WO2012061400A2

WO2012061400A2 - Method for reduction of free polysaccharide in polysaccharide-protein vaccines reactions using ion-exchange matrices

Info

Publication number: WO2012061400A2
Application number: PCT/US2011/058803
Authority: WO
Inventors: Raphael SIMON; Andrew Lees
Original assignee: University Of Maryland, Baltimore
Priority date: 2010-11-01
Filing date: 2011-11-01
Publication date: 2012-05-10
Also published as: WO2012061400A3

Abstract

The present invention relates to methods for removing free carbohydrate from a carbohydrate-protein conjugate composition using ion exchange membranes or monoliths. The present invention further relates to pharmaceutical compositions produced in accordance with the methods of the invention.

Description

Method for Reduction of Free Polysaccharide in Polysaccharide-Protein Vaccines Reactions Using Ion-Exchange Matrices

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Appl. No. 61/408,793, filed November 1, 2010. The content of the aforesaid application is relied upon and incorporated by reference in its entirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with government support under NIH Grant No. AI057168 awarded by the National Institutes of Health. The government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readable sequence listing submitted concurrently herewith and identified as follows: One 4,524 Byte ASCII (Text) file named "sequence_listing.txt," created on November 1, 2011.

FIELD OF THE INVENTION

The field of the invention generally relates to immunology, molecular biology, infectious disease and medicine. In particular, the invention relates to a method for reducing the level of free carbohydrate from a solution of protein- linked carbohydrate (conjugate) and non-linked carbohydrate using ion exchange membranes or monoliths. In addition, the invention relates to compositions and vaccines produced by the methods of the invention.

BACKGROUND OF THE INVENTION

Vaccines of protein covalently linked to carbohydrate have proven remarkably successful in inducing an immune response to the carbohydrate moiety. In order for these vaccines to be effective, it is necessary to minimize the amount of non-linked carbohydrate present. Specifications for conjugate vaccines set maximum amounts of free polysaccharide that can be present. In contrast, there is generally no specification for the amount of unconjugated protein. In fact, combination vaccines contain significant amounts of unconjugated protein. Removal of the unconjugated protein can usually easily be achieved using size exclusion chromatography, tangential flow filtration or the solid phase method described by U.S. Patent No. 6,284,250. Thus, the reduction in the level of the unconjugated carbohydrate is critical. This reduction can be difficult to achieve with good efficiency and yield. The absence of a good method for removing the unconjugated polysaccharide reduces the yield, increases the needed effort and cost of manufacturing conjugate vaccines.

Conjugate vaccines tend to be of high molecular weight. The carbohydrate component itself may be large, and also because combining the protein and carbohydrate increases the size. In addition, there may be additional crosslinking between the components that further increases the molecular weight. Chromatography is a common means of purifying biological substances. One means of separating the conjugate from free polysaccharide is size exclusion chromatography (SEC), which separates molecules on the basis of their size, or more precisely, their hydrodynamic radius. SEC is a diffusion-limited, nonadsorptive form of chromatography. SEC suffers from low resolution and capacity. Furthermore, SEC is only successful if there is a significant difference in size molecular weight between the conjugate and the free polysaccharide. Because each is polydisperse, there can be significant overlap in their elution profiles and thus resolution is poor. In order to obtain material with substantially reduced amounts of free polysaccharide, it is generally necessary to discard part of the conjugate, thereby reducing yields. One solution is to use sized, lower molecular weight polysaccharides and then to crosslink the conjugate sufficiently so that the molecular weight increases enough that it can be separated from the sized polysaccharides. This process requires extra processing and additional losses of material.

Chromatography resins (e.g. sorbents, media) consist of porous particles that may be functionalized with charges, ligands and other binding partners. In adsorptive chromatography, substances are bound to the sorbent via these groups. However, in order to be adsorbed, substances need to enter the pores, a process which is diffusion limited. Most of the surface area of the particles is on the porous interior and large molecules, like conjugates diffuse slowly and due to their size, cannot easily access the pores. These difficulties are further accentuated by the fact that conjugates are polydisperse in size. Thus, some of the smaller conjugate may be able to enter the pores and will chromatograph (i.e., separate) differently than conjugate that does not enter the pores. Due to the fact that the conjugates can generally only access the surface of the particles, their binding capacity for conjugates is severely restricted.

A solution is to use oligosaccharides of a size so that the conjugate formed is still of low enough molecular weight that chromatographic separation can be achieved. Again, this entails additional processing and losses of material. Further purification strategies could make use of binding to immobilized metal affinity chromatography (IMAC) sorbents. IMAC sorbents can interact with protein histidines, tryptophans and cysteines. However, the metal may leach and need to be subsequently removed. This would be undesirable in the manufacture of vaccines. Other methods for separating the conjugate from the free polysaccharide include tangential flow filtration (TFF). In this process, the solution is rapidly passed across a porous membrane with pores of a nominal molecular weight cutoff lower than that of the conjugate and higher than that of the free carbohydrate. The conjugate is retained and the free polysaccharide passes through into the filtrate. This process can be effective if there is a large difference in molecular weight between the conjugate and the free carbohydrate. If they are too close in size, depending on the molecular weight cutoff of the membrane pores, either too much free carbohydrate is retained or too much of the conjugate is found in the filtrate. Furthermore, it has been found that in some cases, even when there is a large difference in size, poor separation and/or recovery is observed. McMaster (U.S. Patent No. 6,146,902) has claimed that the addition of ammonium sulfate can promote separation by TFF. However, this process was unable to be replicated. Another nonchromatographic method of separation which takes advantage of the difference in hydrophobicity between the protein and the carbohydrate is the selective precipitation of the protein component with a lyotropic salt such as ammonium sulfate. As the protein, whether free or conjugated, is more hydrophobic than the polysaccharide it should precipitate at lower salt concentrations than the free carbohydrate. Thus, the conjugate should precipitate while leaving the carbohydrate in solution. The precipitate is separated by centrifugation and then resuspended. In practice, however, some of the free carbohydrate can become entrapped in the precipitate. Furthermore, there can be significant losses associated with the process.

