altered immunogenicity |
GB0301014D0 (en) * | 2003-01-16 | 2003-02-19 | Biocompatibles Ltd | Conjugation reactions |
WO2004074345A2 (en) * | 2003-02-19 | 2004-09-02 | Pharmacia Corporation | Carbonate esters of polyethylene glycol activated by means of oxalate esters |
SI1596887T1 (en) * | 2003-02-26 | 2022-05-31 | Nektar Therapeutics | Polymer-factor viii moiety conjugates |
US20090123367A1 (en) * | 2003-03-05 | 2009-05-14 | Delfmems | Soluble Glycosaminoglycanases and Methods of Preparing and Using Soluble Glycosaminoglycanases |
US7871607B2 (en) | 2003-03-05 | 2011-01-18 | Halozyme, Inc. | Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases |
CA2517145C (en) * | 2003-03-05 | 2017-08-01 | Halozyme, Inc. | Soluble hyaluronidase glycoprotein (shasegp), process for preparing the same, uses and pharmaceutical compositions comprising thereof |
US20060104968A1 (en) | 2003-03-05 | 2006-05-18 | Halozyme, Inc. | Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminogly ycanases |
US7610156B2 (en) * | 2003-03-31 | 2009-10-27 | Xencor, Inc. | Methods for rational pegylation of proteins |
US7642340B2 (en) | 2003-03-31 | 2010-01-05 | Xencor, Inc. | PEGylated TNF-α variant proteins |
AU2004227937B2 (en) * | 2003-03-31 | 2007-09-20 | Xencor, Inc | Methods for rational pegylation of proteins |
US7691603B2 (en) * | 2003-04-09 | 2010-04-06 | Novo Nordisk A/S | Intracellular formation of peptide conjugates |
WO2004091517A2 (en) | 2003-04-15 | 2004-10-28 | Smithkline Beecham Corporation | Conjugates comprising human il-18 and substitution mutants thereof |
US7947261B2 (en) | 2003-05-23 | 2011-05-24 | Nektar Therapeutics | Conjugates formed from polymer derivatives having particular atom arrangements |
ES2725808T3 (en) * | 2003-05-23 | 2019-09-27 | Nektar Therapeutics | PEG derivatives containing two PEG chains |
EP1656410B1 (en) | 2003-07-22 | 2010-03-10 | Nektar Therapeutics | Method for preparing functionalized polymers from polymer alcohols |
JP2007501812A (en) * | 2003-08-08 | 2007-02-01 | ノボ ノルディスク アクティーゼルスカブ | Synthesis and application of new structurally well-defined branched polymers as binders for peptides |
ES2388138T3 (en) * | 2003-08-27 | 2012-10-09 | Ophthotech Corporation | Combination therapy for the treatment of ocular neovascular disorders |
CA2536873C (en) * | 2003-08-29 | 2019-09-10 | Dyax Corp. | Poly-pegylated protease inhibitors |
EP1675871A2 (en) | 2003-10-10 | 2006-07-05 | Xencor Inc. | Protein based tnf-alpha variants for the treatment of tnf-alpha related disorders |
US20050214250A1 (en) | 2003-11-06 | 2005-09-29 | Harris J M | Method of preparing carboxylic acid functionalized polymers |
EP1694347B1 (en) * | 2003-11-24 | 2013-11-20 | BioGeneriX AG | Glycopegylated erythropoietin |
US20060040856A1 (en) * | 2003-12-03 | 2006-02-23 | Neose Technologies, Inc. | Glycopegylated factor IX |
WO2005055950A2 (en) * | 2003-12-03 | 2005-06-23 | Neose Technologies, Inc. | Glycopegylated factor ix |
US7790835B2 (en) * | 2003-12-03 | 2010-09-07 | Nektar Therapeutics | Method of preparing maleimide functionalized polymers |
US20070254836A1 (en) * | 2003-12-03 | 2007-11-01 | Defrees Shawn | Glycopegylated Granulocyte Colony Stimulating Factor |
US20080318850A1 (en) * | 2003-12-03 | 2008-12-25 | Neose Technologies, Inc. | Glycopegylated Factor Ix |
GB0329825D0 (en) * | 2003-12-23 | 2004-01-28 | Celltech R&D Ltd | Biological products |
KR101439880B1 (en) * | 2004-01-08 | 2014-09-12 | 라티오팜 게엠베하 | O-linked glycosylation of peptides |
EP1720892B1 (en) * | 2004-01-26 | 2013-07-24 | BioGeneriX AG | Branched polymer-modified sugars and nucleotides |
US7414028B1 (en) * | 2004-02-04 | 2008-08-19 | Biosurface Engineering Technologies, Inc. | Growth factor analogs |
US20080227696A1 (en) * | 2005-02-22 | 2008-09-18 | Biosurface Engineering Technologies, Inc. | Single branch heparin-binding growth factor analogs |
US7528105B1 (en) | 2004-02-10 | 2009-05-05 | Biosurface Engineering Technologies | Heterodimeric chain synthetic heparin-binding growth factor analogs |
US7671012B2 (en) | 2004-02-10 | 2010-03-02 | Biosurface Engineering Technologies, Inc. | Formulations and methods for delivery of growth factor analogs |
US20060024347A1 (en) * | 2004-02-10 | 2006-02-02 | Biosurface Engineering Technologies, Inc. | Bioactive peptide coatings |
US6887952B1 (en) * | 2004-02-12 | 2005-05-03 | Biosite, Inc. | N-aryl-carbamic acid ester-derived and valeric acid ester-derived cross-linkers and conjugates, and methods for their synthesis and use |
US7803931B2 (en) | 2004-02-12 | 2010-09-28 | Archemix Corp. | Aptamer therapeutics useful in the treatment of complement-related disorders |
WO2005082005A2 (en) | 2004-02-20 | 2005-09-09 | Biosurface Engineering Technologies, Inc., Et Al. | Positive modulator of bone morphogenic protein-2 |
US7351787B2 (en) * | 2004-03-05 | 2008-04-01 | Bioartificial Gel Technologies, Inc. | Process for the preparation of activated polyethylene glycols |
CN101132812A (en) * | 2004-03-15 | 2008-02-27 | 阿拉巴马耐科塔医药公司 | Polymer-based compositions and conjuates of HIV entry inhibitors |
US20070265200A1 (en) * | 2004-03-17 | 2007-11-15 | Eli Lilly And Company | Glycol Linked Fgf-21 Compounds |
AU2005224078B2 (en) * | 2004-03-17 | 2011-01-27 | Anticancer, Inc. | Methods for increasing protein polyethylene glycol (PEG) conjugation |
US9085659B2 (en) * | 2004-05-03 | 2015-07-21 | Nektar Therapeutics | Polymer derivatives comprising an imide branching point |
US7476725B2 (en) * | 2004-06-08 | 2009-01-13 | Alza Corporation | Preparation of macromolecular conjugates by four-component condensation reaction |
ES2355642T3 (en) * | 2004-07-16 | 2011-03-29 | Nektar Therapeutics | CONJUGATES UNDERSTANDING A PORTION OF GM-CSF AND A POLYMER. |
CA2910494C (en) | 2004-07-19 | 2018-10-23 | Biocon Limited | Insulin-oligomer conjugates, formulations and uses thereof |
MX2007000728A (en) * | 2004-07-21 | 2007-03-15 | Ambrx Inc | Biosynthetic polypeptides utilizing non-naturally encoded amino acids. |
US20060040377A1 (en) * | 2004-08-17 | 2006-02-23 | Biocept, Inc. | Protein microarrays |
EP1794588A2 (en) * | 2004-09-09 | 2007-06-13 | Biosite Incorporated | Methods and compositions for measuring canine bnp and uses thereof |
US20060204512A1 (en) | 2004-09-23 | 2006-09-14 | Vasgene Therapeutics, Inc. | Polypeptide compounds for inhibiting angiogenesis and tumor growth |
US7235530B2 (en) | 2004-09-27 | 2007-06-26 | Dyax Corporation | Kallikrein inhibitors and anti-thrombolytic agents and uses thereof |
US7851565B2 (en) | 2004-12-21 | 2010-12-14 | Nektar Therapeutics | Stabilized polymeric thiol reagents |
EP1674113A1 (en) * | 2004-12-22 | 2006-06-28 | F. Hoffmann-La Roche Ag | Conjugates of insulin-like growth factor-1 (IGF-1) and poly(ethylene glycol) |
WO2006078813A2 (en) * | 2005-01-21 | 2006-07-27 | Biosite Incorporated | Arginine analogs, and methods for their synthesis and use |
DE602005015817D1 (en) * | 2005-01-25 | 2009-09-17 | Varian B V | Chromatographic columns |
US7365127B2 (en) * | 2005-02-04 | 2008-04-29 | Enzon Pharmaceuticals, Inc. | Process for the preparation of polymer conjugates |
AU2006213822B2 (en) * | 2005-02-09 | 2011-05-26 | Covidien Lp | Synthetic sealants |
EP1857462B9 (en) | 2005-02-18 | 2013-02-13 | Nof Corporation | Polyoxyalkylene derivative |
EP1861125A2 (en) * | 2005-03-23 | 2007-12-05 | Nektar Therapeutics Al, Corporation | Conjugates of an hgh moiety and peg derivatives |
US20060246544A1 (en) * | 2005-03-30 | 2006-11-02 | Neose Technologies,Inc. | Manufacturing process for the production of peptides grown in insect cell lines |
US20060222596A1 (en) | 2005-04-01 | 2006-10-05 | Trivascular, Inc. | Non-degradable, low swelling, water soluble radiopaque hydrogel polymer |
US9534013B2 (en) * | 2006-04-12 | 2017-01-03 | Horizon Pharma Rheumatology Llc | Purification of proteins with cationic surfactant |
CN101194016B (en) | 2005-04-11 | 2012-09-05 | 萨文特医药公司 | A variant form of urate oxidase and use thereof |
US8148123B2 (en) | 2005-04-11 | 2012-04-03 | Savient Pharmaceuticals, Inc. | Methods for lowering elevated uric acid levels using intravenous injections of PEG-uricase |
PT3321359T (en) | 2005-04-11 | 2021-03-11 | Horizon Pharma Rheumatology Llc | Variant forms of urate oxidase and use thereof |
US20080159976A1 (en) * | 2005-04-11 | 2008-07-03 | Jacob Hartman | Methods for lowering elevated uric acid levels using intravenous injections of PEG-uricase |
WO2006110776A2 (en) | 2005-04-12 | 2006-10-19 | Nektar Therapeutics Al, Corporation | Polyethylene glycol cojugates of antimicrobial agents |
US7833979B2 (en) * | 2005-04-22 | 2010-11-16 | Amgen Inc. | Toxin peptide therapeutic agents |
PT1881850E (en) | 2005-05-13 | 2010-11-26 | Lilly Co Eli | Glp-1 pegylated compounds |
JP2008545665A (en) * | 2005-05-23 | 2008-12-18 | ユニベルシテ ドゥ ジュネーブ | Injectable superparamagnetic nanoparticles for hyperthermic treatment and use to form hyperthermic implants |
US9505867B2 (en) * | 2005-05-31 | 2016-11-29 | Ecole Polytechmique Fédérale De Lausanne | Triblock copolymers for cytoplasmic delivery of gene-based drugs |
EP2279758B1 (en) | 2005-06-16 | 2015-02-25 | Nektar Therapeutics | Conjugates having a degradable linkage and polymeric reagents useful in preparing such conjugates |
US8728493B2 (en) * | 2005-06-17 | 2014-05-20 | Nektar Therapeutics | Polymer based compositions and conjugates of non-steroidal anti-inflammatory drugs |
EP2412744B1 (en) | 2005-07-18 | 2014-01-22 | Nektar Therapeutics | Method for preparing branched functionalised polymers using branched polyol cores |
ES2341285T3 (en) * | 2005-07-19 | 2010-06-17 | Nektar Therapeutics | METHOD TO PREPARE POLYMERIC MALEIMIDS. |
ES2529258T3 (en) | 2005-07-29 | 2015-02-18 | Nektar Therapeutics | Methods for preparing polymer reagents |
US8008453B2 (en) | 2005-08-12 | 2011-08-30 | Amgen Inc. | Modified Fc molecules |
CN103103238B (en) | 2005-08-18 | 2016-08-10 | Ambrx公司 | A kind of manufacture in cell has selected amino acid whose antibody or the method for antibody fragment polypeptide in specific location |
KR20080080081A (en) * | 2005-08-19 | 2008-09-02 | 네오스 테크놀로지스, 인크. | Glycopegylated factor vii and factor viia |
KR100664969B1 (en) | 2005-08-26 | 2007-01-04 | 아이디비켐(주) | A new preparing method of methoxypolyethyleneglycol and its derivatives |
AU2006311568B2 (en) * | 2005-11-08 | 2010-11-11 | Ambrx, Inc. | Accelerants for the modification of non-natural amino acids and non-natural amino acid polypeptides |
DK1968635T3 (en) * | 2005-12-14 | 2014-12-15 | Ambrx Inc | Compositions and Methods of, and uses of non-natural amino acids and polypeptides |
US8293869B2 (en) * | 2005-12-16 | 2012-10-23 | Nektar Therapeutics | Polymer conjugates of GLP-1 |
US7743730B2 (en) * | 2005-12-21 | 2010-06-29 | Lam Research Corporation | Apparatus for an optimized plasma chamber grounded electrode assembly |
EP1981525B1 (en) * | 2005-12-30 | 2015-01-21 | Zensun (Shanghai) Science and Technology Limited | Extended release of neuregulin for improved cardiac function |
EP2319542B1 (en) | 2006-02-21 | 2018-03-21 | Nektar Therapeutics | Segmented degradable polymers and conjugates made therefrom |
US7928058B2 (en) | 2006-02-22 | 2011-04-19 | Merck Sharp & Dohme Corp. | Pharmaceutical composition comprising oxyntomodulin derivatives and a method for reducing body weight using the composition |
DE602007008074D1 (en) * | 2006-02-28 | 2010-09-09 | Reddy S Lab Eu Ltd Dr | METHOD FOR PRODUCING POLYETHYLENE GLYCOL CARBONATES |
PL2596807T3 (en) * | 2006-03-08 | 2016-06-30 | Archemix Llc | Complement binding aptamers and anti-C5 agents useful in the treatment of ocular disorders |
CA2645844C (en) | 2006-03-13 | 2016-04-26 | Liat Mintz | Use of ghrelin splice variant for treating cachexia and/or anorexia and/or anorexia-cachexia and/or malnutrition and/or lipodystrophy and/or muscle wasting and/or appetite-stimulation |
US8795709B2 (en) * | 2006-03-29 | 2014-08-05 | Incept Llc | Superabsorbent, freeze dried hydrogels for medical applications |
BRPI0709427A2 (en) | 2006-03-30 | 2011-07-12 | Palatin Technologies Inc | "Cyclic construct, pharmaceutical composition and use of a compound |
US8580746B2 (en) * | 2006-03-30 | 2013-11-12 | Palatin Technologies, Inc. | Amide linkage cyclic natriuretic peptide constructs |
EP2004633A4 (en) * | 2006-03-30 | 2009-08-26 | Palatin Technologies Inc | Linear natriuretic peptide constructs |
EP2004231A4 (en) | 2006-04-07 | 2013-07-10 | Nektar Therapeutics | Conjugates of an anti-tnf-alpha antibody |
EP2573111A1 (en) | 2006-04-20 | 2013-03-27 | Amgen Inc. | GLP-1 compounds |
EP2010539B1 (en) | 2006-04-21 | 2017-06-14 | Nektar Therapeutics | Stereoselective reduction of a morphinone |
US7872068B2 (en) * | 2006-05-30 | 2011-01-18 | Incept Llc | Materials formable in situ within a medical device |
US7820172B1 (en) | 2006-06-01 | 2010-10-26 | Biosurface Engineering Technologies, Inc. | Laminin-derived multi-domain peptides |
EP2046350A4 (en) | 2006-06-22 | 2011-09-14 | Biosurface Eng Tech Inc | Composition and method for delivery of bmp-2 amplifier/co-activator for enhancement of osteogenesis |
