WO1996020012A2 - Procedes de preparation et de purification de conjugues macromoleculaires - Google Patents

Procedes de preparation et de purification de conjugues macromoleculaires Download PDF

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Publication number
WO1996020012A2
WO1996020012A2 PCT/US1995/016950 US9516950W WO9620012A2 WO 1996020012 A2 WO1996020012 A2 WO 1996020012A2 US 9516950 W US9516950 W US 9516950W WO 9620012 A2 WO9620012 A2 WO 9620012A2
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conjugate
polymers
particles
protein
polymer
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PCT/US1995/016950
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WO1996020012A3 (fr
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Jie Di
James E. Woiszwillo
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Middlesex Sciences, Inc.
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Priority claimed from US08/372,820 external-priority patent/US5554730A/en
Application filed by Middlesex Sciences, Inc. filed Critical Middlesex Sciences, Inc.
Publication of WO1996020012A2 publication Critical patent/WO1996020012A2/fr
Publication of WO1996020012A3 publication Critical patent/WO1996020012A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere

Definitions

  • This relates to the field of biochemistry and more particularly relates to the preparation and purification of macromolecular conjugates.
  • Haemophilus influenzae type b is the etiologic agent for other diseases such as epiglottitis, sepsis, cellulitis, septic arthritis, osteomyelitis, pericarditis, and pneumonia (MMWR 34:201-205 (1985)).
  • T- independent antigens Two types of lymphocytes, B lymphocytes and T lymphocytes, are normally involved in the development of immunity.
  • Some antigens are capable of stimulating B cells to produce antibody in the absence of T lymphocyte involvement.
  • most antigens require the participation of both B lymphocytes and T lymphocytes to produce an effective immune response, a process often referred to as cell mediated immunity, in which antigen- presenting cells present processed antigen to helper T cells, which secrete lymphokines that promote the production of antibodies by B cells and influence other cells of the immune system.
  • a T-dependent antigen is preferred for use as a vaccine because it results in an enhanced antibody response, the production of immunoglobulins of all classes, and the development of immunologic memory.
  • the covalent binding of antigens has been used to convert T-independent antigens into T-dependent antigens, thereby creating more effective vaccines.
  • Conjugation is achieved by oxidizing the polysaccharide with an oxidizing agent, coupling the oxidized polysaccharide to a protein, and reducing the bond with a reducing agent for stability. This process requires lengthy dialysis and incubation steps and chromatography purification and may take up to two to three weeks for completion.
  • a small, non-immunogenic peptide may be conjugated to a large, carrier protein, such as bovine serum albumin, to elicit an immune response to the small, non-immunogenic protein when the conjugate is administered to a human or animal.
  • a small detectable protein such as an enzyme may be coupled by conjugation to a protein, such as an antibody or receptor, for detection of a ligand by an enzymatic reaction that produces a detectable signal.
  • a non- protein chemical such as a fluorescent or radioactive compound or bioun may be coupled to a protein for use as a label to detect a ligand to which the protein has an affinity.
  • conjugates from the unconjugated reagents such as unconjugated protein
  • Methods are provided for preparing and purifying macromolecular conjugates through the formation of conjugate particles.
  • the methods are particularly useful for the preparation and purification of carbohydrate- protein conjugates from chemical reaction mixtures for administration as vaccines to young humans or animals.
  • Conjugate particles are formed in a first preferred embodiment by reacting a reaction mixture containing a molecule having a reactive amine group, a molecule having a reactive aldehyde group, and a polymer or mixture of polymers for a sufficient amount of time to form conjugate particles.
  • the mixture is incubated at temperatures between approximately 20° C and 65° C for a period of time between approximately 15 minutes and 5 hours at a pH near the pi of the resulting conjugate.
  • the components to be conjugated may be any molecules capable of being conjugated, and the reaction mixture further includes a conjugating agent.
  • pre ⁇ formed conjugates are reacted with the polymer to produce conjugate particles.
  • An organic solvent may be added to any of the foregoing reaction mixtures to facilitate particle formation.
  • the preferred organic solvent is an alcohol. The latter two embodiments are useful for purification of conjugates prepared by known conjugation reactions.
  • the molecule having a reactive aldehyde group is one that either contains a reactive aldehyde group or can be modified to expose or otherwise include a reactive aldehyde group.
  • the reactive aldehyde group is one having the ability to react with and couple to an amine group.
  • Such a molecule includes, but is not limited to, a carbohydrate, glycoprotein, peptide, protein, catecholamine, transmitter, hormone, receptor, label, nucleic acid or chemical.
  • the carbohydrate includes, but is not limited to, lipopolysaccharides, polysaccharides, and oligosaccharides derived from microorganisms such as bacteria, fungi, protozal groups, and virally associated lipopolysaccharides, polysaccharides and oligosaccharides.
  • the molecule having a reactive amine group is a molecule having a tertiary structure, preferably a protein or peptide, and includes, but is not limited to, toxoids; toxins; exotoxins; proteins derived from animals, plants, viruses, bacteria, fungi, and protozoa; recombinant proteins; and synthetic proteins.
  • the reactive amine group is one having the ability to react with and couple to an aldehyde group.
  • the polymers used to form the conjugate particles may be water soluble, semi-water soluble, or non-water soluble and can include the following categories of polymers: carbohydrate-based polymers, polyaliphatic alcohols, poly(vinyl) polymers, polyacrylic acids, polyorganic acids, polyamino acids, co-polymers and block co-polymers, tert-polymers, polyethers, naturally occurring polymers, polyimids, surfactants, polyesters, branched and cyclo-polymers, and polyaldehydes, and mixtures thereof
  • the polymer is preferably polyvinylpyrrolidone, polyethylene glycol, dextran, polyoxyethylene-polyoxypropylene copolymer, polyvinyl alcohol, or mixtures thereof.
  • the polymer is a polymer mixture comprising polyvinylpyrrolidone and polyethylene glycol.
  • the preferred molecular weight of the polyvinylpyrrolidone is approximately 40,000; the preferred molecular weight of the polyethylene glycol is approximately 3350; the preferred concentration of the polymer is between approximately 10 and 80%; and the polymer is preferably adjusted to a predetermined pH value near the pi of the resulting conjugate with a buffer.
  • the molecule containing a reactive aldehyde group is a polysaccharide
  • the polysaccharide is first oxidized with an oxidizing agent or hydrolyzed.
  • the oxidizing agent is preferably a glycol cleaving agent such as tetra-acetate, periodic acid, or sodium periodate. Unreacted oxidizing agent may be quenched with a quenching agent such as ethylene glycol.
  • a protein is then added to the oxidized polysaccharide, and the polysaccharide and protein are coupled in the presence of a polymer or mixture of polymers to form polysaccharide-protein microparticles containing stable conjugates that form through a Schiff base intermediate. Additional reactive aldehyde groups on the conjugate may be quenched with a quenching agent such as hydroxamine.
  • Microparticles formed by the methods described herein may be administered to humans and other animals as microparticles by methods well known to those skilled in the art. Alternatively, the microparticles are substantially dissolved, yielding a composition containing soluble conjugate. The microparticles may be dissolved using chemical means, PCIYUS95/16950
  • conjugate particles are formed as described above and are separated from the unreacted components of the conjugation reaction mixture by physical means, yielding purified conjugates.
  • conjugates are formed by methods known to those skilled in the art, the conjugates are formed into particles by reacting the conjugates with a polymer or mixture of polymers as described above, and the particles are separated from the unreacted components of the conjugation reaction mixture by physical means, yielding purified conjugates. Suitable separation means include filtration, centrifugation, decantation, aspiration, and sedimentation. The particles may be washed for removal of additional undesired contaminants. The particles may then be solubilized, yielding soluble conjugates, prior to use.
  • the conjugates are prepared and purified in two or more separate steps or the conjugates are simultaneously prepared and purified.
  • Conjugates are prepared by reacting the components to be conjugated or purified with a polymer or mixture of polymers to form conjugate particles. Conjugates form contemporaneously with the formation of the particles in the absence of reductive amination. Conjugates are purified by forming conjugate particles and separating the particles from non-reacted components. Conjugate particles are formed in the presence or absence of a conjugating reagent, depending on the nature of the components and the type of conjugation reaction employed. If desired, the conjugate particles may be dissolved chemically or physically to produce solubilized conjugates.
