Classifications

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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
  • C07K16/241—Tumor Necrosis Factors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
  • C07K16/244—Interleukins [IL]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/005—Glycopeptides, glycoproteins
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
  • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
  • C07K2317/41—Glycosylation, sialylation, or fucosylation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/72—Increased effector function due to an Fc-modification
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]

Definitions

• the present invention relates to therapeutic glycoproteins and, in particular, arabinosylated glycoproteins.
• the present invention also relates to methods for making and using arabinosylated glycoproteins.
• the present invention also relates to methods for making and using glycoprotein compositions having a reduced amount of core fucose residues and/or a reduced amount of high mannose residues.
• N-linked glycosylation is commonly found in recombinant glycoprotein therapeutics, and follows a reasonably understood series of enzymatic events (Hossler et al., Biotechnol. Bioeng. 95:946-960 (2006)).
• the pathway begins in the endoplasmic reticulum (ER) where there is an en bloc transfer of a 9-mannose core N-glycan oligosaccharide.
• glycoprotein therapeutics expressed from mammalian cells this is essentially constitutive secretion into the extracellular environment.
• glycosidase enzymes glycosyltransferase enzymes
• nucleotide- sugars in the N-glycan biosynthetic pathway.
• This spectrum of N-glycans is termed the glycoform profile.
• Differences in the monosaccharide composition that comprise the N-glycans are termed microheterogeneity.
• Differences in the N-glycan occupancy across the respective glycosylation sites of different protein molecules are termed macroheterogeneity .
• Control over N-glycan content is important for the commercial manufacturing of recombinant glycoprotein therapeutics for optimization of efficacy, ensuring consistency between repeated batches of the protein product, and directing product quality profiles (e.g., directing a product profile towards a comparability fingerprint as that of a desired reference material).
• Mannose and fucose are two types of sugars attached to N-glycans on recombinant proteins.
• a pre-formed dolichol oligosaccharide unit is first added onto a glycoprotein upon translation in the lumen of the ER.
• Glucose and mannose are serially removed from N-glycans while in the ER.
• mannose removal is not complete before the protein exits the ER and eventually makes its way outside the cell through the secretory pathway.
• Mannose is an important sugar that has been implicated in regulating cellular activities in a variety of studies (Sathyamoorthy et al., Mol. Cell Biochem. 102: 139-147 (1991)).
• high mannose N-glycans can resemble the glycosylation profile from proteins of adventitious organisms (Allavena et al., Crit. Rev. Immunol. 24: 179-192 (2004)), and thus potentially induce both an immunogenic response, as well as a reduced pharmacokinetic profile (PK) (Yu, et al., mAbs 4:475-487 (2012); Goetze et al., Glycobiol. 21 :949-959 (2011)).
• the mannose receptor is a receptor principally found on dendritic cells and macrophages and has been found to be principally important towards the recognition of high mannose epitopes and the resulting clearance of the proteins which display them (Allavena et al., Crit. Rev. Immunol. 24: 179-192 (2004); Stahl, Curr. Opin. Immunol. 4:49-52 (1992); Lee et al., Science 295: 1898-1901 (2002)).
• Fucose is one of the rare sugars that is actually added onto an N-glycan as the L- form of the sugar. Fucose has been implicated in various reports suggesting that lower levels of L-fucose attached to an N-glycan can increase antibody-dependent cell cytotoxicity (ADCC) (Shields et al., J. Biol. Chem. 277:26733 (2002); Shinkawa et al., J. Biol. Chem. 278:3466-3473 (2003)) and that this effect is dependent upon activity of natural killer cells (Peipp et al., Blood 112:2390-2399 (2008)). This latter effect is of contemporary interest in oncology-related protein therapeutics in particular, where the ADCC response is typically the molecule's principal method of action (Chan and Carter, Nat. Rev. Immunol. 10:301-316 (2010).
• the mannose and fucose content of a therapeutic glycoprotein composition can impact the efficacy and/or PK profile of the therapeutically relevant glycoproteins.
• PK profile of the therapeutically relevant glycoproteins.
• mannose and fucose have been shown to play an important role towards these physiological responses of the proteins which bear them, having a means with which to control their relative levels would be extremely beneficial for the targeted optimization of therapeutic safety and efficacy.
• an ideal oligosaccharide profile for many glycoprotein therapeutics is one that is high in galactose, low in fucose, and low in mannose.
• it is not easy to control the levels of one of these particular sugars at a time let alone control all three variables at the same time without introducing any adverse changes to the expression system's cell growth performance, recombinant protein productivity, and/or other product quality attributes.
• compositions and methods for regulating the levels of high mannose N-glycans and N-glycan fucose of glycoproteins such as antibodies like monoclonal antibodies (mAbs) and dual variable domain proteins (DVDs), through the cell culture media supplementation of D-arabinose and/or D-altrose during the production of the glycoproteins.
• mAbs monoclonal antibodies
• DVDs dual variable domain proteins
• D-arabinose and D-altrose were able to significantly and selectively reduce fucosylation within N-glycans (e.g., completely replace fucose within core antibody N-glycans) across multiple glycoproteins, as well as significantly reduce mannosylation, reduce dendritic cell uptake, increase FcyRIIIa binding, maintain (preserve) or enhance complement activation, enhance antibody-dependent cell-mediated cytotoxicity (ADCC), reduce immunogenicity, and increase circulatory pharmacokinetics (PK), of the glycoproteins.
• ADCC enhance antibody-dependent cell-mediated cytotoxicity
• PK circulatory pharmacokinetics
• Figure 1 includes 2 panels, identified as panels (A) and (B), which depict an abbreviated N-glycan biosynthetic pathway in mammalian cells.
• Panel A shows an abbreviated N-glycan biosynthetic pathway in mammalian cells using L-fucose as a sugar substrate.
• Panel B shows an abbreviated N-glycan biosynthetic pathway in mammalian cells using D-arabinose as a sugar substrate.
• Figure 2 depicts the chemical structure comparison between D-arabinose
• Figure 3 includes 3 panels, identified as panels (A), (B), and (C), which depict cell culture performance of a mAb-1 -expressing CHO cell line with different concentrations of D-arabinose supplemented into the culture media.
• Panel A shows results regarding viable cell density.
• Panel B shows results regarding cell viability.
• Panel C shows results regarding relative harvest titer.
• Figure 4 includes 2 panels, identified as panels (A) and (B), which depict a reduced LC-MS spectragraph of purified mAb-1 expressed in CHO cell culture under different conditions.
• Panel A shows the results of a control culture with glucose as the principal sugar.
• Panel B shows the results of an experimental culture with glucose and D- arabinose as the principal sugars.
• Figure 5 includes 2 panels, identified as panels (A) and (B), which depict a
• Panel A shows the results of D-arabinose.
• Panel B shows the results of 13 C labeled D-arabinose.
• Figure 6 shows the results of N-glycan glycopeptide mapping of purified mAb-1 expressed from CHO cells supplemented with D-altrose or D-arabinose.
• Figure 7 includes 3 panels, identified as panels (A), (B), and (C), which depict cell culture performance of an adalimumab-expressing CHO cell line with either sucrose or D-arabinose supplemented into the culture media.
• Panel A shows results regarding viable cell density.
• Panel B shows results regarding cell viability.
• Panel C shows results regarding relative harvest titer.
• Figure 8 includes 3 panels, identified as panels (A), (B), and (C), which depict cell culture performance of a DVD- 1 -expressing CHO cell line with different concentrations of D-arabinose supplemented into the culture media.
• Panel A shows results regarding viable cell density.
• Panel B shows results regarding cell viability.
• Panel C shows results regarding relative harvest titer.
• Figure 9 includes 3 panels, identified as panels (A), (B), and (C), which depict cell culture performance of a mAb-2-expressing CHO cell line with different concentrations of D-arabinose supplemented into the culture media.
• Panel A shows results regarding viable cell density.
• Panel B shows results regarding cell viability.
• Panel C shows results regarding relative harvest titer.
• Figure 10 includes 3 panels, identified as panels (A), (B), and (C), which depict cell culture performance of a mAb-3 -expressing CHO cell line with different concentrations of D-arabinose supplemented into the culture media.
• Panel A shows results regarding viable cell density.
• Panel B shows results regarding cell viability.
• Panel C shows results regarding relative harvest titer.
• Figure 11 depicts dendritic cell uptake data of a purified anti-T Fa antibody expressed in CHO cell culture with different sugar supplements.
• Figure 12 includes 2 panels, identified as panels (A) and (B), which depict complement activation elicited by purified mAb-1 expressed in CHO cell culture with different sugar supplements.
• Panel A shows the response from cells from human donor 1.
• Panel B shows the response from cells from human donor 2.
• Figure 13 includes 2 panels, identified as panels (A) and (B), which depict rat PK results of glycomodulated mAb-1 mediated through D-arabinose cell culture media supplementation.
• Panel A shows the results of intravenous administration.
• Panel B shows the results of subcutaneous administration.
• Figure 14 includes 2 panels, identified as panels (A) and (B), which depict
• ADCC measurements of arabinosylated mAb-2 versus predominantly fucosylated control mAb-2 Panel A shows the results of the VI 58 higher affinity allele of FCyRIIIa. Panel B shows the results of the F 158 lower affinity allele of FCyRIIIa.
• Figure 15 includes 2 panels, identified as panels (A) and (B), which depict
• CD 16 report assay results of predominantly fucosylated control mAb-3 versus predominantly arabinosylated mAb-3.
• Panel A shows the results of the VI 58 higher affinity allele of FCyRIIIa.
• Panel B shows the results of the F 158 lower affinity allele of FCyRIIIa.
• Figure 16 includes 3 panels, identified as panels (A), (B), and (C), which depict cell culture performance of a mAb-1 -expressing CHO cell line with different concentrations of D-arabinose and manganese supplemented into the culture media.
• Panel A shows results regarding viable cell density.
• Panel B shows results regarding cell viability.
• Panel C shows results regarding relative harvest titer. DETAILED DESCRIPTION OF THE INVENTION
• the present invention provides a recombinant glycoprotein, such as a therapeutic protein (e.g., antibody, DVD-Ig, TVD-Ig, Half-body or receptor antibody (“RAB”) compositions) having a modulated glycosylation profile.
• a therapeutic protein e.g., antibody, DVD-Ig, TVD-Ig, Half-body or receptor antibody (“RAB”) compositions
• RAB receptor antibody
• the modulated glycosylation profile includes increased arabinosylation, decreased fucosylation, and/or decreased mannosylation relative to a control protein.
• the present invention further provides a pharmaceutical composition comprising a recombinant glycoprotein, such as a therapeutic protein, having a modulated glycosylation profile.
• a recombinant glycoprotein such as a therapeutic protein
• the modulated glycosylation profile includes increased arabinosylation, decreased fucosylation, and/or decreased mannosylation relative to a control protein.
• the therapeutic protein is an antibody.
• the present invention provides cell culture methods and compositions for modulating the glycosylation profile of a protein such as a therapeutic protein.
• the cell culture methods comprise culturing a host cell expressing the protein in cell culture media supplemented with D-arabinose and/or D-altrose.
• the cell culture composition comprises D-arabinose and/or D-altrose.
• an arabinose residue means one arabinose residue or more than one arabinose residue.
• antibody refers to an immunoglobulin molecule and immunologically active portions of immunoglobulin molecules, i.e., polypeptides of the immunoglobulin family, or fragments thereof, that contain an antigen binding site that immunospecifically binds to a specific antigen (e.g., TNFa) and an Fc domain.
• the term “antibody” includes an immunoglobulin molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as "HCVR” or "VH”) and a heavy chain constant region ("CH").
• HCVR heavy chain variable region
• CH heavy chain constant region
• the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
• Each light chain is comprised of a light chain variable region (abbreviated herein as "LCVR” or “VL”) and a light chain constant region.
• the light chain constant region is comprised of one domain, CL.
• the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions ("CDRs"), interspersed with regions that are more conserved, termed framework regions ("FR").
• CDRs complementarity determining regions
• FR framework regions
• Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
• antigen-binding portion or "antigen-binding fragment” of an antibody includes fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., T Fa). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
• antigen-binding fragments encompassed within the terms "antigen-binding portion” or "antigen-binding fragment” of an antibody include (i) a Fab fragment, a monovalent fragment comprising the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment comprising the VH and CHI domains; (iv) a Fv fragment comprising the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546, the entire teaching of which is incorporated herein by reference), which comprises a VH domain; and (vi) an isolated complementarity determining region (CDR).
• CDR complementarity determining region
• the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883, the entire teachings of which are incorporated herein by reference).
• scFv single chain Fv
• single chain antibodies are also intended to be encompassed within the terms “antigen-binding portion” or “antigen-binding fragment” of an antibody.
• Other forms of single chain antibodies, such as diabodies are also encompassed by the terms “antigen-binding portion” or “antigen- binding fragment” of an antibody.
• Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123, the entire teachings of which are incorporated herein by reference).
• an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecule, formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides.
• immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al.
• the term "monoclonal antibody” refers to an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include murine, human, humanized and chimeric monoclonal antibodies.
• isolated antibody includes an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds hTNFa is substantially free of antibodies that specifically bind antigens other than hTNFa).
• An isolated antibody that specifically binds hTNFa may bind TNFa molecules from other species.
• an isolated antibody may be substantially free of other cellular material and/or chemicals.
• a suitable anti-TNFa antibody is adalimumab or an adalimumab-like polypeptide.
• HUMIRA® (Abb Vie) and sometimes referred to as “D2E7", refers to a human IgGl antibody that binds human tumor necrosis factor a (TNFa).
• the light chain variable region of adalimumab and the heavy chain variable region of adalimumab are provided herein as shown in Table A.
• Adalimumab comprises a light chain variable region comprising a CDR1, a CDR2, and a CDR3 as shown in Table A.
• Adalimumab comprises a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 of SEQ ID NO:4.
• the nucleic acid sequence of the light chain variable region and the nucleic acid sequence of the heavy chain variable region are each also set forth in Table A.
• the full length amino acid sequence of the light chain and the full length amino acid sequence of the heavy chain are also set forth in Table A.
• the heavy chain constant domain 2 ("CH2") of the adalimumab IgG- Fc region is glycosylated through covalent attachment of oligosaccharide at asparagine 297 (Asn-297).
• Adalimumab is described in U.S. Pat. Nos.
• CDR1, CDR2, and CDR3 are shown in consequence order from N-terminus to C-terminus (e.g., RASQGIRNYLA represents the VL CDR1 sequence)]
• Constant region sequences Italicized text *
• the CDR HI is GFTFDDYAMH instead of DYAMH due to an alternative and well-known antibody CDR nomenclature system.
• Either CDR HI sequence annotation can be applied according to the present invention.
• adalimumab-like protein refers to a polypeptide having less than 100% identity, but at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78,%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or any range in between, identity to the polypeptide sequence of adalimumab.
• the percentage identities refer to the light chain variable region of adalimumab and/or the heavy chain variable region of adalimumab. In another embodiment, the percentage identities refer to CDR1, CDR2, and/or CDR3 of the light chain variable region of adalimumab and/or the CDR1, CDR2, and/or CDR3 of the heavy chain variable region of adalimumab. In still another embodiment, the percentage identities refer to CDR3 of the light chain variable region of adalimumab and/or the CDR3 of the heavy chain variable region of adalimumab. Non- limiting representative examples of adalimumab-like proteins are shown below in Table B.
• RASQSIRNYLS represents the VL CDR1 sequence of D2E7-SS
• the CDR HI is GFTFDXYAMH instead of XYAMH, where X is D or H as described above, due to an alternative and well-known antibody CDR nomenclature system.
• CDR HI sequence annotation can be applied according to the present invention.
• the hMAK199 heavy chain CDR HI sequence shown uses the long form nomenclature system and the shorter form CDRHl nomenclature sequence corresponding to the sequence, NYGII, is also equally applicable to the present invention.
• any recombinant arabinosylated glycoprotein described herein can have the amino acid sequence of adalimumab, or an antigen-binding fragment thereof.
• any recombinant arabinosylated glycoprotein describd herein can have an amino acid sequence of an adalimumab-like protein such as an amino acid sequence selected from the group consisting of amino acid sequences shown in Table B.
• any recombinant arabinosylated glycoprotein described herein is an antibody comprising a variable light and/or variable heavy chain selected from the group consisting of variable light and/or variable heavy chains shown in Tables A and/or B.
• any recombinant arabinosylated glycoprotein described herein is an antibody comprising a variable light and/or heavy chain of consecutive pairs of variable light and variable heavy chains shown in Tables A and B. These embodiments are applicable to any recombinant arabinosylated glycoprotein, composition comprising a recombinant arabinosylated glycoprotein, method to produce a recombinant arabinosylated glycoprotein, or any other aspect of the present invention described herein.
• recombinant protein or “recombinant polypeptide” refers to a protein molecule which is expressed from a recombinant nucleic acid molecule.
• recombinant nucleic acid molecule referes to a nucleic acid, such as cDNA, which is comprised of segments of DNA joined together by means of molecular biological techniques and used to engineer a cell or other protein expression system to express protein encoded therein.
• host cell includes a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell” as used herein.
• host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life.
• eukaryotic cells include protist, fungal, plant and animal cells.
• host cells include, but are not limited to, the prokaryotic cell line E. coli; mammalian cell lines CHO, HEK 293, COS, NSO, SP2 and PER.C6; the insect cell line Sf9; and the fungal cell Saccharomyces cerevisiae.