Thus, there is a significant need for an efficient method to remove the unconjugated carbohydrate from the conjugated carbohydrate in conjugate vaccines.

SUMMARY OF THE INVENTION

Unconjugated free polysaccharide carbohydrate is recognized as a significantly deleterious problem in the preparation and efficacy of polysaccharide- protein conjugate vaccines that can lead to impaired anti-saccharide immune responses. Removal of unreacted free carbohydrate in conjugation reactions is a significant challenge. This invention overcomes several problems and disadvantages associated with currently used strategies, and provide new methods for reducing the level of free carbohydrate from mixtures of protein linked carbohydrate and non-linked carbohydrate.

According to non-limiting example embodiments, the present inventors have developed a method for removing free carbohydrate from a carbohydrate- protein conjugate composition. In one embodiment, the invention provides a method for removing free carbohydrate from a carbohydrate-protein conjugate composition, wherein the method comprises i) contacting the composition with an ion exchange matrix, wherein the carbohydrate-protein conjugate binds the matrix; ii) optionally eluting free carbohydrate bound to the matrix with one or more wash solutions; and iii) eluting the carbohydrate-protein conjugate from the matrix with one or more eluent solutions, wherein the free carbohydrate is separated from the carbohydrate-protein conjugate, resulting in a carbohydrate-protein conjugate with reduced levels of free carbohydrate, wherein the matrix is selected from the group consisting of an ion exchange membrane and an ion exchange monolith.

In another embodiment, the invention provides a conjugated carbohydrate- protein pharmaceutical composition, such as a vaccine, that contains reduced free carbohydrate. In some embodiments, the pharmaceutical compositions and vaccines are prepared according to the processes of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIG. 1. (A) Fractionation trace at OD280 of CDAP linked COPS:Flagellin conjugates through Superdex 200. (B) Protein concentration in SEC fractions (1 ml) as determined by BCA assay.

FIG. 2. (A) Fractionation trace OD280 of pooled-concentrated CDAP linked COPS:Flagellin protein conjugates containing SEC fractions through Sartobind Nano-Q anion exchange membrane, and relative NaCl concentration (0-1000 mM). (B) Total polysaccharide. (C) Total protein levels in 0 mM NaCl flow through, 80 mM NaCl wash, and 80 mM-1000 mM gradient fractions.

FIG. 3. Binding of high molecular weight conjugates to membranes but not conventional chromatography resins.

FIG. 4 (A) Structure of pneumococcal serotype 14 capsular polysaccharide (Pnl4). (B) Unexpected binding of Pnl4 to an anion exchange membrane and elution with salt. (C) Shows unexpected binding of Pnl4 to the ion exchange membrane under conditions which there should be no binding. The buffer is 25 mM Tris, pH 8, and the gradient is from 0 mM to 1 M NaCl, at 5 ml/min.

FIG. 5. Gradient elution to separate free from conjugated carbohydrate.

FIG. 6. Spiking of conjugate BSA-dextran with unconjugated dextran polysaccharide. DETAILED DESCRIPTION

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found, for example, in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar technical references.

For the purpose of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used). The use of "or" means "and/or" unless stated otherwise. The use of "a" herein means "one or more" unless stated otherwise or where the use of "one or more" is clearly inappropriate. The use of "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term "comprising," those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language "consisting essentially of and/or "consisting of."

As used herein "about" and "approximately" mean ± 10% of the value indicated.

The term "carbohydrate" includes polysaccharides, oligosaccharides and other carbohydrate polymers, including monomeric sugars.

The term "free carbohydrate" means carbohydrate that is not conjugated to protein. "Unconjugated carbohydrate" and "free carbohydrate" are used interchangeably throughout the application. Ion exchange chromatography based purification is based upon the principle of reversible binding between charged chemical groups attached to chromatography media to oppositely charged groups on molecules to be separated. Removal of the molecule of interest from ion-exchange media can be accomplished by competition with increasing levels of charged ionic salts such as NaCl. Conventional bead based resins used in purification chromatography rely on the diffusion through the pores within individual beads for interaction with charged chemical groups contained within.

Polysaccharide-protein conjugates, such as those used as vaccines, are frequently very large molecules, and can fail to efficiently access the pores within commonly used beads, and thus can be excluded from the column. This can cause the conjugates to fail to bind the charged ion-exchange groups present within the beads, greatly reducing the performance and yield of ion-exchange based purification with bead based resins.