  • macromolecule is defined as a biological or chemical molecule or macromolecule, which has been chemically conjugated to one or more biological or chemical molecules or macromolecules.
  • the components of the conjugate may be molecules of the same type, such as protein-protein conjugates, or molecules of different types, such as carbohydrate-protein conjugates.
  • the biological or chemical molecules or macromolecules used as components of the conjugates may be, but are not limited to, proteins, peptides, carbohydrates, glycoproteins, catecholamines, transmitters, hormones, receptors, labels, nucleic acids and chemicals.
  • microparticles as used herein is interchangeable with the terms “microparticles”, “microspheres”, and “microcapsules”, and refers to solid or semi-solid particles having a diameter of less than one millimeter, more preferably less than 100 microns, and most preferably 10 microns or less.
  • Schiff base is defined herein as any of a class of derivatives of the condensation of aldehydes with primary amines. Schiff bases are also referred to as imines. A Schiff base intermediate is a reversible condensation product of an aldehyde with an amine.
  • polymer as used herein includes a single polymer or a mixture of two or more polymers. It will be understood by those skilled in the art that a mixture of polymers may be premixed and added to the reaction mixture or the polymers may be added to the reaction mixture sequentially. Conjugate Particle Formation
  • Conjugate particles are formed by reacting the components to be conjugated with a polymer.
  • a reaction mixture containing a molecule having a reactive amine group, a molecule having a reactive aldehyde group, and a polymer or mixture of polymers is reacted for a sufficient amount of time to form conjugate particles.
  • the components to be conjugated may be any molecules having the ability to be conjugated and are reacted with the polymer in the presence of a conjugating agent or agents to form conjugate particles.
  • pre-formed conjugates are reacted with the polymer to form conjugate particles. The latter two embodiments will be as described in more detail below with regard to conjugate purification.
  • the reaction mixture is incubated at one or more temperatures between approximately 20° C and 65° C for a period of time between approximately 15 minutes and 5 hours at a pH near the pi of the resulting conjugate, preferably within one or two pH units of the pi of the conjugate.
  • the reaction components are incubated at a temperature between room temperature and 58 °C or at a series of different incubation temperatures within this range for a sufficient amount of time to allow formation of particles.
  • the most preferred length of incubation time is between 30 minutes and 2 hours.
  • the particles are then separated from the non-reacted components of the reaction mixture and may be solubilized using either chemical or physical means, yielding soluble conjugate as described in more detail below.
  • An organic solvent may be added to the reaction mixture to facilitate particle formation.
  • the preferred organic solvent is an alcohol, such as ethanol, isopropanol, or methanol. Ethanol is the preferred alcohol.
  • a molecule having a reactive aldehyde group is defined herein as a molecule that either contains a reactive aldehyde group or can be modified to expose or otherwise include a reactive aldehyde group, such as by hydrolysis.
  • a reactive aldehyde group is defined herein as an aldehyde group having the ability to react with and couple to an amine.
  • a molecule having a reactive amine group is defined herein as a molecule that either contains a reactive amine group or can be modified to expose or otherwise include a reactive amine group.
  • a reactive amine group is defined herein as an amine group having the ability to react with and couple to an aldehyde.
  • the preferred molecule having a reactive aldehyde group is a polysaccharide that has been activated with a specified oxidizing agent or by hydrolysis, and the preferred molecule having a reactive amine group is a protein.
  • polysaccharide- protein conjugate particles are formed.
  • the preferred conjugates are both stable and immunogenic and are particularly useful as vaccines. Particles that are collected and washed prior to solubilization produce a purified conjugate vaccine preparation with excellent recovery of polysaccharide.
  • Each particle includes a non-resilient outer surface encapsulating an inner matrix.
  • Both the outer surface and the inner matrix are composed of homogeneously distributed conjugated macromolecules and polymer molecules, which are intertwined.
  • the inner matrix is water soluble, and the solubilized inner matrix diffuses through the outer surface.
  • the particles have a generally uniform size and shape which, under most conditions, is between approximately 1 and 10 ⁇ m in diameter and is spherical.
  • the outer surface of the particle is a hard, rigid, or non-resilient, solid material, most likely composed of denatured or semi-denatured macromolecules or conjugated macromolecules that resist solubilization, while the interior of the microparticle is more readily dissolved by aqueous solutions.
  • the outer surface is penetrable and not only allows aqueous fluids to enter the interior of the particle, but also allows solubilized conjugate and polymer to exit the microparticle, resulting in a slow, sustained release of conjugate and polymer from the interior of the particle when placed in an appropriate aqueous medium.
  • the conjugates may be administered to a human or animal as particles, thereby providing a sustained or delayed release of the conjugate, or the particles may be solubilized prior to administration, yielding soluble conjugates.
  • a preparation containing a mixture of conjugate particles and solubilized conjugate may be formulated, resulting in both an immediate and a delayed or sustained release of conjugate.
  • the conjugate is a conjugate vaccine administered to a human or animal
  • the mixture of solubilized conjugate and conjugate particles is one that preferably contains a sufficient amount of soluble antigen to prime an immune response and contains a sufficient amount of slow-releasing antigenic particles to subsequently boost the immune response in a single administration.
  • the conjugate particles are substantially dissolved with an acid or a base or in a solubilizing solution, such as an acidic or alkaline solution, to produce a soluble conjugate.
  • the conjugate may then be diluted with a physiologically acceptable buffer. If the pi of the conjugate is less than pH 7, then the conjugate particles are dissolved with a base or basic solution.
  • the preferred base is sodium hydroxide.
  • the conjugate particles are dissolved with an acid or acid solution.
  • the preferred acid is hydrochloric acid.
  • the pH of the dissolving solution for each conjugate particle preparation may be determined by titration, using techniques well known to those skilled in the art.
  • the solubilizing solution is preferably dilute or at a minimum concentration, thereby reducing the amount of denaturation or destruction of the conjugate or the components of which the conjugate is made.
  • particles may be dissolved by physical methods such as sonication.
  • conjugation method described herein is superior to methods currently employed, such as reductive amination, in that conjugation is achieved with a shorter production time, resulting in a higher yield of a substantially pure conjugate. Production is therefore significantly less expensive.
  • this conjugate preparation method is particularly useful for the preparation of conjugates from macromolecules unable to withstand the harsh conditions of conventional conjugation procedures.
  • the conjugate purification method described herein is superior to known conjugate purification processes, such as gel filtration, ultrafiltration, ion exchange chromatography or mixtures thereof, in that the reagents are less expensive and the purification procedure is much more rapid, taking only two to three hours rather than three to four days.
  • the purification method described herein also permits purification of larger samples and rapid scale-up because it is not limited by column capacity.
  • the purification method is particularly useful for the preparation, separation and isolation of carbohydrate-protein conjugates from free carbohydrates, especially when the carbohydrate-protein conjugate has a molecular weight very similar to the molecular weight of the carbohydrate prior to conjugation to the protein.
  • the conjugates are particularly useful as vaccines for administration to humans or animals, particularly infants, in which one or both of the unconjugated components have limited immunogenicity.
  • the conjugates are additionally useful for in vitro and in vivo research and for diagnostic procedures wherein a macromolecule is labeled with a detectable label, such as fluorescein or a radiolabel, and the labeled conjugate is added to a biological sample or cell culture for the analysis of macromolecular structure and function.
  • the conjugate particles are formed by a "macromolecular crowding" or "volume exclusion” effect in which the polymers strip water away from macromolecules in the solution and force these molecules together.
  • This crowding lowers the diffusion coefficient and increases the rates and degree of association of these macromolecules.
  • Covalent bonds are then formed between different reactive components, such as the formation of a stabilized Schiff base linkage between the activated aldehyde on the polysaccharide and amino lysine residues on the protein during the formation of a polysaccharide-protein conjugate.
  • the polymer, or mixture of polymers therefore act as a macromolecular crowding agent, which is defined herein as a compound that attracts water and allows molecules to aggregate.
  • the polymer appears to induce formation of multiple Schiff bases, producing a stable conjugate that precipitates as microparticles during phase separation.
  • the conjugates are stable and appear to remain covalently coupled. This stability is evidenced by the fact that solubilized conjugates induce a T- dependent immune response in an animal that could result only from a polysaccharide-protein conjugate and not from the individual, unconjugated components.
