RU2774891C2 - Increase in immunogenicity of streptococcus pneumoniae polysaccharide-protein conjugates - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to immunogenic compositions for the prevention of an invasive disease caused by S. pneumoniae. Proposed compositions essentially consist of or consist of S. pneumoniae polysaccharide of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, each of which is conjugated with CRM197. At the same time, a conjugation reaction for S. pneumoniae of serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F is carried out in an aprotic solvent, where the aprotic solvent is DMSO, and a conjugation reaction of S. pneumoniae of serotypes 1, 3, 4, 5, 9V, 14, 22F and 33F is carried out in an aqueous solvent.

EFFECT: proposed immunogenic compositions have increased immunogenicity.

4 cl, 3 dwg, 6 tbl, 6 ex

Description

FIELD OF THE INVENTION

The present invention relates to immunogenic compositions containing at least one Streptococcus pneumoniae polysaccharide conjugated to CRM ₁₉₇ by reductive amination in an aprotic solvent such as dimethyl sulfoxide (DMSO). The invention also relates to methods for enhancing the immunogenicity of immunogenic compositions comprising one or more polysaccharide-protein conjugates wherein one or more polysaccharides from S. pneumoniae bacterial capsules are conjugated to a carrier protein by reductive amination carried out in an aprotic solvent such as DMSO.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae is a Gram-positive bacterium and the most common cause of invasive bacterial diseases (such as pneumonia, bacteremia, meningitis, and otitis media) in newborns and young children. Pneumococcus is encapsulated in a chemically bound polysaccharide, which imparts serological specificity. There are over 90 known pneumococcal serotypes, and the capsule is a major determinant of pneumococcal virulence, as it the capsule not only protects the internal surface of bacteria from complement, but is also not very immunogenic in itself. Polysaccharides are T-independent antigens and cannot be processed or presented on MHC molecules for interaction with T cells. However, they may stimulate the immune system through an alternative mechanism involving cross-linking of surface receptors on B cells.

Children less than 2 years of age do not develop an immune response to most polysaccharide vaccines, so it is necessary to make polysaccharides immunogenic by chemical conjugation to a carrier protein. Coupling of a polysaccharide, a T-independent antigen, with a protein, a T-dependent antigen, confers T-dependent antigen properties to the polysaccharide, including isotype switching, affinity maturation, and memory induction.

There are many conjugation reactions used to covalently link polysaccharides to proteins. Three of the more commonly used methods include: 1) reductive amination, where an aldehyde or ketone group on one component of the reaction mixture reacts with an amino or hydrazide group on another component and the resulting C=N double bond is then reduced to a C-N single bond with a reducing agent; 2) conjugation by cyanylation, where the polysaccharide is activated with cyanogen bromide (CNBr) or 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) to insert the cyanate group into the hydroxyl group forming a covalent bond with the amino group or hydrazide group after addition of the protein component; and 3) a carbodiimide reaction wherein the carbodiimide activates a carboxyl group on one component of the conjugation reaction mixture and the activated carbonyl group reacts with an amino group or a hydrazide group on the other component. These reactions are also often used to activate the components of the conjugate prior to the conjugation reaction.

Reductive amination is used to conjugate S. pneumoniae polysaccharides. See, for example, US Patent No. 8192746, US Patent Application Publication No. 20170021006, and International Patent Application Publication Nos. WO2011/110381 and WO2015/110941. Reductive amination involves two steps: (1) oxidation of the antigen and (2) reduction of the antigen and carrier protein to produce a conjugate. The reduction step can be carried out in an aqueous solvent or an aprotic solvent such as DMSO. See, for example, International Patent Application Publication No. WO2016/113644.

SUMMARY OF THE INVENTION

The present invention relates to immunogenic compositions containing polysaccharides of one or more of serotypes 1, 2, 3, 4, 5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F and 38 S. pneumoniae conjugated to a carrier protein, wherein the conjugation reaction by which the polysaccharide is conjugated to carrier protein, carried out in an aprotic solvent. In one embodiment, for compositions containing identical serotypes, one or more serotypes prepared in an aprotic solvent have increased immunogenicity compared to the same one or more serotypes prepared under aqueous conditions.

The present invention relates to immunogenic compositions containing polysaccharide-protein conjugates derived from one or more serotypes 1, 2, 3, 4, 5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A , 15C, 16F, 17F, 18C, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F and 38 S. pneumoniae , conjugated with a carrier protein, where the polysaccharide- the protein is obtained by a method including the step of conjugating the polysaccharide with the carrier protein in an aprotic solvent.

The invention also relates to methods for conjugating a polysaccharide serotype 1, 2, 3, 4, 5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F, or 38 S. pneumoniae with a carrier protein comprising the step of conjugating the polysaccharide to the carrier protein in an aprotic solvent.

The invention also relates to methods of treating an individual using an immunogenic composition containing one or more polysaccharides of serotypes 1, 2, 3, 4, 5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F, or 38 S. pneumoniae conjugated to a carrier protein, where the polysaccharide is conjugated to the protein -carrier in an aprotic solvent.

In some embodiments, polysaccharides of one or more S. pneumoniae serotypes 1, 3, 4, 5, 9V, 11A, 12F, and 14 are conjugated to a carrier protein in an aprotic solvent. In some embodiments, polysaccharides of one or more serotypes 2, 6C, 6D, 7B, 7C, 8, 9N, 15A, 15C, 16F, 17F, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34 , 35A, 35B, 35F and 38 S. pneumoniae are conjugated to a carrier protein in an aprotic solvent.

In some embodiments, polysaccharides from one or more S. pneumoniae serotypes 3 and 18C are conjugated to a carrier protein in an aprotic solvent. In one aspect of this embodiment, S. pneumoniae serotype 3 polysaccharides are conjugated to a carrier protein in an aprotic solvent. In one aspect of this embodiment, S. pneumoniae serotype 18C polysaccharides are conjugated to a carrier protein in an aprotic solvent.

In some embodiments, the conjugation reaction used to conjugate the polysaccharide to the carrier protein is reductive amination.

In some embodiments, the aprotic solvent is DMSO.

In some embodiments, the carrier protein is CRM ₁₉₇ .

In some embodiments, DMSO-formed conjugates have a lysine loss greater than 5.0. In one aspect, the conjugates made in DMSO have a lysine loss value between 7.0 and 18.0, inclusive.

In some embodiments, the immunogenic composition further comprises polysaccharides of one or more S. pneumoniae serotypes 6A, 6B, 7F, 10A, 15B, 19A, 19F, 22F, 23F, and 33F conjugated to a carrier protein, wherein the conjugation reaction by which the polysaccharide is conjugated with a carrier protein, carried out in an aprotic solvent. In some aspects of this embodiment of the invention, the conjugation reaction is reductive amination. In some aspects, the aprotic solvent is DMSO. In some aspects, the carrier protein is CRM ₁₉₇ . In one aspect, the immunogenic composition comprises a polysaccharide of S. pneumoniae serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F conjugated to a carrier protein, wherein the conjugation reaction, whereby the polysaccharide of serotypes 6A, 6B, 7F, 18C, 19A, 19F or 23F S. pneumoniae is conjugated to a carrier protein, carried out in an aprotic solvent. In some aspects, the polysaccharide is derived from S. pneumoniae serotypes 18C, 19A, 19F, or 23F.

In some embodiments, the immunogenic compositions of the invention further comprise polysaccharides of one or more serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F , 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F and 38 S. pneumoniae conjugated to a carrier protein, wherein the conjugation reaction by which a polysaccharide of serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F or 38 S. pneumoniae are conjugated to a carrier protein, carried out in an aqueous solvent. In one aspect, 35-100% of the serotypes in the immunogenic composition are obtained by reductive amination under DMSO conditions, and the remaining polysaccharide-protein conjugates are obtained under aqueous conditions.

In one specific embodiment, the invention relates to an immunogenic composition consisting essentially of polysaccharides of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F S. pneumoniae conjugated with the CRM ₁₉₇ polysaccharide, where the conjugation reaction of S. pneumoniae serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F is carried out under DMSO conditions, and the conjugation reaction of serotypes 1, 3, 4, 5, 9V, 14, 22F and 33F S. pneumoniae carried out in an aqueous solvent, optionally additionally containing approximately 0.2% wt./about. PS-20.

The present invention also relates to methods for inducing a protective immune response in a human, comprising administering any of the immunogenic compositions of the invention. In some embodiments, the individual is 50 years of age or older and/or is immunocompromised. In some embodiments, the individual is 2 years old or less. In some embodiments, the individual is immunocompromised.