In contrast to diffusion limited chromatography, convective ion exchange chromatography such as membrane or monolith chromatography utilizes large channels containing attached charged ion-exchange groups along the channel walls. The use of membrane chromatography as compared to conventional bead based resins for the purification of polysaccharide-protein conjugates allows for unhindered interaction of conjugate molecules with the charged ion-exchange groups, as conjugate molecules can bind along the channel walls, and do no need to enter narrow pores within beads. As binding does not rely on diffusion, flow rates can also be significantly increased, resulting in faster overall purification processes.

It has been surprisingly discovered that convective ion-exchange chromatography is effective at reducing the level of free carbohydrate from protein linked carbohydrate conjugates in conjugate vaccines synthesized with neutral or weakly charged carbohydrates. In some embodiments, membrane ion exchange chromatography is used to reduce the level of free carbohydrate in conjugate compositions. In some embodiments, ion exchange monoliths are used to reduce the level of free carbohydrate in conjugate compositions. Ion exchange monoliths are chromatographic stationary phases that are polymerized directly in a column as a single unit. See, e.g., Nordborg et al. Analytical and Bioanalytical Chemistry, 394(l):71-84 (2009).

In one embodiment, the invention provides a method for removing free carbohydrate from a carbohydrate-protein conjugate composition, wherein the method comprises i) contacting the composition with an ion exchange matrix, wherein the carbohydrate-protein conjugate binds the matrix; ii) optionally eluting free carbohydrate bound to the matrix with one or more wash solutions; and iii) eluting the carbohydrate-protein conjugate bound to the matrix with one or more eluent solutions, wherein the free carbohydrate is separated from the carbohydrate- protein conjugate, resulting in a carbohydrate-protein conjugate with reduced levels of free carbohydrate.

In some embodiments, purification by ion-exchange of polysaccharide- protein or carbohydrate-protein conjugate molecules from unreacted neutral or weakly charged polysaccharides or carbohydrates, is accomplished by taking advantage of the differential charge properties of the protein component within the conjugate, as compared to the free polysaccharide or carbohydrate. For example, at pH conditions greater than the isoelectric point of the protein carrier (net overall negative charge), conjugate molecules can bind positively charged anion exchange media through negatively charged groups located in charged pockets on the protein. Uncharged, or weakly negatively charged unconjugated polysaccharide or carbohydrate fail to strongly bind positively charged anion exchange groups, and the majority of unconjugated polysaccharide or carbohydrate will pass directly through the membrane or monolith. In some embodiments, a low concentration ionic wash, set at a level below the salt competition displacement point of the protein in the conjugate, but above the salt competition displacement point of the unconjugated polysaccharide or carbohydrate is generally sufficient to remove any remaining weakly bound polysaccharide or carbohydrate.

In other embodiments, alternative ion exchange purification strategies that can be employed for removal of free neutral or weakly charged polysaccharide include cation based ion exchange, with the pH set below the isoelectric point of the protein, so as to cause the protein to shift to a net positively charged form. Under these conditions, conjugate molecules can bind negatively charged cation exchange media through positively charged groups located in charged pockets on the protein. Negatively charged unconjugated polysaccharide or carbohydrate fail to bind negatively charged cation exchange groups, and the majority of unconjugated polysaccharide or carbohydrate will pass directly through the membrane. In some embodiments, a low concentration ionic wash is generally sufficient to remove any remaining weakly bound polysaccharide or carbohydrate. Manipulation of the isoelectric point to cause either net positive or net-negative overall charge, in tandem with the appropriate ion exchange membrane (anion or cation) for protein binding can be used to remove polysaccharides that contain homologous ionizable groups of only one type (i.e. non-zwitterionic) that will allow the polysaccharide to assume only a single overall charge state (i.e. only neutral or positive; only neutral or negative), that under the appropriate buffer conditions will be opposite to the protein, or net neutral, and thus will fail to bind entirely to the ion exchange groups on the membrane or monolith.

The compositions produced by the methods of the invention can be used in vaccine preparations, for example. In some embodiments, the free carbohydrate and protein linked carbohydrate are in a solution in a buffer with very low ionic strength, which will allow for binding to matrix containing ion-exchange ligands to separate polysaccharide-protein or carbohydrate-protein conjugate molecules from unreacted neutral or weakly charged carbohydrate or polysaccharide. In some embodiments, the solution is applied to the matrix, wherein the conjugate (as well as free protein) will bind to the matrix more tightly than free weakly charged or neutral carbohydrate. In some embodiments, neutral carbohydrate flows through directly or binds weakly. In some embodiments, the weakly bound carbohydrate is removed by washing the matrix with buffer at an ionic strength strong enough to remove the unconjugated carbohydrate, but weak enough to allow the protein linked carbohydrate (and free protein) to remain bound. In some embodiments, protein linked carbohydrate (and free protein) are removed upon washing with buffer at an ionic strength high enough to displace the protein linked carbohydrate (and free protein) by competition for the charged ion-exchange ligands on the membrane. In some embodiments, the ion exchange matrix can be an ion exchange membrane (cationic or anionic) or an ion exchange monolith (cationic or anionic). Such membranes are commercially available by vendors such as Sartorius (Goettingen, Germany) and Pall (Port Washington, New York), for example. Monolith columns are available, e.g., from BIA Separations (Slovenia).

In some embodiments, the free carbohydrate in the carbohydrate-protein conjugate composition does not substantially bind the matrix and flows through. In some embodiments, only a portion of the free carbohydrate binds the matrix and the remainder flows through. In some embodiments, a substantial portion or all of the free carbohydrate binds the matrix. In some embodiments about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% or more of the free carbohydrate binds the matrix.