  • conjugate particles are formed, as described above and in more detail below, and the particles separated from the conjugation reaction mixture, which includes the unconjugated components, by conventional means well known to those skilled in the art yielding purified conjugates.
  • the conjugate should have a pi that is different from the pi of the free components, particularly the protein component of a carbohydrate-protein conjugate, thereby facilitating separation.
  • Suitable separation means include, but are not limited to, physical separation techniques such as filtration, centrifugation, decantation, aspiration, and sedimentation.
  • the particles may be washed with a conventional solvent, such as water or a buffer, after physical separation from the majority of the unreacted components for removal of additional undesired contaminants.
  • a reaction mixture containing the components to be conjugated is reacted with a polymer or mixture of polymers for a sufficient amount of time to form conjugate particles.
  • the components are conjugated and formed into particles either simultaneously or immediately thereafter because the polymer is present as a component of the conjugation reaction mixture.
  • the components may be a molecule containing a reactive aldehyde group and a molecule containing a reactive amine group as described above, or the components may be other molecules capable of being conjugated with a conjugating agent in accordance with methods known to those skilled in the art.
  • a molecule containing the reactive aldehyde group such as a carbohydrate
  • it is provided in excess so that all of the molecule containing the reactive amine group in the reaction mixture is conjugated.
  • pre ⁇ formed conjugates which have been produced by the methods described herein or by methods known to those skilled in the art, are incubated with a polymer, or mixture of polymers, for a sufficient amount of time to form conjugate particles.
  • the polymer and conjugates or conjugation reaction mixture is preferably incubated at one or more temperatures between approximately 20° C and 65° C for a period of time between approximately 15 minutes and 5 hours.
  • conjugation reactions utilized in the preparation of conjugates that are then purified in accordance with the methods described herein are well known to those skilled in the art and include, but are not limited to, diazo-coupling; triazine bridge; thioether bond; amidation; isourea bond; aldimines; thiocarbamoyl formation; reductive amination reactions, such as described in U.S. Patent No. 4,356,170 to Jennings et al., which is incorporated by reference herein; carbodiimide reactions; glutaraldehyde condensation reactions and other reactions including bisdiazotized benzidine; bisdiazotized phenylacetic acid; thioether reactions as described by Marburg et al.
  • the conjugating agents useful for producing conjugates by the conjugation reactions described above utilize or modify naturally-occurring functional groups such as carboxyl, hemiacetal, amino/imino, mercapto/disulfide, hydroxyl, and phenoxyl moieties.
  • Exemplary conjugating agents include, but are not limited to, bromide, thiol, hydrazide, cyanate, carboxyl, aldehydes, esters, boranes, and amines; more specifically cyanoborohydride, diazonium, triazine, isothiocyanate, aldonic acid, adipic acid dihydrazide, pyridyldisulfide, carbodiimide, l-ethyl-3-(3- dimethylaminopropyl-carbodiimide hydrochloride (EDC), hydroxysuccinimide (NHS), sulfonated hydroxysuccinimide (sulfo-NHS), glutaraldehyde, pyridine borane, methyl borane, carbonyldiimidazoles, and derivatives thereof.
  • Conjugating regents are commercially available from chemical companies such as the Sigma Chemical Company (St. Louis, MO). Examples of these reagents and typical coupling reactions are described by Cruse, J.M and Lewis, R.E., Jr. (eds.), Conjugate Vaccines. Contrib. Microbiol. Immunol. 10:48-114, Basel, Karger (1989), which is incorporated by reference herein. It is understood that other conjugating agents and methods not specifically mentioned herein, which are known to one of ordinary skill in the art, may all be used in the practice of this method.
  • Linkers may improve conjugation efficiencies, promote improved antigenicity for the conjugated components, physically separate the conjugated components, and serve as "spacers" to permit increases in translational and rotational characteristics of the components, thereby increasing access of binding sites to soluble ligands.
  • the purification method described herein will separate the conjugates from various coupling reagents including: chemical conjugating agents, chemical cross-linkers, such as heterobifunctional and homobifunctional cross-linkers including activated halogens, maleimides, pyridyl disulfides, and succinimidyls; and photoactivatable cross-linkers such as aryl azides.
  • conjugate particles are separated from the other reagents in the incubation mixture by conventional methods well known to those skilled in the art such as centrifugation, filtration or decantation in combination with established washing procedures.
  • the resulting purified conjugate particles may then be resuspended in a physiologically-acceptable buffer such as a saline solution or phosphate buffered saline.
  • the purified conjugates may be administered as microparticles.
  • the purified particles may be dissolved with solubilizing solvent having a predetermined pH as described above.
  • solubilizing solvent having a predetermined pH as described above.
  • Polymer Characteristics Various polymers may be utilized to form the macromolecular conjugate particles.
  • polymers may be water soluble, semi-water soluble, or non-water soluble and can include the following categories of polymers: 1) carbohydrate-based polymers, such as methylcellulose, carboxymethyl cellulose-based polymers, dextran, polydextrose, chitins, chitosan, and starch, and derivatives thereof; 2) polyaliphatic alcohols such as polyethylene oxide and derivatives thereof including polyethylene glycol (PEG), PEG-acrylates, polyethyleneimine, polyvinyl acetate, and derivatives thereof; 3) poly(vinyl) polymers such as poly(vinyl) alcohol, poly(vinyl)pyrrolidone, poly(vinyl)phosphate, poly(vinyl)phosphonic acid, and derivatives thereof; 4) polyacrylic acids and derivatives thereof; 5) polyorganic acids, such as polymaleic acid, and derivatives thereof; 6) polyamino acids, such as polylysine, and polyimino acids, such as polyimino tyrosine,
  • the polymer utilized to form the conjugate particles is preferably polyvinylpyrrolidone, polyethylene glycol, dextran, polyoxyethylene- polyoxypropylene copolymer, polyvinyl alcohol, or mixtures thereof, the characteristics of which are described in more detail below.
  • the polymer or polymer mixture may be prepared in accordance with the methods set forth in co-pending U.S. Patent Application Serial No. 07/817,610 filed January 7, 1992 by James E. Woiszwillo, or PCT Patent Application No. US93-00073, filed January 7, 1993 by James E.
  • the polymer is dissolved in water or an aqueous solution, such as a buffer, in a concentration between approximately 1 and 50 g/100 ml depending on the molecular weight of the polymer.
  • the preferred total polymer concentration in the polymer solution is between 10% and 80%, expressed as weight weight %.
  • the preferred concentration of each polymer in the polymer solution is between 20% and 30%
  • the preferred pH of the polymer solution depends on the pi of the molecule being conjugated or the final conjugate, preferably within one or two pH units of the pi. For most molecules, the pH of the polymer solution will be approximately between pH 4 and pH 7, most preferably pH 5.
  • the pH may be adjusted during the preparation of the polymer solution by preparing the polymer in a buffer having a predetermined pH. Alternatively, the pH may be adjusted after preparation of the polymer solution with an acid or a base.
  • Polyoxyethylene-polyoxypropylene copolymer also known as poloxamer, is sold by BASF (Parsippany, New Jersey) and is available in a variety of forms with different relative percentages of polyoxyethylene and polyoxypropylene within the copolymer.
  • PVP is a non-ionogenic, hydrophilic polymer having a mean molecular weight ranging from approximately 10,000 to 700,000 and the chemical formula (C6H9NO)n- PVP is also known as poly[l-(2-oxo-l- pyrrolidinyl)ethylene], PovidoneTM, PolyvidoneTM, RP 143TM, KollidonTM, Peregal STTM, PeristonTM, PlasdoneTM, PlasmosanTM, ProtagentTM, SubtosanTM, and VinisilTM.
  • PVP is non-toxic, highly hygroscopic and readily dissolves in water or organic solvents.
  • Polyethylene glycol also known as poly(oxyethylene) glycol, is a condensation polymer of ethylene oxide and water having the general chemical formula HO(CH2CH2 ⁇ ) n H.
  • Dextran is a term applied to polysaccharides produced by bacteria growing on a sucrose substrate.
  • Native dextrans produced by bacteria such as Leuconostoc mesenteroides and Lactobacteria dextranicum usually have a high molecular weight.
  • Polyvinyl alcohol (PVA) is a polymer prepared from polyvinyl acetates by replacement of the acetate groups with hydroxyl groups and has the formula (CH2CHOH) n . Most polyvinyl alcohols are soluble in water.