• peptides or proteins, including antibodies
• peptides comprise carbohydrate or saccharide moieties attached to the peptide via specific linkages to a select number of amino acids along the length of the primary peptide chain.
• Such peptides or proteins can be referred to as "glycopeptides” or “glycosylated” peptides or “glycoproteins” or “glycosylated” proteins.
• oligosaccharide structure attached to the peptide chain is known as a
• glycoproteins are typically divided into two classes, "N-linked glycans” or “N-glycans” and "O-linked glycans” or “O-glycans.”
• Proteins and peptides expressed in eukaryotic cells typically comprise N-glycans.
• an N-glycan is bound to an asparagine or an arginine residue in a protein via an N-acetylglucosamine residue.
• an N- glycan is bound to asparagine 297 (according to the number of Kabat) of an antibody, although an N-glycan can also be linked to other asparagine residues of the antibody.
• an N-glycan is released from a peptide or protein, such as by treatment with PNGase F.
• PNGase F is an enzyme that cleaves between the innermost GlcNAc and asparagine residues to liberate an N-glycan from a glycoprotein.
• glycoproteins include galactose ("Gal" or
• G mannose
• Man fucose
• Fuc fucose
• GalNAc N-acetylgalactosamine
• GlcNAc N- acetylglucosamine
• sialic acid e.g., N-acetylneuraminic acid (“NANA” or “NeuAc)
• N-linked glycosylation sites occur in a peptide primary structure containing, for example, the amino acid sequence asparagine-X-serine/threonine, where X is any amino acid residue except proline and aspartic acid.
• glycoform refers an isoform of a protein (e.g., an antibody) that differs only with respect to the number and/or type of attached glycan(s).
• a composition comprising a glycoprotein often includes of a number of different glycoforms.
• glycosylation profile refers to the spectrum, including the amount and/or type, of N-glycans present on a protein or peptide or population of glycoforms in a glycoprotein composition.
• a glycosylation profile for a glycoprotein composition includes a percentage or a value for an N-glycan species relative to total N-glycans on, for example, a mol/mol basis.
• a percentage for an N-glycan species refers to the number of moles of that particular N-glycan species relative to total moles of N-glycans in a composition.
• a modulated glycosylation profile includes a profile of a composition comprising a protein (e.g., an antibody) which is modulated as compared to the glycosylation profile of a composition comprising that same protein produced under different conditions.
• a modulated glycosylation profile includes an overall increase in the level of arabinosylated N-glycans in the composition.
• a modulated glycosylation profile includes an overall decrease in the level of fucosylated N-glycans in the composition.
• a modulated glycosylation profile includes an overall decrease in the level of mannosylation of N- glycans in the composition.
• a modulated glycosylation profile includes an overall decrease in the level of high mannose N-glycans in the composition. Differences in the monosaccharide composition that comprise an N-glycan are termed “microheterogeneity.” Differences in the N-glycan occupancy across the respective glycosylation sites of a protein are termed “macroheterogeneity.”
• N-glycans have a common pentasaccharide core of Man 3 GlcNAc 2 .
• the pentasaccharide core is also referred to as a "trimannose core” or a "paucimannose core.”
• N-glycans differ with respect to the presence of, and/or in the number of, branches (also called “antennae”) comprising peripheral sugars such as Gal, GalNAc, GlcNAc, NeuAc, and Fuc that are added to the Man 3 GlcNAc 2 core structure.
• this structure may also contain a core fucose molecule and/or a xylose molecule.
• N-glycans are classified according to their branched constituents (e.g., high mannose, complex, or hybrid).
• a "high mannose N-glycan” has five or more mannose residues.
• a high mannose N-glycan having five (5) mannose residues is referred to as "HM5"; a high mannose N-glycan having six (6) mannose residues is referred to as “HM6”; a high mannose N-glycan having seven (7) mannose residues is referred to as “HM7”; a high mannose N-glycan having eight (8) mannose residues is referred to as "HM8”; and a high mannose N-glycan having nine (9) mannose residues is referred to as "HM9.”
• a "complex N-glycan” typically has at least one GlcNAc residue attached to the 1,3 mannose arm and at least one GlcNAc residue attached to the 1,6 mannose arm of a pentasaccharide core.
• Complex-type N-glycans may also have Gal residues or GalNAc residues that are optionally modified with sialic acid or derivatives.
• Complex N-glycans may also have intrachain substitutions comprising "bisecting" GlcNAc residues, and a core fucose residue.
• Complex N-glycans may also have multiple antennae on the pentasaccharide core and are, therefore, also referred to as “multiple antennary-type glycans.”
• Complex N-glycans may contain zero (GO), one (Gl) or two (G2) galactose residues, as well as one fucose attached to the first GlcNAc residue on the reducing end (denoted as GOF, GIF, G2F, respectively).
• GO refers the N-glycan having a GlcNAc 2 Man 3 GlcNAc 2 structure, wherein no terminal sialic acids or terminal Gal residues are present. See Figure 1.
• GEF refers the N-glycan having a GlcNAc 2 Man 3 GlcNAc 2 structure plus one fucose residue attached to the first GlcNAc on the reducing end, wherein no terminal sialic acids or terminal Gal residues are present. See Figure 1.
• Gl refers the N-glycan having a GlcNAc 2 Man 3 GlcNAc 2 structure plus one terminal galactose residue. See Figure 1.
• GEF refers the N-glycan having a GlcNAc 2 Man 3 GlcNAc 2 structure plus one terminal galactose residue and one fucose residue attached to the first GlcNAc residue on the reducing end. See Figure 1.
• G2 refers the N-glycan having a GlcNAc 2 Man 3 GlcNAc 2 structure plus two terminal galactose residues. See Figure 1.
• G2F refers the N-glycan having a GlcNAc 2 Man 3 GlcNAc 2 structure plus two terminal galactose residues and one fucose residue attached to the first GlcNAc residue on the reducing end. See Figure 1.
• G0A refers the N-glycan having a
• GlcNAc 2 Man 3 GlcNAc 2 structure plus one arabinose residue attached to the first GlcNAc on the reducing end, wherein no terminal sialic acids or terminal Gal residues are present. See Figure 1.
• G1A refers the N-glycan having a
• GlcNAc 2 Man 3 GlcNAc 2 structure plus one terminal galactose residue and one arabinose residue attached to the first GlcNAc on the reducing end. See Figure 1.
• G2A refers the N-glycan having a
• GlcNAc 2 Man 3 GlcNAc 2 structure plus two terminal galactose residues and one arabinose residue attached to the first GlcNAc on the reducing end. See Figure 1.
• a “hybrid N-glycan” comprises at least one GlcNAc on the terminal of the
• N-glycans having only one terminal GlcNAc residue can be referred to as "minus GlcNAc", which is represented by appending "-GlcNAc" to the glycan name. See Figure 1.
• the term "GOF-GlcNAc” refers the N-glycan having a Man 3 GlcNAc 2 core structure plus only one terminal GlcNAc residue and one fucose residue attached to the first GlcNAc on the reducing end, wherein no terminal sialic acids or terminal Gal residues are present. See Figure 1.
• GAA-GlcNAc refers the N-glycan having a
• GlcNAc refers the N-glycan having a Man 3 GlcNAc 2 core structure plus only one terminal GlcNAc residue and one fucose residue attached to the first GlcNAc residue on the reducing end. See Figure 1.
• GlA-GlcNAc refers the N-glycan having a
• fucose residue refers to the presence of a fucose residue within an N-linked glycan.
• arabinosylation refers to the presence of an arabinose residue within an N-linked glycan.
• Arabinosylation can be achieved in numerous ways, such as through supplementation of cell culture media with arabinose (e.g., D- arabinose and/or D-altrose) or through chemical modification.
• core fucosylation or “core fucosylated” refers to a fucose residue attached to the first GlcNAc on the reducing end of an N-linked glycan.
• core arabinosylation or “core arabinosylated” refers to an arabinose residue attached to the first GlcNAc on the reducing end of an N-linked glycan.
• Arabinose can exist in acyclic ⁇ i.e., linear or open-chain) form, or in cyclic form. More than one constitutional isomeric form is contemplated for cyclic arabinose, including the 5-membered ring arabinofuranose and the 6-membered ring arabinopyranose. Furthermore, cyclic arabinose can include more than one stereochemical configuration at the anomeric carbon, the alpha or beta anomer of the aldose sugar.
• Arabinose modifications or derivatives are also contemplated as arabinose residues, and are well known in the art (see, for example U.S. Pat. Nos. 8,487, 110 and 6,462,191 and U.S. Pat. Publ. No. 2003/0186402).
• Modifications to arabinose that are contemplated for use in the invention include protection of hydroxyl (-OH) group.
• the hydroxyl group is protected, thereby forming an ether (e.g., alkyl ether, aralkyl ether, aryl ether, silyl ether, alkoxyalkyl ether), an ester (e.g., acetate, perfluoroacetate, alkyl acetate, aralkyl acetate, aryl acetate), sulfonate, carbonate, carbamate, and the like.
• an hydroxyl groups e.g., two adjacent hydroxyl groups
• the arabinose derivative can include an aza-sugar.
• an amino group e.g., - H 2 , - H(alkyl), - H(alkyl) 2
• an arabinose residue is functionalized (i.e., modified at any substitutable position) by covalent attachment of a pharmacophore, either directly, or indirectly such as via a linker.
• the pharmacophore can be a residue of a therapeutically active agent, a prodrug, an antibody, and the like.
• the pharmacophore can be essentially the therapeutically active agent, covalently linked to the arabinose.
• the pharmacophore relates to the parent structure of the therapeutically active agent via truncation at, for example, any one or more hydrolyzable bonds (e.g., an ester bond) present in the parent structure of the therapeutically active agent.
• the pharmacophore can include a core structure or scaffold of a therapeutically active compound, and may include one or more derivatizations or functionalizations.
• Substitutable positions of arabinose residue at which the arabinose residue can be functionalized, e.g., by the pharmacophore include the oxygen of any hydroxyl group present in arabinose, any methylene carbon, or any methine carbon of arabinose.
• a hydroxyl group of arabinose is replaced by a pharmacophore, optionally bound to the arabinose through a linking moiety.
• the pharmacophore can be directly covalently linked (i.e., directly bonded) to the arabinose residue, or alternatively can be covalently linked to the arabinose residue through a linking moiety.
• exemplary linking moieties include alkylene groups, alkenylene groups, alkynylene groups, ethers, esters, amides, amines, polymeric fragments (e.g., polyethyleneimine, polyethyleneoxide), heterocyclic moieties, and so forth.
• a pharmacophore can be linked to an arabinose residue via click chemistry (e.g., alkyne-azide cycloaddition reaction), esterification, amidation, amination, and other bond-forming reactions.
• the pharmacophore is a residue of a therapeutically active agent that is known to increase ADCC and/or maintain (preserve) or increase complement activation.
• the pharmacophore can be any number of well known ADCC and/or complement activation modulatory agents, such as small molecules, proteins, nucleic acids, antibodies, and the like.
• the pharmacophore can be a residue of taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, and analogs or homologs thereof.
• the pharmacophore is a residue of aminoglutethimide, amsacrine, anastrozole, asparaginase, beg, bicalutamide, bleomycin, bortezomib, buserelin, busulfan, campothecin, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, chloroquine, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, demethoxyviridin, dexamethasone, dichloroacetate, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, everolimus, exemestane, fil
• Arabinosylated glycopolypeptides such as arabinosylated antibodies or antigen-binding fragments thereof, can comprise one or more than one pharmacophores of interest.
• pharmacopore loaded arabinosylated glycopolyppetides can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, such as 8, or any range in between, pharmacophores conjugated to the polkypeptide depending upon designated linker chemistry, linker structure, linker number, functionalized positions of arabinose, and the like.
• Compositions and methods for producing such pharmacophore- conjugated arabinosylated glycopolypeptides are well known in the art and include, for example, PCT Publ. No. WO 2014/152199.
• acyl is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.
• Acetate refers to hydrocarbylC(0)0-.
• alkoxy refers to an alkyl group, preferably a lower alkyl group, having an oxygen attached thereto.
• Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like.
• alkoxy alkyl refers to an alkyl group substituted with an alkoxy group and may be represented by the general formula alkyl-O-alkyl.
• alkenyl refers to an aliphatic group containing at least one double bond and is intended to include both "unsubstituted alkenyls" and “substituted alkenyls", the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the alkenyl group. Such substituents may occur on one or more carbons that are included or not included in one or more double bonds. Moreover, such substituents include all those contemplated for alkyl groups, as discussed below, except where stability is prohibitive. For example, substitution of alkenyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.
• alkyl is a straight chain or branched non-aromatic hydrocarbon moiety which is completely saturated. Typically, a straight chain or branched alkyl group has from 1 to about 20 carbon atoms, preferably from 1 to about 10 unless otherwise defined. Examples of straight chained and branched alkyl groups include methyl, ethyl, n- propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl and octyl.
• a Ci-C 6 straight chained or branched alkyl group is also referred to as a "lower alkyl" group.
• alkylamino refers to an amino group substituted with at least one alkyl group.
• alkynyl refers to an aliphatic group containing at least one triple bond and is intended to include both "unsubstituted alkynyls" and “substituted alkynyls", the latter of which refers to alkynyl moieties having substituents replacing a hydrogen on one or more carbons of the alkynyl group. Such substituents may occur on one or more carbons that are included or not included in one or more triple bonds. Moreover, such substituents include all those contemplated for alkyl groups, as discussed above, except where stability is prohibitive. For example, substitution of alkynyl groups by one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.
• amide refers to a group represented by
• each R 10 independently represents a hydrogen or hydrocarbyl group, or two R 10 are taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.
• amine and “amino” are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety represented by
• each R independently represents a hydrogen or a hydrocarbyl group, or two R 10 are taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure.
• amine encompasses cyclic amines, including bicyclic amines.
• amine includes DABCO (l,4-diazabicyclo[2.2.2]octane).
• aminoalkyl refers to an alkyl group substituted with an amino group.
• aralkyl refers to an alkyl group substituted with an aryl group.
• aryl as used herein include substituted or unsubstituted single- ring aromatic groups in which each atom of the ring is carbon.
• the ring is a 5- to 7-membered ring, more preferably a 6-membered ring.
• aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
• Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.
• R 9 and R 10 independently represent hydrogen or a hydrocarbyl group, such as an alkyl group, or R 9 and R 10 taken together with the intervening atom(s) complete a heterocycle having from 4 to 8 atoms in the ring structure.
• carbonate is art-recognized and refers to a group -OCO 2 -R 10 , wherein R 10 represents a hydrocarbyl group.
• esters refers to a group -C(0)OR 10 wherein R 10 represents a hydrocarbyl group.
• ether refers to a hydrocarbyl group linked through an oxygen to another hydrocarbyl group. Accordingly, an ether substituent of a hydrocarbyl group may be hydrocarbyl-O-. Ethers may be either symmetrical or unsymmetrical. Examples of ethers include, but are not limited to, heterocycle-O- heterocycle and aiyl-O-heterocycle. Ethers include "alkoxyalkyl” groups, which may be represented by the general formula alkyl-O-alkyl.
• heteroaryl and “hetaryl” include substituted or unsubstituted aromatic single ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6- membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms.
• heteroaryl and “hetaryl” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
• Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like.
• heteroatom as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.
• heterocyclyl refers to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms.
• heterocyclyl and “heterocyclic” also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
• Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.
• heterocyclylalkyl refers to an alkyl group substituted with a heterocycle group.
• Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.
• hydroxyalkyl refers to an alkyl group substituted with a hydroxy group.
• sil refers to a silicon moiety with three hydrocarbyl moieties attached thereto.
• the present invention provides a recombinant glycoprotein comprising at least one N-linked glycan, wherein the N-linked glycan comprises at least one arabinose residue.
• the protein for which arabinosylated glycosylation is produced is not limiting and includes, for example, peptides, transmembrane proteins, antibodies, cell surface receptors, and the like, comprising an N-glycan.
• any recombinant arabinosylated glycoprotein described herein can have the amino acid sequence of adalimumab, or an antigen-binding fragment thereof.
• any recombinant arabinosylated glycoprotein describd herein can have an amino acid sequence of any amino acid sequence shown in Tables A and/or B.
• any recombinant arabinosylated glycoprotein described herein is an antibody comprising a variable light and/or variable heavy chain selected from the group consisting of variable light and/or variable heavy chains shown in Tables A and/or B.
• any recombinant arabinosylated glycoprotein described herein is an antibody comprising a variable light and/or heavy chain of consecutive pairs of variable light and variable heavy chains shown in Tables A and B.
• the N- linked glycan is selected from the group consisting of GOA, Gl A, G2A, GOA-GlcNAc, and GlA-GlcNAc.
• the at least one arabinose residue is attached to a core GlcNAc residue of the N-linked glycan.
• the recombinant glycoprotein does not comprise a fucose residue (e.g., the recombinant glycoprotein does not comprise a fucose residue attached to a core GlcNAc residue of the N-linked glycan).
• the recombinant glycoprotein is an antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody or antigen-binding fragment thereof). In certain embodiments, the glycoprotein is an anti-TNFa antibody or antigen- binding fragment thereof.
• the present invention provides a composition comprising a population of one or more glycoforms of a recombinant glycoprotein, wherein at least one of the one or more glycoforms in the composition comprises at least one N-linked glycan, wherein the N-linked glycan comprises at least one arabinose residue.
• the protein for which arabinosylated glycosylation is produced is not limiting and includes, for example, peptides, transmembrane proteins, antibodies, cell surface receptors, and the like, comprising an N-glycan.