In some embodiments, the free carbohydrate that binds the matrix is eluted with one or more wash solutions In some embodiments, the one or more wash solutions substantially elute the free carbohydrate that is bound to the matrix. In some embodiments, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about 99% of the free carbohydrate is eluted by the one or more wash solutions.

In some embodiments, the free carbohydrate is eluted from the matrix due to a difference in the ionic strength of the wash solution and the matrix. In some embodiments, the carbohydrate-protein conjugate is eluted from the matrix due to a difference in the ionic strength of the eluent solution and the matrix.

In some embodiments, the ionic strength of the wash solution that elutes the free carbohydrate is different than the ionic strength of the eluent that elutes the carbohydrate-protein conjugate. The ionic strength of the wash or eluent solutions used to elute the matrix is not limiting and will depend on a variety of factors, including the polysaccharide and protein that make up the conjugate, the type of membrane or monolith used and the pH of the solution or buffer.

In some embodiments, the ionic strength of the wash solution that elutes the free carbohydrate is lower than the ionic strength of the eluent solution that elutes the carbohydrate-protein conjugate. In some embodiments, the wash solution that elutes the free carbohydrate comprises a salt concentration of about 1-1000 mM or less. In some embodiments, the wash solution comprises a salt concentration of about 1-250 mM or less, 10-100 mM or less, 10-80 mM or less, 20-60 mM or less or 30-50 mM or less salt. In some embodiments, the free carbohydrate is eluted with a wash solution comprising a gradient of increasing ionic strength or salt concentration that encompasses the above-listed ranges. In some embodiments, a single wash solution that comprises about 80 mM NaCl or less elutes the free carbohydrate.

In some embodiments, the eluent solution that elutes the carbohydrate- protein conjugate comprises a salt concentration of at least about 80-2000 mM. In some embodiments, the eluent solution comprises a salt concentration of at least about 80-1000 mM, at least about 100-1000 mM, at least about 150-1000 mM, or at least about 200-1000 mM salt. In some embodiments, the carbohydrate-protein conjugate is eluted with a solution comprising a gradient of increasing ionic strength that encompasses the above-listed ranges.

In some embodiments, the wash or eluent solutions used to elute the free carbohydrate or the carbohydrate-protein conjugate comprise one or more salts. The type of salts that can be used are not limiting, and can include NaCl, (NH₄)₂S0₄, NH₄C1 and combinations thereof, for example. In some embodiments, the wash or eluent solutions are buffered. The type of buffer than can be used is not limiting and can include Tris, phosphate buffered saline, HEPES, and the like. In some embodiments, the wash or eluent solution comprises a Tris buffer of 5-100 mM, from pH 7-8. In some embodiments, the buffer is 10 mM Tris, pH 7.5, and further comprises a gradient of NaCl concentrations, depending on whether the free carbohydrate or carbohydrate-protein is eluted. In some embodiments, the wash solution used to elute the free carbohydrate is 10 mM Tris/80 mM NaCl, pH 7.5 and the eluent solution used to elute the carbohydrate-protein conjugate is 10 mM Tris, pH 7.5 with a gradient of 80-1000 mM NaCl.

In some embodiments, the carbohydrate-protein conjugate is eluted from the matrix due to a difference in the pH of the solutions that elute the free carbohydrate and the carbohydrate-protein conjugate. In some embodiments, the free carbohydrate or carbohydrate-protein conjugate is eluted with a wash or eluent solution comprising a gradient of increasing or decreasing pH. In some embodiments, the pH gradient is from about 3.0-10.0, from about 4.0-9.0, or from about 5.0-9.0.

In some embodiments, the method results in a free carbohydrate reduction in the conjugate composition of at least about 25%, at least about 35%, at least about 50%), at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

In some embodiments, the carbohydrate-protein conjugate composition is subjected to one or more processing steps to reduce the level of free carbohydrate in the composition before or after the composition is subjected to ion exchange chromatography in accordance with the methods of the invention. In some embodiments, size-exclusion chromatography (SEC) is performed before or after the methods of the invention is performed.

The protein or the carbohydrate of the carbohydrate-protein conjugate is not limiting. In some embodiments, the protein or carbohydrate is from a pathogenic organism or is characteristic of a protein or carbohydrate from the organism. As used herein, a carbohydrate or protein is "characteristic" of the pathogenic organism if it is substantially similar or identical in structure or sequence to a molecule naturally associated with the pathogen. The term is intended to include both molecules which are specific to the organism, as well as molecules which, though present on other organisms, are involved in the virulence or antigenicity of the particular microorganism in a human or animal host.

In some embodiments, the pathogen is from a microorganism selected from the group consisting of Salmonella, Shigella, Vibrio cholera, E. coli, Streptococcus pneumonia, Clostridium tetani, Corynebacterium diphtheria, Neisseria meningitidis and Haemophilus influenza.