The present invention also relates to methods for achieving an enhanced immune response to a pneumococcal polysaccharide (PnPs)-protein conjugate vaccine comprising administering to an animal an immunogenic composition comprising polysaccharide-protein conjugates containing S. pneumoniae capsular polysaccharides from a first set of two or more pneumococcal serotypes, conjugated to one or more carrier proteins, where two or more polysaccharide-protein conjugates from the first set are obtained by reductive amination under DMSO conditions. In one embodiment, the implementation of the specified immune response is increased relative to the control animal, which is injected with an immunogenic composition, where one or more of the two or more conjugates of the polysaccharide-protein from the first set is obtained by reductive amination in aqueous conditions. In one embodiment, the control animal is a mouse. In another embodiment, the control animal is a human. In some embodiments, the methods use a pneumococcal polysaccharide-protein conjugate vaccine comprising additional polysaccharide-protein conjugates comprising S. pneumoniae capsular polysaccharides from a second set of pneumococcal serotypes conjugated to one or more carrier proteins, obtained by aqueous reductive amination , where the serotypes from the second set are different from the serotypes from the first set.

In some embodiments, the methods use a pneumococcal polysaccharide-protein conjugate vaccine wherein the pneumococcal serotypes are selected from serotypes 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V , 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 33F, 34, 35A , 35B, 35F and 38.

In some embodiments, the methods use a pneumococcal polysaccharide-protein conjugate vaccine, wherein the serotype 3 or 18C polysaccharide-protein conjugates are produced by reductive amination under DMSO conditions.

In some embodiments, the methods use a pneumococcal polysaccharide-protein conjugate vaccine wherein the polysaccharide-protein conjugates from the first set of pneumococcal serotypes are selected from serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F.

In some embodiments, the methods use a pneumococcal polysaccharide-protein conjugate vaccine, wherein the polysaccharide-protein conjugates from the first set of pneumococcal serotypes comprise serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F obtained by reductive amination under DMSO conditions, and polysaccharide-protein conjugates from the second set of serotypes are prepared under aqueous conditions.

In one specific embodiment, the methods use a pneumococcal polysaccharide-protein conjugate vaccine, wherein the polysaccharide-protein conjugates of serotypes 6A, 6B, 7F, 18C, 19A, 19F, and 23F are obtained by reductive amination under DMSO conditions, and the polysaccharide-protein conjugates of serotypes 1 , 3, 4, 5, 9V, 14, 22F and 33F are prepared under aqueous conditions.

In some embodiments, the methods use a pneumococcal polysaccharide-protein conjugate vaccine wherein 35-100% serotype polysaccharide-protein conjugates are made by reductive amination under DMSO conditions and the remaining polysaccharide-protein conjugates are made under aqueous conditions. In one aspect, the polysaccharide-protein conjugates of 45-80% of the serotypes are made by reductive amination under DMSO conditions, and the remaining polysaccharide-protein conjugates are made under aqueous conditions. In another aspect, 75-100% serotype polysaccharide-protein conjugates are made by reductive amination under DMSO conditions, and the remaining polysaccharide-protein conjugates are made under aqueous conditions.

In some embodiments, the methods use a pneumococcal polysaccharide-protein conjugate vaccine wherein the carrier protein is selected from the group consisting of Neisseria meningitidis outer membrane protein complex (OMPC), tetanus toxoid, diphtheria toxoid, protein D, and CRM ₁₉₇ . In one aspect, the carrier protein is CRM ₁₉₇ .

In some embodiments, the methods use a vaccine with a pneumococcal polysaccharide-protein conjugate, wherein the conjugates made by reductive amination under DMSO conditions have a higher proportion of glycopeptide bonds formed, as measured by protein lysine loss greater than 5.0. In one aspect of this embodiment, the conjugates made by reductive amination under DMSO conditions have a lysine loss value between 7.0 and 18, inclusive. In another aspect, conjugates made by reductive amination under DMSO conditions have a lysine loss greater than 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, or 10.0.

In another specific embodiment, the invention relates to a method for achieving an enhanced immune response to a pneumococcal polysaccharide (PnPs)-protein conjugate vaccine consisting essentially of polysaccharides of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14 , 18C, 19A, 19F, 22F, 23F, and 33F S. pneumoniae conjugated to a CRM ₁₉₇ polysaccharide, wherein the method comprises administering to a human an immunogenic composition comprising polysaccharide-protein conjugates from a first set and a second set of pneumococcal serotypes, wherein the first set of serotypes consists of 6A, 6B, 7F, 18C, 19A, 19F and 23F and is prepared by reductive amination under DMSO conditions and the second set of serotypes is 1, 3, 4, 5, 9V, 14, 22F and 33F and is prepared in water conditions.

In some embodiments, the increased immune response in animals vaccinated with the immunogenic compositions obtained by the methods of the invention is measured by geometric mean serum IgG or opsonophagocytic antibody titer. In one aspect increased immune response to pneumococcal serotype increased by 10% or more compared to the response to a polysaccharide-protein conjugate of the same pneumococcal serotype obtained in aquatic conditions. In one embodiment, the animal is a mouse. In another embodiment, the animal is a human.

In some embodiments, the methods are used on a human 50 years of age or older. In some embodiments, the methods are used on a human aged 2 years or less. In some embodiments, the methods are used on a person who is immunocompromised.

The invention also relates to methods for producing a pneumococcal polysaccharide-protein conjugate by reductive amination, including:

a) reacting a Streptococcus pneumoniae polysaccharide selected from serotypes 3, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F with an amount of an oxidizing agent (e.g. periodate) to produce an activated polysaccharide having an activation level from 0.05 to 0.22;

b) reacting the activated polysaccharide with a carrier protein in an aprotic solvent, optionally in the presence of a reducing agent, to obtain a polysaccharide-protein conjugate;

where the conjugate has a loss value of lysine from 7.0 to 18.0, inclusive.

In some embodiments, the activation level is between 0.09 and 0.22.

In some embodiments, the oxidizing agent is a periodate.

In some embodiments, the level of activation is measured by derivatization of aldehydes on a polysaccharide using thiosemicarbazide.

In some embodiments, the reducing agent is a cyanoborohydride salt, such as sodium cyanoborohydride.

In some embodiments, the carrier protein is selected from the group consisting of tetanus toxoid, diphtheria toxoid, and CRM ₁₉₇ . In one embodiment, the carrier protein is CRM ₁₉₇ .

The present invention also relates to a quantitative method for determining the level of aldehydes (i.e. the level of periodate activation) in an activated polysaccharide, comprising the steps:

a) derivatizing the activated polysaccharide to produce a derivatized polysaccharide by reaction with the derivatizing agent until it is complete (ie the reaction plateaus);

b) isolating the derivatized polysaccharide by high performance size exclusion chromatography (to remove unreacted derivatizing agent and matrix components);

c) quantitative analysis of UV absorption by the derivatized polysaccharide.

The derivatizing agent may be selected from the group consisting of thiosemicarbazide, thiosemicarbazide structural analogs, hydrazides, hydrazine, semicarbazide, semicarbazide structural analogs, aminooxy compounds, or aromatic amines.

In one embodiment, the quantitative analysis in step c) is carried out by comparison with a derivative standard. In one embodiment, the quantitative analysis in step c) is carried out by measuring relative to a predetermined extinction coefficient.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. The degree of conjugation in different lysine sites CRM ₁₉₇ determined by tryptic peptide mapping. 19A-CRM ₁₉₇ serotype polysaccharide conjugates obtained by reductive amination in aqueous solution or DMSO were digested with trypsin and analyzed by LC-UV-MS. Peptide signal loss was plotted against CRM ₁₉₇ controls with respect to conjugation sites.

Figure 2. Results of electrochemiluminescence (ECL) analysis of immunogenicity in groups in a study in mice comparing polysaccharide conjugates of serotype 3-CRM ₁₉₇ obtained by reductive amination in aqueous solution or DMSO. The conjugates are formulated with an aluminum phosphate (APA) adjuvant. Pre-vaccination (Pre) and post-dose 3 (PD3) results are shown. Also shown are the results for the control with APA only.