• the N- linked glycan is selected from the group consisting of GOA, Gl A, G2A, GOA-GlcNAc, and GlA-GlcNAc.
• the arabinose residue is attached to a core GlcNAc residue of the N-linked glycan.
• at least one of the one or more glycoforms in the composition does not comprise a fucose residue.
• the at least one of the one or more glycoforms in the composition does not comprise a fucose residue attached to a core GlcNAc residue of the N-linked glycan. In certain embodiments, at least one of the one or more glycoforms in the composition comprises an arabinose residue and does not comprise a fucose residue. In certain embodiments, the at least one of the one or more glycoforms in the composition comprises at least one arabinose residue attached to a core GlcNAc residue of the N-linked glycan and does not comprise a fucose residue at the position of an arabinose attached to the core GlcNAc residuce of the N-linked glycan
• the glycoprotein is an antibody or antigen-binding fragment thereof (e.g., a monoclonal antibody or antigen-binding fragment thereof).
• the at least one glycoform is an isoform of an antibody or antigen- binding fragment thereof.
• the at least one glycoform is an isoform of an anti-TNFa antibody or antigen-binding fragment thereof.
• the composition is a pharmaceutical composition.
• the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue.
• at least about 32% of the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue.
• glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue attached to an N-linked glycan selected from the group consisting of G0A, G1A, G2A, GOA-GlcNAc, and G1A- GlcNac.
• At least about 32% of the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue attached to an N-linked glycan selected from the group consisting of G0A, G1A, G2A, GOA-GlcNAc, and G1A- GlcNac. [00125] In certain embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
• glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue attached to a core GlcNAc residue of the N-linked glycan and does not comprise a fucose residue at the position of an arabinose attached to the core GlcNAc residue of the N-linked glycan.
• At least about 32% of the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue attached to a core GlcNAc residue of the N-linked glycan and does not comprise a fucose residue at the position of an arabinose attached to the core GlcNAc residue of the N-linked glycan.
• less than about 57% of the glycoprotein in the composition comprises a glycoform comprising a fucose residue. In certain embodiments, less than about 0.5%, or substantially 0%, of the glycoprotein in the composition comprises a glycoform comprising a fucose residue.
• the glycoprotein in the composition comprises a glycoform comprising a fucose residue attached to a core GlcNAc residue of the N-linked glycan.
• less than about 57% of the glycoprotein in the composition comprises a glycoform comprising a fucose residue attached to a core GlcNAc residue of the N-linked glycan. In certain embodiments, less than about 0.5%, or substantially 0%, of the glycoprotein in the composition comprises a glycoform comprising a fucose residue attached to a core GlcNAc residue of the N-linked glycan.
• the glycoprotein in the composition comprises a glycoform comprising five or more mannose residues. In certain embodiments, less than about 7% of the composition comprises a glycoform comprising five or more mannose residues. In certain embodiments, less than about 7% of the composition comprises a glycoform comprising five or more mannose residues.
• At least about 30%, at least about 40%, at least about 50%), at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue.
• at least about 5%, at least about 6%, at least about 7%), at least about 8%, at least about 9%, or at least about 10% of the glycoprotein in the composition comprises a glycoform comprising GOA-GlcNAc.
• at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%), at least about 70%, or at least about 80% of the glycoprotein in the composition comprises a glycoform comprising G0A.
• At least about 1%, at least about 2%), at least about 3%, at least about 4%, or at least about 5% of the glycoprotein in the composition comprises a glycoform comprising G1A. In certain embodiments, at least about 0.1%), at least about 0.5%, at least about 1%, at least about 2%, or at least about 3% of the glycoprotein in the composition comprises a glycoform comprising G2A.
• the glycoform can comprise at least one arabinose residue attached to a core GlcNAc residue of the N-linked glycan and does not comprise a fucose residue at the position of an arabinose attached to the core GlcNAc residue of the N-linked glycan.
• less than about 60%, 50%, 40%, 30%, 20%, or 10% of the glycoprotein in the composition comprises a glycoform comprising a fucose residue. In certain embodiments, less than about 50%, 40%, 30%, 20%, or 10% of the glycoprotein in the composition comprises a glycoform comprising G0F. In certain embodiments, substantially none of the glycoprotein in the composition comprises a glycoform comprising G0F. In certain embodiments, less than about 4%, 3%, 2%, or 1% of the glycoprotein in the composition comprises a glycoform comprising GIF. In certain embodiments, substantially none of the glycoprotein in the composition comprises a glycoform comprising a GIF.
• less than about 0.5% of the glycoprotein in the composition comprises a glycoform comprising G2F. In certain embodiments, substantially none of glycoprotein in the composition comprises a glycoform comprising G2F.
• the recited glycoform having less than the recited percentage in the composition comprises a fucose residue attached to a core GlcNAc residue of the N-linked glycan.
• less than about 4%, 3%, 2%, or 1%> of the glycoprotein in the composition comprises a glycoform comprising high mannose glycans (e.g., comprises a glycoform comprising five or more mannose residues). In certain embodiments, less than about 4%, less than about 3%, less than about 2%, or less than about 1%) of the glycoprotein in the composition comprises a glycoform comprising HM5.
• less than about 4%, less than about 3%, less than about 2%), or less than about 1% of the glycoprotein in the composition comprises a glycoform comprising high mannose glycans and less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of the glycoprotein in the composition comprises a glycoform comprising a fucose residue.
• less than about 4%, less than about 3%, less than about 2%), or less than about 1% of the glycoprotein in the composition comprises a glycoform comprising HM5 and less than about 50%, less than about 40%, less than about 30%), less than about 20%, or less than about 10% of the glycoprotein in the composition comprises a glycoform comprising G0F.
• less than about 4%, less than about 3%, less than about 2%), or less than about 1% of the glycoprotein in the composition comprises a glycoform comprising HM5 and less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the glycoprotein in the composition comprises a glycoform comprising GIF.
• less than about 4%, less than about 3%, less than about 2%), or less than about 1% of the glycoprotein in the composition comprises a glycoform comprising HM5 and substantially none of the glycoprotein in the composition comprises a glycoform comprising GIF.
• less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the glycoprotein in the composition comprises a glycoform comprising HM5 and less than about 0.5% of the glycoprotein in the composition comprises a glycoform comprising G2F.
• less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the glycoprotein in the composition comprises a glycoform comprising HM5 and substantially none of the glycoprotein in the composition comprises a glycoform comprising G2F.
• At least about 30% of the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue and less than about 60% of the glycoprotein in the composition comprises a glycoform comprising a fucose residue.
• at least about 40% of the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue and less than about 50%) of the glycoprotein in the composition comprises a glycoform comprising a fucose residue.
• at least about 50% of the N-glycans comprise an arabinose residue and less than about 40% of the N-glycans comprise a fucose residue.
• At least about 60% of the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue and less than about 30% of the glycoprotein in the composition comprises a glycoform comprising a fucose residue.
• at least about 70% of the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue and less than about 20% of the glycoprotein in the composition comprises a glycoform comprising a fucose residue.
• at least about 80% of the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue and less than about 10% of the glycoprotein in the composition comprises a glycoform comprising a fucose residue.
• at least about 90% of the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue and less than about 1% of the glycoprotein in the composition comprises a glycoform comprising a fucose residue.
• At least about 4% of the glycoprotein in the composition comprises a glycoform comprising GOA-GlcNAc and less than about 7% of the glycoprotein in the composition comprises a glycoform comprising GOF-GlcNAc.
• at least about 5% of the glycoprotein in the composition comprises a glycoform comprising GOA-GlcNAc and less than about 6% of the glycoprotein in the composition comprises a glycoform comprising GOF-GlcNAc.
• at least about 6% of the glycoprotein in the composition comprises a glycoform comprising GOA-GlcNAc and less than about 5% of the glycoprotein in the composition comprises a glycoform comprising GOF-GlcNAc.
• at least about 7% of the glycoprotein in the composition comprises a glycoform comprising GOA-GlcNAc and less than about 4% of the glycoprotein in the composition comprises a glycoform comprising GOF-GlcNAc.
• At least about 40% of the glycoprotein in the composition comprises a glycoform comprising GOA and less than about 30%> of the glycoprotein in the composition comprises a glycoform comprising GOF.
• at least about 50% of the glycoprotein in the composition comprises a glycoform comprising GOA and less than about 20% of the glycoprotein in the composition comprises a glycoform comprising GOF.
• at least about 60%> of the glycoprotein in the composition comprises a glycoform comprising GOA and less than about 10%) of the glycoprotein in the composition comprises a glycoform comprising GOF.
• at least about 70% of the glycoprotein in the composition comprises a glycoform comprising are GOA and less than about 1%> of the glycoprotein in the composition comprises a glycoform comprising GOF.
• At least about 1%> of the glycoprotein in the composition comprises a glycoform comprising G1A and less than about 4% of the glycoprotein in the composition comprises a glycoform comprising GIF.
• at least about 2% of the glycoprotein in the composition comprises a glycoform comprising G1A and less than about 3% of the glycoprotein in the composition comprises a glycoform comprising GIF.
• at least about 3% of the glycoprotein in the composition comprises a glycoform comprising G1A and less than about 2%) of the glycoprotein in the composition comprises a glycoform comprising GIF.
• at least about 4% of the glycoprotein in the composition comprises a glycoform comprising G1A and less than about 1% of the glycoprotein in the composition comprises a glycoform comprising GIF.
• At least about 30%, at least about 40%, at least about 50%), at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the N-glycans comprise at least one arabinose residue.
• at least about 5%), at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10% of the N-glycans are GOA-GlcNAc.
• at least about 20%), at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%), or at least about 80% of the N-glycans are GOA.
• At least about 1%, at least about 2%, at least about 3%, at least about 4%, or at least about 5% of the N-glycans are G1A. In certain embodiments, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 2%, or at least about 3% of the N-glycans are G2A.
• less than about 60%, less than about 50%, less than about 40%), less than about 30%, less than about 20%, or less than about 10% of the N- glycans comprise a fucose residue.
• less than about 50%, less than about 40%), less than about 30%, less than about 20%, or less than about 10% of the N- glycans are G0F.
• substantially none of the N-glycans are G0F.
• less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the N-glycans are GIF.
• substantially none of the N-glycans are GIF.
• less than about 0.5% of the N-glycans are G2F.
• substantially none of the N-glycans are G2F.
• less than about 4%, less than about 3%, less than about 2%), or less than about 1% of the N-glycans are high mannose glycans. In certain embodiments, less than about 4%, less than about 3%, less than about 2%, or less than about 1%) of the N-glycans are HM5.
• less than about 4%, less than about 3%, less than about 2%), or less than about 1% of the N-glycans are high mannose glycans and less than about 60%), less than about 50%, less than about 40%, less than about 30%, less than about 20%), or less than about 10% of the N-glycans comprise a fucose residue.
• less than about 4%, less than about 3%, less than about 2%), or less than about 1% of the N-glycans are HM5 and less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of the N- glycans are G0F.
• less than about 4%, less than about 3%, less than about 2%), or less than about 1% of the N-glycans are HM5 and less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the N-glycans are GIF.
• less than about 4%, less than about 3%, less than about 2%), or less than about 1% of the N-glycans are HM5 and substantially none of the N- glycans are GIF.
• less than about 4%, less than about 3%, less than about 2%), or less than about 1% of the N-glycans are HM5 and less than about 0.5% of the N-glycans are G2F.
• less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the N-glycans are HM5 and substantially none of the N-glycans are G2F.
• At least about 30% of the N-glycans comprise at least one arabinose residue and less than about 60% of the N-glycans comprise a fucose residue.
• at least about 40% of the N-glycans comprise at least one arabinose residue and less than about 50% of the N-glycans comprise a fucose residue.
• at least about 50% of the N-glycans comprise at least one arabinose residue and less than about 40% of the N-glycans comprise a fucose residue.
• at least about 60% of the N-glycans comprise at least one arabinose residue and less than about 30% of the N-glycans comprise a fucose residue.
• At least about 70% of the N-glycans comprise at least one arabinose residue and less than about 20% of the N-glycans comprise a fucose residue. In certain embodiments, at least about 80% of the N-glycans comprise at least one arabinose residue and less than about 10% of the N-glycans comprise a fucose residue. In certain embodiments, at least about 90% of the N-glycans comprise at least one arabinose residue and less than about 1% of the N-glycans comprise a fucose residue.
• At least about 4% of the N-glycans are G0A- GlcNAc and less than about 7% of the N-glycans are GOF-GlcNAc. In certain embodiments, at least about 5% of the N-glycans are GOA-GlcNAc and less than about 6% of the N- glycans are GOF-GlcNAc. In certain embodiments, at least about 6% of the N-glycans are GOA-GlcNAc and less than about 5% of the N-glycans are GOF-GlcNAc. In certain embodiments, at least about 7% of the N-glycans are GOA-GlcNAc and less than about 4% of the N-glycans are GOF-GlcNAc.
• At least about 40% of the N-glycans are G0A and less than about 30% of the N-glycans are GOF. In certain embodiments, at least about 50% of the N-glycans are GO A and less than about 20% of the N-glycans are GOF. In certain embodiments, at least about 60% of the N-glycans are GO A and less than about 10% of the N-glycans are GOF. In certain embodiments, at least about 70% of the N-glycans are GO A and less than about 1% of the N-glycans are GOF.
• At least about 1% of the N-glycans are Gl A and less than about 4% of the N-glycans are GIF. In certain embodiments, at least about 2% of the N-glycans are G1A and less than about 3% of the N-glycans are GIF. In certain embodiments, at least about 3% of the N-glycans are Gl A and less than about 2% of the N- glycans are GIF. In certain embodiments, at least about 4% of the N-glycans are Gl A and less than about 1% of the N-glycans are GIF.
• the composition comprising recombinant glycoprotein glycoforms exhibits a modulated glycosylation profile compared to a control composition.
• the modulated glycosylation profile includes a differing type of glycan species.
• the recombinant glycoprotein composition comprises N-glycans having at least one arabinose residue whereas the control composition lacks N-glycans having at least one arabinose residue.
• the glycoprotein composition lacks N-glycans having a core fucose residue, whereas the control composition includes N-glycans having a core fucose residue.
• the modulated glycosylation profile includes a differing amount or level of glycan species.
• the amount or level of fucosylation is decreased.
• the level of G0F- GlcNAc species is decreased.
• the level of GOF-GlcNAc species is decreased by at least about 1%; by at least about 5%; by at least about 10%; or by about 10%).
• the level of G0F species is decreased.
• the level of G0F species is decreased by at least about 1%; by at least about 10%>; by at least about 25%; by at least about 50%; by at least about 75%; by about 25%; or by about 75%.
• the level of GIF species is decreased. In certain embodiments, the level of GIF species is decreased by at least about 1%; by at least about 5%; by at least about 10%; or by about 5%. In certain embodiments, the level of G2F species is decreased. In certain embodiments, the level of G2F species is decreased by at least about 0.1%; by at least about 1%; by about 0.5%. All decreases are relative to the control composition.
• the amount or level of mannosylation is decreased.
• the level of Man5 species is decreased.
• the level of Man5 species is decreased by at least about 0.1%; by at least about 1%; by at least about 2%; by at least about 3%; by at least about 4%; or by about 1%).
• the level of Man6 species is decreased.
• the level of Man6 species is decreased by at least about 0.1%; or by about 0.5%.
• the level of Man7 species is decreased.
• the level of Man8 species is decreased. All decreases are relative to the control composition.
• the amount or level of arabinosylation is increased.
• the level of GOA-GlcNAc species is increased.
• the level of GOA species is increased.
• the level of GIA species is increased.
• the level of G2A species is increased. All increases are relative to the control composition.
• the recombinant glycoprotein composition is prepared by (a) providing a mammalian host cell that expresses a protein containing N- linked glycans; and (b) culturing the host cell in a cell culture media containing D-arabinose and/or D-altrose.
• the cell culture media is supplemented with D- arabinose and/or D-altrose.
• control composition is prepared by (a) providing a mammalian host cell that expresses a protein containing N-linked glycans; and (b) culturing the host cell in the absence of D-arabinose and/or D-altrose.
• control composition is prepared by (a) providing a mammalian host cell that expresses a protein containing N-linked glycans; and (b) culturing the host cell without D-arabinose and/or D-altrose supplementation.
• the method can use D-arabinose alone, D-altrose alone, or the combination of D-arabinose and D- altrose.
• the same type of host cell is used to prepare the glycoprotein composition and the control composition.
• the host cell is a rodent cell.
• the host cell is a CHO cell.
• the same cell culture media is used to prepare the glycoprotein composition and the control composition.
• the at least one glycoform is an isoform of an antibody or antigen-binding fragment thereof. In certain embodiments, the at least one glycoform is an isoform of an anti-TNFa antibody or antigen-binding fragment thereof.
• the modulated glycosylation profile includes a differing type of glycan species.
• the glycoprotein composition comprises N-glycans having an arabinose residue whereas the control composition lacks N-glycans having an arabinose residue.
• the glycoprotein composition lacks N-glycans having a core fucose residue whereas the control composition includes N-glycans having a core fucose residue.
• compositions comprising varying levels of glycoforms of a protein such as a human antibody, or antigen-binding fragment thereof, are useful in that one or more desired characteristics, e.g., rate of serum clearance or ADCC activity, may be achieved by varying the glycoform compositions.
• the present invention provides methods for producing a recombinant glycoprotein or a glycoprotein composition, such as a recombinant glycoprotein or glycoprotein composition described herein.