In some embodiments, the carbohydrate is one or more polysaccharides from a pathogenic microorganism. In some embodiments, the one or more polysaccharides are characteristic of a polysaccharide of Salmonella or isolated therefrom, such as Salmonella enterica, or isolated or derived therefrom. In some embodiments, the polysaccharide comprises core-O-polysaccharide (COPS). As used herein, "core-O-polysaccharide" or "COPS," is a polysaccharide in which the lipid A moiety from lipopolysaccharide (LPS) has been removed. In some embodiments, the polysaccharide comprises Vi capsular polysaccharide from S. Typhi. The Vi capsular polysaccharide of S. Typhi is a linear homopolymer of poly-alpha(l-4)GalNAcp variably O acetylated at the C-3 position. See, e.g., Tacket et al., J. Infect. Diseases, 190:565-70 (2004); Szu et al., Infect Immun. 59(12): 4555^561 (1991).

In some embodiments, the carbohydrate can include lipopolysaccharide derived core-OPS (COPS), Salmonella Vi, Streptococcus pneumoniae capsular polysaccharides, Neisseria meningitidis A/C/Y/X/W135 capsular polysaccharides or Haemophilus influenzae group B polysaccharides. In some embodiments, the carbohydrate is a core-OPS polysaccharide (COPS) from a pathogen selected from the group consisting of Salmonella, Shigella, Vibrio cholera and E. coli. In some embodiments, the carbohydrate is a core-OPS polysaccharide (COPS) from a Salmonella enterica serovar selected from the group consisting of S. Typhimurium and S. Enteritidis.

In some embodiments, the protein of the conjugate includes antigenic carriers typically used in conjugate vaccines. Non-limiting examples of a carrier for one or more conjugates include tetanus toxin/toxoid, NTHi high molecular weight protein, diphtheria toxin/toxoid, detoxified Pseudomonas aeruginosa toxin A, cholera toxin/toxoid, pertussis toxin/toxoid, Clostridium perfringens exotoxins/toxoid, hepatitis B surface antigen, hepatitis B core antigen, rotavirus VP 7 protein, respiratory syncytial virus F and G protein, detoxified ETEC LT and genetically engineered mutant derivatives, detoxified Shigella Stxl/2 and genetically engineered mutants thereof. In some embodiments, the protein antigen is selected from the group consisting of CRM197, flagellin, tetanus toxoid, diptheria toxoid, rEPA, Haemophilus influenza group B protein D, and Neisseria meningitides outer membrane proteins (OMP). See, e.g., Blanchard-Rohner et ah, G., and A. J. Pollard. Expert Rev Vaccines 10:673-684 (2011); Pace et al. Meningococcal A, C, Y and W-135 polysaccharide-protein conjugate vaccines. Arch Dis Child 92:909-915 (2007); Pace et al. Quadrivalent meningococcal conjugate vaccines. Vaccine 27 Suppl 2:B30-41 (2009); Pollard et al. Maintaining protection against invasive bacteria with protein-polysaccharide conjugate vaccines. Nat Rev Immunol 9:213-220 (2009); and Zhongwu Guo, Geert-Jan Boons (ed.), Carbohydrate based vaccines and immunotherapies, published by Wiley, John & Sons, Inc., 2009 (ISBN 978-0-470-19756-1).

The protein of the conjugate can also include fragments or derivatives of the protein. For example, the protein can include fragments of the natural protein, including internal sequence fragments of the protein that retain their ability to elicit protective antibodies against the microorganism from which it is derived. By derivative is further meant an amino acid sequence that is not identical to the wild type amino acid sequence, but rather contains at least one or more amino acid changes (deletion, substitutions, inversion, insertions, etc.) that do not essentially affect the immunogenicity or protective antibody responses induced by the modified protein as compared to a similar activity of the wild type amino acid sequence, when used for the desired purpose.

In some embodiments, the protein of the conjugate is isolated from or is characteristic of a protein of Salmonella, such as Salmonella enterica. In some embodiments, the protein of the conjugate is from the same microorganism from which the carbohydrate is derived. In some embodiments, the protein is a phase 1 flagella protein (FliC) of the Salmonella enterica serovar. As used herein, the term "phase 1 flagella" and FliC protein are used interchangeably.

In some embodiments, the protein is a phase I flagella protein from a Salmonella enterica serovar selected from the group consisting of S. Typhimurium and S. Enteritidis.

In some embodiments, the carbohydrate-protein conjugate is selected from the group consisting of a Salmonella enterica serovar conjugate comprising COPS- FliC, a conjugate comprising Vi capsular polysaccharide of S. Typhi and tetanus toxoid, pneumococcal serotype 14 capsular polysaccharide-CRM197, pneumococcal serotype 14 capsular polysacchari de-tetanus toxoid, and pneumococcal capsular polysaccharide-flagellin.

In some embodiments, the carbohydrate-protein conjugate is a Salmonella enterica serovar phase 1 flagellin protein chemically conjugated to a Salmonella enterica serovar core-O-polysaccharide (COPS). See, e.g, Simon et al., Infection and Immunity. 201 1 79(10)4240-4249 and WO2010/083477. In some embodiments, the COPS-FliC conjugate is from a Salmonella enterica serovar selected from the group consisting of S. Typhimurium and S. Ententidis. The fliC gene from S. Enteritidis is 1518 bp in size (GenBank Accession Number NC_011294.1 (1146600..1148117)) (SEQ ID NO: l). The fliC gene of S. Typhimurium is 1488 bp in size (GenBank Accession Number NC_003197.1 (2047658..2049145, complement)) (SEQ ID NO:2).