US8008948B2 (en) * | 2006-07-06 | 2011-08-30 | Denso Corporation | Peak voltage detector circuit and binarizing circuit including the same circuit |
EP2044150B1 (en) * | 2006-07-21 | 2014-01-15 | Nektar Therapeutics | Polymeric reagents comprising a terminal vinylic group and conjugates formed therefrom |
KR101106795B1 (en) * | 2006-08-31 | 2012-01-18 | 에프. 호프만-라 로슈 아게 | Method for the production of insulin-like growth factor-i |
CL2007002502A1 (en) | 2006-08-31 | 2008-05-30 | Hoffmann La Roche | VARIANTS OF THE SIMILAR GROWTH FACTOR TO HUMAN INSULIN-1 (IGF-1) PEGILATED IN LISIN; METHOD OF PRODUCTION; FUSION PROTEIN THAT UNDERSTANDS IT; AND ITS USE TO TREAT ALZHEIMER'S DISEASE. |
NZ597098A (en) | 2006-09-28 | 2013-05-31 | Merck Sharp & Dohme | Use of pegylated il-10 to treat cancer |
WO2008051383A2 (en) * | 2006-10-19 | 2008-05-02 | Amgen Inc. | Use of alcohol co-solvents to improve pegylation reaction yields |
PE20081140A1 (en) * | 2006-10-25 | 2008-09-22 | Amgen Inc | THERAPEUTIC AGENTS BASED ON PEPTIDES DERIVED FROM TOXINS |
WO2008073620A2 (en) * | 2006-11-02 | 2008-06-19 | Neose Technologies, Inc. | Manufacturing process for the production of polypeptides expressed in insect cell-lines |
ATE459659T1 (en) * | 2006-11-07 | 2010-03-15 | Dsm Ip Assets Bv | CARBAMAT, THIOCARBAMAT OR CARBAMIDE WITH A BIOMOLECULAR GROUP |
AU2007325838B2 (en) | 2006-11-22 | 2013-09-19 | Bristol-Myers Squibb Company | Targeted therapeutics based on engineered proteins for tyrosine kinases receptors, including IGF-IR |
CN101583380B (en) * | 2006-11-30 | 2013-07-10 | 尼克塔治疗公司 | Method for preparing a polymer conjugate |
JP5340956B2 (en) * | 2006-12-20 | 2013-11-13 | アーケマ・インコーポレイテッド | Encapsulation and / or binding of polymers |
NZ577728A (en) | 2006-12-27 | 2012-01-12 | Baxter Int | Von willebrand factor- and factor viii-polymer conjugates having a releasable linkage |
US8507653B2 (en) * | 2006-12-27 | 2013-08-13 | Nektar Therapeutics | Factor IX moiety-polymer conjugates having a releasable linkage |
PT2842967T (en) | 2007-01-18 | 2017-01-13 | Lilly Co Eli | Pegylated amyloid beta fab |
CN101677588A (en) * | 2007-02-01 | 2010-03-24 | 加拿大国家研究委员会 | formulations of lipophilic bioactive molecules |
US20100144599A1 (en) | 2007-02-02 | 2010-06-10 | Bristol-Myers Squibb Company | Vegf pathway blockade |
JP2010522583A (en) | 2007-02-27 | 2010-07-08 | トラスティーズ オブ タフツ カレッジ | Silk organs made by tissue engineering |
US20090227981A1 (en) * | 2007-03-05 | 2009-09-10 | Bennett Steven L | Low-Swelling Biocompatible Hydrogels |
US20090227689A1 (en) * | 2007-03-05 | 2009-09-10 | Bennett Steven L | Low-Swelling Biocompatible Hydrogels |
PL2136850T3 (en) * | 2007-04-13 | 2012-07-31 | Kuros Biosurgery Ag | Polymeric tissue sealant |
MX2009012609A (en) | 2007-05-22 | 2009-12-07 | Amgen Inc | Compositions and methods for producing bioactive fusion proteins. |
WO2009027844A2 (en) * | 2007-05-25 | 2009-03-05 | Celtic Pharma Management L.P. | Crf conjugates with extended half-lives |
DK2211876T3 (en) | 2007-05-29 | 2015-01-12 | Tufts College | PROCESS FOR silk fibroin-GELATION USING sonication |
AR067536A1 (en) * | 2007-07-17 | 2009-10-14 | Hoffmann La Roche | METHOD FOR OBTAINING A MONO-PEGILATED ERYTHROPOYETIN IN A SUBSTANTIALLY HOMOGENOUS FORM |
AR067537A1 (en) * | 2007-07-17 | 2009-10-14 | Hoffmann La Roche | PURIFIED POLYPEPTIDES PURIFICATION |
US8067028B2 (en) * | 2007-08-13 | 2011-11-29 | Confluent Surgical Inc. | Drug delivery device |
US20090075887A1 (en) * | 2007-08-21 | 2009-03-19 | Genzyme Corporation | Treatment with Kallikrein Inhibitors |
US8088884B2 (en) * | 2007-09-27 | 2012-01-03 | Serina Therapeutics, Inc. | Multi-armed forms of activated polyoxazoline and methods of synthesis thereof |
EP2205271B1 (en) * | 2007-10-08 | 2014-05-21 | Quintessence Biosciences, Inc. | Compositions and methods for ribonuclease-based therapies |
MX2010003979A (en) | 2007-10-16 | 2010-06-02 | Biocon Ltd | An orally administerable solid pharmaceutical composition and a process thereof. |
EP2214716B1 (en) | 2007-10-23 | 2021-11-17 | Nektar Therapeutics | Hydroxyapatite-targeting multiarm polymers and conjugates made therefrom |
WO2009089542A2 (en) | 2008-01-11 | 2009-07-16 | Serina Therapeutics, Inc. | Multifunctional forms of polyoxazoline copolymers and drug compositions comprising the same |
US8101706B2 (en) | 2008-01-11 | 2012-01-24 | Serina Therapeutics, Inc. | Multifunctional forms of polyoxazoline copolymers and drug compositions comprising the same |
US7862538B2 (en) * | 2008-02-04 | 2011-01-04 | Incept Llc | Surgical delivery system for medical sealant |
TWI395593B (en) | 2008-03-06 | 2013-05-11 | Halozyme Inc | In vivo temporal control of activatable matrix-degrading enzymes |
CA2720478A1 (en) * | 2008-04-03 | 2009-10-08 | F. Hoffmann-La Roche Ag | Pegylated insulin-like-growth-factor assay |
MX2010010495A (en) * | 2008-04-03 | 2010-10-15 | Hoffmann La Roche | Use of pegylated igf-i variants for the treatment of neuromuscular disorders. |
SG187427A1 (en) | 2008-04-14 | 2013-02-28 | Halozyme Inc | Modified hyaluronidases and uses in treating hyaluronan-associated diseases and conditions |
TWI394580B (en) | 2008-04-28 | 2013-05-01 | Halozyme Inc | Super fast-acting insulin compositions |
CA2722426A1 (en) * | 2008-04-30 | 2009-11-05 | Neutron Row | Methods of using corticotropin-releasing factor for the treatment of cancer |
KR20110008075A (en) * | 2008-05-16 | 2011-01-25 | 넥타르 테라퓨틱스 | Conjugates of a cholinesterase moiety and a polymer |
AR071874A1 (en) | 2008-05-22 | 2010-07-21 | Bristol Myers Squibb Co | ARMAZON DOMAIN PROTEINS BASED ON MULTIVALENT FIBRONECTINE |
JP5639585B2 (en) * | 2008-07-31 | 2014-12-10 | ファーマエッセンティア コーポレイション | Peptide-polymer conjugate |
US8575102B2 (en) * | 2008-08-01 | 2013-11-05 | Nektar Therapeutics | Conjugates having a releasable linkage |
KR101671537B1 (en) | 2008-08-11 | 2016-11-01 | 넥타르 테라퓨틱스 | Multi-arm polymeric alkanoate Conjugates |
EP2326351B1 (en) | 2008-08-19 | 2017-12-27 | Nektar Therapeutics | Conjugates of small-interfering nucleic acids |
WO2010030366A2 (en) | 2008-09-11 | 2010-03-18 | Nektar Therapeutics | Polymeric alpha-hydroxy aldehyde and ketone reagents and conjugation method |
US20110171161A1 (en) * | 2008-09-19 | 2011-07-14 | Nektar Therapeutics | Polymer conjugates of protegrin peptides |
US20110171164A1 (en) * | 2008-09-19 | 2011-07-14 | Nektar Therapeutics | Polymer conjugates of glp-2-like peptides |
EP2334338A2 (en) * | 2008-09-19 | 2011-06-22 | Nektar Therapeutics | Polymer conjugates of c-peptides |
WO2010033216A1 (en) | 2008-09-19 | 2010-03-25 | Nektar Therapeutics | Polymer conjugates of nesiritide peptides |
EP2344200A2 (en) * | 2008-09-19 | 2011-07-20 | Nektar Therapeutics | Modified therapeutics peptides, methods of their preparation and use |
WO2010033222A2 (en) * | 2008-09-19 | 2010-03-25 | Netkar Therapeutics | Polymer conjugates of ziconotide peptides |
EP2344199A1 (en) * | 2008-09-19 | 2011-07-20 | Nektar Therapeutics | Polymer conjugates of thymosin alpha 1 peptides |
US20110171166A1 (en) * | 2008-09-19 | 2011-07-14 | Nektar Therapeutics | Polymer conjugates of osteocalcin peptides |
WO2010033205A1 (en) * | 2008-09-19 | 2010-03-25 | Nektar Therapeutics | Polymer conjugates of v681-like peptides |
WO2010033215A2 (en) * | 2008-09-19 | 2010-03-25 | Nektar Therapeutics | Polymer conjugates of aod-like peptides |
WO2010033224A1 (en) * | 2008-09-19 | 2010-03-25 | Nektar Therapeutics | Polymer conjugates of kiss1 peptides |
EP2334337A1 (en) * | 2008-09-19 | 2011-06-22 | Nektar Therapeutics | Polymer conjugates of opioid growth factor peptides |
EP2350118B1 (en) * | 2008-09-19 | 2016-03-30 | Nektar Therapeutics | Carbohydrate-based drug delivery polymers and conjugates thereof |
TWI496582B (en) | 2008-11-24 | 2015-08-21 | 必治妥美雅史谷比公司 | Bispecific egfr/igfir binding molecules |
JP5814793B2 (en) | 2008-11-25 | 2015-11-17 | エコール ポリテクニク フェデラル ド ローザンヌ(エーペーエフエル) | Block copolymer and use thereof |
KR101546563B1 (en) | 2008-12-09 | 2015-08-28 | 할로자임, 아이엔씨 | Extended soluble ph20 polypeptides and uses thereof |
HUE037936T2 (en) | 2008-12-17 | 2018-09-28 | Merck Sharp & Dohme | Mono- and di-peg il-10 production; and uses |
AU2010203712A1 (en) * | 2009-01-06 | 2010-07-15 | Dyax Corp. | Treatment of mucositis with kallikrein inhibitors |
US20110318322A1 (en) | 2009-01-12 | 2011-12-29 | Nektar Therapeutics | Conjugates of a Lysosomal Enzyme Moiety and a Water Soluble Polymer |
EP2396070A4 (en) | 2009-02-12 | 2012-09-19 | Incept Llc | Drug delivery through hydrogel plugs |
CA2754896C (en) * | 2009-03-09 | 2017-11-28 | Molecular Express, Inc. | Methods and compositions for liposomal formulation of antigens and uses thereof |
JP5569787B2 (en) * | 2009-03-31 | 2014-08-13 | 日油株式会社 | Purification method of high molecular weight polyethylene glycol compound |
ES2427627T3 (en) | 2009-04-06 | 2013-10-31 | Novo Nordisk A/S | Targeted delivery of Factor VIII proteins to platelets |
US8067201B2 (en) * | 2009-04-17 | 2011-11-29 | Bristol-Myers Squibb Company | Methods for protein refolding |
CN101870728A (en) | 2009-04-23 | 2010-10-27 | 派格生物医药(苏州)有限公司 | Novel Exendin variant and conjugate thereof |
CA2760704C (en) * | 2009-05-04 | 2017-10-03 | Incept, Llc | Biomaterials for track and puncture closure |
SG10201408480RA (en) | 2009-06-25 | 2015-02-27 | Crealta Pharmaceuticals Llc | Methods for preventing or predicting infusion reactions or antibody-mediated loss of response, by monitoring serum uric acid levels during pegylated uricase therapy |
WO2011003633A1 (en) | 2009-07-06 | 2011-01-13 | Alize Pharma Ii | Pegylated l-asparaginase |
WO2011035065A1 (en) | 2009-09-17 | 2011-03-24 | Nektar Therapeutics | Monoconjugated chitosans as delivery agents for small interfering nucleic acids |
IN2012DN03219A (en) | 2009-09-17 | 2015-10-23 | Baxter Healthcare Sa | |
WO2011038401A2 (en) | 2009-09-28 | 2011-03-31 | Trustees Of Tufts College | Drawn silk egel fibers and methods of making same |
CA2778678A1 (en) | 2009-10-30 | 2011-05-05 | Cns Therapeutics, Inc. | Improved neurturin molecules |
US20110136727A1 (en) * | 2009-11-20 | 2011-06-09 | Sergei Svarovsky | Compositions and methods for rapid selection of pathogen binding agents |
JO2976B1 (en) | 2009-12-22 | 2016-03-15 | ايلي ليلي اند كومباني | Oxyntomodulin peptide analogue |
AR079344A1 (en) | 2009-12-22 | 2012-01-18 | Lilly Co Eli | PEPTIDAL ANALOG OF OXINTOMODULIN, PHARMACEUTICAL COMPOSITION THAT UNDERSTANDS AND USES TO PREPARE A USEFUL MEDICINAL PRODUCT TO TREAT NON-INSULINED INDEPENDENT DIABETES AND / OR OBESITY |
US20110152188A1 (en) * | 2009-12-23 | 2011-06-23 | Hanns-Christian Mahler | Pharmaceutical compositions of igf/i proteins |
SI2521568T1 (en) * | 2010-01-06 | 2019-01-31 | Dyax Corp. | Plasma kallikrein binding proteins |
WO2011098400A1 (en) | 2010-02-11 | 2011-08-18 | F. Hoffmann-La Roche Ag | Igf-i poly (ethylene glycol) conjugates |
US9493543B2 (en) | 2010-02-16 | 2016-11-15 | Novo Nordisk A/S | Factor VIII fusion protein |
WO2011101242A1 (en) | 2010-02-16 | 2011-08-25 | Novo Nordisk A/S | Factor viii molecules with reduced vwf binding |
US8889193B2 (en) | 2010-02-25 | 2014-11-18 | The Johns Hopkins University | Sustained delivery of therapeutic agents to an eye compartment |
AU2011248625B2 (en) | 2010-04-26 | 2017-01-05 | Pangu Biopharma Limited | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of cysteinyl-tRNA synthetase |
JP6294074B2 (en) | 2010-04-27 | 2018-03-14 | エータイアー ファーマ, インコーポレイテッド | Innovative discovery of therapeutic, diagnostic and antibody compositions related to protein fragments of isoleucyl-tRNA synthetase |
WO2011139853A2 (en) | 2010-04-28 | 2011-11-10 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of alanyl trna synthetases |
US9068177B2 (en) | 2010-04-29 | 2015-06-30 | Atyr Pharma, Inc | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glutaminyl-tRNA synthetases |
CA2797374C (en) | 2010-04-29 | 2021-02-16 | Pangu Biopharma Limited | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of asparaginyl trna synthetases |
CA2797393C (en) | 2010-04-29 | 2020-03-10 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of valyl trna synthetases |
CN103096925A (en) | 2010-05-03 | 2013-05-08 | Atyr医药公司 | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of arginyl-tRNA synthetases |
CA2797978C (en) | 2010-05-03 | 2019-12-03 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of methionyl-trna synthetases |
CN103096912A (en) | 2010-05-03 | 2013-05-08 | Atyr医药公司 | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of phenylalanyl-alpha-tRNA synthetases |
CN102985103A (en) | 2010-05-04 | 2013-03-20 | Atyr医药公司 | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of p38 multi-tRNA synthetase complex |
JP6396656B2 (en) | 2010-05-14 | 2018-09-26 | エータイアー ファーマ, インコーポレイテッド | Innovative discovery of therapeutic, diagnostic and antibody compositions related to protein fragments of phenylalanyl βtRNA synthetase |
JP6027965B2 (en) | 2010-05-17 | 2016-11-16 | エータイアー ファーマ, インコーポレイテッド | Innovative discovery of therapeutic, diagnostic and antibody compositions related to protein fragments of leucyl-tRNA synthetase |