• the methods comprise supplementing a cell culture media with arabinose, such as D-arabinose and/or D-altrose.
• the methods comprise culturing a host cell expressing the glycoprotein in a cell culture media supplemented with D-arabinose and/or D-altrose.
• the host cell is a mammalian cell.
• the mammalian cell is a rodent cell.
• the host cell is a CHO cell.
• the cell culture media is supplemented with about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM D-arabinose and/or D-altrose.
• the cell culture media is supplemented with about 10 mM D-arabinose and/or D-altrose.
• the cell culture media is supplemented with about 15 mM D-arabinose and/or D-altrose.
• the glycoprotein produced by the methods described herein is an arabinosylated glycoprotein as described herein.
• the arabinosylated glycoprotein is an arabinosylated antibody or antigen- binding fragment thereof.
• the arabinosylated glycoprotein is an anti-TNFa antibody or antigen-binding fragment thereof.
• the arabinosylated glycoprotein comprises an arabinose residue attached to a core GlcNAc residue.
• the arabinosylated glycoprotein comprises an N-glycan selected from the group consisting of G0A, G1A, G2A, GOA-GlcNAc, and GlA-GlcNAc.
• the arabinosylated glycoprotein lacks a core fucose residue.
• the glycoprotein composition produced by the methods described herein comprises at least one glycoform having an N-linked glycan comprising arabinose.
• the N-linked glycan comprises an arabinose residue attached to a core GlcNAc residue.
• the N-linked glycan is GO A, G1A, G2A, GOA-GlcNAc, or GlA-GlcNAc.
• the N-linked glycan lacks a core fucose residue.
• the at least one glycoform is an isoform of an antibody or antigen-binding fragment thereof. In certain embodiments, the at least one glycoform is an isoform of an anti-TNFa antibody or antigen-binding fragment thereof.
• the glycoprotein composition produced by the methods described herein exhibits a modulated glycosylation profile compared to a control composition.
• the methods described herein can be used to produce a hypofucosylated composition having decreased amounts or levels of fucose residues relative to a control composition. Such altered glycosylation patterns have previously been demonstrated to increase the ADCC ability of antibodies.
• the methods described herein can be used to produce a hypomannosylated composition having decreased amounts or levels of mannose residues relative to a control composition. Such altered glycosylation patterns have previously been demonstrated to reduce immunogenicity and/or improve pharmacokinetic properties of antibodies.
• the modulated glycosylation profile includes a differing type of glycan species.
• the glycoprotein composition comprises N-glycans having an arabinose residue whereas the control composition lacks N-glycans having an arabinose residue.
• the glycoprotein composition lacks N-glycans having a core fucose residue whereas the control composition includes N-glycans having a core fucose residue.
• the modulated glycosylation profile includes a differing amount or level of glycan species.
• the amount or level of fucosylation is decreased.
• the level of G0F- GlcNAc species is decreased.
• the level of GOF-GlcNAc species is decreased by at least about 1%; by at least about 5%; by at least about 10%; or by about 10%.
• the level of G0F species is decreased.
• the level of G0F species is decreased by at least about 1%; by at least about 10%; by at least about 25%; by at least about 50%; by at least about 75%; by about 25%; or by about 75%.
• the level of GIF species is decreased. In certain embodiments, the level of GIF species is decreased by at least about 1%; by at least about 5%; by at least about 10%; or by about 5%. In certain embodiments, the level of G2F species is decreased. In certain embodiments, the level of G2F species is decreased by at least about 0.1%; by at least about 1%; or by about 0.5%. All decreases are relative to a control composition.
• the amount or level of mannosylation is decreased.
• the level of Man5 species is decreased.
• the level of Man5 species is decreased by at least about 0.1%; by at least about 1%; by at least about 2%; by at least about 3%; by at least about 4%; or by about 1%).
• the level of Man6 species is decreased.
• the level of Man6 species is decreased by at least about 0.1%; or by about 0.5%.
• the level of Man7 species is decreased.
• the level of Man8 species is decreased. All decreases are relative to a control composition.
• the amount or level of arabinosylation is increased.
• the level of GOA-GlcNAc species is increased.
• the level of G0A species is increased.
• the level of GIA species is increased.
• the level of G2A species is increased. All increases are relative to a control composition.
• control composition is prepared by (a) providing a mammalian host cell that expresses a protein containing N-linked glycans; and (b) culturing the host cell in the absence of D-arabinose and/or D-altrose.
• control composition is prepared by (a) providing a mammalian host cell that expresses a protein containing N-linked glycans; and (b) culturing the host cell without D-arabinose and/or without D-altrose supplementation.
• the same type of host cell is used to prepare the glycoprotein composition and the control composition.
• the host cell is a rodent cell.
• the host cell is a CHO cell.
• the same cell culture media is used to prepare the glycoprotein composition and the control composition.
• the at least one glycoform is an isoform of an antibody or antigen-binding fragment thereof. In certain embodiments, the at least one glycoform is an isoform of an anti-TNFa antibody or antigen-binding fragment thereof. [00175] In another aspect, the present invention provides methods for producing a glycoprotein composition having a low amount of fucosylated N-glycans and/or high mannose N-glycans.
• the glycoprotein composition has a low amount of core fucosylated N-glycans and/or high mannose N-glycans. In certain embodiments, the glycoprotein composition has a low amount of GIF-GlcNAc, G0F, GIF, G2F, HM5, and/or HM6. In certain embodiments, the glycoprotein composition has a low amount of G1F- GlcNAc. In certain embodiments, the glycoprotein composition has a low amount of G0F. In certain embodiments, the glycoprotein composition has a low amount of GIF. In certain embodiments, the glycoprotein composition has a low amount of G2F. In certain embodiments, the glycoprotein composition has a low amount of high mannose. In certain embodiments, the glycoprotein composition has a low amount of HM5. In certain embodiments, the glycoprotein composition has a low amount of HM6.
• the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue.
• at least about 32% of the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue.
• glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue attached to an N-linked glycan selected from the group consisting of G0A, G1A, G2A, GOA-GlcNAc, and G1A- GlcNac.
• At least about 32% of the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue attached to an N-linked glycan selected from the group consisting of G0A, G1A, G2A, GOA-GlcNAc, and G1A- GlcNac. [00179] In certain embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
• glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue attached to a core GlcNAc residue of the N-linked glycan and does not comprise a fucose residue at the position of an arabinose attached to the core GlcNAc residue of the N-linked glycan.
• At least about 32% of the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue attached to a core GlcNAc residue of the N-linked glycan and does not comprise a fucose residue at the position of an arabinose attached to the core GlcNAc residue of the N-linked glycan.
• less than about 57% of the glycoprotein in the composition comprises a glycoform comprising a fucose residue. In certain embodiments, less than about 0.5%, or substantially 0%, of the glycoprotein in the composition comprises a glycoform comprising a fucose residue.
• the glycoprotein in the composition comprises a glycoform comprising a fucose residue attached to a core GlcNAc residue of the N-linked glycan.
• less than about 57% of the glycoprotein in the composition comprises a glycoform comprising a fucose residue attached to a core GlcNAc residue of the N-linked glycan. In certain embodiments, less than about 0.5%, or substantially 0%, of the glycoprotein in the composition comprises a glycoform comprising a fucose residue attached to a core GlcNAc residue of the N-linked glycan.
• the glycoprotein in the composition comprises a glycoform comprising five or more mannose residues. In certain embodiments, less than about 7% of the composition comprises a glycoform comprising five or more mannose residues. In certain embodiments, less than about 7% of the composition comprises a glycoform comprising five or more mannose residues.
• the glycoprotein composition includes no more than about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% fucosylated N- glycans, e.g., no more than about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%), about 2%, about 1%, about 0.5%, or about 0.1%.
• the glycoprotein composition includes no more than about 50%, about 40%, about 30%, about 20%), or about 10% G0F glycans.
• the glycoprotein composition includes no detectable G0F glycans.
• the glycoprotein composition includes no more than about 4%, about 3%, about 2%, or about 1% GIF glycans. In certain embodiments, the glycoprotein composition includes no GIF glycans. In certain embodiments, the glycoprotein composition includes no more than about 0.5% of the N- glycans are G2F. In certain embodiments, the glycoprotein composition includes no detectable G2F glycans.
• the level of fucose structures present in a glycoprotein composition can be generally measured as the level of glycans containing fucose structures relative to total amount of glycans in a sample, such as a glycoprotein preparation. In some embodiments, the level of fucose present in a glycoprotein composition can be generally reported as % abundance relative to total glycan mass.
• the glycoprotein composition includes no more than about 20%), about 15%, about 12%, or about 10% high mannose glycans, e.g., no more than about 9%), about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%), about 0.5%), or about 0.1%.
• the glycoprotein composition has high mannose structures (e.g., has at least 0.01%, 0.05% or 0.1% high mannose) but has no more than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%), about 1%, about 0.5%, or about 0.1%, e.g., has between 0.05-20%) high mannose, 0.1-15% high mannose 0.1-10% high mannose, between 1-10% high mannose, between 1-5% high mannose.
• the level of high mannose structures present in a glycoprotein composition can be generally measured as the level of glycans containing high mannose structures relative to total amount of glycans in a sample, such as a glycoprotein preparation. In some embodiments, the level of high mannose structures present in a glycoprotein composition can be generally reported as % abundance relative to total glycan mass.
• glycoprotein composition characteristics have been described according to glycan percentages, such percentages also apply to the percentage of glycoprotein represented by glycoforms comprising the recited glycan structure.
• the present invention provides methods for producing a glycoprotein composition having a reduced fucose and/or mannose content.
• the glycoprotein composition has a reduced core fucose and/or high mannose content. In certain embodiments, the glycoprotein composition has a reduced fucose and/or mannose content relative to a control composition. In certain embodiments, the glycoprotein composition has a reduced GlF-GlcNAc, G0F, GIF, G2F, HM5, and/or HM6 content. In certain embodiments, the glycoprotein composition has a reduced GlF-GlcNAc content. In certain embodiments, the glycoprotein composition has a reduced G0F content. In certain embodiments, the glycoprotein composition has a reduced GIF content. In certain embodiments, the glycoprotein composition has a reduced G2F content. In certain embodiments, the glycoprotein composition has a reduced high mannose content. In certain embodiments, the glycoprotein composition has a reduced HM5 content. In certain embodiments, the glycoprotein composition has a reduced HM6 content.
• the present invention provides cell culture methods for obtaining a desired glycosylation profile for a glycoprotein composition.
• the methods comprise supplementing a cell culture media with D-arabinose and/or D-altrose.
• the methods comprise culturing a host cell expressing the glycoprotein in a cell culture media supplemented with arabinose, such as D-arabinose and/or D-altrose.
• the host cell is a mammalian cell.
• the mammalian cell is a rodent cell.
• the host cell is a CHO cell.
• the cell culture media is supplemented with about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM D-arabinose and/or D-altrose.
• the cell culture media is supplemented with about 10 mM D-arabinose and/or D-altrose.
• the cell culture media is supplemented with about 15 mM D- arabinose and/or D-altrose.
• the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue.
• at least about 32% of the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue.
• glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue attached to an N-linked glycan selected from the group consisting of G0A, G1A, G2A, GOA-GlcNAc, and G1A- GlcNac.
• At least about 32% of the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue attached to an N-linked glycan selected from the group consisting of G0A, G1A, G2A, GOA-GlcNAc, and G1A- GlcNac.
• glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue attached to a core GlcNAc residue of the N-linked glycan and does not comprise a fucose residue at the position of an arabinose attached to the core GlcNAc residue of the N-linked glycan.
• At least about 32% of the glycoprotein in the composition comprises a glycoform comprising at least one arabinose residue attached to a core GlcNAc residue of the N-linked glycan and does not comprise a fucose residue at the position of an arabinose attached to the core GlcNAc residue of the N-linked glycan.
• less than about 57% of the glycoprotein in the composition comprises a glycoform comprising a fucose residue. In certain embodiments, less than about 0.5%, or substantially 0%, of the glycoprotein in the composition comprises a glycoform comprising a fucose residue.
• the glycoprotein in the composition comprises a glycoform comprising a fucose residue attached to a core GlcNAc residue of the N-linked glycan.
• less than about 57% of the glycoprotein in the composition comprises a glycoform comprising a fucose residue attached to a core GlcNAc residue of the N-linked glycan. In certain embodiments, less than about 0.5%, or substantially 0%, of the glycoprotein in the composition comprises a glycoform comprising a fucose residue attached to a core GlcNAc residue of the N-linked glycan.
• a desired glycosylation profile includes at least about 30%, at least about 40%, at least about 50%, at least about 60%>, at least about 70%, at least about 80%>, or at least about 90% arabinosylated N-glycans in a glycoprotein composition.
• a desired glycosylation profile includes at least about 5%), at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10%) GOA-GlcNAc glycans.
• a desired glycosylation profile includes at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% G0A glycans. In certain embodiments, a desired glycosylation profile includes at least about 1%, at least about 2%, at least about 3%, at least about 4%, or at least about 5% G1A glycans. In certain embodiments, a desired glycosylation profile includes at least about 0.1%, at least about 0.5%), at least about 1%, at least about 2%, or at least about 3% G2A glycans.
• the level of arabinose structures present in a glycoprotein composition can be generally measured as the level of glycans containing arabinose structures relative to total amount of glycans in a sample, such as a glycoprotein preparation. In some embodiments, the level of arabinose present in a glycoprotein composition can be generally reported as % abundance relative to total glycan mass.
• a desired glycosylation profile includes no more than about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% fucosylated N-glycans in a glycoprotein composition, e.g., no more than about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, or about 0.1%.
• a desired glycosylation profile includes no more than about 50%, about 40%), about 30%, about 20%, or about 10% G0F glycans.
• a desired glycosylation profile includes no detectable G0F glycans.
• a desired glycosylation profile includes no more than about 4%, about 3%, about 2%, or about 1%) GIF glycans. In certain embodiments, a desired glycosylation profile includes no GIF glycans. In certain embodiments, a desired glycosylation profile includes no more than about 0.5%) of the N-glycans are G2F. In certain embodiments, a desired glycosylation profile includes no detectable G2F glycans.
• the level of fucose structures present in a glycoprotein composition can be generally measured as the level of glycans containing fucose structures relative to total amount of glycans in a sample, such as a glycoprotein preparation.
• the level of fucose present in a glycoprotein composition can be generally reported as % abundance relative to total glycan mass.
• a desired glycosylation profile includes no more than about 20%, about 15%, about 12%, or about 10% high mannose glycans in a glycoprotein composition, e.g., no more than about 9%, about 8%, about 7%, about 6%, about 5%, about 4%), about 3%), about 2%, about 1%, about 0.5%, or about 0.1%.
• the glycoprotein composition has high mannose structures (e.g., has at least 0.01%, 0.05% or 0.1%) high mannose) but has no more than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, or about 0.1%, e.g., has between 0.05-20%) high mannose, 0.1-15% high mannose 0.1-10%) high mannose, between 1-10% high mannose, between 1-5% high mannose.
• the level of high mannose structures present in a glycoprotein composition can be generally measured as the level of glycans containing high mannose structures relative to total amount of glycans in a sample, such as a glycoprotein preparation. In some embodiments, the level of high mannose structures present in a glycoprotein composition can be generally reported as % abundance relative to total glycan mass.
• the methods allow for a glycosylation profile of a glycoprotein composition to be controlled.
• the methods are for controlling levels of fucosylated glycans in the glycoprotein composition.
• core fucosylated N-glycan levels are at or below the low limit of detection.
• D-arabinose and/or D-altrose completely replaces fucose within the N-glycans.
• the methods are for controlling levels of high mannose glycans in the glycoprotein composition.
• the methods are for simultaneously controlling levels of fucosylated glycans and levels high mannose glycans in a glycoprotein composition.
• the methods allow for a glycosylation profile of a glycoprotein composition to be modulated relative to a control composition.
• the glycosylation profile of the glycoprotein composition includes one or more different types of glycan species that is absent in the control composition.
• the glycoprotein composition comprises N-glycans having an arabinose residue whereas the control composition lacks N- glycans having an arabinose residue.
• the glycosylation profile of the glycoprotein composition lacks one or more types of glycan species that is present in the control composition.
• the glycoprotein composition lacks N-glycans having a core fucose residue whereas the control composition includes N-glycans having a core fucose residue.
• the glycosylation profile of the glycoprotein composition includes a differing amount or level of glycan species relative to the control composition.
• the amount or level of fucosylation is decreased relative to the control composition.
• the level of G0F- GlcNAc species is decreased relative to the control composition.
• the level of GOF-GlcNAc species is decreased by at least about 1%; by at least about 5%; by at least about 10%; or by about 10% relative to the control composition.
• the level of G0F species is decreased relative to the control composition.
• the level of G0F species is decreased by at least about 1%; by at least about 10%; by at least about 25%; by at least about 50%; by at least about 75%; by about 25%; or by about 75% relative to the control composition.
• the level of GIF species is decreased relative to the control composition.
• the level of GIF species is decreased by at least about 1%; by at least about 5%; by at least about 10%; or by about 5% relative to the control composition.
• the level of G2F species is decreased relative to the control composition.
• the level of G2F species is decreased by at least about 0.1%; by at least about 1%; by about 0.5% relative to the control composition.
• the amount or level of mannosylation is decreased relative to the control composition.
• the level of Man5 species is decreased.
• the level of Man5 species is decreased by at least about 0.1%; by at least about 1%; by at least about 2%; by at least about 3%; by at least about 4%; or by about 1% relative to the control composition.