The nature of the linkage of the protein to the carbohydrate in the conjugate is not limiting. In some embodiments, the protein is covalently linked to the carbohydrate either directly or using a linker. In some embodiments, the linker is selected from the group consisting of l-cyano-4-dimethylaminopyridinium tetrafluoroborate, adipic acid dihydrazide, ε-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone and p-nitrophenylethyl amine.

While the invention has been described with reference to certain particular examples and embodiments herein, those skilled in the art will appreciate that various examples and embodiments can be combined for the purpose of complying with all relevant patent laws (e.g., methods described in specific examples can be used to describe particular aspects of the invention and its operation even though such are not explicitly set forth in reference thereto).

The following examples illustrate the advantage of using membrane chromatography over conventional chromatographic resins for purifying conjugate vaccines.

EXAMPLES

EXAMPLE 1

Purification of Salmonella Core-OPS based conjugates.

Salmonella Core-OPS (COPS) is the polysaccharide portion of lipopolysaccharide with the lipid-A group removed. Salmonella enterica serovar Enteritidis COPS was chemically conjugated with homologous strain Salmonella flagellin protein at a weight: weight polysaccharide to protein ratio of 10: 1 using CDAP chemistry as described by Lees et al. Vaccine 14(3) pp 190-198 (1996). This material was prepared as an unpurified mixture, containing both conjugated and unconjugated COPS. Analysis of the conjugate by SDS-PAGE revealed no detectable unconjugated protein. Protein linked carbohydrate COPS:Flagellin conjugate molecules were separated from unconjugated low molecular weight COPS species initially by size-exclusion chromatography (SEC) under FPLC conditions (AKTA Explorer) using Superdex 200 resin media (GE Amersham) in 10 mM Tris pH 7.5 at 0.5 ml/minute taking 1 ml fractions. FIG. 1A indicates the absorbance trace at OD280, of fractions eluting from the Superdex 200 column. As both protein as well as unconjugated CDAP labeled free polysaccharide absorb strongly at OD280, individual fractions were monitored by BCA assay for protein content (FIG. IB). Fractionation through Superdex 200 resulted in separation of higher molecular weight conjugated species from low molecular weight material. Fractions 9-15 containing protein including high molecular weight conjugated material were pooled and concentrated by 3 OKI) Molecular Weight Cutoff ultrafiltration (Millipore-Amicon). In order to remove unconjugated COPS present in the pooled and concentrated protein containing SEC fractions, 0.5 ml of concentrated protein containing SEC conjugate fractions in 10 mM Tris pH 7.5 were run through an anion-exchange chromatography using quaternary ammonium (Q) based membrane (Sartobind nano-Q, Sarotorius). Absorbance was monitored at OD280 in the flow through (FIG. 2A). Conjugates were loaded onto the column in 10 mM Tris pH 7.5 at 1 ml/minute, and subjected to a primary isocratic flow- through wash step with 5 column volumes (15 ml) of 10 mM Tris pH 7.5 at 1 ml/minute. This flow through and wash was collected as a single fraction and concentrated to 1 ml with a 30KD Molecular Weight Cutoff ultrafiltration (Millipore Amicon). The membrane was then subjected to an isocratic wash step with 10 column volumes of 10 mM Tris pH 7.5/80 mM NaCl (30 ml) at 1 ml/minute. This low salt wash fraction was collected in a single fraction and concentrated to 2.1 ml with a 3 OKI) Molecular Weight Cutoff ultrafiltration (Millipore-Amicon). An 80-1000 mM NaCl gradient in 10 mM Tris pH 7.5 in 8 column volumes (40 ml) at 1 ml/minute was then applied at 1 ml/minute with 1.2 ml fractions collected. Fractions 3-16 were concentrated to 1.6 ml with a 30KD Molecular Weight Cutoff ultrafiltration (Millipore-Amicon). Carbohydrate and protein levels in fractions were analyzed by resorcinol assay nomalized with Salmonella Enteritidis LPS standards (FIG. 2B), and BCA assay normalized to unconjugated flagellin protein standards (FIG. 2C) respectively. Significant amounts of polysaccharide but not protein were detected in the primary and secondary wash steps, with high levels of both protein and polysaccharide detected in the final elution step, indicating binding of conjugates to the membrane and removal of unconjugated polysaccharide in the wash steps at low salt concentrations, with efficient elution of the conjugate from the membrane at high salt concentrations. Thus the unconjugated carbohydrate is separated from the conjugated carbohydrate, resulting in conjugate with substantially reduced levels of free carbohydrate. This experiment has been repeated with CDAP linked COPS:Flagellin conjugates containing 1 :1 weightweight carbohydrate :protein ratios, CDAP linked COPS:CRM197 conjugates containing 1 :1 weightweight carbohydrate :protein ratios, and Aminoxy linked COPS:Flagellin conjugates containing 3 : 1 weightweight carbohydrate:protein ratios with comparable results.

EXAMPLE 2

High molecular weight conjugates do not bind well to conventional chromatography resins.

Bovine serum albumin (BSA) was chemically linked to 2000 kDa dextran using CDAP chemistry See, e.g., Lees, Andrew., Producing immunogenic constructs using soluble carbohydrates activated via organic cyanylating reagents, U.S. Patent Nos. 5,651,971 ; 5,693,326; and 5,849,301.