MX2012013375A (en) | 2010-05-17 | 2013-04-11 | Cebix Inc | Pegylated c-peptide. |
ES2573108T3 (en) | 2010-05-26 | 2016-06-06 | Bristol-Myers Squibb Company | Fibronectin-based framework proteins that have improved stability |
US8962560B2 (en) | 2010-06-01 | 2015-02-24 | Atyr Pharma Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of Lysyl-tRNA synthetases |
BR112012033466A2 (en) * | 2010-07-01 | 2019-09-24 | Horian America Corp | process for the preparation of poly (alkylene oxide) derivatives for modification of biologically active molecules and materials |
CA2804416C (en) | 2010-07-12 | 2020-04-28 | Atyr Pharma, Inc. | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of glycyl-trna synthetases |
EP2595624B1 (en) | 2010-07-20 | 2018-01-31 | Halozyme, Inc. | Methods of treatment or prevention of the adverse side-effects associated with administration of an anti-hyaluronan agent |
AU2011293294B2 (en) | 2010-08-25 | 2016-03-24 | Pangu Biopharma Limited | Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of Tyrosyl-tRNA synthetases |
WO2012039979A2 (en) | 2010-09-10 | 2012-03-29 | The Johns Hopkins University | Rapid diffusion of large polymeric nanoparticles in the mammalian brain |
EP2616101B1 (en) | 2010-09-14 | 2014-07-09 | F.Hoffmann-La Roche Ag | Method for purifying pegylated erythropoietin |
US8961501B2 (en) | 2010-09-17 | 2015-02-24 | Incept, Llc | Method for applying flowable hydrogels to a cornea |
EP4218891A1 (en) | 2010-10-19 | 2023-08-02 | Trustees Of Tufts College | Silk fibroin-based microneedles and methods of making the same |
WO2012054861A1 (en) | 2010-10-22 | 2012-04-26 | Nektar Therapeutics | Glp-1 polymer conjugates having a releasable linkage |
WO2012054822A1 (en) | 2010-10-22 | 2012-04-26 | Nektar Therapeutics | Pharmacologically active polymer-glp-1 conjugates |
LT2637694T (en) | 2010-11-12 | 2021-05-25 | Nektar Therapeutics | Conjugates of an il-2 moiety and a polymer |
EP2657272B1 (en) * | 2010-12-21 | 2016-04-27 | NOF Corporation | Purification method for carboxyl-containing polyoxyethylene derivative |
US20130331443A1 (en) | 2010-12-22 | 2013-12-12 | Nektar Therapeutics | Multi-arm polymeric prodrug conjugates of taxane-based compounds |
US10894087B2 (en) | 2010-12-22 | 2021-01-19 | Nektar Therapeutics | Multi-arm polymeric prodrug conjugates of cabazitaxel-based compounds |
US9943605B2 (en) | 2010-12-23 | 2018-04-17 | Nektar Therapeutics | Polymer-semaxanib moiety conjugates |
EP2654799B1 (en) | 2010-12-23 | 2017-11-08 | Nektar Therapeutics | Polymer-sunitinib conjugates |
JP6009457B2 (en) | 2010-12-23 | 2016-10-19 | ネクター セラピューティクス | Polymer-desethylsunitinib conjugate |
US8163869B1 (en) | 2010-12-27 | 2012-04-24 | Nof Corporation | Purification method of carboxyl group-containing polyoxyethylene derivative |
CN103635489B (en) | 2011-01-06 | 2016-04-13 | 戴埃克斯有限公司 | Blood plasma prekallikrein associated proteins |
US8440309B2 (en) | 2011-01-31 | 2013-05-14 | Confluent Surgical, Inc. | Crosslinked polymers with the crosslinker as therapeutic for sustained release |
US9327037B2 (en) | 2011-02-08 | 2016-05-03 | The Johns Hopkins University | Mucus penetrating gene carriers |
EP2672958A1 (en) | 2011-02-08 | 2013-12-18 | Halozyme, Inc. | Composition and lipid formulation of a hyaluronan-degrading enzyme and the use thereof for treatment of benign prostatic hyperplasia |
JP6309273B2 (en) | 2011-03-02 | 2018-04-11 | ノヴォ・ノルディスク・ヘルス・ケア・アーゲー | Targeting clotting factors to TLT-1 on activated platelets |
CN103930437A (en) | 2011-03-16 | 2014-07-16 | 安姆根有限公司 | Potent and selective inhibitors of Nav1.3 and Nav1.7 |
WO2012145652A1 (en) | 2011-04-20 | 2012-10-26 | Trustees Of Tufts College | Dynamic silk coatings for implantable devices |
EP2709669A1 (en) | 2011-05-17 | 2014-03-26 | Bristol-Myers Squibb Company | Methods for maintaining pegylation of polypeptides |
WO2012166555A1 (en) | 2011-05-27 | 2012-12-06 | Nektar Therapeutics | Water - soluble polymer - linked binding moiety and drug compounds |
EP2720713A2 (en) | 2011-06-17 | 2014-04-23 | Halozyme, Inc. | Continuous subcutaneous insulin infusion methods with a hyaluronan degrading enzyme |
US9993529B2 (en) | 2011-06-17 | 2018-06-12 | Halozyme, Inc. | Stable formulations of a hyaluronan-degrading enzyme |
JP2014518216A (en) | 2011-06-17 | 2014-07-28 | ハロザイム インコーポレイテッド | Stable formulation of hyaluronan degrading enzyme |
US20130090294A1 (en) | 2011-06-28 | 2013-04-11 | Alternative Innovative Technologies Llc | Novel methods of use of hsp70 for increased performance or treatment of hsp70 related disorders |
WO2013020079A2 (en) | 2011-08-04 | 2013-02-07 | Nektar Therapeutics | Conjugates of an il-11 moiety and a polymer |
WO2013033476A1 (en) * | 2011-08-30 | 2013-03-07 | Quanta Biodesign, Ltd. | Branched discrette peg constructs |
US20130071394A1 (en) | 2011-09-16 | 2013-03-21 | John K. Troyer | Compositions and combinations of organophosphorus bioscavengers and hyaluronan-degrading enzymes, and methods of use |
US10226417B2 (en) | 2011-09-16 | 2019-03-12 | Peter Jarrett | Drug delivery systems and applications |
KR20140070612A (en) | 2011-09-23 | 2014-06-10 | 노보 노르디스크 에이/에스 | Novel glucagon analogues |
US9937241B2 (en) | 2011-10-14 | 2018-04-10 | Alternative Innovative Technologies Llc | Degradation resistant HSP70 formulations and uses thereof |
SG11201401797TA (en) | 2011-10-24 | 2014-09-26 | Halozyme Inc | Companion diagnostic for anti-hyaluronan agent therapy and methods of use thereof |
US9522951B2 (en) | 2011-10-31 | 2016-12-20 | Bristol-Myers Squibb Company | Fibronectin binding domains with reduced immunogenicity |
WO2013075117A2 (en) | 2011-11-17 | 2013-05-23 | John Wahren | Pegylated c-peptide |
US9205150B2 (en) | 2011-12-05 | 2015-12-08 | Incept, Llc | Medical organogel processes and compositions |
WO2013101509A2 (en) | 2011-12-15 | 2013-07-04 | Alternative Innovative Technologies Llc | Hsp70 fusion protein conjugates and uses thereof |
PT3130347T (en) | 2011-12-30 | 2019-12-10 | Halozyme Inc | Ph20 polypeptide variants, formulations and uses thereof |
KR101811917B1 (en) | 2012-01-19 | 2017-12-22 | 더 존스 홉킨스 유니버시티 | Nanoparticles formulations with enhanced mucus penetration |
RU2659423C2 (en) | 2012-02-16 | 2018-07-02 | ЭйТИР ФАРМА, ИНК. | Hystidil-trna-synthetase for treatment of autoimmune and inflammatory diseases |
WO2013128003A1 (en) | 2012-03-01 | 2013-09-06 | Novo Nordisk A/S | N-terminally modified oligopeptides and uses thereof |
US9603897B2 (en) | 2012-03-12 | 2017-03-28 | Massachusetts Institute Of Technology | Methods for treating tissue damage associated with ischemia with apolipoprotein D |
US8962577B2 (en) | 2012-03-16 | 2015-02-24 | The Johns Hopkins University | Controlled release formulations for the delivery of HIF-1 inhibitors |
EA030318B1 (en) | 2012-03-16 | 2018-07-31 | Дзе Джонс Хопкинс Юниверсити | Non-linear multiblock copolymer-drug conjugates for the delivery of active agents |
CA2868883C (en) | 2012-03-30 | 2022-10-04 | Sorrento Therapeutics Inc. | Fully human antibodies that bind to vegfr2 |
PT2833905T (en) | 2012-04-04 | 2018-08-06 | Halozyme Inc | Combination therapy with hyaluronidase and a tumor-targeted taxane |
AU2013255880B2 (en) | 2012-05-01 | 2017-07-20 | Novo Nordisk A/S | Pharmaceutical composition |
US9533068B2 (en) | 2012-05-04 | 2017-01-03 | The Johns Hopkins University | Drug loaded microfiber sutures for ophthalmic application |
CA2872519C (en) | 2012-05-04 | 2017-09-05 | The Johns Hopkins University | Lipid-based drug carriers for rapid penetration through mucus linings |
KR102238317B1 (en) | 2012-05-17 | 2021-04-12 | 익스텐드 바이오사이언시즈, 인크. | Carriers for improved drug delivery |
US9844582B2 (en) | 2012-05-22 | 2017-12-19 | Massachusetts Institute Of Technology | Synergistic tumor treatment with extended-PK IL-2 and therapeutic agents |
AU2013267161A1 (en) | 2012-05-31 | 2014-11-20 | Sorrento Therapeutics, Inc. | Antigen binding proteins that bind PD-L1 |
GB201210770D0 (en) | 2012-06-18 | 2012-08-01 | Polytherics Ltd | Novel conjugation reagents |
US9650331B2 (en) | 2012-06-18 | 2017-05-16 | Polytherics Limited | Conjugation reagents |
US10377827B2 (en) | 2012-06-21 | 2019-08-13 | Sorrento Therapeutics, Inc. | Antigen binding proteins that bind c-met |
JP6438391B2 (en) | 2012-06-22 | 2018-12-12 | ソレント・セラピューティクス・インコーポレイテッドSorrento Therapeutics, Inc. | Antigen binding protein that binds to CCR2 |
US10034945B2 (en) | 2012-07-13 | 2018-07-31 | Trustees Of Tufts College | Silk powder compaction for production of constructs with high mechanical strength and stiffness |
MY172863A (en) | 2012-09-13 | 2019-12-13 | Bristol Myers Squibb Co | Fibronectin based scaffold domain proteins that bind to myostatin |
CA2883833C (en) * | 2012-10-11 | 2020-10-27 | Ascendis Pharma A/S | Hydrogel prodrugs |
WO2014062856A1 (en) | 2012-10-16 | 2014-04-24 | Halozyme, Inc. | Hypoxia and hyaluronan and markers thereof for diagnosis and monitoring of diseases and conditions and related methods |
JP6426107B2 (en) | 2012-12-20 | 2018-11-21 | アムジエン・インコーポレーテツド | APJ receptor agonists and uses thereof |
EP2947111B1 (en) * | 2013-01-17 | 2018-03-07 | Xiamen Sinopeg Biotech Co., Ltd. | Monofunctional branched polyethyleneglycol and bio-related substance modified by same |
EP2951206A2 (en) | 2013-02-01 | 2015-12-09 | Bristol-Myers Squibb Company | Fibronectin based scaffold proteins |
US10568975B2 (en) | 2013-02-05 | 2020-02-25 | The Johns Hopkins University | Nanoparticles for magnetic resonance imaging tracking and methods of making and using thereof |
UY35397A (en) | 2013-03-12 | 2014-10-31 | Amgen Inc | POWERFUL AND SELECTIVE INHIBITORS OF NaV1.7 |
WO2014152657A1 (en) | 2013-03-14 | 2014-09-25 | Alere San Diego, Inc. | 6-acetylmorphine analogs, and methods for their synthesis and use |
US11376329B2 (en) | 2013-03-15 | 2022-07-05 | Trustees Of Tufts College | Low molecular weight silk compositions and stabilizing silk compositions |
CN105209497B (en) | 2013-03-15 | 2021-09-07 | 诺和诺德股份有限公司 | Antibodies capable of specifically binding two epitopes on tissue factor pathway inhibitor |
JP6457482B2 (en) | 2013-03-15 | 2019-01-23 | トラスティーズ オブ タフツ カレッジ | Low molecular weight silk composition and stabilization of silk composition |
EP2986306A4 (en) | 2013-04-18 | 2016-12-07 | Armo Biosciences Inc | Methods of using interleukin-10 for treating diseases and disorders |
WO2014170496A1 (en) | 2013-04-18 | 2014-10-23 | Novo Nordisk A/S | Stable, protracted glp-1/glucagon receptor co-agonists for medical use |
WO2014176458A2 (en) | 2013-04-24 | 2014-10-30 | Trustees Of Tufts College | Bioresorbable biopolymer anastomosis devices |
CN105683217B (en) | 2013-05-31 | 2019-12-10 | 索伦托治疗有限公司 | Antigen binding proteins that bind to PD-1 |
CA2914837A1 (en) | 2013-06-17 | 2014-12-24 | Armo Biosciences, Inc. | Method for assessing protein identity and stability |
TW201534726A (en) | 2013-07-03 | 2015-09-16 | Halozyme Inc | Thermally stable PH20 hyaluronidase variants and uses thereof |
CN105431204A (en) | 2013-07-12 | 2016-03-23 | 奥普索特克公司 | Methods for treating or preventing ophthalmological conditions |
WO2015013510A1 (en) | 2013-07-25 | 2015-01-29 | Ecole Polytechnique Federale De Lausanne Epfl | High aspect ratio nanofibril materials |
EP3038642A4 (en) | 2013-08-30 | 2017-01-18 | Armo Biosciences, Inc. | Methods of using interleukin-10 for treating diseases and disorders |
US10988745B2 (en) | 2013-10-31 | 2021-04-27 | Resolve Therapeutics, Llc | Therapeutic nuclease-albumin fusions and methods |
JP6660297B2 (en) | 2013-11-11 | 2020-03-11 | アルモ・バイオサイエンシーズ・インコーポレイテッド | Methods of using interleukin-10 to treat diseases and disorders |
CN112043835B (en) | 2013-12-06 | 2022-10-21 | 韩捷 | Bioreversible introducing group for nitrogen and hydroxyl-containing drugs |
CU20140003A7 (en) | 2014-01-08 | 2015-08-27 | Ct De Inmunología Molecular Biofarmacuba | CONJUGATE UNDERSTANDING ERYTHROPOYETIN AND A RAMIFIED POLYMER STRUCTURE |
WO2015127368A1 (en) | 2014-02-23 | 2015-08-27 | The Johns Hopkins University | Hypotonic microbicidal formulations and methods of use |
CN106459210A (en) | 2014-03-27 | 2017-02-22 | 戴埃克斯有限公司 | Compositions and methods for treatment of diabetic macular edema |
MA39711A (en) | 2014-04-03 | 2015-10-08 | Nektar Therapeutics | Conjugates of an il-15 moiety and a polymer |
CN103980494B (en) * | 2014-04-21 | 2016-04-13 | 国家纳米科学中心 | A kind of polypeptide polymer with anti-tumor activity and its preparation method and application |
JP7059003B2 (en) | 2014-05-14 | 2022-04-25 | トラスティーズ・オブ・ダートマス・カレッジ | Deimmunized lysostaphin and how to use |
WO2015187295A2 (en) | 2014-06-02 | 2015-12-10 | Armo Biosciences, Inc. | Methods of lowering serum cholesterol |
US10570184B2 (en) | 2014-06-04 | 2020-02-25 | Novo Nordisk A/S | GLP-1/glucagon receptor co-agonists for medical use |
JP6803236B2 (en) | 2014-06-10 | 2020-12-23 | アムジェン インコーポレイテッド | Aperin polypeptide |
US20170216403A1 (en) | 2014-08-12 | 2017-08-03 | Massachusetts Institute Of Technology | Synergistic tumor treatment with il-2, a therapeutic antibody, and an immune checkpoint blocker |