  • the polymer is a polymer mixture containing a solution of polyvinylpyrrolidone and polyethylene glycol.
  • the polyvinylpyrrolidone has a molecular weight between approximately 10,000 and 360,000, most preferably 40,000.
  • the polyethylene glycol has a molecular weight between approximately 2,000 and 8,000, most preferably 3,350.
  • 25 g/100 ml of a 40,000 MW solution of polyvinylpyrrolidone (PVP) is mixed with a 25 g/100 ml of a 3,350 MW solution of polyethylene glycol (PEG) to form a PVP/PEG polymer mixture.
  • PVP polyvinylpyrrolidone
  • Equal concentrations of PVP and PEG generally provide the most favorable polymer mixture for the formation of a polysaccharide-protein conjugate.
  • the volume of polymer added to the polysaccharide varies depending on the sizes and quantities of the polysaccharide and protein. Preferably, approximately three volumes of the polymer mixture are added to one volume of a solution containing the polysaccharide and protein.
  • the pH of the macromolecular crowding agent is preferably between 4 and 9, most preferably pH 5.
  • the preferred molecule containing a reactive amine group is a peptide or protein having a tertiary structure or a molecule that can be coupled or coordinated with other molecules to create a molecule having a tertiary structure.
  • the protein portion of the such a conjugate may be any type of protein that can be conjugated and specifically includes, but is not limited to, proteins derived from animals, plants, viruses, bacteria, fungi, and protozoa; including toxoids, toxins, exotoxins, antibodies, both monoclonal and polyclonal, receptor proteins, protein hormones, transporter proteins, such as hemoglobin, myoglobin and transferrin, enzymes, repressor proteins, stimulator proteins, growth factor proteins, structural proteins, such as muscle proteins, collagen, elastin, proteoglycans and fibronectin, recombinant proteins, and synthetic proteins.
  • the molecule to which the protein is conjugated can be any molecule that will chemically conjugate to the protein in the presence of one or more conjugating reagents, including a second protein, a carbohydrate, glycoprotein, peptide, catecholamine, transmitter, hormone, receptor, label or chemical.
  • Functional groups at which a peptide or protein may be conjugated include, but are not limited to, amino, imino, amide, phenolic, carboxyl, imidazo, phenyl, guanidino, indolyl, sulfhydral, thiol, alcohols, and hydroxyl.
  • coupling reagents for protein-protein and peptide- protein conjugates may be selected from the following list, which is not limiting: dialdehydes such as glutaraldehyde and formaldehyde, carbodiimide, bisdiazotized benzidine, bisdiazotized phenylacetic acid, toluene and diisocyanate.
  • the preferred molecule containing a reactive aldehyde group is a carbohydrate.
  • the carbohydrate portion of the carbohydrate-protein conjugate includes lipopolysaccharides, polysaccharides and oligosaccharides derived from organisms, such as, but not limited to, bacterial groups including Micrococcaceae, particularly the genera Sarcina, Micrococcus and Staphylococcus; Streptococcaceae, particularly the genera Streptococcaceae more particularly S.
  • pneumoniae and other pneumococci and Leuconostoc and more particularly the organisms classified as beta-hemolytic streptococci and Group B streptococci; Lactobacillaceae, particularly the genera Lactobacillus; Propionibacteriaceae, particularly the genera Lactobacillus, Propionibacterium, Corynebacterium, Listeria, and Erysipelothrix, Bacillaceae, particularly the genera Bacillus and Clostridium; Neisseriaceae, particularly the genera Neisseria, more particularly the organisms N. meningitidis and N.
  • gonorrhoeae and other meningococci Brucellaceae, particularly the genera Brucella, Bordetella, Pasteur ella, Hemophilus, more particularly the organisms H. influenzae and H. influenzae type b; Escherichia, more particularly the organism E. coli; Erwinia, Shigella, Salmonella, Proteus, Yersinia, Enterobacter, Klebsiellae, more particularly the organism K. pneumoniae, and Serratia; Pseudomonadaceae, particularly the genera Pseudomonas, more particularly P.
  • Spirillaceae particularly the genera Photobacterium, Zymomonas, Aeromonas, Vibrio, more particularly the organism, Vibrio cholerae, Desulfovibrio, and Spirillum
  • the order Actinomycetales particularly the genera, Mycobacterium, more particularly the organisms M. tuberculosis, M. leprae, M. kansasii, M.
  • carbohydrate portion of a carbohydrate-protein conjugate includes lipopolysaccharides, polysaccharides and oligosaccharides derived from organisms such as, but not limited to, fungal groups, represented by the following organisms, Cryptococcus, particularly Crytococcus neoformans, Coccidiodes immi ⁇ s, Histoplasma capsulatum, Blastomyces dermatitidis, Candida, particularly C. albicans, Aspergillus, particularly A. fumigatus, Phycomycetes, particularly Mucor and Rhizopus, Sporothrix schenckii, Microsporum species, Trichophyton species, and Epidermophyton floccosum.
  • organisms such as, but not limited to, fungal groups, represented by the following organisms, Cryptococcus, particularly Crytococcus neoformans, Coccidiodes immi ⁇ s, Histoplasma capsulatum, Blastomyces dermatitidis,
  • the carbohydrate portion of the carbohydrate-protein conjugate includes lipopolysaccharides, polysaccharides and oligosaccharides derived from organisms such as, but not limited to, protozal groups and virally associated lipopolysaccharides, polysaccharides and oligosaccharides.
  • Suitable polysaccharides include, but are not limited to, Pn23F, a pneumococcal type 23 F polysaccharide and Pnl4, a pneumococcal type 14 polysaccharide, both available from the American Type Culture Collection (ATCC, Rockville, MD); and the polyribosylribitol phosphate (PRP) of Haemophilus influenzae type b (Hib).
  • Additional polysaccharides include pneumococcal type 9V polysaccharide (Pn9V), pneumococcal type 4 polysaccharide (Pn4), pneumococcal type 18F polysaccharide (Pnl8F) and pneumococcal type 18C polysaccharide (Pnl8C).
  • the protein portion includes, but is not limited to, tetanus toxoid, diphtheria toxoid, Neisseria meningitidis outer membrane protein, nontoxic cross-reacting mutant of diphtheria toxin, proteins derived from bacteria, exotoxins, and recombinant derivatives thereof, and synthetic proteins, preferably those containing lysine residues.
  • the cross-reactive material or "CRM" protein of diphtheria toxin specifically the CRM 197 protein
  • CRM 197 is a native mutant form of the diphtheria toxin.
  • CRM197 is non-toxic due to a single amino acid change in the fragment A of the molecule.
  • CRM 197 is commercially available from commercial sources such as Sigma Chemical Co. (St. Louis, MO).
  • the conjugate formed by the method is a polysaccharide-protein conjugate.
  • the polysaccharide of the polysaccharide-protein conjugate is one that includes an oxidizable terminal aldehyde group capable of reacting with an amino group of a protein to form a Schiff base intermediate.
  • the polysaccharide is one onto which an aldehyde group can be introduced, preferably by hydrolysis.
  • the polysaccharide is preferably a bacterial antigen capable of inducing an immune response when coupled to a protein carrier.
  • Useful polysaccharides include, but are not limited to, those derived from Haemophilus influenza, pneumococci, meningococci, ⁇ - hemolytic streptococci, Escherichia coli, Pseudomonas aeruginosa, Klebsiella, and Vibrio cholerae. Most preferably, the polysaccharide is one that is incapable of inducing an effective immune response in infants when administered alone, but produces a T lymphocyte-dependent immune response when coupled to a protein carrier and administered to infants.
  • the protein may be any physiologically tolerated protein having a free amino group.
  • Preferred proteins include, but are not limited to, tetanus toxoid, diphtheria toxoid, Neisseria meningitidis outer membrane protein, nontoxic cross-reacting mutant of diphtheria toxin, a protein derived from bacteria, or a synthetic protein containing lysine residues.
  • the protein may be derived from the same source as the polysaccharide.
  • the polysaccharide is oxidized by incubating the polysaccharide with an oxidizing agent.
  • the incubation is performed at room temperature for a sufficient amount of time to cause oxidation, most preferably between 15 and 45 minutes.
  • the oxidizing agent may be any glycol cleaving agent capable of introducing an aldehyde.