Figure 3. Results of analysis of opsophagocytic activity (OPA) after dose 3 in groups in a study in mice comparing polysaccharide conjugates of serotype 3-CRM ₁₉₇ obtained by reductive amination in aqueous solution or DMSO. Also shown are pre-vaccination (pre-immunization) OPA results and for APA-only controls.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to immunogenic compositions containing pneumococcal polysaccharide-protein conjugates, wherein the conjugates of at least one pneumococcal serotype are prepared by reductive amination in an aprotic solvent such as DMSO. The present invention is based in part on the discovery that the use of DMSO as solvent during reductive amination of polysaccharide-protein conjugates results in unexpectedly superior stability and increased immunogenicity for these serotypes relative to the same conjugates prepared under aqueous conditions. The present invention relates to the advantages of the DMSO solvent in increasing the covalent bonds of the polysaccharide to the protein through the direct consumption of lysine residues on the surface of the carrier protein. For most of the serotypes tested, "lysine loss" providing good immunogenicity (≥7.0) can be achieved with a lower level of polysaccharide activation (0.05 to 0.22) when conjugated in an aprotic solvent than in an aqueous buffer. Increased covalent binding has the immediate benefit of enhancing the stability of the polysaccharide-protein conjugate and enhancing the immune response to these particular DMSO-conjugated polysaccharide antigens.

Without wishing to be bound by any theory, it is believed that one possible mechanism for the increase in immunogenicity observed with DMSO-formed glycoconjugates involves an increase in the number of bonds between the carbohydrate (capsular polysaccharide) and lysine residues on the surface of the carrier protein, which would result in additional points of attachment between the protein and the polysaccharide to impart stability and counteract chemical depolymerization or breakdown of the peptide-carbohydrate bond. See, for example, Hsieh, Characterization of Saccharide-CRM ₁₉₇ Conjugate Vaccines in Brown F, Corbel M, Griffiths E (eds): Physico-Chemical Procedures for the Characterization of Vaccines. dev. Biol. Basel, Karger, 2000, vol. 103, pp. 93-104. An additional advantage of increasing the polysaccharide-protein bonds formed by conjugation in the DMSO solvent may be additional opportunities for successful presentation of the peptide-carbohydrate conjugate to T cells. It should be understood that as a result of genetic variability in the human population resulting in different properties and sensitivities of loading or binding to specific peptide sequences conjugated to carbohydrate antigens, additional attachment points on the carrier protein will increase the chances of successful presentation of the antigen on the APC surface, which makes possible T-dependent response to an otherwise T-independent antigen. Another possible mechanism for increasing the observed immunogenicity via conjugation in the DMSO solvent may be related to the denaturation of CRM ₁₉₇ in an organic solvent, which leads to the exposure of additional lysines to bind to the polysaccharide, increasing the chances of glycopeptide presentation on the APC surface for a T-dependent response to different peptide epitopes. See Avci et al ., 2011, Nature Medicine 17:1602-1610.

Another advantage of organic solvent conjugation, whereby denatured CRM ₁₉₇ is obtained in conjugates, may be reduced immune interference of antibodies against native CRM ₁₉₇ epitopes. An additional benefit of increasing the polysaccharide-protein bonds formed by conjugation in the DMSO solvent may be the formation of larger polysaccharide-protein conjugates, resulting in increased immunogenicity. The compositions of the invention are believed to provide significant benefits in terms of response induction in humans.

As shown in Example 5, a polysaccharide-protein conjugate prepared from S. pneumoniae serotype 3 by reductive amination in DMSO exhibits increased immunogenicity (compared to the same conjugate prepared by reductive amination in water) in a mouse model as measured by opsophagocytic activity (OPA). In addition, as shown in Example 6, a 15-valent pneumococcal conjugate vaccine containing seven serotypes obtained by reductive amination in DMSO (the remaining eight were obtained in aqueous solvent) tends to be better immunogenic in humans (with a superiority of 4 serotypes is statistically significant) for all seven serotypes prepared in DMSO compared to the corresponding 15-valent PCV in which all 15 serotypes were prepared in aqueous vehicle.

As shown in Example 4, plotting the reduction in peptide signal for lysine positions on the CRM ₁₉₇ protein in serotype 19A conjugates relative to possible conjugation sites (compared to control CRM197) revealed additional conjugation sites located in previously identified common human T cell peptide epitopes. (See Raju et al ., 1995, Eur. J. Immunol. 25:3207-3214, located at peptide 411-430 and peptide 431-450 of the CRM ₁₉₇ sequence). Thus, in some embodiments, the present invention also relates to immunogenic compositions comprising one or more polysaccharide-CRM ₁₉₇ conjugates, wherein at least one of the polysaccharide-CRM ₁₉₇ conjugates is prepared in an aprotic solvent, and wherein the conjugate prepared in an aprotic solvent is shows higher availability of lysine residues at amino acids 411-430 or 431-450 of CRM ₁₉₇ compared to the same conjugate prepared in an aqueous solvent. In some embodiments, the present invention also provides immunogenic compositions comprising one or more polysaccharide-CRM ₁₉₇ conjugates, wherein at least one of the polysaccharide-CRM ₁₉₇ conjugates is prepared in an aprotic solvent, and wherein one or more lysine residues at amino acids 411-430 or 431-450 CRM ₁₉₇ in the conjugate obtained in an aprotic solvent, more than 10% conjugated. In some embodiments, the present invention relates to methods for increasing the availability of lysine residues in CRM ₁₉₇ , particularly at amino acids 411-430 or 431-450 of CRM ₁₉₇ , comprising conjugating a polysaccharide to CRM ₁₉₇ in an aprotic solvent. In some aspects of this embodiment of the invention, one or more lysine residues at amino acids 411-430 or 431-450 of CRM ₁₉₇ in the conjugate prepared in an aprotic solvent are more than 10% conjugated. In these embodiments, the implementation of the polysaccharide can be obtained from any organism suitable for obtaining immunogenic compositions. In some aspects, the polysaccharide is obtained from N. meningitidis or S. pneumoniae . The polysaccharide can be obtained from any serotype of these organisms.

Within the scope of the invention, the terms "aqueous solvent" or "aqueous conditions" when used in relation to conjugation, such as reductive amination, refer to the use of water as a solvent for the conjugation reaction. Water may contain buffers and other components, with the exception of the organic solvent.

As used herein, the term "aprotic solvent" when used in relation to conjugation such as reductive amination refers to the use of a polar aprotic solvent or a combination of polar aprotic solvents as the solvent for the conjugation reaction. Non-limiting examples of polar aprotic solvents include dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and hexamethylphosphamide (HMPA). The aprotic solvent may contain some water, for example up to 1%, 2%, 5%, 10% or 20%.

Within the scope of the invention, the terms "DMSO solvent" and "DMSO conditions" are used interchangeably.

Within the scope of the invention, the term "comprises" when used in relation to an immunogenic composition of the invention refers to the inclusion of any other components (to be limited to the phrase "consisting of" in the case of a mixture of antigens), such as adjuvants and excipients. The term "consisting of" when used in relation to a multivalent mixture of polysaccharide-protein conjugates refers to a mixture containing specific S. pneumoniae polysaccharide-protein conjugates, but not other S. pneumoniae polysaccharide-protein conjugates of a different serotype.

Within the scope of the invention, the term "loss of lysine" refers to the consumption of lysine during conjugation, and it is determined by the difference between the average measured amount of lysine in the conjugate and the expected amount of lysine in the original protein. Example 4 describes one way to define "loss of lysine".

As used herein, the term "polysaccharide" is intended to include any antigenic saccharide element (or antigenic unit) commonly used in the field of immunological and bacterial vaccines, including, but not limited to, "saccharide", "oligosaccharide", "polysaccharide", "liposaccharide" , "lipooligosaccharide (LOS)", "lipopolysaccharide (LPS)", "glycosylate", "glycoconjugate" and the like.

With respect to the percentages of serotypes in an immunogenic composition prepared in an aprotic solvent (e.g., DMSO), if the remaining polysaccharide-protein conjugates are prepared under aqueous conditions, the percentages simply refer to the number of serotypes prepared in the aprotic solvent divided by the total number of serotypes in the composition. .

Within the scope of the invention, all ranges, such as pH, temperature and concentration, should be inclusive. For example, a pH range of 5.0 to 9.0 is intended to include pH 5.0 and pH 9.0. Similarly, the temperature range of 4 to 25°C is intended to include the outer limits of the range, i.e. 4°C and 25°C.

Polysaccharide

Capsular polysaccharides of S. pneumoniae, which can be obtained by the methods of the invention, i. by reductive amination in an aprotic solvent include, but are not limited to, serotypes: 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F and 38. The polysaccharides can be used in the form of oligosaccharides. They are conveniently obtained by fragmentation of the purified polysaccharide (for example, by hydrolysis), after which, as a rule, will carry out the purification of fragments of the desired size.