DK3180018T3 (en) | 2014-08-12 | 2019-10-28 | Massachusetts Inst Technology | Synergistic tumor treatment with IL-2 and integrin-binding Fc fusion protein |
CN106794152A (en) | 2014-08-13 | 2017-05-31 | 约翰霍普金斯大学 | For preventing the nano particle for being loaded with glucocorticoid that corneal allograft repels and new vessels is formed |
MX2017002380A (en) | 2014-08-22 | 2017-09-15 | Sorrento Therapeutics Inc | Antigen binding proteins that bind cxcr3. |
EP3186281B1 (en) | 2014-08-28 | 2019-04-10 | Halozyme, Inc. | Combination therapy with a hyaluronan-degrading enzyme and an immune checkpoint inhibitor |
WO2016060996A2 (en) | 2014-10-14 | 2016-04-21 | Armo Biosciences, Inc. | Interleukin-15 compositions and uses thereof |
NZ730563A (en) | 2014-10-14 | 2019-05-31 | Halozyme Inc | Compositions of adenosine deaminase-2 (ada2), variants thereof and methods of using same |
US9789197B2 (en) | 2014-10-22 | 2017-10-17 | Extend Biosciences, Inc. | RNAi vitamin D conjugates |
WO2016065052A1 (en) | 2014-10-22 | 2016-04-28 | Extend Biosciences, Inc. | Insulin vitamin d conjugates |
WO2016065042A1 (en) | 2014-10-22 | 2016-04-28 | Extend Biosciences, Inc. | Therapeutic vitamin d conjugates |
JP6675394B2 (en) | 2014-10-22 | 2020-04-01 | アルモ・バイオサイエンシーズ・インコーポレイテッド | Use of interleukin-10 for the treatment of diseases and disorders |
KR102569907B1 (en) | 2014-10-23 | 2023-08-24 | 엔지엠 바이오파마슈티컬스, 아이엔씨. | Pharmaceutical Compositions Comprising Peptide Variants and Methods of Use Thereof |
KR20230079520A (en) | 2014-11-06 | 2023-06-07 | 파마에센시아 코퍼레이션 | Dosage Regimen for PEGylated Interferon |
EP3223866B1 (en) | 2014-11-25 | 2023-03-08 | Bristol-Myers Squibb Company | Methods and compositions for 18f-radiolabeling of the fibronectin type (iii) domain |
EP3224277B1 (en) | 2014-11-25 | 2020-08-26 | Bristol-Myers Squibb Company | Novel pd-l1 binding polypeptides for imaging |
EP3247406A1 (en) | 2015-01-20 | 2017-11-29 | The Johns Hopkins University | Compositions for the sustained release of anti-glaucoma agents to control intraocular pressure |
AU2016211696B2 (en) | 2015-01-27 | 2018-05-10 | The Johns Hopkins University | Hypotonic hydrogel formulations for enhanced transport of active agents at mucosal surfaces |
WO2016126615A1 (en) | 2015-02-03 | 2016-08-11 | Armo Biosciences, Inc. | Methods of using interleukin-10 for treating diseases and disorders |
JP6917902B2 (en) | 2015-02-13 | 2021-08-11 | ソレント・セラピューティクス・インコーポレイテッド | Antibody drug that binds to CTLA4 |
US9951144B2 (en) | 2015-04-08 | 2018-04-24 | Sorrento Therapeutics, Inc. | Antibody therapeutics that bind CD38 |
WO2016171980A1 (en) | 2015-04-24 | 2016-10-27 | Bristol-Myers Squibb Company | Polypeptides targeting hiv fusion |
EP4014985A1 (en) | 2015-05-01 | 2022-06-22 | Allysta Pharmaceuticals, Inc. | Adiponectin peptidomimetics for treating ocular disorders |
AU2016268403A1 (en) | 2015-05-28 | 2017-12-07 | Armo Biosciences, Inc. | Pegylated interleukin-10 for use in treating cancer |
CA2986774A1 (en) | 2015-05-29 | 2016-12-08 | Armo Biosciences, Inc. | Methods of using interleukin-10 for treating diseases and disorders |
ES2902467T3 (en) | 2015-06-15 | 2022-03-28 | Univ Leland Stanford Junior | TIMP2 for use in the treatment of conditions associated with aging |
EP3341012A4 (en) | 2015-08-25 | 2019-03-20 | Armo Biosciences, Inc. | Methods of using interleukin-10 for treating diseases and disorders |
EP3733698A1 (en) | 2015-09-23 | 2020-11-04 | Bristol-Myers Squibb Company | Glypican-3 binding fibronectin based scafflold molecules |
CA2998708C (en) | 2015-10-01 | 2019-09-03 | Elysium Therapeutics, Inc. | Polysubunit opioid prodrugs resistant to overdose and abuse |
US10335406B2 (en) | 2015-10-01 | 2019-07-02 | Elysium Therapeutics, Inc. | Opioid compositions resistant to overdose and abuse |
EP3377090B1 (en) | 2015-11-09 | 2021-04-07 | NGM Biopharmaceuticals, Inc. | Methods for treatment of bile acid-related disorders |
WO2017094897A1 (en) | 2015-12-04 | 2017-06-08 | 全薬工業株式会社 | Anti-il-17 aptamer having improved retention in blood |
EA201891388A1 (en) | 2015-12-11 | 2018-11-30 | Дайэкс Корп. | PLASMA KALLIKREIN INHIBITORS AND THEIR APPLICATION FOR THE TREATMENT OF THE EXPOSURE OF HEREDITARY ANGIONEUROTIC DOMESTIC |
JP6883590B2 (en) | 2016-01-29 | 2021-06-09 | ソレント・セラピューティクス・インコーポレイテッドSorrento Therapeutics, Inc. | Antigen-binding protein that binds to PD-L1 |
WO2017147298A1 (en) | 2016-02-23 | 2017-08-31 | The Regents Of The University Of Colorado, A Body Corporate | Peptide-based methods for treating neurological injury |
US10821160B2 (en) | 2016-03-01 | 2020-11-03 | The Board Of Trustees Of The University Of Illinois | L-asparaginase variants and fusion proteins with reduced L-glutaminase activity and enhanced stability |
US20190022016A1 (en) | 2016-03-02 | 2019-01-24 | The Johns Hopkins University | Compositions for sustained release of anti-glaucoma agents to control intraocular pressure |
WO2017160599A1 (en) | 2016-03-14 | 2017-09-21 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Use of cd300b antagonists to treat sepsis and septic shock |
EP3458439B1 (en) | 2016-05-18 | 2021-12-08 | Alere San Diego, Inc. | 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine analogs and methods for their synthesis and use |
US10994033B2 (en) | 2016-06-01 | 2021-05-04 | Bristol-Myers Squibb Company | Imaging methods using 18F-radiolabeled biologics |
CN109415427A (en) | 2016-07-15 | 2019-03-01 | 豪夫迈·罗氏有限公司 | Method for purifying the hematopoietin of Pegylation |
WO2018017923A1 (en) | 2016-07-22 | 2018-01-25 | Nektar Therapeutics | Conjugates of a factor viii moiety having an oxime-containing linkage |
US11248313B2 (en) | 2016-08-01 | 2022-02-15 | Trustees Of Tufts College | Biomimetic mechanical tension driven fabrication of nanofibrillar architecture |
WO2018031361A2 (en) | 2016-08-09 | 2018-02-15 | Eli Lilly And Company | Combination therapy |
CN109891237B (en) | 2016-10-24 | 2022-08-23 | 诺和诺德股份有限公司 | Bioassay of insulin preparations |
US10603358B2 (en) | 2017-01-10 | 2020-03-31 | Nodus Therapeutics | Combination tumor treatment with an integrin-binding-Fc fusion protein and immune stimulator |
US10350266B2 (en) | 2017-01-10 | 2019-07-16 | Nodus Therapeutics, Inc. | Method of treating cancer with a multiple integrin binding Fc fusion protein |
EP3595663A4 (en) | 2017-03-17 | 2021-01-13 | Elysium Therapeutics, Inc. | Polysubunit opioid prodrugs resistant to overdose and abuse |
JP6935059B2 (en) | 2017-03-30 | 2021-09-15 | 日油株式会社 | A method for purifying polyethylene glycol having one carboxyl group |
US20200115324A1 (en) | 2017-06-11 | 2020-04-16 | Molecular Express, Inc. | Methods and compositions for substance use disorder vaccine formulations and uses thereof |
US10174302B1 (en) | 2017-06-21 | 2019-01-08 | Xl-Protein Gmbh | Modified L-asparaginase |
CN111107870A (en) | 2017-06-22 | 2020-05-05 | 催化剂生物科学公司 | Modified membrane serine protease 1(MTSP-1) polypeptides and methods of use thereof |
CN111050784B (en) | 2017-08-11 | 2024-03-19 | 伊利诺伊大学理事会 | Truncated guinea pig L-asparaginase variants and methods of use thereof |
EP3684811A2 (en) | 2017-08-17 | 2020-07-29 | Massachusetts Institute of Technology | Multiple specificity binders of cxc chemokines and uses thereof |
SG11202001311VA (en) | 2017-08-22 | 2020-03-30 | Sanabio Llc | Soluble interferon receptors and uses thereof |
US11897917B2 (en) | 2017-09-27 | 2024-02-13 | The University Of York | Bioconjugation of polypeptides |
KR102020995B1 (en) | 2017-10-30 | 2019-09-16 | 한국코러스 주식회사 | A method of preparing gcsf and polyol_conjugated conjugates with high yield |
WO2019094268A1 (en) | 2017-11-10 | 2019-05-16 | Armo Biosciences, Inc. | Compositions and methods of use of interleukin-10 in combination with immune checkpoint pathway inhibitors |
WO2019129877A1 (en) | 2017-12-29 | 2019-07-04 | F. Hoffmann-La Roche Ag | Process for providing pegylated protein composition |
KR102497097B1 (en) | 2017-12-29 | 2023-02-06 | 에프. 호프만-라 로슈 아게 | Methods of Providing Pegylated Protein Compositions |
JP7137625B2 (en) | 2017-12-29 | 2022-09-14 | エフ.ホフマン-ラ ロシュ アーゲー | Methods for providing PEGylated protein compositions |
US20190351031A1 (en) | 2018-05-16 | 2019-11-21 | Halozyme, Inc. | Methods of selecting subjects for combination cancer therapy with a polymer-conjugated soluble ph20 |
KR102167755B1 (en) | 2018-05-23 | 2020-10-19 | 주식회사 큐어바이오 | Fragmented GRS polypeptide, mutants thereof and use thereof |
CN112533629A (en) | 2018-06-19 | 2021-03-19 | 阿尔莫生物科技股份有限公司 | Compositions and methods for combined use of IL-10 agents with chimeric antigen receptor cell therapy |
CN110964116A (en) | 2018-09-26 | 2020-04-07 | 北京辅仁瑞辉生物医药研究院有限公司 | GLP1-Fc fusion proteins and conjugates thereof |
JP2022502037A (en) | 2018-09-28 | 2022-01-11 | マサチューセッツ インスティテュート オブ テクノロジー | Immunomodulatory molecules localized to collagen and their methods |
US20200262887A1 (en) | 2018-11-30 | 2020-08-20 | Opko Ireland Global Holdings, Ltd. | Oxyntomodulin peptide analog formulations |
US11613744B2 (en) | 2018-12-28 | 2023-03-28 | Vertex Pharmaceuticals Incorporated | Modified urokinase-type plasminogen activator polypeptides and methods of use |
KR20210110848A (en) | 2018-12-28 | 2021-09-09 | 카탈리스트 바이오사이언시즈, 인코포레이티드 | Modified urokinase type plasminogen activator polypeptides and methods of use |
MX2021008081A (en) | 2019-01-04 | 2021-08-05 | Resolve Therapeutics Llc | Treatment of sjogren's disease with nuclease fusion proteins. |
EP3911668A1 (en) | 2019-01-18 | 2021-11-24 | The Regents of the University of Colorado, a body corporate | Amphipathic alpha-helical antimicrobial peptides treat infections by gram-negative pathogens |
WO2020154032A1 (en) | 2019-01-23 | 2020-07-30 | Massachusetts Institute Of Technology | Combination immunotherapy dosing regimen for immune checkpoint blockade |
CN112175088B (en) | 2019-07-02 | 2023-03-28 | 江苏晟斯生物制药有限公司 | FIX fusion proteins, conjugates and uses thereof |
WO2021038296A2 (en) | 2019-08-27 | 2021-03-04 | Tonix Pharma Holdings Limited | Modified tff2 polypeptides |
US20220409697A1 (en) | 2019-10-19 | 2022-12-29 | Ramea Llc | Extended Half-life G-CSF and GM-CSF Vitamin D Conjugates |
JP2023505256A (en) | 2019-12-05 | 2023-02-08 | ソレント・セラピューティクス・インコーポレイテッド | Compositions and methods comprising anti-CD47 antibodies in combination with tumor-targeting antibodies |
CN115023444A (en) | 2019-12-20 | 2022-09-06 | 再生元制药公司 | Novel IL2 agonists and methods of use thereof |
CA3168986A1 (en) | 2020-02-26 | 2021-09-02 | Sorrento Therapeutics, Inc. | Activatable antigen binding proteins with universal masking moieties |
WO2021174045A1 (en) | 2020-02-28 | 2021-09-02 | Bristol-Myers Squibb Company | Radiolabeled fibronectin based scaffolds and antibodies and theranostic uses thereof |
WO2021195089A1 (en) | 2020-03-23 | 2021-09-30 | Sorrento Therapeutics, Inc. | Fc-coronavirus antigen fusion proteins, and nucleic acids, vectors, compositions and methods of use thereof |
KR20230004682A (en) | 2020-04-22 | 2023-01-06 | 머크 샤프 앤드 돔 엘엘씨 | Human interleukin-2 conjugates biased against the interleukin-2 receptor beta gamma c dimer and conjugated to non-peptide water soluble polymers |
BR112022022045A2 (en) | 2020-04-30 | 2023-01-10 | Sairopa B V | ANTIGEN-BINDING ANTIBODY OR FRAGMENT OF THE SAME WHICH BINDS HUMAN CD103, ONE OR MORE NUCLEIC ACIDS, EXPRESSION SYSTEM, HOST CELL, COMPOSITION, METHODS OF PRODUCTION OF AN ANTIBODY OR ANTIGEN-BINDING FRAGMENT, TO DETECT THE PRESENCE OF CD103 IN A BIOLOGICAL SAMPLE, TO TREAT OR PREVENT A CONDITION MEDIATED BY CD103 SIGNALING IN AN INDIVIDUAL IN NEED, TO INHIBIT CD103 SIGNALING IN A CELL, TO INHIBIT THE BINDING OF CD103 TO E-CADHERIN PRESENT IN A CELL, TO REMOVE THE CD103-EXPRESSING CELLS IN AN INDIVIDUAL, TO TREAT OR PREVENT A DISEASE AND IMAGING AGENT |
US11673930B2 (en) | 2020-05-12 | 2023-06-13 | Regeneran Pharmaceuticals, Inc. | IL10 agonists and methods of use thereof |
CN116209677A (en) | 2020-06-26 | 2023-06-02 | 索伦托药业有限公司 | anti-PD 1 antibodies and uses thereof |
EP4171614A1 (en) | 2020-06-29 | 2023-05-03 | Resolve Therapeutics, LLC | Treatment of sjogren's syndrome with nuclease fusion proteins |
CA3128035A1 (en) | 2020-08-13 | 2022-02-13 | Bioasis Technologies, Inc. | Combination therapies for delivery across the blood brain barrier |
WO2022211829A1 (en) | 2021-03-30 | 2022-10-06 | Jazz Pharmaceuticals Ireland Ltd. | Dosing of recombinant l-asparaginase |
WO2023022965A2 (en) | 2021-08-16 | 2023-02-23 | Regeneron Pharmaceuticals, Inc. | Novel il27 receptor agonists and methods of use thereof |
US20230279153A1 (en) | 2021-11-11 | 2023-09-07 | Regeneron Pharmaceuticals, Inc. | Cd20-pd1 binding molecules and methods of use thereof |
GB202117727D0 (en) | 2021-12-08 | 2022-01-19 | Univ Edinburgh | Fap detection |
WO2024015529A2 (en) | 2022-07-14 | 2024-01-18 | Jazz Pharmaceuticals Ireland Ltd. | Combination therapies involving l-asparaginase |
CN116178733B (en) * | 2023-03-03 | 2023-08-01 | 浙江博美生物技术有限公司 | Branched monodisperse PEG derivative based on trifunctional amino acid, preparation method and application |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0400472A2 (en) * | 1989-05-27 | 1990-12-05 | Sumitomo Pharmaceuticals Company, Limited | Process for preparing polyethylene glycol derivatives and modified protein. |