  • the glycol cleaving agent is an oxidizing agent such as lead tetra- acetate, periodic acid, or sodium periodate.
  • the glycol cleaving agent is sodium periodate.
  • the glycol cleaving agent is then quenched by the addition of a quenching reagent.
  • the quenching reagent is an alcohol containing a vicinal-hydroxyl group such as ethylene glycol.
  • the protein is added to the oxidized polysaccharide reaction mixture and incubated in the presence of a polymer or mixture of polymers.
  • the polymer may be added to the polysaccharide either before or after the addition of the protein.
  • a frequently utilized conjugation procedure involves the labeling of a protein with a detectable biomolecule or chemical for purposes such as in vivo and in vitro diagnostics and laboratory research.
  • Various types of labels and methods of conjugating the labels to the proteins are well known to those skilled in the art.
  • the labels are particularly useful when conjugated to a protein such as an antibody or receptor.
  • the protein can be conjugated to a radiolabel such as, but not restricted to, 32 P, 3 H, 1 C, 35 S, 125 I, or 131 I.
  • Detection of a label can be by methods such as scintillation counting, gamma ray spectrometry or autoradiography.
  • Bioluminescent labels such as derivatives of firefly luciferin, are also useful.
  • the bioluminescent substance is covalently bound to the protein by conventional methods, and the labeled protein is detected when an enzyme, such as luciferase, catalyzes a reaction with ATP causing the bioluminescent molecule to emit photons of light.
  • Fluorogens may also be used to label proteins.
  • fluorogens include fluorescein and derivatives, phycoerythrin, allo-phycocyanin, phycocyanin, rhodamine, and Texas Red.
  • the fluorogens are generally detected by a fluorescence detector.
  • the protein can alternatively be labeled with a chromogen to provide an enzyme or affinity label.
  • the protein can be biotinylated so that it can be utilized in a biotin-avidin reaction, which may also be coupled to a label such as an enzyme or fluorogen.
  • the protein can be labeled with peroxidase, alkaline phosphatase or other enzymes giving a chromogenic or fluorogenic reaction upon addition of substrate.
  • Additives such as 5-amino-2,3-dihydro-l,4-phthalazinedione (also known as LuminolTM) (Sigma Chemical Company, St.
  • rate enhancers such as p-hydroxybiphenyl (also known as p-phenylphenol) (Sigma Chemical Company, St. Louis, MO) can be used to amplify enzymes such as horseradish peroxidase through a luminescent reaction; and luminogeneic or fluorogenic dioxetane derivatives of enzyme substrates can also be used.
  • enzymes such as horseradish peroxidase through a luminescent reaction
  • luminogeneic or fluorogenic dioxetane derivatives of enzyme substrates can also be used.
  • Such labels can be detected using enzyme-linked immunoassays (ELISA) or by detecting a color change with the aid of a spectrophotometer.
  • proteins may be labeled with colloidal gold for use in immunoelectron microscopy in accordance with methods well known to those skilled in the art.
  • the location of a ligand in cells can be determined by labeling an antibody as described above and detecting the label in accordance with methods well known to those skilled in the art, such as immunofluorescence microscopy using procedures such as those described by Warren and Nelson, Mol. Cell. Biol. 1: 1326-1337 (1987).
  • Formulations Macromolecular conjugates, prepared or purified by the methods described herein, which are useful for administration as vaccines, can be formulated and packaged, alone or in combination with other antigens, using methods and materials known to those skilled in the art for vaccines.
  • a carbohydrate-protein conjugate prepared or purified as described herein is preferably added to the composite vaccine normally administered to infants.
  • the carbohydrate-protein conjugate may be administered with an adjuvant in an amount effective to enhance the immunogenic response against the conjugate.
  • an adjuvant approved for use in humans has been alum (aluminum phosphate or aluminum hydroxide).
  • chemically defined preparations such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates, such as those described by Goodman-Snitkoff et al. J. Immunol.
  • encapsulation of the conjugate within a proteoliposome as described by Miller et al., J. Exp. Med. 176:1739-1744 (1992) and incorporated by reference herein, and encapsulation of the protein in lipid vesicles, such as NovasomeTM lipid vesicles (Micro Vescular Systems, Inc., Nashua, NH) may also be useful.
  • lipid vesicles such as NovasomeTM lipid vesicles (Micro Vescular Systems, Inc., Nashua, NH) may also be useful.
  • the conjugate may be packaged as a vaccine in a single dosage for immunization by parenteral (i. e., intramuscular, intradermal or subcutaneous) administration or nasopharyngeal (i.e., intranasal) administration.
  • parenteral i. e., intramuscular, intradermal or subcutaneous
  • nasopharyngeal i.e., intranasal
  • the vaccine may confer immunity upon a single administration or upon multiple administrations including a primary administration and one or more subsequent administrations or boosts.
  • the conjugate is most preferably injected intramuscularly into the deltoid muscle.
  • the conjugate is preferably combined with a pharmaceutically acceptable carrier to facilitate administration.
  • the preferred carrier is water or a buffered saline, with or without a preservative.
  • the antigen may be lyophilized for resuspension at the time of administration or in solution.
  • the carrier may also be a polymeric delayed release system.
  • Synthetic polymers are particularly useful in the formulation of a vaccine to effect the controlled release of antigens.
  • the polymerization of methyl methacrylate into spheres having diameters less than one micron has been reported by Kreuter, J., MICROCAPSULES AND NANOPARTICLES IN MEDICINE AND PHARMACOLOGY, M. Donbrow (Ed). CRC Press, p. 125-148.
  • Microencapsulation of the conjugate or conjugate particle will provide a controlled release. A number of factors contribute to the selection of a particular polymer for microencapsulation.
  • the preferred dose for a human infant of average weight is a 0.5 ml injection containing a carbohydrate concentration of between 5 and 25 ⁇ g of the carbohydrate-protein conjugate. Based on this range, equivalent dosages for heavier body weights can be determined. It is possible that, due to the increased purity of the conjugate contained in the vaccine, a smaller dose may be administered without sacrificing immunogenicity. It will be understood by those skilled in the art that a conjugate vaccine may additionally contain stabilizers such as thimerosal (ethyl(2- mercaptobenzoato-S)mercury sodium salt) (Sigma Chemical Company, St. Louis, MO) or physiologically acceptable preservatives.
  • stabilizers such as thimerosal (ethyl(2- mercaptobenzoato-S)mercury sodium salt) (Sigma Chemical Company, St. Louis, MO) or physiologically acceptable preservatives.
  • Example 1 Preparation of Haemophilus Influenza Type b Polysaccharide-Tetanus Conjugate This experiment describes the preparation of Haemophilus influenzae type b-tetanus toxoid conjugates in the absence of reductive amination by forming Schiff base intermediate conjugate particles using a mixture of polymers. The effects of the pH of the polymer solution on conjugate recovery and ratio of the conjugated components, immunogenicity of both the conjugate particles and solubilized conjugate, and the effects of solubilizing base concentration on immunogenicity were analyzed.
  • PRP polyribosylribitol phosphate
  • the reaction mixture was incubated either a) at room temperature for 30 minutes; b) at room temperature for 30 minutes and at 37"C for 30 minutes; or c) at room temperature for 30 minutes, at 37 ⁇ C for 30 minutes, and at 58°C for 30 minutes.
  • Microparticles formed.
  • the mixture was centrifuged at 13,200 rpm for 20 minutes to pellet the microparticles.
  • the supernatant was decanted and the microparticles washed twice with 1.0 ml of 25% ethanol and 1.0 ml of deionized water.
  • the resulting Haemophilus influenzae type b-tetanus toxoid (Hib-TT or PRP-TT) conjugate microparticles were injected into mice as described below.
  • microparticles were suspended in 1.0 ml of deionized water, substantially dissolved in 0.1 N NaOH, and diluted in phosphate buffer to produce a soluble Schiff base intermediate Haemophilus influenzae type b-tetanus toxoid (Hib-TT) conjugate and injected as follows.
  • the conjugates were analyzed for Hib polysaccharide content using the Orcinol assay and for protein content using the BCATM protein assay (Pierce, Rockford, IL).
  • the Orcinol assay is specific for pentose type carbohydrates.