In some embodiments, one or more of serotypes 1, 2, 3, 4, 5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 20 , 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F and 38 are prepared by reductive amination in an aprotic solvent. In some aspects, pneumococcal polysaccharides of one or more of serotypes 1, 3, 4, 5, 9V, 11A, 12F, and 14 are obtained by reductive amination in an aprotic solvent. In some aspects, pneumococcal polysaccharides of one or more of serotypes 2, 6C, 6D, 7B, 7C, 8, 9N, 15A, 15C, 16F, 17F, 19F, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F and 38 are prepared by reductive amination in an aprotic solvent. In some aspects, pneumococcal polysaccharides from one or both of serotypes 3 or 18C are conjugated to a carrier protein by reductive amination in an aprotic solvent. Polysaccharides of other serotypes in a multivalent composition can be conjugated by reductive amination in an aprotic solvent or in an aqueous solvent. The polysaccharides of other serotypes in the multivalent composition may also be conjugated using other chemicals, which may be in an aprotic solvent or in an aqueous solvent.

Streptococcus pneumoniae capsular polysaccharides can be prepared by standard methods known to those skilled in the art. For example, polysaccharides can be isolated from bacteria and ground to some extent by known methods (see, for example, European Patent Nos. EP497524 and EP497525), preferably by microfluidization carried out using a homogenizer or chemical hydrolysis. In one embodiment, each pneumococcal polysaccharide serotype is grown in a soy medium. The individual polysaccharides are then purified using standard steps including centrifugation, settling and ultrafiltration. See, for example, US Patent Application Publication No. 2008/0286838 and US Patent No. 5,847,112. Polysaccharides can be milled to reduce the viscosity of polysaccharide samples and/or improve the filterability of conjugate products using methods such as mechanical or chemical milling. Chemical hydrolysis can be carried out using acetic acid. Mechanical grinding can be carried out using fragmentation through high pressure homogenization.

In some embodiments, the purified polysaccharides have a molecular weight of 5 kDa to 4000 kDa prior to conjugation. Molecular weight can be calculated by size exclusion chromatography (SEC) in combination with a multi-angle light scattering (MALS) detector and a refractive index detector (RI). In other such embodiments, the polysaccharide has a molecular weight of 10 kDa to 4000 kDa, 50 kDa to 4000 kDa, 50 kDa to 3000 kDa, 50 kDa to 2000 kDa, 50 kDa to 1500 kDa, 50 kDa to 1000 kDa , 50 kDa to 750 kDa, 50 kDa to 500 kDa, 100 kDa to 4000 kDa, 100 kDa to 3000 kDa, 100 kDa to 2000 kDa, 100 kDa to 1500 kDa, 100 kDa to 1000 kDa, 100 kDa to 750 kDa 100 kDa to 500 kDa 100 to 400 kDa 200 kDa to 4,000 kDa 200 kDa to 3000 kDa 200 kDa to 2000 kDa 200 kDa to 1500 kDa 200 kDa to 200 kDa kDa up to 1000 kDa or from 200 kDa to 500 kDa.

Purified polysaccharides can be chemically activated to render the saccharides reactive with the carrier protein. Purified polysaccharides can be combined with a linker. After activation or connection to a linker, each capsular polysaccharide is separately conjugated to a carrier protein to form a glycoconjugate. Polysaccharide conjugates can be prepared by known coupling methods.

The polysaccharide can be combined with a linker to form a polysaccharide-linker intermediate in which the free end of the linker is an ester group. Thus, a linker is a linker in which at least one end is an ester group. The other end is chosen such that it can react with the polysaccharide to form a polysaccharide-linker intermediate.

The polysaccharide can be connected to the linker using the primary amino group in the polysaccharide. In this case, the linker typically contains an ester group at both ends. This allows attachment by reaction of one of the ester groups with a primary amino group in the polysaccharide via nucleophilic substitution of the acyl. The reaction results in a polysaccharide-linker intermediate in which the polysaccharide is linked to the linker by an amide bond. Thus, the linker is a bifunctional linker providing a first ester group for reaction with the primary amino group on the polysaccharide and a second ester group for reaction with the primary amino group on the carrier molecule. A typical linker is N-hydroxysuccinimide ester of adipic acid (SIDEA).

The connection can also be made indirectly, i. e. with an additional linker used to derivatize the polysaccharide prior to linking to the linker.

The polysaccharide is coupled to an additional linker using a carbonyl group at the reducing end of the polysaccharide. This addition involves two steps: (a1) reaction of the carbonyl group with the additional linker and (a2) reaction of the free end of the additional linker with the linker. In these embodiments, the additional linker typically contains a primary amino group at both ends, thus allowing step (a1) to be performed by reacting the primary amino groups with the carbonyl group in the polysaccharide via reductive amination. A primary amino group is used which can react with the carbonyl group in the polysaccharide. Hydrazide groups or hydroxylamino groups are suitable for this. As a rule, the same primary amino group is located at both ends of the additional linker. The reaction results in a polysaccharide-additional linker intermediate in which the polysaccharide is coupled to the additional linker via a C-N bond.

The polysaccharide can be coupled to an additional linker using another group on the polysaccharide, in particular a carboxyl group. This addition involves two steps: (a1) reacting the group with an additional linker and (a2) reacting the free end of the additional linker with a linker. In this case, the additional linker typically contains a primary amino group at both ends, thus allowing step (a1) to be carried out by reacting one of the primary amino groups with a carboxyl group in the polysaccharide via EDAC activation. A primary amino group is used that can react with an EDAC-activated carboxyl group in the polysaccharide. A hydrazide group is suitable for this. As a rule, the same primary amino group is located at both ends of the additional linker. The reaction results in a polysaccharide-additional linker intermediate in which the polysaccharide is linked to the additional linker by an amide bond.

In one embodiment, chemical activation of polysaccharides and subsequent conjugation to a carrier protein via reductive amination can be achieved by the methods described in US Patent Nos. 0231340 and 2007/0184071 and International Patent Application Publication Nos. WO2006/110381, WO2008/079653 and WO2008/143709. The chemical method can cause activation of the pneumococcal polysaccharide by reaction with any oxidizing agent that oxidizes the hydroxyl group to an aldehyde, such as periodate (including sodium periodate, potassium periodate, or iodic acid). The reaction results in random oxidative cleavage of the vicinal hydroxyl groups of carbohydrates to form reactive aldehyde groups.

In one embodiment, the polysaccharide reacts with 0.01 to 10.0, 0.05 to 5.0, 0.1 to 1.0, 0.5 to 1.0, 0.7 to 0 ,8, 0.05 to 0.5, 0.1 to 0.3 molar equivalents of the oxidant. In an embodiment, the polysaccharide reacts with about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6 , 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 molar equivalents of oxidant. In general, it is preferable to use lower amounts of periodate for activation, for example 0.1 to 0.3 mEq, to achieve limited activation of the polysaccharide (for example, 0.05 to 0.22 or 0.09 to 0, 22 mol aldehyde/mol repeating polysaccharide subunit). Within the scope of the invention, the term "level of activation" refers to moles of aldehyde/moles of a repeating polysaccharide subunit. Less activation of the polysaccharide results in a polysaccharide that is more similar to the native polysaccharide, ie. a smaller number of hydroxyl groups is converted to aldehydes.

In another embodiment, the duration of the oxidation reaction is 1 to 50 hours, 10 to 30 hours, 15 to 20 hours, 15 to 17 hours, or about 16 hours.

In another embodiment, the oxidation reaction temperature is maintained at 15°C to 45°C, 15°C to 30°C, 20°C to 25°C. In another embodiment, the reaction temperature is maintained at about 23°C.

Attachment to the carrier protein is carried out by reductive amination by direct amination using lysyl groups of the protein. For example, conjugation is carried out by reacting a mixture of activated polysaccharide and carrier protein with a reducing agent such as sodium cyanoborohydride in the presence of nickel. The conjugation reaction can be carried out in an aqueous solution or in the presence of dimethyl sulfoxide (DMSO). See, for example, US Patent Application Publication Nos. 2015/0231270 and 2011/0195086 and European Patent No. EP0471177 B1. The unreacted aldehydes are then blocked by adding a strong reducing agent such as sodium borohydride.