• the level of Man6 species is decreased relative to the control composition.
• the level of Man6 species is decreased by at least about 0.1%; or by about 0.5% relative to the control composition.
• the level of Man7 species is decreased relative to the control composition.
• the level of Man8 species is decreased relative to the control composition.
• the amount or level of arabinosylation is increased relative to the control composition.
• the level of GOA-GlcNAc species is increased relative to the control composition.
• the level of GOA species is increased relative to the control composition.
• the level of G1A species is increased relative to the control composition.
• the level of G2A species is increased relative to the control composition.
• control composition is prepared by (a) providing a mammalian host cell that expresses a protein containing N-linked glycans; and (b) culturing the host cell in the absence of D-arabinose and/or in the absence of D-altrose.
• control composition is prepared by (a) providing a mammalian host cell that expresses a protein containing N-linked glycans; and (b) culturing the host cell without D-arabinose supplementation and/or without D-altrose supplementation.
• the same type of host cell is used to prepare the glycoprotein composition and the control composition.
• the host cell is a rodent cell.
• the host cell is a CHO cell.
• the same cell culture media is used to prepare the glycoprotein composition and the control composition.
• adverse effects may include changes in protein folding, solubility, susceptibility to proteases, trafficking, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity, or allergenicity. Accordingly, using the methods of the art may modulate the glycosylation profile of a protein (e.g., an antibody) to achieve a desired activity such as increased or decreased rate of clearance, maintained (preserved) or increased complement activity, and/or increased ADCC activity.
• a protein e.g., an antibody
• the present invention provides methods for producing a recombinant glycoprotein (e.g., an antibody, or antigen binding fragment thereof) with a modulated glycosylation profile.
• the methods involve modification of the conditions used during upstream protein production, such as recombinant cell culture conditions.
• the methods comprise supplementing the recombinant cell culture media with D-arabinose and/or D-altrose to modulate the glycosylation profile of the protein.
• the host cell culture conditions can be modified as compared to conditions during production of the same protein without modulation of the glycosylation profile.
• a protein with a modulated glycosylation profile is produced by culturing cells expressing the protein in a cell culture media supplemented with D-arabinose and/or supplementaed with D-altrose.
• one or more DNAs encoding the protein such as DNAs encoding partial or full- length light and heavy chains in the case of antibodies, are inserted into one or more expression vector such that the genes are operatively linked to transcriptional and translational control sequences.
• the term "operatively linked” is intended to mean that a gene encoding the protein is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the gene.
• the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
• the protein or protein composition with a modulated glycosylation profile will comprise multiple polypeptides, such as the heavy and light chains of an antibody.
• genes encoding multiple polypeptides, such as antibody light chain genes and antibody heavy chain genes can be inserted into a separate vector or, more typically, the genes are inserted into the same expression vector.
• Genes are inserted into expression vectors by standard methods (e.g., ligation of complementary restriction sites on the gene fragment and vector, or blunt end ligation if no restriction sites are present).
• the expression vector may already carry additional polypeptide sequences, such as, but not limited to, antibody constant region sequences.
• additional polypeptide sequences such as, but not limited to, antibody constant region sequences.
• one approach to converting the anti-TNFa antibody or anti-TNFa antibody-related VH and VL sequences to full-length antibody genes is to insert them into expression vectors already encoding heavy chain constant and light chain constant regions, respectively, such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector.
• the recombinant expression vector can encode a signal peptide that facilitates secretion of the protein from a host cell.
• the gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the gene.
• the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).
• a recombinant expression vector can carry one or more regulatory sequence that controls the expression of the protein coding genes in a host cell.
• the term "regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the protein coding genes.
• Such regulatory sequences are described, e.g., in Goeddel; Gene Expression Technology Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990), the entire teaching of which is incorporated herein by reference.
• Suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma.
• CMV cytomegalovirus
• SV40 Simian Virus 40
• AdMLP adenovirus major late promoter
• a recombinant expression vector may also carry one or more additional sequences, such as a sequence that regulates replication of the vector in host cells (e.g., origins of replication) and/or a selectable marker gene.
• the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5, 179,017, all by Axel et al., the entire teachings of both of which are incorporated herein by reference).
• the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
• Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
• DHFR dihydrofolate reduc
• An antibody, or antigen binding fragment thereof, to be used in the method of preparing a protein or protein composition can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell.
• a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and secreted into the medium in which the host cells are cultured, from which medium the antibodies can be recovered.
• Standard recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat. Nos. 4,816,397 & 6,914,128, the entire teachings of both of which are incorporated herein.
• the expression vector(s) encoding the protein is (are) transfected into a host cell by standard techniques.
• the various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
• Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above.
• Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, e.g., Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B.
• Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus
• Salmonella e.g., Salmonella typhimurium
• Serratia e.
• E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.
• eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for polypeptide encoding vectors.
• Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
• a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24, 178), K.
• waltii ATCC 56,500
• K. drosophilarum ATCC 36,906
• K. thermotolerans K. marxianus
• yarrowia EP 402,226
• Pichia pastoris EP 183,070
• Candida Trichoderma reesia
• Neurospora crassa Schwanniomyces such as Schwanniomyces occidentalis
• filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
• Suitable host cells for the expression of a protein or protein composition are derived from multicellular organisms.
• invertebrate cells include plant and insect cells.
• Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified.
• a variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
• Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
• Mammalian cells can be used for expression and production of the protein compositions of the invention, however other eukaryotic cell types can also be employed in the context of the instant invention. See, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987).
• Suitable mammalian host cells for expressing recombinant proteins according to the invention include Chinese Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin, (1980) PNAS USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol.
• NSO myeloma cells NSO myeloma cells
• COS cells COS cells
• SP2 cells SP2 cells.
• the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown.
• useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol.
• monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383 :44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), the entire teachings of which are incorporated herein by reference.
• CV1 ATCC CCL 70 African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo
• Host cells are transformed with the above-described expression or cloning vectors for protein production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
• the host cells used to produce a protein or a protein composition may be cultured in a variety of media which are supplemented or modified in accordance with the present invention.
• Commercially available media such as Ham's F10TM (Sigma), Minimal Essential MediumTM (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's MediumTM (DMEM), (Sigma), Iscove's Modified Dulbecco's Medium, Minimal Essential Medium-alpha.
• MEM-alpha DME/F12, alpha MEM, Basal Medium Eagle with Earle's BSS, DMEM high Glucose, with L-Glutamine, DMEM high glucose, without L- Glutamine, DMEM low Glucose, without L-Glutamine, DMEM:F12 1 : 1, with L-Glutamine, GMEM (Glasgow's MEM), GMEM with L-glutamine, Grace's Complete Insect Medium, Grace's Insect Medium, without FBS, Ham's F-10, with L-Glutamine, Ham's F-12, with L- Glutamine, IMDM with HEPES and L-Glutamine, IMDM with HEPES and without L- Glutamine, IPL-41 Insect Medium, L-15 (Leibovitz)(2.
• any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used as culture media for the host cells, the entire teachings of all of which are incorporated herein by reference.
• any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamycin drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
• the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
• Host cells can also be used to produce portions of intact proteins, for example, antibodies, including Fab fragments or scFv molecules. It is understood that variations on the above procedure are within the scope of the present invention. For example, in certain embodiments it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an antibody. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to an antigen. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention.
• bifunctional antibodies may be produced in which one heavy and one light chain are an antibody of the invention and the other heavy and light chain are specific for an antigen other than the target antibody, depending on the specificity of the antibody of the invention, by crosslinking an antibody of the invention to a second antibody by standard chemical crosslinking methods.
• a recombinant expression vector encoding the protein for example, both an antibody heavy chain and an antibody light chain, is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection.
• the protein gene(s) are each operatively linked to CMV enhancer/ AdMLP promoter regulatory elements to drive high levels of transcription of the gene(s).
• the recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification.
• the selected transformant host cells are cultured to allow for expression of the protein, for example, the antibody heavy and light chains, and intact protein, for example, an antibody, is recovered from the culture medium.
• Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the protein from the culture medium.
• the protein for example, antibodies or antigen binding fragments thereof, can be produced intracellularly, in the periplasmic space, or directly secreted into the medium.
• the particulate debris either host cells or lysed cells (e.g., resulting from homogenization)
• supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, e.g., an AmiconTM or Millipore PelliconTM ultrafiltration unit.
• the first step of a purification process typically involves: lysis of the cell, which can be done by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments. Such disruption releases the entire contents of the cell into the homogenate, and in addition produces subcellular fragments that are difficult to remove due to their small size. These are generally removed by differential centrifugation or by filtration.
• supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, e.g., an AmiconTM or Millipore PelliconTM ultrafiltration unit.
• the recombinant host cells can also be separated from the cell culture medium, e.g., by tangential flow filtration. Antibodies can be further recovered from the culture medium using antibody purification methods.
• modulation of the glycosylation profile of the protein or protein composition produced by recombinant cell culture can be achieved by supplementation of the cell culture media with arabinose, such as L-arabinose, D-arabinose, L-altrose, and/or D-altrose.
• arabinose such as L-arabinose, D-arabinose, L-altrose, and/or D-altrose.
• Specific host cell culture conditions can be used with various cultivation methods including, but not limited to, batch, fed-batch, chemostat and perfusion, and with various cell culture equipment including, but not limited to, shake flasks with or without suitable agitation, spinner flasks, stirred bioreactors, airlift bioreactors, membrane bioreactors, reactors with cells retained on a solid support or immobilized/entrapped as in microporous beads, and any other configuration appropriate for optimal growth and productivity of the desired host cell line.
• the upstream process technologies may be used alone or in combination with the downstream process technologies described below.
• the protein compositions described herein may be purified using downstream process technologies (e.g., purification or concentration), following production using the upstream process technologies of the present invention. For example, once a clarified solution or mixture comprising the protein has been obtained, separation of the protein from process-related impurities, such as the other proteins produced by the host cell, as well as product-related substances, such acidic or basic variants, is performed.
• process-related impurities such as the other proteins produced by the host cell
• product-related substances such as acidic or basic variants
• the initial steps of the purification methods involve the clarification and primary recovery of an antibody from a sample matrix by methods such as centrifugation, depth filtration and/or viral inactivation/reduction.
• further separation is performed using cation exchange chromatography, anion exchange chromatography, and/or multi-mode chromatography.
• a combination of one or more different purification techniques including affinity separation step(s), ion exchange separation step(s), mixed-mode step(s), and/or hydrophobic interaction separation step(s) can also be employed.
• additional purification steps separate mixtures of proteins on the basis of their charge, degree of hydrophobicity, and/or size.
• Continuous and recycle chromatography are also applicable to chromatography methods where the protein is collected in the unbound faction during chromatography or where the protein is first bound to the chromatography resin and subsequently recovered by washing the media with conditions that elute the bound component.
• chromatography resins are commercially available for each of these techniques, allowing accurate tailoring of the purification scheme to the particular protein involved.
• Each of the separation methods allow proteins to either traverse at different rates through a column, achieving a physical separation that increases as they pass further through the column, or to adhere selectively to a separation resin (or medium). The proteins are then differentially eluted using different eluents.
• the protein is separated from impurities when the impurities specifically adhere to the column's resin and the protein does not, i.e., the protein is contained in the effluent, while in other cases the protein will adhere to the column's resin, while the impurities and/or product-related substances are extruded from the column's resin during a wash cycle.
• the protein compositions of the invention may be further purified using viral filtration. Ultrafiltration and/or diafiltration may be used to further concentrate and formulate the protein.
• glycosylation profile of the recombinant glycoproteins and glycoprotein compositions prepared by the methods of the invention can be analyzed using methods well known to those skilled in the art, e.g., removal and derivatization of N-glycans followed by NP-HPLC analysis, weak cation exchange chromatography (WCX), capillary isoelectric focusing (cIEF), size-exclusion chromatography, Poros A HPLC Assay, Host cell Protein ELISA, DNA assay, and western blot analysis.
• efficient methods are available for (i) the splitting of glycosidic bonds either by chemical cleavage such as hydrolysis, acetolysis, hydrazinolysis, or by nitrous deamination; (ii) complete methylation followed by hydrolysis or methanolysis and by gas-liquid chromatography and mass spectroscopy of the partially methylated monosaccharides; and (iii) the definition of anomeric linkages between monosaccharides using exoglycosidases, which also provide insight into the primary glycan structure by sequential degradation.
• HPLC high performance liquid chromatography
• P-HPLC normal phase HPLC
• MR nuclear magnetic resonance
• Kits and equipment for carbohydrate analysis are also commercially available.
• Fluorophore Assisted Carbohydrate Electrophoresis (FACE) is available from Glyko, Inc. (Novato, Calif).
• FACE Fluorophore Assisted Carbohydrate Electrophoresis
• glycoconjugates are released from the peptide with either Endo H or N-glycanase (PNGase F) for N-linked glycans, or hydrazine for Ser/Thr linked glycans.
• PNGase F N-glycanase
• hydrazine for Ser/Thr linked glycans.
• the glycan is then labeled at the reducing end with a fluorophore in a non-structure discriminating manner.
• the fluorophore labeled glycans are then separated in polyacrylamide gels based on the charge/mass ratio of the saccharide as well as the hydrodynamic volume. Images are taken of the gel under UV light and the composition of the glycans is determined by the migration distance as compared with the standards. Oligosaccharides can be sequenced in this manner by analyzing migration shifts due to the sequential removal of saccharides by exoglycosidase digestion.
• glycoprotein compositions described herein including, but not limited to, glycoprotein compositions having increased arabinosylation, reduced fucosylation, and/or reduced mannosylation relative to a control composition, may be used to treat any disorder in a subject for which the therapeutic protein (e.g., an antibody) comprised in the composition is appropriate for treating.
• therapeutic protein e.g., an antibody
• a “disorder” is any condition that would benefit from treatment with the therapeutic protein. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the subject to the disorder in question.
• a therapeutically effective amount of a glycoprotein composition may be administered to treat a disorder in which TNFa activity is detrimental.
• the term "subject" is intended to include living organisms, e.g., prokaryotes and eukaryotes. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In some embodiments, the subject is a human.
• treatment refers to both therapeutic treatment and prophylactic or preventative measures.
• Those in need of treatment include those already with the disorder, as well as those in which the disorder is to be prevented.
• the invention provides a method of administering a glycoprotein composition comprising, for example, an arabinosylated antibody or antigen binding fragment thereof to a subject such that the activity of the target containing the antigen is inhibited or a disorder in which such targetactivity is detrimental is treated.
• a glycoprotein composition comprising, for example, an arabinosylated antibody or antigen binding fragment thereof to a subject such that the activity of the target containing the antigen is inhibited or a disorder in which such targetactivity is detrimental is treated.
• the T Fa is human TNFa and the subject is a human subject.
• glycoprotein compositions can be administered by a variety of methods known in the art. Exemplary routes/modes of administration include subcutaneous injection, intravenous injection or infusion. In certain aspects, a glycoprotein composition may be orally administered. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
• Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In certain embodiments it is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
• Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit comprising a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of a glycoprotein composition is 0.01-20 mg/kg, or 1-10 mg/kg, or 0.3-1 mg/kg.
• dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
• the present invention further provides preparations and formulations comprising a glycoprotein composition with a glycosylation profile described herein, for example an arabinosylated glycoprotein composition, optionally with a decreased fucosylation level and/or decreased mannosylation level relative to a control composition.
• a glycoprotein composition with a glycosylation profile described herein, for example an arabinosylated glycoprotein composition, optionally with a decreased fucosylation level and/or decreased mannosylation level relative to a control composition.
• the glycoprotein composition comprises an antibody or antigen-binding portion thereof, such as an anti-TNFa antibody or antigen-binding portion thereof.
• the glycoprotein compositions may be formulated with a pharmaceutically acceptable carrier as pharmaceutical (therapeutic) compositions, and may be administered by a variety of methods known in the art.
• pharmaceutically acceptable carrier means one or more nontoxic materials that do not interfere with the effectiveness of the biological activity of the active ingredients.
• Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
• Such pharmaceutically acceptable preparations may also routinely contain compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human.
• carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
• the components of the pharmaceutical compositions also are capable of being co-mingled with the glycoprotein composition, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
• a formulation comprising a glycoprotein composition is a liquid formulation.
• a formulation comprising a glycoprotein composition is a lyophilized formulation.
• a formulation comprising a glycoprotein composition is a reconstituted liquid formulation.
• a formulation comprising a glycoprotein composition is a stable liquid formulation.
• a liquid formulation comprising a glycoprotein composition is an aqueous formulation.
• the liquid formulation is non-aqueous.
• a liquid formulation comprising a glycoprotein composition is an aqueous formulation wherein the aqueous carrier is distilled water.
• the formulations comprise a glycoprotein composition in a concentration resulting in a w/v appropriate for a desired dose.
• the glycoprotein composition may be present in the formulation at a concentration of about 1 mg/mL to about 500 mg/mL, e.g., at a concentration of at least 1 mg/mL, at least 5 mg/mL, at least 10 mg/mL, at least 15 mg/mL, at least 20 mg/mL, at least 25 mg/mL, at least 30 mg/mL, at least 35 mg/mL, at least 40 mg/mL, at least 45 mg/mL, at least 50 mg/mL, at least 55 mg/mL, at least 60 mg/mL, at least 65 mg/mL, at least 70 mg/mL, at least 75 mg/mL, at least 80 mg/mL, at least 85 mg/mL, at least 90 mg/mL, at least 95 mg/mL, at least 100 mg/mL, at least 105 mg/mL
• a formulation comprises at least about 50 mg/mL, at least about 100 mg/mL, at least about 125 mg/mL, at least 130 mg/mL, or at least about 150 mg/mL of the glycoprotein composition.