The conjugate was loaded onto a 5 ml Q fast flow column (GE Healthcare) or a Nano Q device (Sartorius), each equilibrated with 50 mM Tris, pH 8. Absorbance at 280 nm was monitored. Each was then eluted with buffer containing 1 M NaCl . As shown in FIG. 3, the first peak is the unbound material. The second peak is material eluted with NaCl. FIG. 3 indicates that the conjugate was poorly adsorbed to the resin but bound to the membrane. EXAMPLE 3

Binding of Pneumococcal Serotype 14 capsular polysaccharide to an anion exchange membrane.

Pneumococcal Serotype 14 capsular polysaccharide (Pnl4) is an example of a neutral polysaccharide (FIG. 4A). Unexpectedly, this neutral polysaccharide was found to bind to an anion exchange membrane and co-eluted with the conjugate (FIGS. 4B and C). Elution with sodium chloride suggests that this supposedly neutral polysaccharide is bound ionically to the membrane.

EXAMPLE 4

Separation of free carbohydrate from conjugate on an anion exchange membrane using a salt gradient.

A tetanus toxoid-PN14 conjugate, containing free polysaccharide, was applied to Nano Q ion exchange membrane device (Sartorius), equilibrated with 50 mM Tris, pH 8. The membrane was successfully eluted by increasing the sodium chloride concentration. FIG. 5 shows the elution profile. Protein, assayed using the Coomassie Plus assay (Pierce), is indicated in red (squares) and the carbohydrate, assayed using a resorcinol/sulfuric acid method (Monsigny et al, Anal Biochem 175:525 (1998)), is indicated in blue (diamonds). It is seen that much more carbohydrate is eluted at low salt (100 mM NaCl or less) and most of the protein conjugate is eluted at 150 mM or more NaCl. This provides a conjugate with significantly reduced free carbohydrate.

EXAMPLE 5

Spiking of conjugate with unconjugated polysacchride.

A 3 ml Sartobind Q device is equilibrated with 50 mM Tris pH 8 at a flow rate of 5 ml/min. 3 mg of BS A-dextran is applied, with and without the addition of 1.8 mg of unconjugated dextran. In each case, the membrane was eluted with a gradient to 1 M NaCl. The membrane was cleaned with 1 M NaOH between runs. Fractions were collected and each tube assayed for dextran using the resorcinol/sulfuric acid assay. The sample containing additional free dextran shows a large peak of dextran in the flow through. The eluant profile of the conjugate with and without free dextran overlapped, indicating that there was no additional unconjugated dextran eluting along with the conjugate.

While there have been shown and described what are presently believed to be the preferred embodiments of the present invention, those skilled in the art will realize that other and further embodiments can be made without departing from the spirit and scope of the invention described in this application, and this application includes all such modifications that are within the intended scope of the claims set forth herein. All patents and publications mentioned and/or cited herein are incorporated by reference to the same extent as if each individual publication was specifically and individually indicated as having been incorporated by reference in its entirety.

Claims

WHAT IS CLAIMED IS:

1. A method for removing free carbohydrate from a carbohydrate-protein conjugate composition, wherein the method comprises

i) contacting the composition with an ion exchange matrix, wherein the carbohydrate-protein conjugate binds the matrix;

ii) optionally eluting free carbohydrate bound to the matrix with one or more wash solutions; and

iii) eluting the carbohydrate-protein conjugate bound to the matrix with one or more eluent solutions,

wherein the free carbohydrate is separated from the carbohydrate-protein conjugate, resulting in a carbohydrate-protein conjugate with reduced levels of free carbohydrate, wherein the matrix is selected from the group consisting of an ion exchange membrane and an ion exchange monolith.

2. The method of claim 1 , wherein the free carbohydrate is eluted from the matrix due to a difference in the ionic strength of the wash solution of ii) and the matrix.

3. The method of any of claims 1 or 2, wherein the carbohydrate-protein conjugate is eluted from the matrix due to a difference in the ionic strength of the eluent solution of iii) and the matrix.

4. The method of any of claims 1-3, wherein the ionic strength of the wash solution of ii) is different than the ionic strength of the eluent solution of iii).

5. The method of any of claims 1-4, wherein the wash solution of ii) has an ionic strength that is lower than the eluent solution of iii).

6. The method of any of claims 1-5, wherein the free carbohydrate is eluted by a wash solution comprising a gradient of increasing ionic strength.

7. The method of any of claims 1-6, wherein the carbohydrate-protein conjugate is eluted by a solution comprising a gradient of increasing ionic strength.

8. The method of any of claims 1-7, wherein the wash solution of ii) or the eluent solution of iii) comprises a salt selected from the group consisting of NaCl, (NH₄)₂S04, NH₄C1 and combinations thereof.

9. The method of any of claims 1-8, wherein the one or more wash solutions of ii) comprises a salt concentration that ranges from about 1 mm to about 1000 mM.

10. The method of any of claims 1-9, wherein the one or more wash solutions of ii) comprises a solution of about 80 mM or less of NaCl.

11. The method of any of claims 1-10, wherein the one or more eluent solutions of iii) comprises a salt concentration of at least about 80 mM.

12. The method of any of claims 1-11, wherein the one or more eluent solutions of iii) comprises a salt concentration of at least about 80 mM NaCl.

13. The method of any of claims 1-12, wherein the one or more eluent solutions of iii) comprises a gradient of increasing salt concentration of NaCl of about 80 mM to about 1000 mM.