Reductive amination involves two steps: (1) oxidation of the polysaccharide to produce reactive aldehydes and (2) reduction of the imine (Schiff base) formed between the activated polysaccharide and the carrier protein to produce a stable bond in the amine conjugate. The polysaccharide is optionally ground prior to oxidation. Mechanical methods (eg homogenization) or chemical hydrolysis can be used. Chemical hydrolysis can be carried out using acetic acid. The oxidation step may include reaction with periodate. Within the scope of the invention, the term "periodate" includes periodate and iodic acid; the term also includes metaperiodate (IO ₄ ^- ), orthoperiodate (IO ₆ ^- ) and various periodate salts (eg, sodium periodate and potassium periodate). In an embodiment, the capsular polysaccharide is oxidized in the presence of metaperiodate, preferably in the presence of sodium periodate (NaIO ₄ ). In another embodiment, the capsular polysaccharide is oxidized in the presence of orthoperiodate, preferably in the presence of iodic acid.

In an embodiment, the oxidizing agent is a stable compound with a nitroxyl or nitroxide radical, such as piperidine-N-hydroxy or pyrrolidine-N-hydroxy compounds, in the presence of an oxidizing agent to selectively oxidize primary hydroxyls (as described in WO 2014/097099). In this reaction, the particular oxidizing agent is the N-oxoammonium salt in the catalyst cycle. In one aspect, said stable compound with a nitroxyl or nitroxide radical is a piperidine-N-oxy or pyrrolidine-N-oxy compound. In one aspect, said stable compound with a nitroxyl or nitroxide radical carries a TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy group) or PROXYL (2,2,5,5-tetramethyl-1-pyrrolidinyloxy group) moiety. In one aspect, said stable compound with a nitroxyl radical is TEMPO or a derivative thereof. In one aspect, said oxidizing agent is a molecule bearing an N-halogen moiety. In one aspect, said oxidizing agent is selected from the group consisting of N-chlorosuccinimide, N-bromosuccinimide, N-iodosuccinimide, dichloroisocyanuric acid, 1,3,5-trichloro-1,3,5-triazinan-2,4,6-trione , dibromoisocyanuric acid, 1,3,5-tribromo-1,3,5-triazinan-2,4,6-trione, diiodioisocyanuric acid and 1,3,5-triiodo-1,3,5-triazinan-2,4 ,6-trion.

In some aspects, the oxidizing agent is a free 2,2,6,6-tetramethyl-1-piperidinoxy radical (TEMPO) and N-chlorosuccinimide (NCS) is used as the co-oxidizing agent (as described in International Patent Application Publication No. WO2014/097099 ). Thus, in one aspect, S. pneumoniae glycoconjugates are prepared by a process comprising the steps of: a) reacting a saccharide with a 2,2,6,6-tetramethyl-1-piperidinyloxy group (TEMPO) and N-chlorosuccinimide (NCS) in an aqueous solvent to obtain the activated saccharide; and b) reacting the activated saccharide with a carrier protein containing one or more amino groups (this method is hereinafter referred to as "TEMPO/NCS reductive amination").

Optionally, the oxidation reaction is quenched by adding an quencher. The quencher can be selected from vicinal diols, 1,2-amino alcohols, amino acids, glutathione, sulfite, bisulfates, dithionite, metabisulfite, thiosulfate, phosphites, hypophosphites or phosphorous acid, glycerin, ethylene glycol, propane-1,2-diol, butane-1, 2-diol or butane-2,3-diol, ascorbic acid.

The second step of the conjugation process is the reduction of the imine (Schiff base) bond between the activated polysaccharide and the carrier protein to obtain a stable bond in the conjugate (so-called reductive amination) using a reducing agent. Suitable reducing agents include cyanoborohydrides (such as sodium cyanoborohydride or sodium borohydride). In one embodiment, the reducing agent is sodium cyanoborohydride.

In some embodiments of the methods of the invention, the reductive amination reaction is carried out in an aprotic solvent (or a mixture of aprotic solvents). In an embodiment, the reduction reaction is carried out in a DMSO or DMF (dimethylformamide) solvent. DMSO or DMF solvent can be used to reconstitute the activated polysaccharide and carrier protein if lyophilized. In one embodiment, the aprotic solvent is DMSO.

At the end of the reduction reaction, unreacted aldehyde groups may remain in the conjugates, which can be blocked using a suitable blocking agent. In one embodiment, this blocking agent is sodium borohydride (NaBH ₄ ). Suitable alternatives include sodium triacetoxyborohydride or sodium or zinc borohydride in the presence of a Bronsted or Lewis acid, aminoboranes such as pyridine-borane, 2-picoline-borane, 2,6-diborane-methanol, dimethylamine-borane, t-BuMe'PrN- BH ₃ , benzylamine-BH ₃ , 5-ethyl-2-methylpyridine-borane (PEMB) or borohydride ion exchange resin. After conjugation (reduction and optionally blocking reactions), glycoconjugates can be purified (enriched in terms of the amount of polysaccharide-protein conjugate) by various methods known to the person skilled in the art. These methods include dialysis, concentration/diafiltration methods, tangential filtration, precipitation/elution, column chromatography (ion exchange chromatography, multimodal ion exchange chromatography, DEAE or hydrophobic interaction chromatography) and depth filtration. In an embodiment, the glycoconjugates are purified by diafiltration or ion exchange chromatography or size exclusion chromatography.

Glycoconjugates prepared by reductive amination in an aprotic solvent are generally used in pneumococcal conjugate multivalent vaccines. Thus, in some embodiments, in the case of multivalent compositions in which not all serotypes are prepared in an aprotic solvent, the reduction reaction of the remaining serotypes is carried out in an aqueous solvent (for example, selected from PBS (phosphate-buffered saline), MES (2-( N - morpholino)ethanesulfonic acid), HEPES, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), bis-tris, ADA (N-(2-acetamido)iminodiacetic acid), PIPES (piperazine-N,N'-bis (2-ethanesulfonic acid)), MOPSO (3-morpholino-2-hydroxypropanesulfonic acid), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid ), DIPSO (3-bis(2-hydroxyethyl)amino-2-hydroxypropane-1-sulfonic acid), MOBS (4-(N-morpholino)butanesulfonic acid), HEPPSO (N-(2-hydroxyethyl)piperazine-N- (2-hydroxypropanesulfonic acid)), POPSO (piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid)), TEA (triethanolamine), EPPS (4-(2-hydroxyethyl)piperazine-1 -propanesulfonic acid), bicine or HEPB at pH 6.0 to 8.5, 7.0 to 8.0, or 7.0 to 7.5).

In some embodiments, the implementation of the glycoconjugates of the present invention contain a polysaccharide having a molecular weight from 10 kDa to 10,000 kDa. In other such embodiments, the implementation of the polysaccharide has a molecular weight from 25 kDa to 5000 kDa. In other such embodiments, the implementation of the polysaccharide has a molecular weight from 50 kDa to 1000 kDa. In other such embodiments, the implementation of the polysaccharide has a molecular weight from 70 kDa to 900 kDa. In other such embodiments, the implementation of the polysaccharide has a molecular weight from 100 kDa to 800 kDa. In other such embodiments, the implementation of the polysaccharide has a molecular weight from 200 kDa to 600 kDa. In further such embodiments, the polysaccharide has a molecular weight of 100 kDa to 1000 kDa, 100 kDa to 900 kDa, 100 kDa to 800 kDa, 100 kDa to 700 kDa, 100 kDa to 600 kDa, 100 kDa to 500 kDa , 100 kDa to 400 kDa, 100 kDa to 300 kDa, 150 kDa to 1000 kDa, 150 kDa to 900 kDa, 150 kDa to 800 kDa, 150 kDa to 700 kDa, 150 kDa to 600 kDa, 150 kDa to 500 kDa 150 kDa to 400 kDa 150 kDa to 300 kDa 200 kDa to 1000 kDa 200 kDa to 900 kDa 200 kDa to 800 kDa 200 kDa to 700 kDa 200 kDa to 600 kDa, 200 kDa to 500 kDa, 200 kDa to 400 kDa, 200 kDa to 300 kDa, 250 kDa to 1000 kDa, 250 kDa to 900 kDa, 250 kDa to 800 kDa, 250 kDa to 800 kDa kDa to 700 kDa, 250 kDa to 600 kDa, 250 kDa to 500 kDa, 250 kDa to 400 kDa, 250 kDa to 350 kDa, 300 kDa to 1000 kDa, 300 kDa to 900 kDa, 300 kDa up to 800 kDa, 300 kDa to 700 kDa, 300 kDa to 600 kDa, 300 kDa to 500 kDa, 300 kDa to 400 kDa, 400 kDa to 1000 kDa, 400 kDa to 900 kDa, 400 kDa to 800 kDa, 400 kDa to 700 kDa, 400 kDa to 600 kDa, 500 kDa to 600 kDa.