EP0400486A2 (en) * | 1989-05-26 | 1990-12-05 | Sumitomo Pharmaceuticals Company, Limited | Polyethylene glycol derivatives, modified peptides and production thereof |
EP0632082A1 (en) * | 1993-06-29 | 1995-01-04 | "HEYLECINA", Société Anonyme | Preparation of activated carbamates of poly(alkylene glycol) and their use |
WO1995011924A1 (en) * | 1993-10-27 | 1995-05-04 | Enzon, Inc. | Non-antigenic branched polymer conjugates |
Family Cites Families (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US40076A (en) * | 1863-09-22 | Improvement in molds for | ||
US52430A (en) * | 1866-02-06 | Improved heel-cutter | ||
US52443A (en) * | 1866-02-06 | Improved brad-awl handle | ||
US4179337A (en) * | 1973-07-20 | 1979-12-18 | Davis Frank F | Non-immunogenic polypeptides |
US4722906A (en) * | 1982-09-29 | 1988-02-02 | Bio-Metric Systems, Inc. | Binding reagents and methods |
US4650909A (en) * | 1984-11-28 | 1987-03-17 | Yoakum George H | Polyethylene glycol (PEG) reagent |
EP0310361A3 (en) * | 1987-09-30 | 1989-05-24 | Beckman Instruments, Inc. | Tridentate conjugate and method of use thereof |
US5324844A (en) * | 1989-04-19 | 1994-06-28 | Enzon, Inc. | Active carbonates of polyalkylene oxides for modification of polypeptides |
US5122614A (en) * | 1989-04-19 | 1992-06-16 | Enzon, Inc. | Active carbonates of polyalkylene oxides for modification of polypeptides |
JP3051145B2 (en) * | 1990-08-28 | 2000-06-12 | 住友製薬株式会社 | Novel polyethylene glycol derivative modified peptide |
US5359030A (en) * | 1993-05-10 | 1994-10-25 | Protein Delivery, Inc. | Conjugation-stabilized polypeptide compositions, therapeutic delivery and diagnostic formulations comprising same, and method of making and using the same |
US5919455A (en) * | 1993-10-27 | 1999-07-06 | Enzon, Inc. | Non-antigenic branched polymer conjugates |
US5605976A (en) * | 1995-05-15 | 1997-02-25 | Enzon, Inc. | Method of preparing polyalkylene oxide carboxylic acids |
US5932462A (en) | 1995-01-10 | 1999-08-03 | Shearwater Polymers, Inc. | Multiarmed, monofunctional, polymer for coupling to molecules and surfaces |
US5756593A (en) * | 1995-05-15 | 1998-05-26 | Enzon, Inc. | Method of preparing polyalkyene oxide carboxylic acids |
US5747639A (en) * | 1996-03-06 | 1998-05-05 | Amgen Boulder Inc. | Use of hydrophobic interaction chromatography to purify polyethylene glycols |
ES2307865T3 (en) * | 1998-03-12 | 2008-12-01 | Nektar Therapeutics Al, Corporation | METHOD FOR PREPARING POLYMERIC CONJUGATES. |
CA2330451A1 (en) * | 1998-04-28 | 1999-11-04 | Applied Research Systems Ars Holding N.V. | Polyol-ifn-beta conjugates |
-
1995
- 1995-05-17 US US08/443,383 patent/US5932462A/en not_active Expired - Lifetime
-
1996
- 1996-01-11 AU AU47555/96A patent/AU4755596A/en not_active Abandoned
- 1996-01-11 WO PCT/US1996/000474 patent/WO1996021469A1/en active Application Filing
-
1998
- 1998-08-27 US US09/140,907 patent/US20010007765A1/en not_active Abandoned
-
2002
- 2002-04-10 US US10/119,546 patent/US20030114647A1/en not_active Abandoned
-
2003
- 2003-08-05 US US10/634,970 patent/US7419600B2/en not_active Expired - Fee Related
-
2008
- 2008-09-20 US US12/284,357 patent/US7786221B2/en not_active Expired - Fee Related
-
2010
- 2010-08-03 US US12/849,683 patent/US8354477B2/en not_active Expired - Fee Related
-
2012
- 2012-12-14 US US13/714,917 patent/US8546493B2/en not_active Expired - Fee Related
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0400486A2 (en) * | 1989-05-26 | 1990-12-05 | Sumitomo Pharmaceuticals Company, Limited | Polyethylene glycol derivatives, modified peptides and production thereof |
EP0400472A2 (en) * | 1989-05-27 | 1990-12-05 | Sumitomo Pharmaceuticals Company, Limited | Process for preparing polyethylene glycol derivatives and modified protein. |
EP0632082A1 (en) * | 1993-06-29 | 1995-01-04 | "HEYLECINA", Société Anonyme | Preparation of activated carbamates of poly(alkylene glycol) and their use |
WO1995011924A1 (en) * | 1993-10-27 | 1995-05-04 | Enzon, Inc. | Non-antigenic branched polymer conjugates |
Non-Patent Citations (1)
Title |
---|
MONFARDINI C. ET AL: "A branched monomethoxypolyethyleneglycol for protein modifications", BIOCONJUGATE CHEMISTRY, vol. 06, no. 01, January 1995 (1995-01-01), WASHINGTON D.C., pages 62 - 69, XP002004192 * |
Cited By (355)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6147204A (en) * | 1990-06-11 | 2000-11-14 | Nexstar Pharmaceuticals, Inc. | Nucleic acid ligand complexes |
US6465188B1 (en) | 1990-06-11 | 2002-10-15 | Gilead Sciences, Inc. | Nucleic acid ligand complexes |
US6011020A (en) * | 1990-06-11 | 2000-01-04 | Nexstar Pharmaceuticals, Inc. | Nucleic acid ligand complexes |
US6168778B1 (en) | 1990-06-11 | 2001-01-02 | Nexstar Pharmaceuticals, Inc. | Vascular endothelial growth factor (VEGF) Nucleic Acid Ligand Complexes |
US8071737B2 (en) | 1995-05-04 | 2011-12-06 | Glead Sciences, Inc. | Nucleic acid ligand complexes |
US5859228A (en) * | 1995-05-04 | 1999-01-12 | Nexstar Pharmaceuticals, Inc. | Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes |
US6025325A (en) * | 1995-05-05 | 2000-02-15 | Hoffman-La Roche Inc. | Pegylated obese (ob) protein compositions |
US6582918B2 (en) | 1995-06-07 | 2003-06-24 | Gilead Sciences, Inc. | Platelet derived growth factor (PDGF) nucleic acid ligand complexes |
US7879993B2 (en) | 1995-06-07 | 2011-02-01 | Gilead Sciences, Inc. | Platelet derived growth factor (PDGF) nucleic acid ligand complexes |
US6229002B1 (en) | 1995-06-07 | 2001-05-08 | Nexstar Pharmaceuticlas, Inc. | Platelet derived growth factor (PDGF) nucleic acid ligand complexes |
US8377466B2 (en) | 1995-12-18 | 2013-02-19 | Angiotech Pharmaceuticals (Us), Inc. | Adhesive tissue repair patch |
EP1704878A2 (en) | 1995-12-18 | 2006-09-27 | AngioDevice International GmbH | Crosslinked polymer compositions and methods for their use |
EP1704878A3 (en) * | 1995-12-18 | 2007-04-25 | AngioDevice International GmbH | Crosslinked polymer compositions and methods for their use |
US8617584B2 (en) | 1995-12-18 | 2013-12-31 | Angiodevice International Gmbh | Adhesive tissue repair patch and collagen sheets |
US6025324A (en) * | 1996-05-15 | 2000-02-15 | Hoffmann-La Roche Inc. | Pegylated obese (ob) protein compositions |
US7201897B2 (en) | 1996-05-31 | 2007-04-10 | Hoffmann-La Roche Inc. | Interferon conjugates |
US8088365B2 (en) | 1996-09-26 | 2012-01-03 | Nektar Therapeutics | Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution |
US7655747B2 (en) * | 1996-09-26 | 2010-02-02 | Nektar Therapeutics | Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution |
US8404222B2 (en) | 1996-09-26 | 2013-03-26 | Nektar Therapeutics | Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution |
US6962784B2 (en) | 1996-10-25 | 2005-11-08 | Gilead Sciences, Inc. | Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes |
US6258351B1 (en) | 1996-11-06 | 2001-07-10 | Shearwater Corporation | Delivery of poly(ethylene glycol)-modified molecules from degradable hydrogels |
US7018624B2 (en) | 1996-11-06 | 2006-03-28 | Debio Recherche Pharmaceutique S.A. | Delivery of poly(ethylene glycol)-modified molecules from degradable hydrogels |
US6432397B1 (en) | 1996-11-06 | 2002-08-13 | Debio Recherche Pharmaceutique S. A. | Delivery of poly(ethylene glycol)-modified molecules from degradable hydrogels |
US6558658B2 (en) | 1996-11-06 | 2003-05-06 | Debio Recherche Pharmaceutique S.A. | Delivery of poly (ethylene glycol)-modified molecules from degradable hydrogels |
US6051698A (en) * | 1997-06-06 | 2000-04-18 | Janjic; Nebojsa | Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes |
WO1999006071A1 (en) * | 1997-07-30 | 1999-02-11 | The Procter & Gamble Company | Modified polypeptides with high activity and reduced allergenicity |
US6284246B1 (en) | 1997-07-30 | 2001-09-04 | The Procter & Gamble Co. | Modified polypeptides with high activity and reduced allergenicity |
US6583251B1 (en) | 1997-09-08 | 2003-06-24 | Emory University | Modular cytomimetic biomaterials, transport studies, preparation and utilization thereof |
US6426335B1 (en) | 1997-10-17 | 2002-07-30 | Gilead Sciences, Inc. | Vascular endothelial growth factor (VEGF) nucleic acid ligand complexes |
WO1999022770A1 (en) * | 1997-11-05 | 1999-05-14 | Shearwater Polymers, Inc. | Delivery of poly(ethylene glycol)-conjugated molecules from degradable hydrogels |
US6869932B2 (en) | 1997-12-03 | 2005-03-22 | Applied Research Systems Ars Holding N.V. | Site-specific preparation of polyethlene glycol-GRF conjugates |
WO1999027897A1 (en) * | 1997-12-03 | 1999-06-10 | Applied Research Systems Ars Holding N.V. | Site-specific preparation of polyethylene glycol-grf conjugates |
US7317002B2 (en) | 1997-12-03 | 2008-01-08 | Applied Research Systems Ars Holding N.V. | Site-specific preparation of polyethylene glycol-GRF conjugates |
EP1040151A4 (en) * | 1997-12-12 | 2003-05-21 | Macromed Inc | Heterofunctionalized star-shaped poly(ethylene glycols) for protein modification |
EP1040151A1 (en) * | 1997-12-12 | 2000-10-04 | MacroMed, Inc. | Heterofunctionalized star-shaped poly(ethylene glycols) for protein modification |
US7939654B2 (en) | 1997-12-16 | 2011-05-10 | Gilead Sciences, Inc. | Platelet derived growth factor (PDGF) nucleic acid ligand complexes |
US6664331B2 (en) | 1998-03-12 | 2003-12-16 | Nektar Therapeutics Al, Corporation | Poly(ethylene glycol) derivatives with proximal reactive groups |
WO1999045964A1 (en) * | 1998-03-12 | 1999-09-16 | Shearwater Polymers, Incorporated | Poly(ethylene glycol) derivatives with proximal reactive groups |
US7223803B2 (en) | 1998-03-12 | 2007-05-29 | Nektar Therapeutics Al, Corporation | Polyethylene (glycol) derivatives with proximal reactive groups |
EP1411075A2 (en) * | 1998-03-12 | 2004-04-21 | Nektar Therapeutics Al, Corporation | Method for preparing polymer conjugates |
US7528202B2 (en) | 1998-03-12 | 2009-05-05 | Nektar Therapeutics Al, Corporation | Poly (ethylene glycol) derivatives with proximal reactive groups |
US6437025B1 (en) | 1998-03-12 | 2002-08-20 | Shearwater Corporation | Poly(ethylene glycol) derivatives with proximal reactive groups |
EP1411075A3 (en) * | 1998-03-12 | 2004-07-28 | Nektar Therapeutics Al, Corporation | Method for preparing polymer conjugates |
US8003742B2 (en) | 1998-03-12 | 2011-08-23 | Nektar Therapeutics | Polymer derivatives with proximal reactive groups |
US7030278B2 (en) | 1998-03-12 | 2006-04-18 | Nektar Therapeutics Al, Corporation | Polyethylene(Glycol) derivatives with proximal reactive groups |
US6541543B2 (en) | 1998-03-12 | 2003-04-01 | Shearwater Corporation | Poly (ethylene glycol) derivatives with proximal reactive groups |
US7714088B2 (en) | 1998-03-12 | 2010-05-11 | Nektar Therapeutics | Poly(ethylene glycol) derivatives with proximal reactive groups |
US7268210B2 (en) | 1998-04-28 | 2007-09-11 | Applied Research Systems Ars Holding N.V. | PEG-LHRH analog conjugates |
US6638500B1 (en) | 1998-04-28 | 2003-10-28 | Applied Research Systems Ars Holding N.V. | Polyol-IFN-βconjugates modified at Cys-17 and composition containing same |
US7700314B2 (en) | 1998-04-28 | 2010-04-20 | Merck Serono Sa | Method for producing polyol-IFN-β conjugate |
WO1999055376A1 (en) * | 1998-04-28 | 1999-11-04 | Applied Research Systems Ars Holding N.V. | Peg-lhrh analog conjugates |
US6914121B2 (en) | 1998-04-28 | 2005-07-05 | Applied Research Systems Ars Holding N.V. | PEG-LHRH analog conjugates |
EP1588717A1 (en) * | 1998-04-28 | 2005-10-26 | Applied Research Systems ARS Holding N.V. | PEG-LHRH analog conjugates |
US6433135B1 (en) | 1998-04-28 | 2002-08-13 | Applied Research Systems Ars Holding N.V. | PEG-LHRH analog conjugates |
US7357925B2 (en) | 1998-04-28 | 2008-04-15 | Laboratoires Seronosa | Method for treating disorders and diseases treatable with human fibroblast interferon |
EP1107813A1 (en) * | 1998-08-26 | 2001-06-20 | Neomend, Inc. | Compositions, systems, and methods for creating in situ, chemically cross-linked, mechanical barriers or covering structures |
EP1107813A4 (en) * | 1998-08-26 | 2003-09-10 | Neomend Inc | Compositions, systems, and methods for creating in situ, chemically cross-linked, mechanical barriers or covering structures |
US6410017B1 (en) | 1998-09-22 | 2002-06-25 | The Procter & Gamble Company | Personal care compositions containing active proteins tethered to a water insoluble substrate |
US7247314B2 (en) | 1998-11-06 | 2007-07-24 | Neomend, Inc | Biocompatible material composition adaptable to diverse therapeutic indications |
US6899889B1 (en) | 1998-11-06 | 2005-05-31 | Neomend, Inc. | Biocompatible material composition adaptable to diverse therapeutic indications |
US8383144B2 (en) | 1998-11-06 | 2013-02-26 | Neomend, Inc. | Tissue adhering compositions |
US8409605B2 (en) | 1998-11-06 | 2013-04-02 | Neomend, Inc. | Biocompatible material composition adaptable to diverse therapeutic indications |
US6303119B1 (en) | 1999-09-22 | 2001-10-16 | The Procter & Gamble Company | Personal care compositions containing subtilisin enzymes bound to water insoluble substrates |