• the concentration of a glycoprotein in the glycoprotein composition that is included in the formulation is between about 1 mg/mL and about 25 mg/mL, between about 1 mg/mL and about 200 mg/mL, between about 25 mg/mL and about 200 mg/mL, between about 50 mg/mL and about 200 mg/mL, between about 75 mg/mL and about 200 mg/mL, between about 100 mg/mL and about 200 mg/mL, between about 125 mg/mL and about 200 mg/mL, between about 150 mg/mL and about 200 mg/mL, between about 25 mg/mL and about 150 mg/mL, between about 50 mg/mL and about 150 mg/mL, between about 75 mg/mL and about 150 mg/mL, between about 100 mg/mL and about 150 mg/mL, between about 125 mg/mL and about 150 mg/mL, between about 25 mg/mL and about 125 mg/mL, between about 50 mg/mL and about 125 mg/m/mL, between about 125
• a formulation comprises between about 40 mg/mL and about 110 mg/mL or between about 50 mg/mL and about 210 mg/mL of a glycoprotein composition.
• the formulations comprising a glycoprotein composition may further comprise one or more active compounds as necessary for the particular indication being treated, typically those with complementary activities that do not adversely affect each other. Such additional active compounds are suitably present in combination in amounts that are effective for the purpose intended.
• the formulations comprising a glycoprotein composition may be prepared for storage by mixing a glycoprotein having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers, including, but not limited to buffering agents, saccharides, salts, surfactants, solubilizers, polyols, diluents, binders, stabilizers, salts, lipophilic solvents, amino acids, chelators, preservatives, or the like (Goodman and Gilman's The Pharmacological Basis of Therapeutics, 12th edition, L. Brunton, et al. and Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed.
• Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as histidine, phosphate, citrate, glycine, acetate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3- pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilionate, glycine, acetate and other organic acids; antioxidants including ascorbic acid and me
• the buffering agent may be histidine, citrate, phosphate, glycine, or acetate.
• the saccharide excipient may be trehalose, sucrose, mannitol, maltose or raffinose.
• the surfactant may be polysorbate 20, polysorbate 40, polysorbate 80, or Pluronic F68.
• the salt may be NaCl, KC1, MgC12, or CaC12.
• the formulations comprising a glycoprotein composition may include a buffering or pH adjusting agent to provide improved pH control.
• a buffering or pH adjusting agent to provide improved pH control.
• Such a formulation may have a pH of between about 3.0 and about 9.0, between about 4.0 and about 8.0, between about 5.0 and about 8.0, between about 5.0 and about 7.0, between about 5.0 and about 6.5, between about 5.5 and about 8.0, between about 5.5 and about 7.0, or between about 5.5 and about 6.5.
• such a formulation has a pH of about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0.
• a formulation has a pH of about 6.0.
• the pH of a formulation generally should not be equal to the isoelectric point of the particular a glycoprotein (e.g., antibody) to be used in the formulation.
• the buffering agent is a salt prepared from an organic or inorganic acid or base.
• Representative buffering agents include, but are not limited to, organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers.
• amino acid components can also function in a buffering capacity.
• Representative amino acid components which may be utilized in the formulations as buffering agents include, but are not limited to, glycine and histidine. In certain embodiments, the buffering agent is chosen from histidine, citrate, phosphate, glycine, and acetate.
• the buffering agent is histidine. In another specific embodiment, the buffering agent is citrate. In yet another specific embodiment, the buffering agent is glycine.
• the purity of the buffering agent should be at least 98%, or at least 99%, or at least 99.5%.
• the term "purity" in the context of histidine and glycine refers to chemical purity of histidine or glycine as understood in the art, e.g., as described in The Merck Index, 13th ed., O'Neil et al. ed. (Merck & Co., 2001).
• Buffering agents are typically used at concentrations between about 1 mM and about 200 mM or any range or value therein, depending on the desired ionic strength and the buffering capacity required.
• concentrations of conventional buffering agents employed in parenteral formulations can be found in: Pharmaceutical Dosage Form: Parenteral Medications, Volume 1, 2nd Edition, Chapter 5, p. 194, De Luca and Boylan, "Formulation of Small Volume Parenterals", Table 5: Commonly used additives in Parenteral Products.
• the buffering agent is at a concentration of at least about 1 mM, or of at least about 5 mM, or of at least about 10 mM, or of at least about 15 mM, or of at least about 20 mM, or of at least about 25 mM, or of at least about 30 mM, or of at least about 35 mM, or of at least about 40 mM, or of at least about 45 mM, or of at least about 50 mM, or of at least about 60 mM, or of at least about 70 mM, or of at least about 80 mM, or of at least about 90 mM, or of at least about 100 mM.
• the buffering agent is at a concentration of about 1 mM, or of about 5 mM, or of about 10 mM, or of about 15 mM, or of about 20 mM, or of about 25 mM, or of about 30 mM, or of about 35 mM, or of about 40 mM, or of about 45 mM, or of about 50 mM, or of about 60 mM, or of about 70 mM, or of about 80 mM, or of about 90 mM, or of about 100 mM.
• the buffering agent is at a concentration of between about 5 mM and about 50 mM.
• the buffering agent is at a concentration of between 5 mM and 20 mM.
• the formulation comprising a glycoprotein composition comprises histidine as a buffering agent.
• the histidine is present in the formulation at a concentration of at least about 1 mM, at least about 5 mM, at least about 10 mM, at least about 20 mM, at least about 30 mM, at least about 40 mM, at least about 50 mM, at least about 75 mM, at least about 100 mM, at least about 150 mM, or at least about 200 mM histidine.
• a formulation comprises between about 1 mM and about 200 mM, between about 1 mM and about 150 mM, between about 1 mM and about 100 mM, between about 1 mM and about 75 mM, between about 10 mM and about 200 mM, between about 10 mM and about 150 mM, between about 10 mM and about 100 mM, between about 10 mM and about 75 mM, between about 10 mM and about 50 mM, between about 10 mM and about 40 mM, between about 10 mM and about 30 mM, between about 20 mM and about 75 mM, between about 20 mM and about 50 mM, between about 20 mM and about 40 mM, or between about 20 mM and about 30 mM histidine.
• the formulation comprises about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 150 mM, or about 200 mM histidine.
• a formulation may comprise about 10 mM, about 25 mM, or no histidine.
• the formulations comprising a glycoprotein composition may comprise a carbohydrate excipient.
• Carbohydrate excipients can act, e.g., as viscosity enhancing agents, stabilizers, bulking agents, solubilizing agents, and/or the like.
• Carbohydrate excipients are generally present at between about 1% to about 99% by weight or volume, e.g., between about 0.1%) to about 20%>, between about 0.1%> to about 15%>, between about 0.1%> to about 5%), between about 1%> to about 20%>, between about 5%> to about 15%>, between about 8%> to about 10%), between about 10%> and about 15%>, between about 15%> and about 20%>, between 0.1% to 20%, between 5% to 15%, between 8% to 10%, between 10% and 15%, between 15%> and 20%>, between about 0.1%> to about 5%>, between about 5%> to about 10%>, or between about 15%> to about 20%>.
• the carbohydrate excipient is present at 1%>, or at 1.5%, or at 2%, or at 2.5%, or at 3%, or at 4%, or at 5%, or at 10%, or at 15%, or at 20%.
• Carbohydrate excipients suitable for use in the formulations include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and the like.
• monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like
• disaccharides such as lactose, sucrose, trehalose, cellobio
• the carbohydrate excipients for use in the present invention are chosen from sucrose, trehalose, lactose, mannitol, and raffinose.
• the carbohydrate excipient is trehalose.
• the carbohydrate excipient is mannitol.
• the carbohydrate excipient is sucrose.
• the carbohydrate excipient is raffinose.
• the purity of the carbohydrate excipient should be at least 98%, or at least 99%, or at least 99.5%.
• the formulations comprising a glycoprotein composition may comprise trehalose.
• a formulation comprises at least about 1%, at least about 2%, at least about 4%, at least about 8%, at least about 20%, at least about 30%, or at least about 40% trehalose.
• a formulation comprises between about 1% and about 40%, between about 1% and about 30%, between about 1% and about 20%, between about 2% and about 40%, between about 2% and about 30%), between about 2% and about 20%, between about 4% and about 40%, between about 4% and about 30%, or between about 4% and about 20% trehalose.
• a formulation comprises about 1%, about 2%, about 4%, about 6%, about 8%, about 15%), about 20%, about 30%, or about 40% trehalose. In a specific embodiment, a formulation comprises about 4%, about 6% or about 15% trehalose.
• a formulation comprising a glycoprotein composition comprises an excipient.
• a formulation comprises at least one excipient chosen from: sugar, salt, surfactant, amino acid, polyol, chelating agent, emulsifier and preservative.
• a formulation comprises a salt, e.g., a salt selected from: NaCl, KC1, CaC12, and MgC12. In a specific embodiment, the formulation comprises NaCl.
• a formulation comprising a glycoprotein composition may comprise at least about 10 mM, at least about 25 mM, at least about 50 mM, at least about 75 mM, at least about 80 mM, at least about 100 mM, at least about 125 mM, at least about 150 mM, at least about 175 mM, at least about 200 mM, or at least about 300 mM sodium chloride (NaCl).
• the formulation may comprise between about 10 mM and about 300 mM, between about 10 mM and about 200 mM, between about 10 mM and about 175 mM, between about 10 mM and about 150 mM, between about 25 mM and about 300 mM, between about 25 mM and about 200 mM, between about 25 mM and about 175 mM, between about 25 mM and about 150 mM, between about 50 mM and about 300 mM, between about 50 mM and about 200 mM, between about 50 mM and about 175 mM, between about 50 mM and about 150 mM, between about 75 mM and about 300 mM, between about 75 mM and about 200 mM, between about 75 mM and about 175 mM, between about 75 mM and about 150 mM, between about 100 mM and about 300 mM, between about 100 mM and about 200 mM, between about 100 mM and about 200 mM
• the formulation may comprise about 10 mM, about 25 mM, about 50 mM, about 75 mM, about 80 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, or about 300 mM sodium chloride.
• a formulation comprising a glycoprotein composition may also comprise an amino acid, e.g., lysine, arginine, glycine, histidine or an amino acid salt.
• the formulation may comprise at least about 1 mM, at least about 10 mM, at least about 25 mM, at least about 50 mM, at least about 100 mM, at least about 150 mM, at least about 200 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, or at least about 400 mM of an amino acid.
• the formulation may comprise between about 1 mM and about 100 mM, between about 10 mM and about 150 mM, between about 25 mM and about 250 mM, between about 25 mM and about 300 mM, between about 25 mM and about 350 mM, between about 25 mM and about 400 mM, between about 50 mM and about 250 mM, between about 50 mM and about 300 mM, between about 50 mM and about 350 mM, between about 50 mM and about 400 mM, between about 100 mM and about 250 mM, between about 100 mM and about 300 mM, between about 100 mM and about 400 mM, between about 150 mM and about 250 mM, between about 150 mM and about 300 mM, or between about 150 mM and about 400 mM of an amino acid.
• a formulation comprises about 1 mM, 1.6 mM, 25 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, or about 400 mM of an amino acid.
• the formulations comprising a glycoprotein composition may further comprise a surfactant.
• surfactant refers to organic substances having amphipathic structures; namely, they are composed of groups of opposing solubility tendencies, typically an oil-soluble hydrocarbon chain and a water-soluble ionic group. Surfactants can be classified, depending on the charge of the surface-active moiety, into anionic, cationic, and nonionic surfactants. Surfactants are often used as wetting, emulsifying, solubilizing, and dispersing agents for various pharmaceutical compositions and preparations of biological materials.
• surfactants like polysorbates (e.g., polysorbates 20 or 80); polyoxamers (e.g., poloxamer 188); Triton; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine
• a formulation comprises Polysorbate 20, Polysorbate 40, Polysorbate 60, or Polysorbate 80.
• Surfactants are particularly useful if a pump or plastic container is used to administer the formulation.
• the presence of a pharmaceutically acceptable surfactant mitigates the propensity for the protein to aggregate.
• the formulations may comprise a polysorbate which is at a concentration ranging from between about 0.001% to about 1%, or about 0.001%> to about 0.1%), or about 0.01%> to about 0.1%>.
• the formulations comprise a polysorbate which is at a concentration of 0.001%>, or 0.002%), or 0.003%>, or 0.004%, or 0.005%, or 0.006%, or 0.007%, or 0.008%, or 0.009%, or 0.01%, or 0.015%, or 0.02%.
• the formulations comprising a glycoprotein composition may optionally further comprise other common excipients and/or additives including, but not limited to, diluents, binders, stabilizers, lipophilic solvents, preservatives, adjuvants, or the like.
• Pharmaceutically acceptable excipients and/or additives may be used in the formulations of the invention.
• Commonly used excipients/additives, such as pharmaceutically acceptable chelators for example, but not limited to, EDTA, DTPA or EGTA
• EDTA for example, but not limited to, EDTA, DTPA or EGTA
• Preservatives such as phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (for example, but not limited to, hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof can optionally be added to the formulations at any suitable concentration such as between about 0.001%> to about 5%, or any range or value therein.
• concentration of preservative used in the formulations is a concentration sufficient to yield a microbial effect. Such concentrations are dependent on the preservative selected and are readily determined by the skilled artisan.
• contemplated excipients/additives which may be utilized in the formulations include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids such as phospholipids or fatty acids, steroids such as cholesterol, protein excipients such as serum albumin (human serum albumin (HSA), recombinant human albumin (rHA)), gelatin, casein, salt-forming counterions such as sodium and the like.
• HSA human serum albumin
• rHA recombinant human albumin
• salt-forming counterions such as sodium and the like.
• compositions suitable for use in the formulations are known in the art, e.g., as listed in "Remington: The Science & Practice of Pharmacy", 21st ed., Lippincott Williams & Wilkins, (2005), and in the “Physician's Desk Reference", 60th ed., Medical Economics, Montvale, N.J. (2005).
• Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and/or stability of the glycoprotein of the glycoprotein composition, as well known those in the art or as described herein.
• the formulations comprising a glycoprotein composition may be isotonic with human blood, wherein the formulations have essentially the same osmotic pressure as human blood.
• Such isotonic formulations will generally have an osmotic pressure from about 250 mOSm to about 350 mOSm.
• Isotonicity can be measured by, for example, using a vapor pressure or ice-freezing type osmometer.
• Tonicity of a formulation is adjusted by the use of tonicity modifiers.
• “Tonicity modifiers” are those pharmaceutically acceptable inert substances that can be added to the formulation to provide an isotonity of the formulation.
• Tonicity modifiers suitable for this invention include, but are not limited to, saccharides, salts and amino acids.
• the formulations comprising a glycoprotein composition have an osmotic pressure from about 100 mOSm to about 1200 mOSm, or from about 200 mOSm to about 1000 mOSm, or from about 200 mOSm to about 800 mOSm, or from about 200 mOSm to about 600 mOSm, or from about 250 mOSm to about 500 mOSm, or from about 250 mOSm to about 400 mOSm, or from about 250 mOSm to about 350 mOSm.
• the concentration of any one component or any combination of various components, of the formulations comprising a glycoprotein composition is adjusted to achieve the desired tonicity of the final formulation.
• the ratio of the carbohydrate excipient to glycoprotein may be adjusted according to methods known in the art (e.g., U.S. Pat. No. 6,685,940).
• the molar ratio of the carbohydrate excipient to glycoprotein may be from about 100 moles to about 1000 moles of carbohydrate excipient to about 1 mole of glycoprotein, or from about 200 moles to about 6000 moles of carbohydrate excipient to about 1 mole of glycoprotein, or from about 100 moles to about 510 moles of carbohydrate excipient to about 1 mole of glycoprotein, or from about 100 moles to about 600 moles of carbohydrate excipient to about 1 mole of glycoprotein.
• the desired isotonicity of the final formulation may also be achieved by adjusting the salt concentration of the formulations.
• Pharmaceutically acceptable salts and those suitable as tonicity modifiers include, but are not limited to, sodium chloride, sodium succinate, sodium sulfate, potassuim chloride, magnesium chloride, magnesium sulfate, and calcium chloride.
• formulations comprise NaCl, MgCl 2 , and/or CaCl 2 .
• concentration of NaCl is between about 75 mM and about 150 mM.
• concentration of MgCl 2 is between about 1 mM and about 100 mM.
• Pharmaceutically acceptable amino acids including those suitable as tonicity modifiers include, but are not limited to, proline, alanine, L-arginine, asparagine, L-aspartic acid, glycine, serine, lysine, and histidine.
• the formulations comprising a glycoprotein composition are pyrogen-free formulations which are substantially free of endotoxins and/or related pyrogenic substances.
• Endotoxins include toxins that are confined inside a microorganism and are released only when the microorganisms are broken down or die.
• Pyrogenic substances also include fever-inducing, thermostable substances from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, even low amounts of endotoxins must be removed from intravenously administered pharmaceutical drug solutions.
• FDA Food & Drug Administration
• EU endotoxin units
• the endotoxin and pyrogen levels in the composition are less than 10 EU/mg, or less than 5 EU/mg, or less than 1 EU/mg, or less than 0.1 EU/mg, or less than 0.01 EU/mg, or less than 0.001 EU/mg.