  • Conjugate particles were prepared according to the protocol set forth above with the polymer solution containing sodium acetate buffer, pH 5.0, and the reactants incubated at room temperature for 30 minutes, at 37 * C for 30 minutes, and at 58°C for 30 minutes. Conjugates were also prepared with a polymer solution containing sodium acetate buffer, pH 4.0. The percent recovery and ratio of polysaccharide to protein in the conjugate particles are set forth in Table 1 below.
  • Conjugate particles were prepared as described in the general protocol set forth above.
  • Sample 49-71 was incubated at room temperature for 30 minutes.
  • Sample 49-72 was incubated at room temperature for 30 minutes and at 37°C for 30 minutes.
  • Sample 49-73 was incubated at room temperature for 30 minutes, at 37°C for 30 minutes, and at 58°C for 30 minutes.
  • Samples C29 and C30 were prepared in the absence of sodium periodate.
  • Sample C29 was prepared in the presence of glutaraldehyde.
  • the controls contained two different doses of a Hib-TT conjugate prepared using the conjugating reagent l-ethyl-3-(3-dimethylaminopropyl- carbod ⁇ mide hydrochloride (a conjugation method known to those skilled in the art as the "EDC” or "EDAC” reaction).
  • the PRP+TT sample was prepared in the absence of polymer. The samples were injected into mice as described above.
  • the immunogenicity of the particles was determined by measuring the geometric mean concentration (GMC) of IgG antibodies to PRP and TT in mouse blood samples taken two weeks after administration of the boost dose (secondary injection). The results are set forth in Table 2 below.
  • the PRP-TT particles prepared above as Sample 49-72 were dissolved in 0.1 N NaOH and neutralized with phosphate buffer (15 mM) to produce solubilized PRP-TT conjugate.
  • the solubilized conjugate was injected into groups of ten mice as described above in accordance with the general protocol.
  • the immunogenicity of the solubilized conjugate is set forth in Table 3 below.
  • PRP-TT conjugate particles prepared as described above, using incubations at room temperature for 30 minutes, at 37°C for 30 minutes, and at 58°C for 30 minutes, were dissolved with different concentrations of sodium hydroxide ranging from 0.2 N to 0.001 N and injected into groups of ten mice as described above.
  • the immunogenicity results are shown below in Table 4.
  • the Pn6B polysaccharide is a pneumococcal type 6 B polysaccharide, available from the American Type Culture Collection (ATCC, Rockville, MD).
  • Pn6B-TT conjugates were prepared in accordance with the general procedure set forth in Example 1 above for the preparation of PRP-TT conjugates.
  • a 1 : 1 ratio of polysaccharide to protein was reacted by combining 5 mg of Pn6B (30 mg/ml) with 5 mg of TT (28 mg/ml). Oxidation of Pn6B Polysaccharide
  • Pn6B-TT conjugate particles were prepared according to the protocol set forth above with the polymer solution containing sodium acetate buffer, pH 5.0. Conjugates were also prepared with a polymer solution containing sodium acetate buffer, pH 4.0. The percent recovery and ratio of polysaccharide to protein in the conjugate particles are set forth in Table 6 below. Table 6: Effect of pH Polymer Solution on Recovery and Ratio of Polysaccharide and Protein in the Conjugation
  • Pn6B-TT conjugates were prepared according to the protocol set forth above with the polymer solution containing sodium acetate buffer, pH
  • the Pn6B-TT conjugates were also prepared with the polymer solution adjusted to pH 4.0, using the following starting ratios of Pn6B to
  • Pn6B-TT Conjugate Preparation Procedure Three different lots of Pn6B-TT conjugates were prepared as described above using 4 mM sodium periodate as the oxidizing agent, a polymer solution adjusted to a pH of 4.0, and a 2:1 starting ratio of Pn6B to TT. The different lots were then analyzed for percent recovery and ratio of polysaccharide to protein in the conjugate particles. The results are set forth in Table 8 below.
  • Pn6B-TT conjugate particles were prepared as described above using 10 mM or 4 mM sodium periodate as the oxidizing agent and a polymer solution adjusted to a pH of 4.0 or a pH of 5.0 as specified below. The particles were dissolved in 0.1 N sodium hydroxide and neutralized in 0.02 M phosphate buffer to produce solubilized Pn6B-TT conjugate. The solubilized conjugate was injected subcutaneously into CD-I female mice (13-15 g) (groups of 10 mice). A primary boost was injected on day 28. A secondary boost was injected on day 42. Blood samples were taken for immunogenicity analysis on day 0, day 28 (4w), day 42 (6w) and day 56 (8w).
  • Sample Pn6B+TT contained unconjugated polysaccharide and protein and was administered in a dose containing 2.0 ⁇ g Pn6B and 2.0 ⁇ g TT.
  • Sample 6433-1 was prepared by oxidizing the polysaccharide with 10 mM sodium periodate and forming particles with a polymer solution at pH 5.0 and was administered in a dose containing 2.0 ⁇ g Pn6B and 5.1 ⁇ g TT.
  • Sample 6433-2 was prepared by oxidizing the polysaccharide with 4 mM sodium periodate and forming particles with a polymer solution at pH 5.0 and was administered in a dose containing 2.0 ⁇ g Pn6B and 5.1 ⁇ g TT.
  • Sample 6433-3 was prepared by oxidizing the polysaccharide with 4 mM sodium periodate and forming particles with a polymer solution at pH 4.0 and was administered in a dose containing 2.0 ⁇ g Pn6B and 1.2 ⁇ g TT.
  • Sample 6433-4 was prepared by oxidizing the polysaccharide with 4 mM sodium periodate and forming particles with a polymer solution at pH 4.0 and was administered in a dose containing 2.0 ⁇ g Pn6B and 2.7 ⁇ g TT.
  • Sample 9411 contained Pn6B-TT conjugates prepared without polymers and without the formation of particles.
  • conjugates were made using cyanogen bromide and the conjugating reagent l-ethyl-3-(3- dimethylaminopropyl-carbodiimide hydrochloride (a conjugation method known to those skilled in the art as the "EDC” or “EDAC” reaction).
  • Sample 9411 was administered in a dose containing 2.0 ⁇ g Pn6B and 5.1 ⁇ g TT.
  • the immunogenicities of the solubilized conjugates are set forth below in Table 9.
  • the abbreviation R/T in the table represents the number of "responder mice” over the number of total mice in the group.
  • This experiment describes the preparation of pneumococcal polysaccharide type 23F-CRMJ97 conjugates in the absence of reductive amination by forming Schiff base intermediate conjugate particles using a mixture of polymers.
  • the immunogenicity of the conjugates were analyzed in mice.
  • Pn23F polysaccharide is a high molecular weight polysaccharide having an approximate molecular weight of from 1 to 3 million.
  • the Pn23F polysaccharide is a pneumococcal type 23 F polysaccharide and is available from the American Type Culture Collection (ATCC, Rockville, MD).
  • the CRM 197 protein is a native, non- toxic, mutant form of the cross- reactive material of diphtheria toxin.
  • CRM 197 is commercially available from Sigma Chemical Co. (St. Louis, MO).
  • Pn23F-CRMi97 conjugates were prepared by first combining, a
  • Pn23F polysaccharide solution (11.3 mg/ml) with a sodium periodate solution (2.5 mM) and incubating the reaction at room temperature for one hour. Ethylene glycol was then added and the reactants incubated a further 30 minutes at room temperature to give a quenched, oxidized polysaccharide solution.
  • the mixture was centrifuged at 13,200 rpm for 10 minutes and the pellet was washed three times with 0.5 ml of 30% ethanol.
  • the pellet was resuspended in phosphate buffered saline to provide a polysaccharide concentration of 12.5 ⁇ g/ml.
  • mice (4-6 weeks of age) were injected with two doses of the samples (2.5 ⁇ g polysaccharide) with the booster dose being given on day 28. As additional controls, mice were also injected with the Pn23F polysaccharide alone and the oxidized Pn23F polysaccharide alone. Blood samples were taken on day 42. The sera was diluted 200 and 800 fold and analyzed by ELISA for Pn23F. The results are presented in Table 10 below.
  • This experiment describes the preparation of pneumococcal polysaccharide type 18C-tetanus toxoid conjugates in the absence of reductive amination by forming Schiff base intermediate conjugate particles using a mixture of polymers.
  • the effects of the starting ratio of the components, the sodium periodate concentration, and the incubation times and temperatures on conjugate recovery and ratio of the conjugated components were analyzed.