WO2001048052A1 (en) | 1999-12-24 | 2001-07-05 | Kyowa Hakko Kogyo Co., Ltd. | Branched polyalkylene glycols |
EP2133098A1 (en) | 2000-01-10 | 2009-12-16 | Maxygen Holdings Ltd | G-CSF conjugates |
EP1982732A2 (en) | 2000-02-11 | 2008-10-22 | Maxygen Holdings Ltd. | Factor VII or VIIA-like molecules |
EP2319541A1 (en) | 2000-02-11 | 2011-05-11 | Bayer HealthCare LLC | Factor VII or VIIA-like conjugates |
US6936298B2 (en) | 2000-04-13 | 2005-08-30 | Emory University | Antithrombogenic membrane mimetic compositions and methods |
WO2001093914A3 (en) * | 2000-06-08 | 2002-09-12 | Jolla Pharma | Multivalent platform molecules comprising high molecular weight polyethylene oxide |
WO2001093914A2 (en) * | 2000-06-08 | 2001-12-13 | La Jolla Pharmaceutical Company | Multivalent platform molecules comprising high molecular weight polyethylene oxide |
US7713544B2 (en) | 2000-07-28 | 2010-05-11 | Emory University | Biological component comprising artificial membrane |
US7244830B2 (en) | 2001-01-12 | 2007-07-17 | Emory University | Glycopolymers and free radical polymerization methods |
EP2080771A2 (en) | 2001-02-27 | 2009-07-22 | Maxygen Aps | New interferon beta-like molecules |
US8071678B2 (en) | 2001-10-09 | 2011-12-06 | Nektar Therapeutics | Thioester-terminated water soluble polymers and method of modifying the N-terminus of a polypeptide therewith |
US8329876B2 (en) | 2001-10-09 | 2012-12-11 | Nektar Therapeutics | Thioester-terminated water soluble polymers and method of modifying the N-terminus of a polypeptide therewith |
US7834088B2 (en) | 2001-10-09 | 2010-11-16 | Nektar Therapeutics | Thioester-terminated water soluble polymers and method of modifying the N-terminus of a polypeptide therewith |
EP1434589A2 (en) * | 2001-10-09 | 2004-07-07 | Nektar Therapeutics Al, Corporation | Thioester-terminated water soluble polymers and method of modifying the n-terminus of a polypeptide therewith |
US7511095B2 (en) | 2001-10-09 | 2009-03-31 | Nektar Therapeutics Al, Corporation | Thioester-terminated water soluble polymers and method of modifying the N-terminus of a polypeptide therewith |
EP1434589A4 (en) * | 2001-10-09 | 2006-06-21 | Nektar Therapeutics Al Corp | Thioester-terminated water soluble polymers and method of modifying the n-terminus of a polypeptide therewith |
EP2042196A2 (en) | 2001-10-10 | 2009-04-01 | Neose Technologies, Inc. | Remodelling and glycoconjugation of Granulocyte Colony Stimulating Factor (G-CSF) |
EP2305313A2 (en) | 2001-10-10 | 2011-04-06 | BioGeneriX AG | Remodelling and glycoconjugation of interferon-alpha (IFNa) |
EP2305312A2 (en) | 2001-10-10 | 2011-04-06 | BioGeneriX AG | Remodelling and glycoconjugation of follicle-stimulating hormone (FSH) |
EP2305314A2 (en) | 2001-10-10 | 2011-04-06 | BioGeneriX AG | Remodelling and glycoconjugation of antibodies |
US8716240B2 (en) | 2001-10-10 | 2014-05-06 | Novo Nordisk A/S | Erythropoietin: remodeling and glycoconjugation of erythropoietin |
US8716239B2 (en) | 2001-10-10 | 2014-05-06 | Novo Nordisk A/S | Granulocyte colony stimulating factor: remodeling and glycoconjugation G-CSF |
US7795210B2 (en) | 2001-10-10 | 2010-09-14 | Novo Nordisk A/S | Protein remodeling methods and proteins/peptides produced by the methods |
EP2279753A2 (en) | 2001-10-10 | 2011-02-02 | Novo Nordisk A/S | Remodeling and glycoconjugation of peptides |
EP2080525A1 (en) | 2001-10-10 | 2009-07-22 | BioGeneriX AG | Remodeling and Glycoconjugation of Peptides |
EP2279754A2 (en) | 2001-10-10 | 2011-02-02 | BioGeneriX AG | Remodelling and glycoconjugation of human growth hormone (hGH) |
EP2322229A2 (en) | 2001-10-10 | 2011-05-18 | Novo Nordisk A/S | Remodelling and glycoconjugation of Granulocyte Colony Stimulating Factor (G-CSF) |
US8008252B2 (en) | 2001-10-10 | 2011-08-30 | Novo Nordisk A/S | Factor VII: remodeling and glycoconjugation of Factor VII |
EP2298354A2 (en) | 2001-10-10 | 2011-03-23 | BioGeneriX AG | Remodelling and glycoconjugation of interferon-beta |
US7696163B2 (en) | 2001-10-10 | 2010-04-13 | Novo Nordisk A/S | Erythropoietin: remodeling and glycoconjugation of erythropoietin |
EP2305311A2 (en) | 2001-10-10 | 2011-04-06 | BioGeneriX AG | Glycoconjugation of peptides |
EP2279755A2 (en) | 2001-10-10 | 2011-02-02 | BioGeneriX AG | Remodelling and glycoconjugation of Fibroblast Growth Factor (FGF) |
WO2003032990A3 (en) * | 2001-10-18 | 2004-02-26 | Nektar Therapeutics Al Corp | Polymer conjugates of opioid antagonists |
US8349307B2 (en) | 2001-10-18 | 2013-01-08 | Nektar Therapeutics | Polymer conjugates of opioid antagonists |
US7662365B2 (en) | 2001-10-18 | 2010-02-16 | Nektar Therapeutics | Polymer conjugates of opioid antagonists |
KR101009309B1 (en) * | 2001-10-18 | 2011-01-18 | 넥타르 테라퓨틱스 | Polymer conjugates of opioid antagonists |
AU2002360284B2 (en) * | 2001-10-18 | 2006-11-02 | Nektar Therapeutics | Polymer conjugates of opioid antagonists |
US7056500B2 (en) | 2001-10-18 | 2006-06-06 | Nektar Therapeutics Al, Corporation | Polymer conjugates of opioid antagonists |
WO2003032990A2 (en) * | 2001-10-18 | 2003-04-24 | Nektar Therapeutics Al, Corporation | Polymer conjugates of opioid antagonists |
US8617530B2 (en) | 2001-10-18 | 2013-12-31 | Nektar Therapeutics | Polymer conjugates of opioid antagonists |
EP2236161A1 (en) * | 2001-10-18 | 2010-10-06 | Nektar Therapeutics | Polymer conjugates of opioid antagonists |
KR100974842B1 (en) | 2001-10-18 | 2010-08-11 | 넥타르 테라퓨틱스 | Polymer conjugates of opioid antagonists |
WO2003093346A1 (en) * | 2002-05-06 | 2003-11-13 | Universita' Degli Studi Di Trieste | Multifunctional polyethylene glycol derivatives: preparation and use |
WO2004000366A1 (en) | 2002-06-21 | 2003-12-31 | Novo Nordisk Health Care Ag | Pegylated factor vii glycoforms |
US8853376B2 (en) | 2002-11-21 | 2014-10-07 | Archemix Llc | Stabilized aptamers to platelet derived growth factor and their use as oncology therapeutics |
US7648962B2 (en) | 2002-11-26 | 2010-01-19 | Biocon Limited | Natriuretic compounds, conjugates, and uses thereof |
US7662773B2 (en) | 2002-11-26 | 2010-02-16 | Biocon Limited | Natriuretic compounds, conjugates, and uses thereof |
WO2004061094A1 (en) | 2002-12-30 | 2004-07-22 | Gryphon Therapeutics, Inc. | Water-soluble thioester and selenoester compounds and methods for making and using the same |
US8034900B2 (en) | 2002-12-30 | 2011-10-11 | Amylin Pharmaceuticals, Inc. | Water-soluble thioester and selenoester compounds and methods for making and using the same |
US8247381B2 (en) | 2003-03-14 | 2012-08-21 | Biogenerix Ag | Branched water-soluble polymers and their conjugates |
US7803777B2 (en) | 2003-03-14 | 2010-09-28 | Biogenerix Ag | Branched water-soluble polymers and their conjugates |
US8063015B2 (en) | 2003-04-09 | 2011-11-22 | Novo Nordisk A/S | Glycopegylation methods and proteins/peptides produced by the methods |
EP2338333A2 (en) | 2003-04-09 | 2011-06-29 | BioGeneriX AG | Glycopegylation methods and proteins/peptides produced by the methods |
US8853161B2 (en) | 2003-04-09 | 2014-10-07 | Novo Nordisk A/S | Glycopegylation methods and proteins/peptides produced by the methods |
US8791070B2 (en) | 2003-04-09 | 2014-07-29 | Novo Nordisk A/S | Glycopegylated factor IX |
EP2055189A1 (en) | 2003-04-09 | 2009-05-06 | Neose Technologies, Inc. | Glycopegylation methods and proteins/peptides produced by the methods |
US7932364B2 (en) | 2003-05-09 | 2011-04-26 | Novo Nordisk A/S | Compositions and methods for the preparation of human growth hormone glycosylation mutants |
US9005625B2 (en) | 2003-07-25 | 2015-04-14 | Novo Nordisk A/S | Antibody toxin conjugates |
EP2322569A2 (en) | 2003-10-09 | 2011-05-18 | Ambrx, Inc. | Polymer derivates |
US8008428B2 (en) | 2003-10-09 | 2011-08-30 | Ambrx, Inc. | Azide- or acetylene-terminated poly(alkylene oxide, oxyethylated polyol or olefinic Alcohol) |
WO2005035727A2 (en) | 2003-10-09 | 2005-04-21 | Ambrx, Inc. | Polymer derivatives |
US7737226B2 (en) | 2003-10-09 | 2010-06-15 | Ambrx, Inc. | Acetylene group-containing poly(alkylene oxide, oxyethylated polyol or olefinic alcohol) |
US9574048B2 (en) | 2003-10-09 | 2017-02-21 | Ambrx, Inc. | Polymer derivatives |
US7230068B2 (en) | 2003-10-09 | 2007-06-12 | Ambrx, Inc. | Polymer derivatives |
US7820766B2 (en) * | 2003-10-09 | 2010-10-26 | Ambrx, Inc. | Branched acetylene-containing poly(alkylene oxides, oxyethylated polyols or olefinic alcohols) |
EP2263684A1 (en) | 2003-10-10 | 2010-12-22 | Novo Nordisk A/S | IL-21 derivatives |
EP2633866A2 (en) | 2003-10-17 | 2013-09-04 | Novo Nordisk A/S | Combination therapy |
EP2641611A2 (en) | 2003-10-17 | 2013-09-25 | Novo Nordisk A/S | Combination therapy |
US7842661B2 (en) | 2003-11-24 | 2010-11-30 | Novo Nordisk A/S | Glycopegylated erythropoietin formulations |
US8633157B2 (en) | 2003-11-24 | 2014-01-21 | Novo Nordisk A/S | Glycopegylated erythropoietin |
US8916360B2 (en) | 2003-11-24 | 2014-12-23 | Novo Nordisk A/S | Glycopegylated erythropoietin |
US7956032B2 (en) | 2003-12-03 | 2011-06-07 | Novo Nordisk A/S | Glycopegylated granulocyte colony stimulating factor |
US8034825B2 (en) | 2003-12-16 | 2011-10-11 | Nektar Therapeutics | Chemically modified small molecules |
US11129794B2 (en) | 2003-12-16 | 2021-09-28 | Nektar Therapeutics | Chemically modified small molecules |
US8067431B2 (en) | 2003-12-16 | 2011-11-29 | Nektar Therapeutics | Chemically modified small molecules |
US9388104B2 (en) | 2003-12-16 | 2016-07-12 | Nektar Therapeutics | Chemically modified small molecules |
US7786133B2 (en) | 2003-12-16 | 2010-08-31 | Nektar Therapeutics | Chemically modified small molecules |
US9920106B2 (en) | 2003-12-18 | 2018-03-20 | Novo Nordisk A/S | GLP-1 compounds |
US8232371B2 (en) | 2004-02-02 | 2012-07-31 | Ambrx, Inc. | Modified human interferon polypeptides and their uses |
US8097702B2 (en) | 2004-02-02 | 2012-01-17 | Ambrx, Inc. | Modified human interferon polypeptides with at least one non-naturally encoded amino acid and their uses |
EP2327724A2 (en) | 2004-02-02 | 2011-06-01 | Ambrx, Inc. | Modified human growth hormone polypeptides and their uses |
US8906676B2 (en) | 2004-02-02 | 2014-12-09 | Ambrx, Inc. | Modified human four helical bundle polypeptides and their uses |
US8907064B2 (en) | 2004-02-02 | 2014-12-09 | Ambrx, Inc. | Modified human four helical bundle polypeptides and their uses |
US9260472B2 (en) | 2004-02-02 | 2016-02-16 | Ambrx, Inc. | Modified human four helical bundle polypeptides and their uses |
US7833978B2 (en) | 2004-02-20 | 2010-11-16 | Emory University | Thrombomodulin derivatives and conjugates |
US7824672B2 (en) | 2004-03-26 | 2010-11-02 | Emory University | Method for coating living cells |
US9393319B2 (en) | 2004-04-13 | 2016-07-19 | Quintessence Biosciences, Inc. | Non-natural ribonuclease conjugates as cytotoxic agents |
WO2005108463A2 (en) * | 2004-05-03 | 2005-11-17 | Nektar Therapeutics Al, Corporation | Branched polyethylen glycol derivates comprising an acetal or ketal branching point |
WO2005108463A3 (en) * | 2004-05-03 | 2006-03-30 | Nektar Therapeutics Al Corp | Branched polyethylen glycol derivates comprising an acetal or ketal branching point |
US8562965B2 (en) | 2004-05-03 | 2013-10-22 | Nektar Therapeutics | Polymer derivatives comprising an acetal or ketal branching point |
US9308273B2 (en) | 2004-05-03 | 2016-04-12 | Nektar Therapeutics | Polymer derivatives comprising an acetal or ketal branching point |
EP1776132A2 (en) * | 2004-05-26 | 2007-04-25 | Nobex Corporation | Natriuretic compounds, conjugates, and uses thereof |
JP2008500375A (en) * | 2004-05-26 | 2008-01-10 | バイオコン・リミテッド | Natriuretic compounds, complexes, and uses thereof |
EP1776132A4 (en) * | 2004-05-26 | 2009-07-01 | Biocon Ltd | Natriuretic compounds, conjugates, and uses thereof |
WO2006009901A2 (en) | 2004-06-18 | 2006-01-26 | Ambrx, Inc. | Novel antigen-binding polypeptides and their uses |
US9175083B2 (en) | 2004-06-18 | 2015-11-03 | Ambrx, Inc. | Antigen-binding polypeptides and their uses |
US8791066B2 (en) | 2004-07-13 | 2014-07-29 | Novo Nordisk A/S | Branched PEG remodeling and glycosylation of glucagon-like peptide-1 [GLP-1] |
US8268967B2 (en) | 2004-09-10 | 2012-09-18 | Novo Nordisk A/S | Glycopegylated interferon α |
EP2586456A1 (en) | 2004-10-29 | 2013-05-01 | BioGeneriX AG | Remodeling and glycopegylation of fibroblast growth factor (FGF) |
US10874714B2 (en) | 2004-10-29 | 2020-12-29 | 89Bio Ltd. | Method of treating fibroblast growth factor 21 (FGF-21) deficiency |
EP3061461A1 (en) | 2004-10-29 | 2016-08-31 | ratiopharm GmbH | Remodeling and glycopegylation of fibroblast growth factor (fgf) |
US9200049B2 (en) | 2004-10-29 | 2015-12-01 | Novo Nordisk A/S | Remodeling and glycopegylation of fibroblast growth factor (FGF) |
US8178494B2 (en) | 2004-12-22 | 2012-05-15 | Ambrx, Inc. | Modified human growth hormone formulations with an increased serum half-life |
US7736872B2 (en) | 2004-12-22 | 2010-06-15 | Ambrx, Inc. | Compositions of aminoacyl-TRNA synthetase and uses thereof |