• the formulations comprising a glycoprotein composition should be sterile.
• the formulations may be sterilized by various sterilization methods, including sterile filtration, radiation, etc.
• formulation is filter-sterilized with a presterilized 0.22-micron filter.
• Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in "Remington: The Science & Practice of Pharmacy", 21st ed., Lippincott Williams & Wilkins, (2005).
• Formulations comprising proteins of interest e.g., antibody or other glycoprotein
• proteins of interest e.g., antibody or other glycoprotein
• sterile compositions comprising proteins of interest (e.g., antibody or other glycoprotein) are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierceable by a hypodermic injection needle.
• a composition is provided as a pre-filled syringe.
• a formulation comprising a glycoprotein composition is a lyophilized formulation.
• lyophilized or “freeze-dried” includes a state of a substance that has been subjected to a drying procedure such as lyophilization, where at least 50% of moisture has been removed.
• the phrase "bulking agent” includes a compound that is pharmaceutically acceptable and that adds bulk to a lyo cake.
• Bulking agents known to the art include, for example, carbohydrates, including simple sugars such as dextrose, ribose, fructose and the like, alcohol sugars such as mannitol, inositol and sorbitol, disaccharides including trehalose, sucrose and lactose, naturally occurring polymers such as starch, dextrans, chitosan, hyaluronate, proteins (e.g., gelatin and serum albumin), glycogen, and synthetic monomers and polymers.
• carbohydrates including simple sugars such as dextrose, ribose, fructose and the like, alcohol sugars such as mannitol, inositol and sorbitol, disaccharides including trehalose, sucrose and lactose, naturally occurring polymers such as starch, dextrans, chitosan,
• a "lyoprotectant” is a molecule which, when combined with a glycoprotein composition, significantly prevents or reduces chemical and/or physical instability of the protein upon lyophilization and subsequent storage.
• Lyoprotectants include, but are not limited to, sugars and their corresponding sugar alcohols; an amino acid or salt thereof such as monosodium glutamate or histidine; a methylamine such as betaine; a lyotropic salt such as magnesium sulfate; a polyol such as trihydric or higher molecular weight sugar alcohols, e.g., glycerin, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol; PLURONICS®; and combinations thereof.
• lyoprotectants include, but are not limited to, glycerin and gelatin, and the sugars mellibiose, melezitose, raffinose, mannotriose and stachyose.
• reducing sugars include, but are not limited to, glucose, maltose, lactose, maltulose, iso- maltulose and lactulose.
• non-reducing sugars include, but are not limited to, non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other straight chain polyalcohols.
• sugar alcohols include, but are not limited to, monoglycosides, compounds obtained by reduction of disaccharides such as lactose, maltose, lactulose and maltulose.
• the glycosidic side group can be either glucosidic or galactosidic.
• Additional examples of sugar alcohols include, but are not limited to, glucitol, maltitol, lactitol and iso-maltulose.
• trehalose or sucrose is used as a lyoprotectant.
• the lyoprotectant is added to the pre-lyophilized formulation in a
• lyoprotecting amount which means that, following lyophilization of the protein in the presence of the lyoprotecting amount of the lyoprotectant, the protein essentially retains its physical and chemical stability and integrity upon lyophilization and storage.
• the molar ratio of a lyoprotectant (e.g., trehalose) and a glycoprotein in the formulation is at least about 10, at least about 50, at least about 100, at least about 200, or at least about 300. In another embodiment, the molar ratio of a lyoprotectant (e.g., trehalose) and a glycoprotein in the formulation is about 1, is about 2, is about 5, is about 10, about 50, about 100, about 200, or about 300.
• a "reconstituted" formulation is one which has been prepared by dissolving a lyophilized glycoprotein composition in a diluent such that the glycoprotein molecules are dispersed in the reconstituted formulation.
• the reconstituted formulation is suitable for administration (e.g., parenteral administration) to a patient to be treated with the glycoprotein composition and, in certain embodiments of the invention, may be one which is suitable for intravenous administration.
• diluent of interest herein is one which is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is useful for the preparation of a liquid formulation, such as a formulation reconstituted after lyophilization.
• diluents include, but are not limited to, sterile water, bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution or dextrose solution.
• BWFI bacteriostatic water for injection
• a pH buffered solution e.g., phosphate-buffered saline
• sterile saline solution e.g., Ringer's solution or dextrose solution.
• diluents can include aqueous solutions of salts and/or buffers.
• a formulation comprising a glycoprotein composition is a lyophilized formulation, wherein at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% of the glycoprotein molecules may be recovered from a vial upon shaking the vial for 4 hours at a speed of 400 shakes per minute wherein the vial is filled to half of its volume with the formulation.
• a formulation is a lyophilized formulation, wherein at least about 90%, at least about 95%), at least about 97%, at least about 98%, or at least about 99% of the glycoprotein molecules may be recovered from a vial upon subjecting the formulation to three freeze/thaw cycles wherein the vial is filled to half of its volume with the formulation.
• a formulation is a lyophilized formulation, wherein at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% of the glycoprotein molecules may be recovered by reconstituting a lyophilized cake generated from the formulation.
• a reconstituted liquid formulation may comprise a glycoprotein composition at the same concentration as the pre-lyophilized liquid formulation.
• a reconstituted liquid formulation may comprise a glycoprotein composition at a higher concentration than the pre-lyophilized liquid formulation, e.g., about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, or about 10 fold higher concentration of a glycoprotein composition than the pre-lyophilized liquid formulation.
• a reconstituted liquid formulation may comprise a glycoprotein composition at a lower concentration than the pre-lyophilized liquid formulation, e.g., about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold or about 10 fold lower concentration of a glycoprotein composition than the pre-lyophilized liquid formulation.
• the pharmaceutical formulations comprising a glycoprotein composition are typically stable formulations, e.g., stable at room temperature.
• a reference formulation may be a reference standard frozen at -70° C. consisting of 10 mg/mL of a glycoprotein in PBS.
• Therapeutic formulations comprising a glycoprotein composition may be formulated for a particular dosage. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms are dictated by and directly dependent on (a) the unique characteristics of the glycoprotein, and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such a glycoprotein for the treatment of sensitivity in individuals.
• compositions comprising a glycosylation profile described herein, can be formulated for particular routes of administration, such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
• routes of administration such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
• the formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy.
• the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration.
• the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect.
• glycoprotein compositions are formulated for intravenous administration.
• glycoprotein compositions are formulated for local delivery to the cardiovascular system, for example, via catheter, stent, wire, intramyocardial delivery, intrapericardi
• Formulations comprising a glycoprotein composition which are suitable for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
• the glycoprotein may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required (U.S. Pat. Nos. 7,378, 110; 7,258,873; 7,135, 180; 7,923,029; and US Publication No. 20040042972).
• parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
• compositions comprising a glycosylation profile described herein
• dosage levels of the glycoprotein in the pharmaceutical compositions may be varied so as to obtain an amount of the glycoprotein which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
• the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
• glycoprotein compositions can be formulated to ensure proper distribution in vivo.
• the blood-brain barrier excludes many highly hydrophilic compounds.
• the therapeutic compounds can cross the BBB (if desired)
• they can be formulated, for example, in liposomes.
• liposomes For methods of manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; 5,399,331.
• the liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685).
• Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Pat. No. 5,416,016); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153 : 1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant Protein A receptor (Briscoe et al. (1995) Am. J. Physiol.
• the glycoproteins are formulated in liposomes; in another embodiment, the liposomes include a targeting moiety. In another embodiment, the glycoproteins in the liposomes are delivered by bolus injection to a site proximal to the desired area.
• the composition When administered in this manner, the composition must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi. Additionally or alternatively, the proteins with modulated glycosylation profiles (e.g., antibodies) may be delivered locally to the brain to mitigate the risk that the blood brain barrier slows effective delivery.
• the proteins with modulated glycosylation profiles e.g., antibodies
• the glycoprotein compositions may be administered with medical devices known in the art.
• the glycoprotein composition is administered locally via a catheter, stent, wire, or the like.
• a therapeutic composition can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Pat. Nos. 5,399, 163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; 4,596,556.
• Examples of well-known implants and modules include: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S.
• Pat. No. 4,486,194 which discloses a therapeutic device for administering medicants through the skin
• U.S. Pat. No. 4,447,233 which discloses a medication infusion pump for delivering medication at a precise infusion rate
• U.S. Pat. No. 4,447,224 which discloses a variable flow implantable infusion apparatus for continuous drug delivery
• U.S. Pat. No. 4,439,196 which discloses an osmotic drug delivery system having multi-chamber compartments
• U.S. Pat. No. 4,475, 196 which discloses an osmotic drug delivery system.
• Many other such implants, delivery systems, and modules are known to those skilled in the art.
• compositions comprising a glycosylation profile described herein depend on the disease or condition to be treated and can be determined by the persons skilled in the art. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
• kits comprising the compositions comprising a glycosylation profile described herein, for example an arabinosylated glycoprotein such as an antibody or antigen-binding portion thereof, optionally with a decreased fucosylation level or amount and/or an decreased mannosylation level or amount and instructions for use.
• kit refers to a packaged product comprising components with which to administer the glycoprotein composition for treatment of a disease or disorder.
• the kit may comprise a box or container that holds the components of the kit.
• the box or container is affixed with a label or a Food and Drug Administration approved protocol.
• the box or container holds components which may be contained within plastic, polyethylene, polypropylene, ethylene, or propylene vessels.
• the vessels can be capped-tubes or bottles.
• the kit can also include instructions for administering a glycoprotein composition.
• the kit can further contain one more additional reagents, such as an immunosuppressive reagent, a cytotoxic agent or a radiotoxic agent or one or more additional proteins of interest (e.g., an antibody having a complementary activity which binds to an epitope in the TNFa antigen distinct from a first anti-TNFa antibody).
• Kits typically include a label indicating the intended use of the contents of the kit.
• the term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.
• the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with a liquid formulation or lyophilized formulation of a glycoprotein composition.
• a container filled with a liquid formulation is a pre-filled syringe.
• the formulations are formulated in single dose vials as a sterile liquid.
• the formulations may be supplied in 3 cc USP Type I borosilicate amber vials (West Pharmaceutical Services—Part No. 6800-0675) with a target volume of 1.2 mL.
• Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
• a container filled with a liquid formulation is a pre- filled syringe.
• Any pre-filled syringe known to one of skill in the art may be used in combination with a liquid formulation of the invention.
• Pre-filled syringes that may be used are described in, for example, but not limited to, PCT Publications WO05032627, WO08094984, W09945985, WO03077976, U.S. Pat. No. 6,792,743, U.S. Pat. No. 5,607,400, U.S. Pat. No. 5,893,842, U.S. Pat. No. 7,081, 107, U.S. Pat. No. 7,041,087, U.S. Pat.
• Pre-filled syringes may be made of various materials. In certain embodiments a pre-filled syringe is a glass syringe. In another embodiment a pre-filled syringe is a plastic syringe.
• a pre-filled syringe comprises a silicone based lubricant.
• a pre-filled syringe comprises baked on silicone.
• a pre-filled syringe is free from silicone based lubricants.
• a pre-filled syringe may comprise tungsten at a level above 500 ppb.
• a pre-filled syringe is a low tungsten syringe.
• a pre- filled syringe may comprise tungsten at a level between about 500 ppb and about 10 ppb, between about 400 ppb and about 10 ppb, between about 300 ppb and about 10 ppb, between about 200 ppb and about 10 ppb, between about 100 ppb and about 10 ppb, between about 50 ppb and about 10 ppb, between about 25 ppb and about 10 ppb.
• kits comprising an arbinosylated glycoprotein composition, optionally having a decreased fucosylation level or amount and/or a decreased mannosylation level or amount, are also provided that are useful for various purposes, e.g., research and diagnostic including for purification or immunoprecipitation from cells or detection in vitro or in vivo.
• the kit may contain an antibody coupled to beads (e.g., sepharose beads).
• Kits may be provided which contain the antibodies for detection and quantitation of glycoprotein molecules in vitro, e.g., in an ELISA or a Western blot.
• the kit comprises a container and a label or package insert on or associated with the container.
• the container holds a composition comprising at least one glycoprotein composition.
• Additional containers may be included that contain, e.g., diluents and buffers, control compositions.
• the label or package insert may provide a description of the composition as well as instructions for the intended in vitro or diagnostic use.
• the present invention also encompasses a finished packaged and labeled pharmaceutical product.
• This article of manufacture includes the appropriate unit dosage form in an appropriate vessel or container such as a glass vial, pre-filled syringe or other container that is hermetically sealed.
• the unit dosage form is provided as a sterile particulate free solution comprising a glycoprotein composition that is suitable for parenteral administration.
• the unit dosage form is provided as a sterile lyophilized powder comprising a glycoprotein composition that is suitable for reconstitution.
• the unit dosage form is suitable for intravenous, intramuscular, intranasal, oral, topical or subcutaneous delivery.
• the present invention encompasses sterile solutions comprising an arabinosylated glycoprotein suitable for each delivery route.
• the present invention further encompasses sterile lyophilized powders comprising an arabinosylated glycoprotein that are suitable for reconstitution.
• the packaging material and container are designed to protect the stability of the product during storage and shipment.
• the products include instructions for use or other informational material that advise the physician, technician or patient on how to appropriately prevent or treat the disease or disorder in question, as well as how and how frequently to administer the pharmaceutical.
• the article of manufacture includes instruction means indicating or suggesting a dosing regimen including, but not limited to, actual doses, monitoring procedures, and other monitoring information.
• the present invention provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, pre-filled syringe, sprayer, insufflator, intravenous (i.v.) bag, envelope and the like; and at least one unit dosage form of a pharmaceutical agent contained within the packaging material, wherein the pharmaceutical agent comprises a liquid formulation containing a glycoprotein composition.
• packaging material includes instruction means which indicate how that the glycoprotein composition can be used to prevent, treat and/or manage one or more symptoms associated with a disease or disorder.
• Table 1 Summary of novel sugar supplementation details Sugar 0.1 , 1 ,1 0,20,50
• Viable cell density (VCD) and cell viability values were measured through trypan blue exclusion via Cedex automated cell counters (Roche Applied Science, Indianapolis, IN), glucose and lactate values were measured with an ABL-805 (Radiometer Medical, Denmark) blood gas analyzer. Media pH measurements were also performed with an ABL-805 (Radiometer Medical, Denmark) blood gas analyzer. Media osmolality was measured on a Multi-Osmette 2430 osmometer (Precision Systems, Natick, MA).
• Protein A Affinity Chromatography Antibody titers were measured from clarified cell culture harvests on a Poros ATM (Life Technologies, Carlsbad, CA) affinity column using an HPLC system operating with a low pH, step elution gradient with detection at 280 nm. Absolute concentrations were assigned with respect to reference standard calibration curves. Purified antibodies subjected to additional analytical characterization were purified using MabSelectTM Protein A (GE Healthcare, Piscataway, NJ) using a low pH, step elution gradient, followed by buffer exchange using Spin-X® UF Concentrator columns (Corning Lifesciences, Tewksbury, MA), or equivalent, according to the manufacturer's recommended procedure.
• N-glycan Oligosaccharide Profiling-LC/MS The heavy chain of protein samples were analyzed using an HPLC (Agilent 1260, Santa Clara, CA) with a reversed phase column (Vydac, C4, 1 x 150 mm, 5 ⁇ particle size) coupled to a Q-TOF mass spectrometer (Agilent, 6510). Protein A-purified samples from cell culture harvests were first reduced using DTT at 37°C for 30 minutes.
• the samples were then loaded using 95% mobile phase A (0.08% formic acid and 0.02% TFA in water) and 5% mobile phase B (0.08% formic acid and 0.02% TFA in acetonitrile), and then eluted using a gradient of increasing levels of mobile phase B.
• the flow rate was 50 ⁇ / ⁇ and the column temperature was set to 60°C.
• the mass spectrometer was operated in positive ion mode with an ion spray voltage of 4500 volts and a source temperature of 350°C. Resolved peaks centered on the known masses of different N-glycans were subsequently used to identify the different types of N-glycans, and the relative peak heights were used to quantitate their respective levels.
• N-glycan Glycopeptide Mapping-LC/MS/MS Protein samples were denatured with 6M guanidine-HCl at pH 8.0. The proteins were reduced with 10 mM DTT for 30 minutes at 37 °C. The proteins were alkylated with 25 mM iodoacetic acid for 30 minutes at 37 °C in the dark. The proteins were buffer exchanged to 10 mM tris pH 8.0 using NAP-5 columns. The proteins were digested with trypsin (1 :20 trypsimantibody) for 4 hours at 37 °C. The digest was quenched by adding 5 ⁇ ⁇ of formic acid.
• Peptide mapping was performed by LC/MS/MS using a Waters Acuity UPLC coupled to a Thermo Orbitrap VelosTM. The summed spectrum was extracted for the chromatographic region where all the glycopeptides eluted. The relative peak intensites of all the glycovariants were determined as a percent of the total.
• Serum concentrations of mAb-1 were measured using a GYROS method employing biotinylated antibodies to capture mAb-1, and Alexa Fluor 647 goat, anti-human IgG detection. Concentrations were calculated using logistic fit using XLfit4, and PK parameters calculated using non-compartmental analysis using PLASMA v2.6.12 (SParCS).
• PBMC Peripheral blood mononuclear cells
• GE Healthcare Pittsburgh, PA
• Monocytes were isolated by magnetic sorting using CD14 microbeads (Miltenyi Biotec, San Diego, CA). The purity of the resulting monocytes, as assessed by flow cytometric analysis, was typically greater than 98%.