  • the Pnl8C polysaccharide is a pneumococcal type 18 C polysaccharide, available from the American Type Culture Collection (ATCC, Rockville, MD).
  • Pn6B-TT conjugates were prepared in accordance with the general procedure set forth in Example 1 above for the preparation of PRP-TT conjugates.
  • Pnl8C was mixed with sodium periodate for 30 minutes and quenched with ethylene glycol.
  • TT was added to the quenched mixture along with a polymer mixture containing 20% PVP and 20% PEG, pH 4.
  • the mixture was incubated at room temperature, 37°C, and 58°C as indicated below in Table 11. Particles formed and were centrifuged and washed with deionized water. The particles were dissolved with sodium hydroxide and neutralized with phosphate buffer (pH 7).
  • the polysaccharide content was determined using the Anthrone free polysaccharide assay described in METHODS IN IMMUNOLOGY .AND IMMUNOCHEMISTRY, Vol. II, Williams, C.A. and Chase, M.W.(eds.), 1968, pp. 288 - 289, Academic Press, NY.
  • the protein content was determined using the BCATM protein assay (Pierce, Rockford, IL).
  • the HPLC analysis was performed on a TSK-4000 column. The results are shown below in Table 11. Table 11: Recovery Yield and Ratio of Polysaccharide to Protein in Pnl8C-TT Conjugates Prepared Under Various Conditions
  • Toxoid Conjugates This experiment describes the preparation of Group A meningococcal polysaccharide (GAMP)-tetanus toxoid conjugates in the absence of reductive amination by forming Schiff base intermediate conjugate particles using a mixture of polymers. Several physicochemical characteristics of two preparations of the GAMP-TT conjugates were determined.
  • GAMP meningococcal polysaccharide
  • Group A meningococcal polysaccharide having a size of 96,000 D (Shanghai Institute of Biological Production, P.R. China) was dissolved in 0.1 M MES (2-[N-morpholino]ethanesulfonic acid) buffer, pH 5.0, and incubated at 60°C for one hour to produce hydrolyzed GAMP having a size of 64,000 D.
  • MES 2-[N-morpholino]ethanesulfonic acid
  • the mixture was incubated at room temperature for two hours and dialyzed against deionized water for three days.
  • the resulting reduced GAMP had a size of 50,000 daltons and a concentration of 8.7 mg/ml.
  • Sodium periodate was added to provide a concentration of 8 mM.
  • the mixture was incubated in the dark at room temperature for 30 minutes, then quenched by adding ethylene glycol (to provide a final concentration of 50 mM).
  • TT (10 mg/ml) was added, followed by the addition of three volumes of a polymer solution containing 25% PVP, 25% PEG, and sodium acetate buffer, pH 4.0. The reaction mixture was incubated at room temperature for thirty minutes and 37°C for thirty minutes. The pellet was collected by centrifugation and divided into two parts.
  • Pellet 1 was resuspended in deionized water, allowed to stand for one hour, washed with deionized water three times, dissolved in 0.1 N sodium hydroxide, and neutralized with 0.1 N HC1 to provide GAMP-TT conjugate solution 1.
  • Pellet 2 was suspended in 0.1 M hydroxamine (NH2OH) at room temperature for one hour to quench unreacted aldehyde groups and washed with saturated ammonia sulfate three times. The pellet was dissolved in 9.2
  • the polysaccharide content of the conjugates was determined using the phosphorus assay (Anal. Chem. 28:1756-1759 (1956)).
  • the protein content of the conjugates was determined using the well known Bradford protein assay.
  • the free polysaccharide content was determined using an acid-detergent precipitation method.
  • Meningococcal C polysaccharide (Connaught Laboratories, Swiftwater, PA) was dissolved in deionized water (20 mg/ml). To this solution (0.3 ml) was added sodium periodate (8 mM, 0.3 ml) in deionized water. The mixture was incubated at room temperature for 30 minutes. 12 ⁇ l of ethylene glycol was added and mixed well. The mixture was incubated at room temperature for 30 minutes. Diphtheria toxoid (3.9 mg/ml, 0.6 ml, Connaught Laboratories) was added while vortexing.
  • the particles were resuspended in deionized water (0.8 ml) and analyzed for protein content using the BCATM protein assay (Pierce, Rockford, IL) and polysaccharide content using the sialic acid assay (Biochimica Biophysica Acta 24:604-611 (1957). The ratio of polysaccharide to protein was 0.14.
  • the particles were redissolved in 0.1 N NaOH and neutralized with phosphate buffer. Immunogenicity of the Conjugate in Rabbits
  • Pnl4-CRM197 were purified as described below by forming conjugate particles and separating the particles from unreacted components and reagents in the conjugate reaction mixture.
  • Pn23F polysaccharide and Pnl4 polysaccharide are high molecular weight polysaccharides, each having an approximate molecular weight of from 1 to 3 million.
  • the Pn23F polysaccharide is a pneumococcal type 23 F polysaccharide.
  • the Pnl4 polysaccharide is a pneumococcal type 14 polysaccharide. Both polysaccharides are available from the American Type Culture Collection (ATCC, Rockville, MD).
  • the CRM 197 protein is a native, non-toxic, mutant form of the cross-reactive material of diphtheria toxin. CRM197 is commercially available from Sigma Chemical Co. (St. Louis, MO).
  • Each polysaccharide was independently conjugated to the CRM 197 protein using a reductive amination conjugation reaction.
  • the Pn23F-CRMi97 conjugate reaction mixture was carried out using dimethylsulfoxide (DMSO).
  • the Pnl4-CRMi97 conjugate reaction mixture was carried out using phosphate buffer.
  • Purification of the Pn23F-CRMi97 and Pnl4-CRMi97 conjugates from free polysaccharide was achieved in two to three hours by incubating the conjugates with a polymer mixture of polyvinylpyrrolidone and polyethylene glycol to form conjugate particles and the particles separated from the reaction mixture by centrifugation and decantation. Purification of Pn23F-CRM 197 Conjugate
  • the conjugate reaction mixture containing the Pn23F-CRMi97 conjugate in dimethylsulfoxide (DMSO) was diluted with deionized water to give a 50% DMSO solution.
  • the diluted solution was then concentrated with a Centricon 30 filter (Amicon, Beverly, MA) to yield a solution containing 2.84 mg/ml CRM 197, determined by the BCATM protein assay (Pierce, Rockford, IL).
  • To the mixture (1.8 ml) was added a polymer solution containing 25% PVP (40 kD), 25% PEG (3.35 kD) in 0.1M sodium acetate, pH 5.0 (5.4 ml), while vortexing.
  • To the polymer/conjugate mixture was added 2.7 ml of a 95% ethanol solution while vortexing. The mixture was incubated for 30 minutes each at 20°C,
  • Conjugate particles were formed. The mixture was centrifuged at approximately 14,000 x g in a microfuge at room temperature to precipitate the particles, and the supernatant was removed.
  • PBS phosphate buffered saline
  • Polysaccharide content was determined by the Anthrone free polysaccharide assay described in METHODS EN IMMUNOLOGY AND IMMUNOCHEMISTRY, Vol. ⁇ , Williams, C.A. and Chase, M.W.(eds.), 1968, pp. 288 - 289, Academic Press, NY, which is incorporated herein by reference.
  • Protein content was determined by the BCATM protein assay (Pierce, Rockford, IL). The polysaccharide content was calculated to be 1.47 mg/ml. The protein content was calculated to be 1.26 mg/ml. Recovery of the conjugate was calculated to be 86%, as determined by comparing the protein content of the conjugate reaction mixture after purification to the protein levels in the conjugate reaction mixture before purification. Free polysaccharide content in the purified conjugate mixture was determined by first passing the mixture through a phenyl-SepharoseTM column (Pharmacia Biotech, Piscataway, NY), which binds the conjugate.
  • the residue was washed with 25% ethanol (2.4 ml) and then washed twice with deionized water (2.4 ml).
  • the pellet was then resuspended in 5.9 ml of phosphate buffered saline with sonication to yield the conjugate in solubilized form.
  • the recovery of the conjugate was 90% as determined by comparing the protein content of the conjugate reaction mixture before and after purification.
  • the free polysaccharide content was approximately 14% as determined by the Anthrone free polysaccharide assay described above.