14. The method of claim 1 , wherein the free carbohydrate is eluted from the matrix due to a difference in the pH of the wash solution of ii) and the matrix.

15. The method of any of claims 1 or 14, wherein the carbohydrate-protein conjugate is eluted from the matrix due to a difference in the pH of the eluent solution of iii) and the matrix.

16. The method of any of claims 1, 14 or 15, wherein the pH of the wash solution of ii) is different than the pH of the eluent solution of iii).

17. The method of any of claims 1 or 14-16, wherein the wash solution of ii) comprises a gradient of increasing or decreasing pH.

18. The method of any of claims 1 or 14-17, wherein the eluent solution of iii) comprises a gradient of increasing or decreasing pH.

19. The method of any of claims 1-18, wherein the ion exchange matrix is an anionic membrane.

20. The method of any of claims 1-19, wherein the ion exchange matrix is a cationic membrane.

21. The method of any of claims 1-20, wherein the composition that contacts the matrix is a solution comprising 10 mM Tris buffer at pH 7.5.

22. The method of any of claims 1-21, wherein the matrix is washed and equilibrated with a solution prior to performing step ii).

23. The method of any of claims 1-13, wherein the wash solution of part ii) comprises 10 mM Tris/ 80 mM NaCl at pH 7.5.

24. The method of any one of claims 1-13 and 23, wherein the eluent solution of part iii) comprises 10 mM Tris/80-1000 mM NaCl at pH 7.5.

25. The method of any of claims 1-24, wherein the carbohydrate-protein conjugate composition is subjected to size-exclusion chromatography (SEC) before being contacted with the ion exchange matrix.

26. The method of any one of claims 1-25, wherein the carbohydrate-protein conjugate comprises a protein antigen from a pathogen.

27. The method of any one of claims 1-26, wherein the pathogen is selected from the group consisting of Salmonella, Shigella, Vibrio cholera, E. coli, Streptococcus pneumonia, Clostridium tetani, Corynebacterium diphtheria, Neisseria meningitidis and Haemophilus influenza.

28. The method of any one of claims 1-27, wherein the protein is selected from the group consisting of CRM197, flagellin, tetanus toxoid, diptheria toxoid, rEPA, Haemophilus influenza group B protein D, and Neisseria meningitides outer membrane proteins (OMP).

29. The method of any one of claims 1 -28, wherein the protein is from a

Salmonella enterica serovar.

30. The method of any one of claims 1-29, wherein the protein antigen is from a Salmonella enterica serovar selected from the group consisting of S. Typhimurium and S. Enteritidis.

31. The method of any of claims 1 -30, wherein the protein is a phase 1 flagella protein (FliC) or an antigenic fragment or derivative thereof from Salmonella enterica.

32. The method of any one of claims 1-31, wherein the carbohydrate-protein conjugate comprises a polysaccharide from a pathogen.

33. The method of any one of claims 1-32, wherein the polysaccharide is from a pathogen is selected from the group consisting of Salmonella, Shigella, Vibrio cholera, E. coli, Streptococcus pneumonia, Clostridium tetani, Corynebacterium diphtheria, Neisseria meningitidis and Haemophilus influenza.

34. The method of any of claims 1-33, wherein the carbohydrate is selected from the group consisting of lipopolysaccharide derived core-OPS (COPS), Salmonella Vi, Streptococcus pneumoniae capsular polysaccharides, Neisseria meningitidis A/C/Y/X/W135 capsular polysaccharides and Haemophilus influenzae group B polysaccharides.

35. The method of any of claims 1-34, wherein the carbohydrate is a core-OPS polysaccharide (COPS) from a pathogen selected from the group consisting of Salmonella, Shigella, Vibrio cholera and E. coli.

36. The method of any of claims 1-35, wherein the carbohydrate is a core-OPS polysaccharide (COPS) from a Salmonella enterica serovar selected from the group consisting of S. Typhimurium and S. Enteritidis.

37. The method of any one of claims 1-36 wherein the carbohydrate-protein conjugate is selected from the group consisting of a Salmonella enterica serovar conjugate comprising COPS-FliC, pneumococcal serotype 14 capsular polysaccharide-CRM197, pneumococcal serotype 14 capsular polysaccharide-tetanus toxoid, and pneumococcal capsular polysaccharide- flagellin.

38. The method of claim 37, wherein the COPS-FliC conjugate is from a Salmonella enterica serovar selected from the group consisting of S. Typhimurium and S. Enteritidis.

39. The method of any of claims 1-38, wherein the protein is covalently linked to the carbohydrate either directly or using a linker.

40. The method of any of claims 1-39, wherein said linker is selected from the group consisting of l -cyano-4-dimethylaminopyridinium tetrafluoroborate, adipic acid dihydrazide, ε-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone and p-nitrophenylethyl amine.

41. The method of any of claims 1 -40, wherein said linker is l-cyano-4- dimethylaminopyridinium tetrafluoroborate.

42. The method of any of claims 1 -41 , wherein the level of free carbohydrate in the conjugate composition is reduced by an amount selected from the group consisting of at least about 25%, at least about 35%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%o, and at least about 99%>.

43. A pharmaceutical composition comprising the carbohydrate-protein conjugate produced according to the method of any one of claims 1-42 and a pharmaceutically acceptable carrier.