In some embodiments, the implementation of the glycoconjugates of the present invention have a molecular weight from 1000 kDa to 10000 kDa. In other such embodiments, the implementation of the polysaccharide has a molecular weight from 1000 kDa to 7000 kDa. In other such embodiments, the implementation of the polysaccharide has a molecular weight from 1000 kDa to 6000 kDa.

In some embodiments, the conjugation reaction is carried out by reductive amination, where nickel is used to increase the efficiency of the conjugation reaction and to facilitate the removal of free cyanide. Transition metals are known to form stable cyanide complexes and improve reductive methylation of protein amino groups and formaldehyde when sodium cyanoborohydride is used (S Gidley et al ., Biochem J. 1982, 203: 331-334; Jentoft et al . Anal Biochem. 1980, 106 : 186-190). By complexing the residual inhibitory cyanide, the addition of nickel increases protein consumption during conjugation and results in larger, potentially more immunogenic conjugates.

Differences in initial levels of cyanide in batches of sodium cyanoborohydride reagent also result in inconsistent conjugation performance resulting in inconsistent product characteristics such as conjugate size and the ratio of polysaccharide to CRM ₁₉₇ in the conjugate. Nickel addition reduced conjugation variability due to cyanide complexing, eliminating batch differences in sodium cyanoborohydride.

Suitable alternative chemistries include activation of the saccharide with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form the cyanate ester. Thus, the activated saccharide can be linked to the amino group on the carrier protein directly or via a spacer (linker) group. For example, the spacer may be a cystamine or cysteamine to form a thiolated polysaccharide that can be coupled to the carrier via a thioether bond formed after reaction with a maleimide-activated carrier protein (for example, using GMBS) or a haloacetylated carrier protein (for example, with using iodoacetimide [eg ethyliodoacetimide HCl] or N-succinimidyl bromoacetate or SIAB or SIA or SBAP). For example, a cyanate ester (optionally produced by CDAP) is coupled to hexanediamine or adipic acid dihydrazide (ADH) and the amino-derivatized saccharide is conjugated to a carrier protein using a carbodiimide reaction (e.g., EDAC or EDC) through the carboxyl group on the protein -carrier. Such conjugates are described in International Patent Application Publication Nos. WO 93/15760, WO 95/08348 and WO 96/29094; and Chu et al ., 1983, Infect. Immunity 40:245-256.

Other suitable methods use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S--NHS, EDC, TSTU. Many of these are described in International Patent Publication No. WO 98/42721. The conjugation may include a carbonyl linker which can be prepared by reacting the free hydroxyl group of the saccharide with CDI (See Bethell et al ., 1979, J. Biol. Chem. 254:2572-4; Hearn et al ., 1981, J. Chromatogr. 218:509-18) followed by reaction with the protein to form a carbamate bond. This may include reduction of the anomeric end to the primary hydroxyl group, optional addition/deprotection of the primary hydroxyl group, reaction of the primary hydroxyl group with CDI to produce a CDI carbamate intermediate, and coupling the CDI carbamate intermediate to an amino group on the protein.

After the capsular polysaccharide has been conjugated to a carrier protein, the polysaccharide-protein conjugates are purified (enriched in terms of the amount of polysaccharide-protein conjugate) by one or more of various methods. Examples of these methods are well known to those skilled in the art and include concentration/diafiltration, ultrafiltration, precipitation/elution, column chromatography, and depth filtration. See, for example, US Pat. No. 6,146,902.

The glycoconjugates of the invention can also be characterized by the number of lysine residues in the carrier protein (eg CRM ₁₉₇ ) conjugated to the saccharide, which can be characterized as a range of conjugated lysines (degree of conjugation). Signs of lysine modification in the carrier protein due to covalent attachment to polysaccharides can be determined by amino acid analysis by conventional methods known to those skilled in the art. The conjugation results in a reduction in the amount of reduced lysine residues compared to the carrier protein starting material used to prepare the conjugate materials. In an embodiment, the degree of conjugation of the glycoconjugate of the invention is 2 to 18, 2 to 13, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, 3 to 15, 3 to 13 3 to 10 3 to 8 3 to 6 3 to 5 3 to 4 5 to 18 5 to 13 7 to 18 7 to 13 8 to 18, 8 to 13, 10 to 18, or 10 to 13. In an embodiment, the degree of conjugation of the glycoconjugate of the invention is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15. In an embodiment, the degree of conjugation of the glycoconjugate of the invention is from 7 to 18. In some such embodiments, the carrier protein is CRM ₁₉₇ .

The glycoconjugates of the invention can also be characterized by the ratio (w/w) of saccharide to carrier protein. In some embodiments, the ratio of polysaccharide to carrier protein in the glycoconjugate (w/w) is 0.5 to 3.0 (e.g., about 0.5, about 0.6, about 0.7, about 0.8 , about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8 , about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8 , about 2.9 or about 3.0). In other embodiments, the ratio of saccharide to carrier protein (w/w) is 0.5 to 2.0, 0.5 to 1.5, 0.8 to 1.2, 0.5 to 1.0, 1.0 to 1.5, or 1.0 to 2.0. In additional embodiments, the ratio of saccharide to carrier protein (w/w) is 0.8 to 1.2. In an embodiment, the ratio of capsular polysaccharide to carrier protein in the conjugate is between 0.9 and 1.1. In some such embodiments, the carrier protein is CRM ₁₉₇ . The glycoconjugates and immunogenic compositions of the invention may contain a free saccharide not covalently conjugated to the carrier protein but still present in the glycoconjugate composition. The free saccharide may be non-covalently bound (ie, non-covalently bound, adsorbed or entrapped) to the glycoconjugate.

In an embodiment, the glycoconjugate contains less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 15% free polysaccharide relative to total polysaccharide. In an embodiment, the glycoconjugate contains less than about 25% free polysaccharide relative to the total polysaccharide. In an embodiment, the glycoconjugate contains less than about 20% free polysaccharide relative to the total polysaccharide. In an embodiment, the glycoconjugate contains less than about 15% free polysaccharide relative to the total polysaccharide.

Multivalent vaccines with a polysaccharide-protein conjugate

Multivalent pneumococcal immunogenic compositions may contain capsular polysaccharides of S. pneumoniae serotype selected from at least one of 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7B, 7C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19A, 19F, 20, 21, 22A, 22F, 23A, 23B, 23F, 24F, 27, 28A, 31, 33F, 34, 35A, 35B, 35F and 38 conjugated to one or more carrier proteins, wherein the at least one serotype polysaccharide is obtained by reductive amination in an aprotic solvent such as DMSO. The present invention includes multivalent pneumococcal immunogenic compositions containing polysaccharides of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24 or 25 serotypes. Preferably, saccharides of a particular serotype are not conjugated to multiple carrier proteins.

In some embodiments, polysaccharides of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 serotypes obtained by reductive amination in an aprotic solvent such as DMSO.

In some embodiments, one or more of serotypes 3, 6A, 6B, 7F, 18C, 19A, 19F or 23F is obtained by reductive amination in an aprotic solvent. In some aspects of this embodiment of the invention, one or both of serotypes 3 or 18C is obtained by reductive amination in an aprotic solvent.

In some embodiments, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the serotypes in a multivalent composition are prepared in an aprotic solvent. The remaining serotypes are obtained using an alternative reaction and/or in an aqueous solvent.

In some embodiments, one or more of serotypes 1, 2, 3, 4, 5, 6C, 6D, 7B, 7C, 8, 9N, 9V, 11A, 12F, 14, 15A, 15C, 16F, 17F, 18C, 20 , 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35A, 35B, 35F and 38 are prepared by reductive amination in an aprotic solvent. In some aspects, one or more of serotypes 1, 3, 4, 5, 9V, 11A, 12F and 14 is obtained by reductive amination in an aprotic solvent. In some aspects, one or more of serotypes 2, 6C, 6D, 7B, 7C, 8, 9N, 15A, 15C, 16F, 17F, 19F, 20, 21, 22A, 23A, 23B, 24F, 27, 28A, 31, 34, 35B, 35F and 38 are prepared by reductive amination in an aprotic solvent.