  • adipic acid dihydrazide derivative (AH) of the polyribosylribitol phosphate (PRP) of Haemophilus influenzae type b (Hib), one of the major causative organisms of bacterial meningitis, referred to as PRP-AH, was prepared by coupling PRP to adipic acid (Sigma Chemical Co., St. Louis, MO) in the presence of cyanogen bromide (Sigma Chemical Co., St. Louis, MO) (The PRP was obtained from the Massachusetts Public Health Biologic Laboratory (Jamaica Plain, MA)).
  • PRP-AH was coupled to tetanus toxoid (TT) in the presence of l-3-ethyl-3-(3 dimethyl aminopropyl) carbodiimide (EDC, Sigma Chemical Co., St. Louis, MO) to generate a PRP-AH-TT conjugate.
  • TT tetanus toxoid
  • EDC l-3-ethyl-3-(3 dimethyl aminopropyl) carbodiimide
  • a reaction mixture (0.2 ml) containing PRP-AH (200,000 D) (10 mg/ml) and TT (10 mg ml) conjugates was combined with a polymer solution (0.6 ml) containing 25% PVP and 25% PEG in 0.1M sodium acetate pH 5.0, while vortexing. The mixture was incubated sequentially at room temperature, 37 °C, and 58°C for 30 minutes each. Conjugate particles were formed. The mixture was centrifuged in a microfuge at 14,000 x g and washed three times with deionized water (0.5 ml). The pellet was resuspended in 0.5 ml of deionized water.
  • Kd as used herein refers to a distribution coefficient, which is defined as the fraction of the stationary phase available for diffusion of a given solute species.
  • Kd the elution volume (V e ) minus the void volume (Vo) divided by the total volume of the packed bed (V ).
  • Recoveries of TT and PRP were 37% and 24%, respectively. In both cases HPLC analysis revealed no significant PRP peak.
  • Example 9 were formed and purified in a single continuous process in the same reaction vessel using the following method.
  • Various conjugation reaction conditions were studied in which the polymers were added either at the beginning of the conjugation reaction or at a particular time during the conjugation reaction.
  • a conjugation reaction mixture was prepared containing tetanus toxoid (TT, 10 mg/ml) in 0.2 M MES buffer (pH 5.0), an adipic acid dihydrazide derivative (AH) of the polyribosylribitol phosphate (PRP) of Haemophilus influenzae type b (Hib), (PRP-AH, 10 mg/ml, prepared as described in Example 3) in 0.2 M MES buffer (pH 5.0), and 0.1 M of the carbodiimide conjugating reagent, l-ethyl-3-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC), Sigma Chemical Co., St. Louis, MO) in MES buffer (pH 5.0).
  • TT tetanus toxoid
  • AH adipic acid dihydrazide derivative
  • PRP polyribosylribitol phosphate
  • Hib Haemophilus influenza
  • Example 1 a polymer solution consisting of three volumes of conjugation mixture containing 25% PVP (40,000 D), 25% PEG (3,350 D) in 0.1 M MES buffer pH 5.0 was added at the beginning of the conjugation reaction. The mixture was incubated at room temperature for 30 minutes, at 37°C for 30 minutes, and at 58°C for 30 minutes. The particles were recovered and resuspended in 25% ethanol and centrifuged at 13,200 rpm for 5 minutes, and resuspended in dH2 ⁇ twice and centrifuged at 13,200 rpm for 5 minutes. The washed particles were then dissolved in 0.1 N NaOH and neutralized with phosphate buffer.
  • the resulting conjugate was analyzed for PRP by Ribose assay, TT by BCA assay and unconjugated PRP and TT by HPLC using a TSK column, and the molecular distribution of the conjugate was analyzed by gel filtration chromatography (CL-4B).
  • sample 2 In the second sample (Sample 2), a similar procedure was followed as for the first sample except the polymer solution was added after the conjugation mixture was incubated at room temperature for 1 hour. In the third sample (Sample 3), the polymer solution was added after the conjugation mixture was incubated at room temperature for 3 hours.
  • the results of samples 1-3, as listed in Table 15, indicate that the polymer solution containing PVP and PEG can be used to separate PRP- AH-TT conjugates from the reaction mixture even though approximately 5% unconjugated PRP may exist in the conjugate preparation.
  • Fluorescein isothiocyanate (FITC, Sigma Chemical Company, St.
  • HSA human serum albumin
  • FITC (6.2 ⁇ g) was dissolved in 2 ml of carbonate buffer (pH 10).
  • the dissolved FTTC was combined with 1 ml of HSA (25%) and 6 ml of a polymer solution containing 25% PVP and 25% PEG in 0.1M sodium acetate pH 5.0, while vortexing.
  • the mixture was incubated sequentially at room temperature, 37°C, and 58°C for 30 minutes each. Conjugate particles were formed.
  • the mixture was centrifuged in a microfuge at 14,000 x g, the supernatant removed, and the particles washed three times with deionized water (10 ml each). The particles were resuspended in 10 ml deionized water.

Abstract

Procédés de préparation et de purification de conjugués par réaction d'une molécule contenant un groupe aldéhyde avec une molécule contenant un groupe amine en présence d'un polymère pendant une durée suffisante pour obtenir des particules de conjugués. On prépare les conjugués rapidement et efficacement par oxydation de l'aldéhyde et par combinaison de l'aldéhyde oxydé avec une solution à base de protéines et de polymère, afin d'obtenir des particules de conjugués intermédiaires de base de Schiff en l'absence d'une animation réductrice. La séparation subséquente des particules du mélange réactionnel de conjugaison permet d'obtenir des conjugués purifiés qu'on a séparé des constituants libres non conjugués. De préférence, le polymère est un mélange de polymères comprenant polyvinylpyrrolidone et polyéthylène glycol. On peut ajouter un solvant organique, tel qu'un alcool, au mélange d'incubation, afin de faciliter la formation des particules. On peut également ajouter un agent de conjugaison au mélange réactionnel. On peut laver les particules afin de supprimer les contaminants supplémentaires indésirables et les solubiliser au moyen d'une base, ce qui produit des conjugués solubles. Les conjugués préparés à partir d'un ou plusieurs antigènes sont utiles en tant que vaccins quand on les administre à l'homme ou à l'animal.
PCT/US1995/016950 1994-12-23 1995-12-22 Procedes de preparation et de purification de conjugues macromoleculaires WO1996020012A2 (fr)

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US08/372,820 US5554730A (en) 1993-03-09 1994-12-23 Method and kit for making a polysaccharide-protein conjugate
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US08/538,817 1995-10-04

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WO2001070272A1 (fr) * 2000-03-21 2001-09-27 Igeneon Krebs-Immuntherapie Forschungs- Und Entwicklungs- Ag Conjugue de polysaccharide et de polypeptide
US6573245B1 (en) 1998-04-28 2003-06-03 Galenica Pharmaceuticals, Inc. Modified polysaccharide adjuvant-protein antigen conjugates, the preparation thereof and the use thereof
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US6956119B2 (en) 2000-03-21 2005-10-18 Hans Loibner Polysaccharide-polypeptide conjugate
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US7964217B2 (en) 1996-09-13 2011-06-21 Nektar Therapeutics Degradable poly(ethylene glycol) hydrogels with controlled half-life and precursors therefor
US6960344B2 (en) 1997-10-03 2005-11-01 Galenica Pharmaceuticals, Inc. Use of imine-forming polysaccharides as adjuvants and immunostimulants
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WO1999034833A1 (fr) * 1998-01-07 1999-07-15 Shearwater Polymers, Incorporated Acrylates de polyethyleneglycol heterobifonctionnel degradables, et gels et conjugues derives de ces acrylates
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US7214388B2 (en) 1998-01-07 2007-05-08 Debio Recherche Pharmaceutique S.A. Degradable heterobifunctional poly(ethylene glycol) acrylates
EP1047725B1 (fr) * 1998-01-16 2003-06-04 Unilever N.V. Conjugue de polysaccharides capable de se fixer a la cellulose
US6573245B1 (en) 1998-04-28 2003-06-03 Galenica Pharmaceuticals, Inc. Modified polysaccharide adjuvant-protein antigen conjugates, the preparation thereof and the use thereof
US6956119B2 (en) 2000-03-21 2005-10-18 Hans Loibner Polysaccharide-polypeptide conjugate
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