EP2399893A2 (en) | 2004-12-22 | 2011-12-28 | Ambrx, Inc. | Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides |
US7858344B2 (en) | 2004-12-22 | 2010-12-28 | Ambrx, Inc. | Compositions of aminoacyl-tRNA synthetase and uses thereof |
US7939496B2 (en) | 2004-12-22 | 2011-05-10 | Ambrx, Inc. | Modified human growth horomone polypeptides and their uses |
US7846689B2 (en) | 2004-12-22 | 2010-12-07 | Ambrx, Inc. | Compositions of aminoacyl-tRNA synthetase and uses thereof |
US7959926B2 (en) | 2004-12-22 | 2011-06-14 | Ambrx, Inc. | Methods for expression and purification of recombinant human growth hormone mutants |
US7838265B2 (en) | 2004-12-22 | 2010-11-23 | Ambrx, Inc. | Compositions of aminoacyl-tRNA synthetase and uses thereof |
US8143216B2 (en) | 2004-12-22 | 2012-03-27 | Ambrx, Inc. | Modified human growth hormone |
WO2006069246A2 (en) | 2004-12-22 | 2006-06-29 | Ambrx, Inc. | Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides |
US8163695B2 (en) | 2004-12-22 | 2012-04-24 | Ambrx | Formulations of human growth hormone comprising a non-naturally encoded amino acid |
US8080391B2 (en) | 2004-12-22 | 2011-12-20 | Ambrx, Inc. | Process of producing non-naturally encoded amino acid containing high conjugated to a water soluble polymer |
US7947473B2 (en) | 2004-12-22 | 2011-05-24 | Ambrx, Inc. | Methods for expression and purification of pegylated recombinant human growth hormone containing a non-naturally encoded keto amino acid |
US8178108B2 (en) | 2004-12-22 | 2012-05-15 | Ambrx, Inc. | Methods for expression and purification of recombinant human growth hormone |
US7829310B2 (en) | 2004-12-22 | 2010-11-09 | Ambrx, Inc. | Compositions of aminoacyl-tRNA synthetase and uses thereof |
US7816320B2 (en) | 2004-12-22 | 2010-10-19 | Ambrx, Inc. | Formulations of human growth hormone comprising a non-naturally encoded amino acid at position 35 |
EP2284191A2 (en) | 2004-12-22 | 2011-02-16 | Ambrx, Inc. | Process for the preparation of hGH |
US7883866B2 (en) | 2004-12-22 | 2011-02-08 | Ambrx, Inc. | Compositions of aminoacyl-tRNA synthetase and uses thereof |
US9029331B2 (en) | 2005-01-10 | 2015-05-12 | Novo Nordisk A/S | Glycopegylated granulocyte colony stimulating factor |
EP2514757A2 (en) | 2005-01-10 | 2012-10-24 | BioGeneriX AG | Glycopegylated granulocyte colony stimulating factor |
EP2314320A2 (en) | 2005-04-05 | 2011-04-27 | Istituto di Richerche di Biologia Molecolare P. Angeletti S.p.A. | Method for shielding functional sites or epitopes on proteins |
EP2279756A2 (en) | 2005-04-05 | 2011-02-02 | Instituto di Ricerche di Biologia Molecolare p Angeletti S.P.A. | Method for shielding functional sites or epitopes on proteins |
EP2386571A2 (en) | 2005-04-08 | 2011-11-16 | BioGeneriX AG | Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants |
US9187546B2 (en) | 2005-04-08 | 2015-11-17 | Novo Nordisk A/S | Compositions and methods for the preparation of protease resistant human growth hormone glycosylation mutants |
EP1891231A2 (en) * | 2005-05-25 | 2008-02-27 | Neose Technologies, Inc. | Glycopegylated factor ix |
JP2008542285A (en) * | 2005-05-25 | 2008-11-27 | ネオス テクノロジーズ インコーポレイテッド | Glycopegylated factor IX |
EP1891231A4 (en) * | 2005-05-25 | 2011-06-22 | Novo Nordisk As | Glycopegylated factor ix |
EP2975135A1 (en) | 2005-05-25 | 2016-01-20 | Novo Nordisk A/S | Glycopegylated factor IX |
US8093356B2 (en) | 2005-06-03 | 2012-01-10 | Ambrx, Inc. | Pegylated human interferon polypeptides |
WO2006134173A2 (en) | 2005-06-17 | 2006-12-21 | Novo Nordisk Health Care Ag | Selective reduction and derivatization of engineered proteins comprising at least one non-native cysteine |
EP2360170A2 (en) | 2005-06-17 | 2011-08-24 | Novo Nordisk Health Care AG | Selective reduction and derivatization of engineered proteins comprinsing at least one non-native cysteine |
US8911967B2 (en) | 2005-08-19 | 2014-12-16 | Novo Nordisk A/S | One pot desialylation and glycopegylation of therapeutic peptides |
US8841439B2 (en) | 2005-11-03 | 2014-09-23 | Novo Nordisk A/S | Nucleotide sugar purification using membranes |
US9488660B2 (en) | 2005-11-16 | 2016-11-08 | Ambrx, Inc. | Methods and compositions comprising non-natural amino acids |
WO2007062610A3 (en) * | 2005-11-30 | 2007-09-20 | Ct Ingenieria Genetica Biotech | Four branched dendrimer-peg for conjugation to proteins and peptides |
AU2006319636B2 (en) * | 2005-11-30 | 2012-10-18 | Centro De Ingenieria Genetica Y Biotecnologia | Four branched dendrimer-peg for conjugation to proteins and peptides |
US8703893B2 (en) | 2005-11-30 | 2014-04-22 | Centro De Ingenienia Genetica Y Biotecnologia | Four branched dendrimer-PEG for conjugation to proteins and peptides |
JP2009517414A (en) * | 2005-11-30 | 2009-04-30 | セントロ デ インジエニエリア ジエネテイカ イ バイオテクノロジア | 4-branch dendrimer-PEG for conjugation to proteins and peptides |
KR101134983B1 (en) * | 2005-11-30 | 2012-04-09 | 센트로 데 인제니에리아 제네티카 와이 바이오테크놀로지아 | Four branched dendrimer-peg for conjugation to proteins and peptides |
US7632492B2 (en) | 2006-05-02 | 2009-12-15 | Allozyne, Inc. | Modified human interferon-β polypeptides |
US10407482B2 (en) | 2006-05-02 | 2019-09-10 | Allozyne, Inc. | Amino acid substituted molecules |
US7829659B2 (en) | 2006-05-02 | 2010-11-09 | Allozyne, Inc. | Methods of modifying polypeptides comprising non-natural amino acids |
EP2581450A2 (en) | 2006-05-02 | 2013-04-17 | Allozyne, Inc. | Non-natural amino acid substituted polypeptides |
EP2395099A2 (en) | 2006-05-02 | 2011-12-14 | Allozyne, Inc. | Amino acid substituted molecules |
EP2444499A2 (en) | 2006-05-02 | 2012-04-25 | Allozyne, Inc. | Amino acid substituted molecules |
EP2213733A2 (en) | 2006-05-24 | 2010-08-04 | Novo Nordisk Health Care AG | Factor IX analogues having prolonged in vivo half life |
US8840882B2 (en) | 2006-06-23 | 2014-09-23 | Quintessence Biosciences, Inc. | Modified ribonucleases |
JP2009541333A (en) * | 2006-06-23 | 2009-11-26 | クインテセンス バイオサイエンシーズ インコーポレーティッド | Modified ribonuclease |
US9192656B2 (en) | 2006-07-17 | 2015-11-24 | Quintessence Biosciences, Inc. | Methods and compositions for the treatment of cancer |
US9187532B2 (en) | 2006-07-21 | 2015-11-17 | Novo Nordisk A/S | Glycosylation of peptides via O-linked glycosylation sequences |
WO2008030558A2 (en) | 2006-09-08 | 2008-03-13 | Ambrx, Inc. | Modified human plasma polypeptide or fc scaffolds and their uses |
US9133495B2 (en) | 2006-09-08 | 2015-09-15 | Ambrx, Inc. | Hybrid suppressor tRNA for vertebrate cells |
US8053560B2 (en) | 2006-09-08 | 2011-11-08 | Ambrx, Inc. | Modified human plasma polypeptide or Fc scaffolds and their uses |
US8420792B2 (en) | 2006-09-08 | 2013-04-16 | Ambrx, Inc. | Suppressor tRNA transcription in vertebrate cells |
US7919591B2 (en) | 2006-09-08 | 2011-04-05 | Ambrx, Inc. | Modified human plasma polypeptide or Fc scaffolds and their uses |
US8022186B2 (en) | 2006-09-08 | 2011-09-20 | Ambrx, Inc. | Modified human plasma polypeptide or Fc scaffolds and their uses |
US8618257B2 (en) | 2006-09-08 | 2013-12-31 | Ambrx, Inc. | Modified human plasma polypeptide or Fc scaffolds and their uses |
US8846624B2 (en) | 2006-09-11 | 2014-09-30 | Emory University | Modified protein polymers |
EP2548967A2 (en) | 2006-09-21 | 2013-01-23 | The Regents of The University of California | Aldehyde tags, uses thereof in site-specific protein modification |
US8969532B2 (en) | 2006-10-03 | 2015-03-03 | Novo Nordisk A/S | Methods for the purification of polypeptide conjugates comprising polyalkylene oxide using hydrophobic interaction chromatography |
WO2008060780A2 (en) | 2006-10-04 | 2008-05-22 | Novo Nordisk A/S | Glycerol linked pegylated sugars and glycopeptides |
US9079971B2 (en) | 2007-03-30 | 2015-07-14 | Ambrx, Inc. | Modified FGF-21 polypeptides comprising non-naturally occurring amino acids |
US9975936B2 (en) | 2007-03-30 | 2018-05-22 | Ambrx, Inc. | Nucleic acids encoding modified FGF-21 polypeptides comprising non-naturally occurring amino acids |
US8012931B2 (en) | 2007-03-30 | 2011-09-06 | Ambrx, Inc. | Modified FGF-21 polypeptides and their uses |
US10377805B2 (en) | 2007-03-30 | 2019-08-13 | Ambrx, Inc. | Modified FGF-21 polypeptides comprising non-naturally encoding amino acids and their uses |
US10961291B2 (en) | 2007-03-30 | 2021-03-30 | Ambrx, Inc. | Modified FGF-21 polypeptides and their uses |
US8383365B2 (en) | 2007-03-30 | 2013-02-26 | Ambrx, Inc. | Methods of making FGF-21 mutants comprising non-naturally encoded phenylalanine derivatives |
US9517273B2 (en) | 2007-03-30 | 2016-12-13 | Ambrx, Inc. | Methods of treatment using modified FGF-21 polypeptides comprising non-naturally occurring amino acids |
US9050304B2 (en) | 2007-04-03 | 2015-06-09 | Ratiopharm Gmbh | Methods of treatment using glycopegylated G-CSF |
US8114630B2 (en) | 2007-05-02 | 2012-02-14 | Ambrx, Inc. | Modified interferon beta polypeptides and their uses |
US9493499B2 (en) | 2007-06-12 | 2016-11-15 | Novo Nordisk A/S | Process for the production of purified cytidinemonophosphate-sialic acid-polyalkylene oxide (CMP-SA-PEG) as modified nucleotide sugars via anion exchange chromatography |
EP4129343A1 (en) * | 2007-08-16 | 2023-02-08 | PharmaEssentia Corp. | Protein-polymer conjugates |
EP2205281A4 (en) * | 2007-08-16 | 2014-10-08 | Pharmaessentia Corp | Protein-polymer conjugates |
EP2205281A1 (en) * | 2007-08-16 | 2010-07-14 | Pharmaessentia Corp. | Protein-polymer conjugates |
EP2205281B1 (en) * | 2007-08-16 | 2022-06-29 | PharmaEssentia Corp. | Protein-polymer conjugates |
WO2009023826A1 (en) | 2007-08-16 | 2009-02-19 | Pharmaessentia Corp. | Protein-polymer conjugates |
US8207112B2 (en) | 2007-08-29 | 2012-06-26 | Biogenerix Ag | Liquid formulation of G-CSF conjugate |
US8946148B2 (en) | 2007-11-20 | 2015-02-03 | Ambrx, Inc. | Modified insulin polypeptides and their uses |
WO2009067636A2 (en) | 2007-11-20 | 2009-05-28 | Ambrx, Inc. | Modified insulin polypeptides and their uses |
EP2930182A1 (en) | 2007-11-20 | 2015-10-14 | Ambrx, Inc. | Modified insulin polypeptides and their uses |
EP3103880A1 (en) | 2008-02-08 | 2016-12-14 | Ambrx, Inc. | Modified leptin polypeptides and their uses |
US9938333B2 (en) | 2008-02-08 | 2018-04-10 | Ambrx, Inc. | Modified leptin polypeptides and their uses |
US9150848B2 (en) | 2008-02-27 | 2015-10-06 | Novo Nordisk A/S | Conjugated factor VIII molecules |
EP3225248A1 (en) | 2008-07-23 | 2017-10-04 | Ambrx, Inc. | Modified bovine g-csf polypeptides and their uses |
WO2010011735A2 (en) | 2008-07-23 | 2010-01-28 | Ambrx, Inc. | Modified bovine g-csf polypeptides and their uses |
US10138283B2 (en) | 2008-07-23 | 2018-11-27 | Ambrx, Inc. | Modified bovine G-CSF polypeptides and their uses |
EP3216800A1 (en) | 2008-09-26 | 2017-09-13 | Ambrx, Inc. | Modified animal erythropoietin polypeptides and their uses |
US9121025B2 (en) | 2008-09-26 | 2015-09-01 | Ambrx, Inc. | Non-natural amino acid replication-dependent microorganisms and vaccines |
US9644014B2 (en) | 2008-09-26 | 2017-05-09 | Ambrx, Inc. | Modified animal erythropoietin polypeptides and their uses |
US8278418B2 (en) | 2008-09-26 | 2012-10-02 | Ambrx, Inc. | Modified animal erythropoietin polypeptides and their uses |
US9156899B2 (en) | 2008-09-26 | 2015-10-13 | Eli Lilly And Company | Modified animal erythropoietin polypeptides and their uses |
US10428333B2 (en) | 2008-09-26 | 2019-10-01 | Ambrx Inc. | Non-natural amino acid replication-dependent microorganisms and vaccines |
US8569233B2 (en) | 2008-09-26 | 2013-10-29 | Eli Lilly And Company | Modified animal erythropoietin polypeptides and their uses |
US9121024B2 (en) | 2008-09-26 | 2015-09-01 | Ambrx, Inc. | Non-natural amino acid replication-dependent microorganisms and vaccines |
US9006407B2 (en) | 2008-10-01 | 2015-04-14 | Quintessence Biosciences, Inc. | Therapeutic ribonucleases |
US9579365B2 (en) | 2008-10-01 | 2017-02-28 | Quintessence Biosciences, Inc. | Therapeutic ribonucleases |
WO2010144629A1 (en) | 2009-06-09 | 2010-12-16 | Prolong Pharmaceuticals, LLC | Hemoglobin compositions |
EP3266463A1 (en) | 2009-06-09 | 2018-01-10 | Prolong Pharmaceuticals, LLC | Hemoglobin compositions |
EP2805964A1 (en) | 2009-12-21 | 2014-11-26 | Ambrx, Inc. | Modified bovine somatotropin polypeptides and their uses |
EP2805965A1 (en) | 2009-12-21 | 2014-11-26 | Ambrx, Inc. | Modified porcine somatotropin polypeptides and their uses |
WO2011107591A1 (en) | 2010-03-05 | 2011-09-09 | Rigshospitalet | Chimeric inhibitor molecules of complement activation |
EP3815708A1 (en) | 2010-03-05 | 2021-05-05 | Omeros Corporation | Chimeric inhibitor molecules of complement activation |
WO2011143274A1 (en) | 2010-05-10 | 2011-11-17 | Perseid Therapeutics | Polypeptide inhibitors of vla4 |
US11786578B2 (en) | 2010-08-17 | 2023-10-17 | Ambrx, Inc. | Modified relaxin polypeptides and their uses |
US10253083B2 (en) | 2010-08-17 | 2019-04-09 | Ambrx, Inc. | Therapeutic uses of modified relaxin polypeptides |
WO2012024452A2 (en) | 2010-08-17 | 2012-02-23 | Ambrx, Inc. | Modified relaxin polypeptides and their uses |
US9962450B2 (en) | 2010-08-17 | 2018-05-08 | Ambrx, Inc. | Method of treating heart failure with modified relaxin polypeptides |