• Monocytes were cultured in RPMI1640 medium supplemented 2 mM L-glutamine, 100 ng/mL of recombinant human GM-CSF and 5 ng/mL of human IL- 4 (Peprotech, Rocky Hill, NJ), 100 ⁇ g/mL penicillin, and streptomycin, and 10% fetal bovine serum at a density of 1 x 10 6 cells/mL at 37°C with 5% C0 2 for 5 days.
• Dendritic cell differentiation and stimulation Dendritic cells were generated by culturing monocytes in RPMI1640 medium supplemented with 100 ng/mL of recombinant human GM-CSF and 5 ng/mL of human IL-4 (Peprotech, Rocky Hill, NJ) for 4 days. To investigate the TNFoc production, dendritic cells were stimulated with 1 mg/mL LPS (from Salmonella typhimurium, Sigma-Aldrich, St. Louis, MO) for 1 hour.
• LPS from Salmonella typhimurium, Sigma-Aldrich, St. Louis, MO
• Antigen Binding Biacore® T100 (GE Healthcare Bio-Sciences, Pittsburgh,
• PA PA
• HBS-EP buffer GE Healthcare
• Dissociation was then monitored for 900s. Regeneration was performed after each run. Purified cell culture samples were compared to mAb-1 reference standard. All samples were assayed in triplicate.
• HMW High molecular weight
• LMW low molecular weight
• Viscosity Measurement Taylor dispersion analysis (TDA) was performed with the Viscosizer 200 (Malvern) to determine sample viscosities. The specific viscosity for the sample solution was calculated using Poiseuille's Law and relied upon the accurate timing of UV absorbance migrating past two separate points within a capillary tube. Both the total length of the capillary and distance between the two observation points were known. By comparing the migration of a known sample against that of a reference buffer spiked with caffeine, the specific viscosity of protein formulations were determined. Ten of samples were injected into a cellulose coated capillary under a continuous flow of buffer (15mM histidine, pH 6.0) at 2,000 mBar of pressure.
• the same formulation buffer was utilized as the running buffer.
• Two detector heads were positioned in the capillary system and temperature was controlled at 25°C.
• the capillary was washed between experiments with buffer at 2,000 mBar of pressure as per the manufacturer's protocol.
• the UV detection wavelength utilized was 280 nm.
• Cytokine Release Assay 96-well propylene plates were coated with 1 ⁇ g of purified protein from experimental samples in 60 ⁇ of DPBS per well for 1.5 hrs at room temperature. After rinsing, lxlO 5 human peripheral blood mononuclear cells (PBMC) were added and incubated for approximately 2 days at 37°C. Well plate supernatants were then assayed for IL- ⁇ , IL-6, IL-8, IL-2, T Fa, and IFNy using MSD (Meso Scale Discovery, Rockville, MD) technology. Values were measured using a SECTOR® Imager 6000 reader (Meso Scale Discovery, Rockville, MD). Experimental samples were compared to both positive and negative controls. A cytokine response was considered positive when the levels were greater than twice the level released from the negative control.
• PBMC peripheral blood mononuclear cells
• Complement Activation 96-well EIA (Corning, Corning, NY) ELISA plates were coated with various concentrations of purified antibody protein from the experimental samples. Plate wells were then incubated with human serum from healthy donors as a source of complement for 1 hr at 37°C. Complement activation was determined by measuring the levels of human iC3 bound to the plate using mouse, anti-human iC3b antibody (Quidel, San Diego, CA), followed by incubation with anti-mouse IgG-HRP. Absorbance at 450 nm was read on a SpectraMax® 340PC plate reader (Molecular Devices, Sunnyvale, CA).
• FcyR, FcR n Binding Biacore® was used to evaluate for differences in binding of purified mAb-1 towards both human FcyRs and human FcRn between control and experimental samples.
• Biacore® was used to evaluate for differences in binding of purified mAb-1 towards both human FcyRs and human FcRn between control and experimental samples.
• the following FcyRs were immobilized on a CM5 chip using immobilized goat anti-histidine antibody: huFcyRIIal38H-His, huFcyRIIal31R-His, huFcyRIIb-His, huFcyRIIIal58F-His, huFcyRIIIal58V-His.
• ADCC activity was assessed using a calceinAM-based assay. Briefly, DoHH2 target cells were incubated for 30 minutes with 10-15 ⁇ calceinAm in DMSO at a concentration of 4 x 10 6 cell/mL at 37°C, washed two times with culture medium, and then incubated with antibodies for 15 minutes on ice. The cells were then plated at 10,000/well in a 96-well v-bottom plate. K-92 V158 or K- 92 F158 effector cells (E) were added at a 9: 1 E:T ratio. After a 2.5-hour incubation at 37°C, 100 ⁇ .
• CD 16 (FcyRIIIa) Reporter Assay CD 16 signaling was assessed using CD 16 transfected reporter cells (Promega, Madison, WI). Reporter cells and human, transmembrane-TNFa expressing HEK293 cells were incubated in a 96-well plate with increasing concentrations of antibody for 18 hours at a ratio of 1 reporter to 2 targets. After incubation, CD16-mediated luciferase induction was measured by adding Bio-GloTM luciferase substrate (Promega, Madison, WI). Relative luciferase units were measured on an
• CHO cell line 1 expressing mAb-1 (adalimumab; see Table A) was evaluated in shaker flasks in fed-batch operation mode. Variable amounts of D-arabinose were supplemented into both the chemically-defined basal and feed medias.
• Agents for increasing levels of galactosylated N-glycans such as supplementation of media with trace metals (e.g., manganese, cobalt, iron, and the like) and/or with sugars other than arabinose (e.g., altrose, galactose, turanose, melezitose, and the like), as well as methods for supplementation thereof (e.g., amounts, concentrations, and the like), are well known in the art (see, for example, U.S. Pat. Publ. Nos. 2015/0050693, 2014/0314745, 2014/0271633, and 2012/0195885; the entire contents of each of which published application is incorporated herein by this reference).
• trace metals e.g., manganese, cobalt, iron, and the like
• sugars other than arabinose e.g., altrose, galactose, turanose, melezitose, and the like
• methods for supplementation thereof e.g.,
• Arabinose' s ability to serve as a replacement for fucose is likely attributable to its structural similarity to fucose ( Figure 2).
• the stereochemistry at the C-2 to C-4 position is exactly the same. Due to the close chemical similarity between these two sugars, it is apparent that the 5 carbon containing arabinose is able to be used as an alternative substrate for this addition reaction onto the N-glycan as that of the 6 carbon containing fucose.
• the capability of using arabinose in this regard is not immediately obvious, even more so when considering that the native fucose is unique and exists preferentially in the L-form before addition onto the N- glycan.
• Arabinose is more commonly found in nature in its L-form.
• the arabinose that was utilized in the present work was the D-form.
• Table 4 highlights that both D-arabinose and sucrose were very effective at eliciting changes in the protein oligosaccharide profile compared to the unsupplemented control.
• Arabinose had a similar impact as that which was reported above.
• Sucrose had a very significant impact on high mannose type N-glycans, and was evaluated as a control condition for high mannose to be tested alongside the low mannose condition elicited through the arabinose treatment..
• DVD-1 (ABT-482) was evaluated in shaker flasks in fedbatch operation mode.
• DVD-1 (ABT-482) is a TNF/IL-17-binding DVD-Ig having the following sequence:
• VCD profiles indicate that across the range of tested concentrations of 0.1 mM to 50 mM, the majority of the cultures supported peak VCD's either on par, on only nominally lower, with that of the unsupplemented control.
• the expressed DVD was harvested from each of the respective cultures when cell viability dropped below 70%. Only one of the D-arabinose supplemented cultures had a harvest date earlier than the unsupplemented control, with the higher supplemented conditions of 20 mM and 50 mM actually supporting a longer culture duration before the harvest criteria was triggered.
• CHO cell line 3 expressing mAb-2 (anti-CD20 mAb) was evaluated in shaker flasks in fed-batch operation mode.
• mAb-2 has the following sequence: mAb-2 (Light Chain)
• CHO cell line 4 expressing mAb-3 (MAK-199-AM1-QL mAb; see Table A) was evaluated in shaker flasks in fed-batch operation mode. Variable amounts of D- arabinose were supplemented into both the chemically-defined basal and feed medias. The cell culture performance results are highlighted in Figure 10. In this particular experiment only one concentration of D-arabinose was evaluated in the basal and feed media. The 30 mM D-arabinose supplemented cultures grow comparably to the unsupplemented control, with cell viabilities trending similarly up till harvest on Day 13. Titers measured from the harvests suggests a modest 15% reduction in the D-arabinose supplemented cultures.
• Viscosity Measurements Purified samples of mAb-1, DVD-1, and mAb-2 were subjected to viscosity measurements after concentrating all to 50 mg/mL in the same formulation buffer (15 mM histidine, pH 6.0). The results are shown in Table 9.
• Dendritic Cell Uptake Dendritic cells are potent and professional antigen presenting cells that have a very important role towards both the innate and adaptive immune responses. Uptake of antigens by dendritic cells is important for the activation of their contribution to immunogenic responses. A specific type of dendritic cell, called an inflammatory dendritic cell, has been shown to appear during inflammation (Segura and Amigorena, Trends Immunol. 34:440-445 (2013)). Thus, any antigen capable of enhancing or reducing dendritic cell uptake in vitro may be potentially correlated to the activation or attenuation of their immune response towards the antigen in vivo.
• this material reduce dendritic cell antigen display activity, including any associated attenuation of immunogenic and inflammatory responses.
• the results indicate that the utility of the present methods to control protein mannosylation levels provides for methods to tune the therapeutic profile, and by decreasing the respective levels, enables the manufacturing of a more efficacious therapeutic.
• FcyR FcR n Binding Therapeutic proteins have been documented to bind to antigens on cell surfaces and elicit their ability to kill target cells either through antibody dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and direct apoptosis. Fey receptors serve a variety of in vivo immune functions and are important for therapeutic proteins to elicit their desired effect. In particular, the ability to bind to FcyRIIIa receptors is important for therapeutic proteins to elicit ADCC activity. FcyRIIIa is the binding receptor on natural killer (NK) effector cells that antibodies bind to through their Fc region. Upon antigen binding these same antibodies are then able to bind the receptors on NK cells to elicit ADCC activity.
• NK natural killer
• mAb-1 is a neutralizing antibody that binds to its target antigen with strong affinity. Ordinarily mAb-1 is fucosylated, and in the 15 mM arabinose/70 mM sucrose experiment, it was found that the oligosaccharides were significantly impacted as highlighted above. To further characterize the functional impact of these oligosaccharide changes towards the antibody, antigen binding assays were performed via Biacore®. The results from this study are shown in Table 12.
• Cytokines are a broad class of proteins produced by immune cells, as well as non-immune cells such as fibroblasts. These proteins serve a great deal of functions, including humoral and cell-based immune response, and frequently modulate other target cells, or the cells that secrete the cytokines. Many cytokines are categorized as pro-inflammatory cytokines for their role associated with inflammation. Thus, monitoring the levels of these particular cytokines upon exposure to a specific compound provides an indication of the resulting levels of inflammatory responses associated with this exposure. In the present example, a series of inflammatory cytokines were monitored upon administration of various glycomodulated mAb-1 samples. The results using PBMC cells from 3 different donors are shown below in Table 13. Positive results were assigned when the measured cytokine levels exceeded two times (2X) the levels elicited with the negative control (Herceptin).
• Complement Activation The complement system is an important part of the innate immune system that is comprised of a series of proteins which get activated by a variety of triggers, including the classical, alternative, and lectin complement pathways. Upon activation, proteases in these pathways degrade pathway-specific proteins which leads to further molecular events which cascade and amplify leading to cell lysis or opsonization of foreign cells, as well as other immune defensive responses.
• Rodent Pharamacokinetics Rat PK studies were conducted to evaluate if there was any impact towards the in vivo clearance rate of either of the arabinosylated antibodies purified from the 15 mM arabinose supplemented cell culture samples. Clearance rates were compared to the antibody purified from the unsupplemented experimental control and the results are shown in Figure 13 and Table 14.
• ADCC Antibody Dependent Cellular Cytotoxicity
• mAb-2 is an oncology relevant recombinant protein that has ADCC as a principal component of its method of action. Since it was proven that mAb-2 was very responsive towards a high degree of arabinosylation, and this facilitated an equal amount of overall afucosylation, mAb-2 was purified and analyzed with a cell based ADCC assay following the procedure as outlined in Example 1 ( Figure 14). The results indicate a very similar behavior between both the higher affinity and lower affinity allele of FcyRIIIa evaluated as part of the assay. That is, with the exception of the lowest arabinose supplemented culture, the mAb-2 generated from the cultures supplemented with more than 0.1 mM arabinose increased ADCC activity in a very significant manner.
• mAb-1 also unexpectedly preserved the ability of the therapeutic antibody to activate complement in contrast to the well-known problem that removal of fucosyl residues compromises the ability of a therapeutic antibody to activate complement.
• mAb-3 binds T Fa and is an immunology relevant recombinant protein.
• the antibody was proven to be very responsive towards protein arabinosylation, and was thus purified and analyzed for CD 16 (FcyRIIIa) signaling (Figure 15).
• An elevated signal from the assay would indicate a higher binding towards CD 16, and would reinforce the previous results that glycoproteins whose mechanism of action relies on CD 16 binding (e.g., ADCC), could be enhanced through arabinosylation.
• Arabinosylated mAb-3 and predominantly fucosylated mAb-3 (control) were purified and compared alongside reference material from mAbl that was both predominantly fucosylated, as well as afucosylated through the addition of kifunensine, a small molecule which inhibits the Manl enzyme and facilitates high mannose N-glycans.
• arabinose monosaccharide as a cell culture media supplement for the targeted shifting of protein glycosylation profiles was demonstrated to provide for numerous changes towards the protein glycosylation profile of proteins, including recombinant proteins.
• Arabinose was shown to lead to a significant reduction in high mannose type N-glycans, an increase in GO type N-glycans, and either the complete or predominant removal of fucose sugars on N-glycans with arabinose being inserted in its place.
• purified recombinant proteins utilizing these glycomodulation tools provides for therapeutic benefits as described above.
• mAb-1 purified from arabinose supplemented cultures demonstrated a significant decrease in dendritic cell uptake, an increase in FcyRIIIa binding consistent with the mechanism for an increased ADCC response, as well as a comparable circulatory PK in a rat model.
• the demonstrated utility of this approach towards recombinant protein production, as well as therapeutic benefit associated thereof, enable the manufacturing of more efficacious and effective protein based biologies.
• Fucose is unique amongst the sugars that comprise a typical N-glycan in the sense that it is an L-sugar, whereas the others are D- sugars.
• D-arabinose was effective at eliciting changes in the protein glycosylation profile, including the replacement of L-fucose. Since arabinose is more typically found in nature in its L-form, the utility of the D- arabinose sugar in the present methods is unexpected.
• D-arabinose as a novel substrate for the FucT enzyme for addition onto N-glycans is also unexpected given the state of the art since protein glycosylation enzymes in mammalian cells are generally accepted to exhibit strong specificity for both the glycosyl donor and the protein acceptor substrates (Breton et al., Glycobiol. 16:29R-37R (2006)). The results described herein show that this is not necessarily always true when it comes to the enzymatic activity of the FucT enzyme, which is capable of recognizing its native 6-carbon fucose substrate, in addition to the smaller 5- carbon D-arabinose substrate.
• glycoproteins such as adalimumab
• the cell line used to express this aforementioned recombinant glycoprotein was a clonally derived Chinese Hamster Ovary (CHO) cell line of DUKX-B 11 origin, transfected with a proprietary plasmid vector encoding adalimumab (mAb-1) using standard promoters and selection systems such as dihydrofolate reductase (dhfr).
• Cryopreserved cells from a research cell bank of Cell Line 1 are thawed and expanded into increasingly larger volumes using sterile, plastic shaker flasks.
• Cell 1 was evaluated in shaker flasks in fed- batch operation mode.
• Variable amounts of D-arabinose were supplemented into both the chemically-defined basal and feed medias.
• Cell growth and antibody productivity were compared between these cultures with that of the unsupplemented control ( Figure 16). The results indicate that between the evaluated concentrations of 0.1 mM to 10 mM there was no adverse impact towards cell growth, or cell viability.
• Cell viability results between the D-arabinose supplemented cultures and the control indicate that cell viability was also not adversely impacted.
• the D-arabinose titration experiment also highlights that somewhere between 0.1 and 1.0 mM is the effective concentration at which arabinosylation begins to be observed. At higher concentrations than this, the N-glycans were fully arabinosylated, with no appreciable presence of fucose on any of the N-glycans.
• the media supplementation of manganese in addition to the 10 mM D-arabinose facilitated the largest overall increase of galactosylated N-glycans.
• Both the production bioreactor growth media and feed media are supplemented using manganese and D-arabinose to elicit the desired impact, and optimize the resulting oligosaccharide profile in the expressed protein.
• the means with which this oligosaccharide optimization can be accomplished included the supplementation of manganese levels in the range of 0 to 100 ⁇ , the supplementation of galactose levels in the range of 0 to 50 mM, and the supplementation of arabinose in the 0 to 50 mM range.
• the cumulative high mannose type N-glycans i.e., Man5, Man6, Man7, Man8, and Man9
• the levels of galactosylated N-glycans Gl A, G2A
• the levels of afucosylation can be increased to levels > 99%.

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