In one embodiment, the multivalent composition consists of serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F polysaccharides obtained by reductive amination in an aprotic solvent such as DMSO and serotypes 1, 3, 4, 5, 9V polysaccharides, 14, 22F and 33F obtained by reductive amination in an aqueous solvent.

After purification of individual glycoconjugates, they are mixed to formulate the immunogenic composition of the present invention. These pneumococcal conjugates are prepared by separate methods and formulated together in unit dosage form.

carrier protein

In a specific embodiment of the present invention, CRM ₁₉₇ is used as a carrier protein. CRM ₁₉₇ is a non-toxic variant (ie, toxoid) of diphtheria toxin. In one embodiment, it is isolated from cultures of Corynebacterium diphtheria strain C7 (β197) grown in a medium based on casamino acids and yeast extract. In another embodiment, CRM ₁₉₇ is produced recombinantly by the methods described in US Pat. No. 5,614,382. Typically, CRM ₁₉₇ is purified using a combination of ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography. In some embodiments, CRM ₁₉₇ is produced in Pseudomonas fluorescens using Pfenex Expression Technology™ (Pfenex Inc., San Diego, CA).

Other suitable carrier proteins include additional inactivated bacterial toxins such as DT (diphtheria toxoid) or fragment B of DT (DTFB), TT (tetanus toxoid) or fragment C of TT, pertussis toxoid, cholera toxoid (e.g., as described in International Patent Application No. WO 2004/083251), E. coli LT, E. coli ST, and Pseudomonas aeruginosa exotoxin A. Bacterial outer membrane proteins such as outer membrane protein complex c (OMPC), porins, transferrin binding proteins, pneumococcal surface protein A (PspA; see International Patent Application Publication No. WO 02/091998), pneumococcal protein adhesin can also be used. (PsaA), peptidase C5a of group A or group B streptococci, or Haemophilus influenzae protein D, pneumococcal pneumolysin (Kuo et al ., 1995, Infect Immun 63; 2706-13), including ply detoxified in some way, e.g. dPLY-GMBS (see International Patent Application Publication No. WO 04/081515) or dPLY-formol, PhtX, including PhtA, PhtB, PhtD, PhtE and Pht protein fusion products, e.g. PhtDE fusion products, PhtBE fusion products (see international patent publications Application Nos. WO 01/98334 and WO 03/54007). Other proteins can also be used as carrier proteins, such as ovalbumin, saucer hemocyanin (KLH), bovine serum albumin (BSA) or purified tuberculin protein derivative (PPD), PorB (from N. meningitidis ), PD ( Haemophilus protein D influenzae ; see, for example, European Patent No. EP0594610 B) or their immunological functional equivalents, synthetic peptides (see European Patent Nos. EP0378881 and EP0427347), heat shock proteins (see International Patent Publication Nos. WO 93/17712 and WO 94/03208), pertussis proteins (see International Patent Publication No. WO 98/58668 and European Patent No. EP0471177), cytokines, lymphokines, growth factors or hormones (see International Patent Publication No. WO 91/01146 ), artificial proteins containing multiple human CD4+ T cell epitopes of various pathogen-derived antigens (see Falugi et al ., 2001, Eur J Immunol 31:3816-3824), such as the N19 protein (see Baraldoi et al ., 200 4, Infect Immun 72:4884-7), iron capture proteins (see International Patent Application Publication No. WO 01/72337), C. difficile toxin A or B (see International Patent Application Publication No. WO 00/61761) and flagellin (see Ben-Yedidia et al ., 1998, Immunol Lett 64 :9).

Other DT mutants such as CRM ₁₇₆ , CRM ₂₂₈ , CRM ₄₅ can be used as the second carrier protein (Uchida et al., 1973, J Biol Chem 218:3838-3844); CRM ₉ , CRM ₄₅ , CRM ₁₀₂ , CRM ₁₀₃ and CRM ₁₀₇ and other mutants described in Nicholls and Youle, Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc, 1992; variants with deletion or mutation of Glu-148 in Asp, Gln or Ser and/or Ala 158 in Gly and other mutations described in US patent No. 4709017 or US patent No. 4950740; variants with a mutation at least one or more of the residues of Lys 516, Lys 526, Phe 530 and/or Lys 534 and other mutations described in US patent No. 5917017 or US patent No. 6455673; or the fragment described in US Pat. No. 5,843,711. Such DT mutants can also be used to generate DTFB variants containing fragment B containing epitope regions.

When using multivalent vaccines, a second carrier may be used for one or more of the antigens in the multivalent vaccine. The second carrier protein is preferably a non-toxic and non-reactogenic protein that can be obtained in sufficient quantity and with sufficient purity. The second carrier protein is also conjugated or coupled to an antigen, such as a S. pneumoniae polysaccharide, to increase the immunogenicity of the antigen. Carrier proteins should be suitable for standard conjugation methods. In one embodiment, each capsular polysaccharide not conjugated to a first carrier protein is conjugated to the same second carrier protein (eg, each capsular polysaccharide molecule is conjugated to one carrier protein). In another embodiment, the capsular polysaccharides unconjugated to the first carrier protein are conjugated to two or more carrier proteins (each capsular polysaccharide molecule is conjugated to one carrier protein). In such embodiments, each capsular polysaccharide of one serotype is typically conjugated to one carrier protein.

Pharmaceutical/vaccine compositions

The present invention further relates to compositions, including pharmaceutical, immunogenic and vaccine compositions, comprising, essentially consisting of, or alternatively consisting of, any combination of the polysaccharide serotypes described above, together with a pharmaceutically acceptable carrier and adjuvant.

The formulation of the polysaccharide-protein conjugates of the present invention can be carried out by methods known in the art. For example, to obtain a composition, individual pneumococcal conjugates can be formulated with a physiologically acceptable carrier. Non-limiting examples of such carriers include water, buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol), and dextrose solutions.

In an embodiment, the vaccine composition is formulated in L-histidine buffer with sodium chloride.

In the context of the invention, an "adjuvant" is a substance used to increase the immunogenicity of an immunogenic composition of the invention. An immunological adjuvant may increase the immune response to an antigen that is weakly immunogenic when administered alone, such as non-inducing or inducing low antibody titers or a cell-mediated immune response, increase antibody titers against the antigen, and/or allow a reduction in the dose of the antigen that is effective in achieving an immune response. at the individual. Thus, adjuvants are often administered to enhance the immune response and are well known to those skilled in the art. Suitable adjuvants to improve the effectiveness of the composition include, as non-limiting examples:

(1) aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.;

(2) oil-in-water emulsion formulations (with or without other specific immunostimulatory agents such as muramyl peptides (defined below) or bacterial cell wall components), such as, for example, (a) MF59 (International Publication patent No. WO 90/14837) containing 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing varying amounts of MTP-PE) formulated in submicron particles using a microfluidizer such as the model microfluidizer 110Y (Microfluidics, Newton, MA), (b) SAF containing 10% squalene, 0.4% Tween 80, 5% Pluronic L121 and thr-MDP block copolymer microfluidized into a submicron emulsion or vortexed to form an emulsion with larger particles, (c) Ribi™ Adjuvant System (RAS) (Corixa, Hamilton, MT) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of 3-O- deacylated monophosphoryl lipid A (MPL™) described about in US patent No. 4912094, trehalose dimycolate (TDM) and cell wall cytoskeleton (CWS), preferably MPL+CWS (Detox™); and (d) montanide ISA;

(3) saponin adjuvants such as Quil A or STIMULON™ QS-21 (Antigenics, Framingham, MA) (see e.g. US Pat. No. 5,057,540) or particles derived therefrom such as ISCOM (immunostimulatory complexes, obtained by combining cholesterol, saponin, phospholipid and amphipathic proteins) and ^Iscomatrix® (having essentially the same structure as ISCOM, but without protein);

(4) bacterial lipopolysaccharides, synthetic analogs of lipid A such as aminoalkylglucosamine phosphate (AGP) compounds or derivatives or analogs thereof, available from Corixa and described in US Pat. No. 6,113,918; one such AGP is 2-[(R)-3-tetradecanoyloxytetradecanoylamino]ethyl-2-deoxy-4-O-phosphono-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-[(R)-3- -tetradecanoyloxytetradecanoylamino]-b-D-glucopyranoside, also known as 529 (formerly known as RC529), formulated in aqueous form or stable emulsion;

(5) synthetic polynucleotides such as oligonucleotides containing CpG motifs (US Pat. No. 6,207,646);

(6) cytokines such as interleukins (eg, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc. .), interferons (for example, interferon gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), costimulatory molecules B7-1 and B7-2, etc. d.; and

(7) a complement component, such as a C3d complement component trimer.