US11439710B2 (en) | 2010-08-17 | 2022-09-13 | Ambrx, Inc. | Nucleic acids encoding modified relaxin polypeptides |
US9567386B2 (en) | 2010-08-17 | 2017-02-14 | Ambrx, Inc. | Therapeutic uses of modified relaxin polypeptides |
US9452222B2 (en) | 2010-08-17 | 2016-09-27 | Ambrx, Inc. | Nucleic acids encoding modified relaxin polypeptides |
US10751391B2 (en) | 2010-08-17 | 2020-08-25 | Ambrx, Inc. | Methods of treatment using modified relaxin polypeptides comprising a non-naturally encoded amino acid |
US10702588B2 (en) | 2010-08-17 | 2020-07-07 | Ambrx, Inc. | Modified relaxin polypeptides comprising a non-naturally encoded amino acid in the A chain |
EP4302783A2 (en) | 2010-08-17 | 2024-01-10 | Ambrx, Inc. | Modified relaxin polypeptides and their uses |
US11311605B2 (en) | 2010-08-17 | 2022-04-26 | Ambrx, Inc. | Methods of treating heart failure and fibrotic disorders using modified relaxin polypeptides |
US8735539B2 (en) | 2010-08-17 | 2014-05-27 | Ambrx, Inc. | Relaxin polypeptides comprising non-naturally encoded amino acids |
US11273202B2 (en) | 2010-09-23 | 2022-03-15 | Elanco Us Inc. | Formulations for bovine granulocyte colony stimulating factor and variants thereof |
WO2013004607A1 (en) | 2011-07-01 | 2013-01-10 | Bayer Intellectual Property Gmbh | Relaxin fusion polypeptides and uses thereof |
US9382305B2 (en) | 2011-07-01 | 2016-07-05 | Bayer Intellectual Property Gmbh | Relaxin fusion polypeptides and uses thereof |
EP3088005A1 (en) | 2011-07-05 | 2016-11-02 | biOasis Technologies Inc | P97-antibody conjugates |
WO2013006706A1 (en) | 2011-07-05 | 2013-01-10 | Bioasis Technologies Inc. | P97-antibody conjugates and methods of use |
WO2013185115A1 (en) | 2012-06-08 | 2013-12-12 | Sutro Biopharma, Inc. | Antibodies comprising site-specific non-natural amino acid residues, methods of their preparation and methods of their use |
EP3505534A1 (en) | 2012-06-08 | 2019-07-03 | Sutro Biopharma, Inc. | Antibodies comprising sitespecific nonnatural amino acid residues, methods of their preparation and methods of their use |
EP3135690A1 (en) | 2012-06-26 | 2017-03-01 | Sutro Biopharma, Inc. | Modified fc proteins comprising site-specific non-natural amino acid residues, conjugates of the same, methods of their preparation and methods of their use |
WO2014022515A1 (en) | 2012-07-31 | 2014-02-06 | Bioasis Technologies, Inc. | Dephosphorylated lysosomal storage disease proteins and methods of use thereof |
EP3584255A1 (en) | 2012-08-31 | 2019-12-25 | Sutro Biopharma, Inc. | Modified amino acids comprising an azido group |
WO2014036492A1 (en) | 2012-08-31 | 2014-03-06 | Sutro Biopharma, Inc. | Modified amino acids comprising an azido group |
EP4074728A1 (en) | 2012-08-31 | 2022-10-19 | Sutro Biopharma, Inc. | Modified peptides comprising an azido group |
US9579390B2 (en) | 2012-11-12 | 2017-02-28 | Redwood Bioscience, Inc. | Compounds and methods for producing a conjugate |
US9833515B2 (en) | 2012-11-16 | 2017-12-05 | Redwood Bioscience, Inc. | Hydrazinyl-indole compounds and methods for producing a conjugate |
US9310374B2 (en) | 2012-11-16 | 2016-04-12 | Redwood Bioscience, Inc. | Hydrazinyl-indole compounds and methods for producing a conjugate |
US10888623B2 (en) | 2012-11-16 | 2021-01-12 | Redwood Bioscience, Inc. | Hydrazinyl-indole compounds and methods for producing a conjugate |
US10314919B2 (en) | 2012-11-16 | 2019-06-11 | Redwood Bioscience, Inc. | Hydrazinyl-indole compounds and methods for producing a conjugate |
US9605078B2 (en) | 2012-11-16 | 2017-03-28 | The Regents Of The University Of California | Pictet-Spengler ligation for protein chemical modification |
US11426465B2 (en) | 2012-11-16 | 2022-08-30 | Redwiid Bioscience, Inc. | Hydrazinyl-indole compounds and methods for producing a conjugate |
WO2014118382A1 (en) | 2013-02-04 | 2014-08-07 | W. L. Gore & Associates, Inc. | Coating for substrate |
US9272075B2 (en) | 2013-02-04 | 2016-03-01 | W.L. Gore & Associates, Inc. | Coating for substrate |
WO2014160438A1 (en) | 2013-03-13 | 2014-10-02 | Bioasis Technologies Inc. | Fragments of p97 and uses thereof |
EP3336103A1 (en) | 2013-07-10 | 2018-06-20 | Sutro Biopharma, Inc. | Antibodies comprising multiple site-specific non-natural amino acid residues, methods of their preparation and methods of their use |
WO2015006555A2 (en) | 2013-07-10 | 2015-01-15 | Sutro Biopharma, Inc. | Antibodies comprising multiple site-specific non-natural amino acid residues, methods of their preparation and methods of their use |
WO2015031673A2 (en) | 2013-08-28 | 2015-03-05 | Bioasis Technologies Inc. | Cns-targeted conjugates having modified fc regions and methods of use thereof |
WO2015054658A1 (en) | 2013-10-11 | 2015-04-16 | Sutro Biopharma, Inc. | Modified amino acids comprising tetrazine functional groups, methods of preparation, and methods of their use |
WO2015081282A1 (en) | 2013-11-27 | 2015-06-04 | Redwood Bioscience, Inc. | Hydrazinyl-pyrrolo compounds and methods for producing a conjugate |
US10189883B2 (en) | 2014-10-24 | 2019-01-29 | Bristol-Myers Squibb Company | Therapeutic uses of modified FGF-21 polypeptides |
US10377806B2 (en) | 2014-10-24 | 2019-08-13 | Bristol-Myers Squibb Company | Methods of treating diseases associated with fibrosis using modified FGF-21 polypeptides and uses thereof |
US9631004B2 (en) | 2014-10-24 | 2017-04-25 | Bristol-Myers Squibb Company | Modified FGF-21 polypeptides comprising an internal deletion and uses thereof |
US9434778B2 (en) | 2014-10-24 | 2016-09-06 | Bristol-Myers Squibb Company | Modified FGF-21 polypeptides comprising an internal deletion and uses thereof |
US11248031B2 (en) | 2014-10-24 | 2022-02-15 | Bristol-Myers Squibb Company | Methods of treating diseases associated with fibrosis using modified FGF-21 polypeptides |
US10131688B2 (en) | 2014-11-19 | 2018-11-20 | NZP UK Limited | 5.beta.-6-alkyl-7-hydroxy-3-one steroids as intermediates for the production of steroidal FXR modulators |
US10597423B2 (en) | 2014-11-19 | 2020-03-24 | NZP UK Limited | 6.alpha.-alkyl-6,7-dione steroids as intermediates for the production of steroidal FXR modulators |
US10538550B2 (en) | 2014-11-19 | 2020-01-21 | NZP UK Limited | 6.alpha.-alkyl-3,7-dione steroids as intermediates for the production of steroidal FXR modulators |
US10301350B2 (en) | 2014-11-19 | 2019-05-28 | NZP UK Limited | 6-alkyl-7-hydroxy-4-en-3-one steroids as intermediates for the production of steroidal FXR modulators |
WO2017132617A1 (en) | 2016-01-27 | 2017-08-03 | Sutro Biopharma, Inc. | Anti-cd74 antibody conjugates, compositions comprising anti-cd74 antibody conjugates and methods of using anti-cd74 antibody conjugates |
WO2017132615A1 (en) | 2016-01-27 | 2017-08-03 | Sutro Biopharma, Inc. | Anti-cd74 antibody conjugates, compositions comprising anti-cd74 antibody conjugates and methods of using anti-cd74 antibody conjugates |
US10766921B2 (en) | 2016-05-18 | 2020-09-08 | NZP UK Limited | Process and intermediates for the 6,7-alpha-epoxidation of steroid 4,6-dienes |
US11479577B2 (en) | 2016-05-18 | 2022-10-25 | NZP UK Limited | Intermediates for the synthesis of bile acid derivatives, in particular of obeticholic acid |
US10968250B2 (en) | 2016-05-18 | 2021-04-06 | NZP UK Limited | Intermediates for the synthesis of bile acid derivatives, in particular of obeticholic acid |
EP3848382A1 (en) | 2016-05-18 | 2021-07-14 | NZP UK Limited | Intermediates for the synthesis of bile acid derivatives, in particular of obeticholic acid |
WO2017199033A1 (en) | 2016-05-18 | 2017-11-23 | NZP UK Limited | Intermediates for the synthesis of bile acid derivatives, in particular of obeticholic acid |
US11185570B2 (en) | 2017-02-08 | 2021-11-30 | Bristol-Myers Squibb Company | Method of treating cardiovascular disease and heart failure with modified relaxin polypeptides |
US10266578B2 (en) | 2017-02-08 | 2019-04-23 | Bristol-Myers Squibb Company | Modified relaxin polypeptides comprising a pharmacokinetic enhancer and uses thereof |
US11364281B2 (en) | 2017-02-08 | 2022-06-21 | Bristol-Myers Squibb Company | Modified relaxin polypeptides comprising a pharmacokinetic enhancer and pharmaceutical compositions thereof |
WO2019023316A1 (en) | 2017-07-26 | 2019-01-31 | Sutro Biopharma, Inc. | Methods of using anti-cd74 antibodies and antibody conjugates in treatment of t-cell lymphoma |
WO2019055909A1 (en) | 2017-09-18 | 2019-03-21 | Sutro Biopharma, Inc. | Anti-folate receptor alpha antibody conjugates and their uses |
WO2019133399A1 (en) | 2017-12-26 | 2019-07-04 | Becton, Dickinson And Company | Deep ultraviolet-excitable water-solvated polymeric dyes |
WO2019191482A1 (en) | 2018-03-30 | 2019-10-03 | Becton, Dickinson And Company | Water-soluble polymeric dyes having pendant chromophores |
WO2020023300A1 (en) | 2018-07-22 | 2020-01-30 | Bioasis Technologies, Inc. | Treatment of lymmphatic metastases |
WO2020056066A1 (en) | 2018-09-11 | 2020-03-19 | Ambrx, Inc. | Interleukin-2 polypeptide conjugates and their uses |
WO2020060944A1 (en) | 2018-09-17 | 2020-03-26 | Sutro Biopharma, Inc. | Combination therapies with anti-folate receptor antibody conjugates |
WO2020061670A1 (en) * | 2018-09-25 | 2020-04-02 | Universidade Federal Do Rio De Janeiro | Liposomal formulation, pharmaceutical composition, use of a liposomal formulation, method for treating cancer, and process for preparing a liposomal formulation |
WO2020082057A1 (en) | 2018-10-19 | 2020-04-23 | Ambrx, Inc. | Interleukin-10 polypeptide conjugates, dimers thereof, and their uses |
WO2020168017A1 (en) | 2019-02-12 | 2020-08-20 | Ambrx, Inc. | Compositions containing, methods and uses of antibody-tlr agonist conjugates |
WO2020227105A1 (en) | 2019-05-03 | 2020-11-12 | Sutro Biopharma, Inc. | Anti-bcma antibody conjugates |
WO2020252043A1 (en) | 2019-06-10 | 2020-12-17 | Sutro Biopharma, Inc. | 5H-PYRROLO[3,2-d]PYRIMIDINE-2,4-DIAMINO COMPOUNDS AND ANTIBODY CONJUGATES THEREOF |
WO2020257235A1 (en) | 2019-06-17 | 2020-12-24 | Sutro Biopharma, Inc. | 1-(4-(aminomethyl)benzyl)-2-butyl-2h-pyrazolo[3,4-c]quinolin-4-amine derivatives and related compounds as toll-like receptor (tlr) 7/8 agonists, as well as antibody drug conjugates thereof for use in cancer therapy and diagnosis |
EP4083108A4 (en) * | 2019-12-27 | 2024-02-14 | Nof Corp | Refinement method for polyethylene glycol compound |
WO2021178597A1 (en) | 2020-03-03 | 2021-09-10 | Sutro Biopharma, Inc. | Antibodies comprising site-specific glutamine tags, methods of their preparation and methods of their use |
WO2021183832A1 (en) | 2020-03-11 | 2021-09-16 | Ambrx, Inc. | Interleukin-2 polypeptide conjugates and methods of use thereof |
WO2021236526A1 (en) | 2020-05-18 | 2021-11-25 | Bioasis Technologies, Inc. | Compositions and methods for treating lewy body dementia |
WO2021255524A1 (en) | 2020-06-17 | 2021-12-23 | Bioasis Technologies, Inc. | Compositions and methods for treating frontotemporal dementia |
WO2022040596A1 (en) | 2020-08-20 | 2022-02-24 | Ambrx, Inc. | Antibody-tlr agonist conjugates, methods and uses thereof |
WO2022103983A2 (en) | 2020-11-11 | 2022-05-19 | Sutro Biopharma, Inc. | Fluorenylmethyloxycarbonyl and fluorenylmethylaminocarbonyl compounds, protein conjugates thereof, and methods for their use |
WO2022212899A1 (en) | 2021-04-03 | 2022-10-06 | Ambrx, Inc. | Anti-her2 antibody-drug conjugates and uses thereof |
US11931420B2 (en) | 2021-04-30 | 2024-03-19 | Celgene Corporation | Combination therapies using an anti-BCMA antibody drug conjugate (ADC) in combination with a gamma secretase inhibitor (GSI) |
EP4155349A1 (en) | 2021-09-24 | 2023-03-29 | Becton, Dickinson and Company | Water-soluble yellow green absorbing dyes |
WO2024006272A1 (en) | 2022-06-27 | 2024-01-04 | Sutro Biopharma, Inc. | β-GLUCURONIDE LINKER-PAYLOADS, PROTEIN CONJUGATES THEREOF, AND METHODS THEREOF |
WO2024006542A1 (en) | 2022-06-30 | 2024-01-04 | Sutro Biopharma, Inc. | Anti-ror1 antibodies and antibody conjugates, compositions comprising anti-ror1 antibodies or antibody conjugates, and methods of making and using anti-ror1 antibodies and antibody conjugates |
WO2024007016A2 (en) | 2022-07-01 | 2024-01-04 | Beckman Coulter, Inc. | Novel fluorescent dyes and polymers from dihydrophenanthrene derivatives |
WO2024015229A1 (en) | 2022-07-15 | 2024-01-18 | Sutro Biopharma, Inc. | Protease/enzyme cleavable linker-payloads and protein conjugates |
WO2024044327A1 (en) | 2022-08-26 | 2024-02-29 | Beckman Coulter, Inc. | Dhnt monomers and polymer dyes with modified photophysical properties |
WO2024044780A1 (en) | 2022-08-26 | 2024-02-29 | Sutro Biopharma, Inc. | Interleukin-18 variants and uses thereof |
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US20100298496A1 (en) | 2010-11-25 |
US20090054590A1 (en) | 2009-02-26 |
US20010007765A1 (en) | 2001-07-12 |
US8354477B2 (en) | 2013-01-15 |
US20050090650A1 (en) | 2005-04-28 |
AU4755596A (en) | 1996-07-31 |
US7786221B2 (en) | 2010-08-31 |
US20030114647A1 (en) | 2003-06-19 |
US7419600B2 (en) | 2008-09-02 |
US20130177961A1 (en) | 2013-07-11 |
US8546493B2 (en) | 2013-10-01 |
US5932462A (en) | 1999-08-03 |
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