In another embodiment, the adjuvant is a mixture of 2, 3 or more of the above adjuvants, eg SBAS2 (oil-in-water emulsion also containing 3-deacylated monophosphoryl lipid A and QS21).

Muramyl peptides include, as non-limiting examples, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2-(1'-2'-dipalmitoyl-sn- glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

In some embodiments, the adjuvant is an aluminum salt. The aluminum salt adjuvant may be an alum-precipitated vaccine or an alum-adsorbed vaccine. Aluminum salt adjuvants are well known in the art and are described, for example, in Harlow, E. and D. Lane (1988; Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory) and Nicklas, W. (1992; Aluminum salts. Research in Immunology 143:489-493). The aluminum salt includes, but is not limited to, aluminum hydroxide, aluminum oxide hydrate, aluminum oxide trihydrate (ATH), aluminum hydrate, aluminum trihydrate, Alhydrogel, Superfos, Amphogel, aluminum(III) hydroxide, aluminum hydroxyphosphate sulfate, phosphate based adjuvant alumina (APA), amorphous alumina, alumina trihydrate, or aluminum trihydroxide.

APA is an aqueous suspension of aluminum hydroxyphosphate. APA is made by mixing aluminum chloride and sodium phosphate in a 1:1 volume ratio to precipitate aluminum hydroxyphosphate. After mixing, grind with a high shear mixer to obtain a monodisperse particle size distribution. The product is then diafiltered against saline and steam sterilized.

In some embodiments, commercially available Al(OH) ₃ (eg, Alhydrogel or Superfos Denmark/Accurate Chemical and Scientific Co., Westbury, NY) is used to adsorb proteins at a ratio of 50-200 g protein/mg aluminum hydroxide. In another embodiment, protein adsorption depends on the pI (isoelectric point) of the protein and the pH of the medium. A protein with a lower pI adsorbs to a positively charged aluminum ion more strongly than a protein with a higher pI. Aluminum salts can form a depot for the antigen, slowly released over 2-3 weeks, are involved in non-specific macrophage and complement activation, and/or stimulate the innate immune mechanism (possibly through stimulation of uric acid formation). See, for example, Lambrecht et al ., 2009, Curr Opin Immunol 21:23.

Monovalent large aqueous conjugates are typically mixed and diluted to a target concentration of 8 μg/ml for all serotypes except 6B, which will be diluted to a target concentration of 16 μg/ml. After reconstitution, the batch will be sterile filtered and an equal volume of aluminum phosphate adjuvant added aseptically to a target final aluminum concentration of 250 µg/mL. The formulated batch with adjuvant will be filled into disposable vials at 0.5 ml/dose.

In some embodiments, the adjuvant is a CpG-containing nucleotide sequence, for example, a CpG-containing oligonucleotide, in particular a CpG-containing oligodeoxynucleotide (CpG ODN). In another embodiment, the adjuvant is ODN 1826 available from Coley Pharmaceutical Group.

"CpG containing nucleotide", "CpG containing oligonucleotide", "CpG oligonucleotide" and similar terms refer to a 6-50 nucleotide long molecule containing an unmethylated CpG residue. See, for example, Wang et al ., 2003, Vaccine 21:4297. In another embodiment, any other definition of terms accepted in this area is meant. CpG-containing oligonucleotides include modified oligonucleotides that use any synthetic internucleoside linkages, modified bases and/or modified sugar.

Methods for using CpG oligonucleotides are well known in the art and are described, for example, in Sur et al ., 1999, J Immunol. 162:6284-93; Verthelyi, 2006, Methods Mol Med. 127:139-58; and Yasuda et al ., 2006, Crit Rev Ther Drug Carrier Syst. 23:89-110.

Administration/Dosage

Compositions and compositions of the present invention can be used to prevent or treat a person susceptible to infections, such as pneumococcal infection, by administering a vaccine by the systemic or transmucosal route. In one embodiment, the present invention relates to a method of inducing an immune response to a S. pneumoniae capsular polysaccharide conjugate comprising administering to a human an immunologically effective amount of an immunogenic composition of the present invention. In another embodiment, the present invention relates to a method of vaccinating a human against pneumococcal disease, comprising the step of administering to a human an immunologically effective amount of an immunogenic composition of the present invention.

The optimal amounts of components for a particular vaccine can be determined using standard studies, including the observation of appropriate immune responses in individuals. For example, in another embodiment, the human vaccination dose is determined by extrapolating animal data to human data. In another embodiment, the dose is determined empirically. The present inventors have shown that the vaccine is immunogenic using data from neonatal rhesus monkeys.

The term "effective amount" of a composition of the invention refers to the dose required to induce the production of antibodies significantly reducing the likelihood or degree of infectivity of a microorganism, eg S. pneumoniae , upon subsequent challenge with an antigen.

The methods of the invention can be used to prevent and/or reduce primary clinical syndromes caused by microorganisms such as S. pneumoniae , including invasive infections (meningitis, pneumonia and bacteremia) and non-invasive infections (acute otitis media and sinusitis).

Administration of the compositions of the invention may include one or more of: injection by intramuscular, intraperitoneal, intradermal, or subcutaneous routes, or transmucosal administration to the oral/alimentary, respiratory, or genitourinary tracts. In one embodiment, intranasal administration is used to treat pneumonia or otitis media (because nasopharyngeal carriage of pneumococci can be more effectively prevented, thus attenuating infection at a very early stage).

The amount of conjugate in each vaccine dose is chosen as the amount that elicits an immunoprotective response without significant adverse effects. This number may vary depending on the serotype of pneumococcus. In general, in the case of polysaccharide-based conjugates, each dose will contain from 0.1 to 100 micrograms of each polysaccharide, in particular from 0.1 to 10 micrograms, and more specifically from 1 to 5 micrograms. For example, each dose may contain 100, 150, 200, 250, 300, 400, 500 or 750 ng or 1, 1.5, 2, 3, 4, 5, 6, 7, 7.5, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 18, 20, 22, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mcg.

The optimal amounts of components for a particular vaccine can be determined using standard studies, including the observation of appropriate immune responses in individuals. For example, in another embodiment, the human vaccination dose is determined by extrapolating animal data to human data. In another embodiment, the dose is determined empirically.

In one embodiment, the aluminum salt dose is 10, 15, 20, 25, 30, 50, 70, 100, 125, 150, 200, 300, 500, or 700 μg, or 1, 1.2, 1.5, 2, 3.5 mg or more. In yet another embodiment, the dose of the aluminum salt described above is determined per μg of the recombinant protein.

In any of the methods of the present invention, and in one embodiment, the individual is a human. In some embodiments, the human is a neonate (less than 1 year of age), a toddler (about 12 to 24 months of age), or a toddler (about 2 to 5 years of age). In other embodiments, the human is an elderly individual (eg, >50 years of age or >65 years of age). The compositions of the present invention are also suitable for use in older children, adolescents and adults (eg 18 to 45 years of age or 18 to 65 years of age).

In one of the embodiments of the methods of the present invention, the composition of the present invention is administered as a single vaccination. In another embodiment, the vaccine is administered two, three or four or more times at sufficient time intervals. For example, the composition can be administered at intervals of 1, 2, 3, 4, 5, or 6 months, or any combination thereof. The immunization schedule may be similar to those for pneumococcal vaccines. For example, a common regimen for neonates and toddlers against invasive S. pneumoniae disease is administration at 2, 4, 6, and 12-15 months of age. Thus, in an embodiment, the composition is administered as a sequence of 4 doses at 2, 4, 6, and 12-15 months of age.

Compositions of the present invention may also include one or more S. pneumoniae proteins. Examples of S. pneumoniae proteins suitable for inclusion in compositions include those defined in International Patent Application Publication Nos. WO 02/083855 and WO 02/053761.

Lineups

Compositions of the invention may be administered to an individual by one or more routes known to those skilled in the art, such as parenteral, transdermal, transdermal, intramuscular, intravenous, intradermal, intranasal, subcutaneous, intraperitoneal, and formulated as appropriate.

In one embodiment, the compositions of the present invention are administered by epidermal liquid formulation injection, intramuscular injection, intravenous, intra-arterial, subcutaneous, or respiratory mucosal injection. Liquid formulations for injection include solutions and the like.

The composition of the invention can be formulated in disposable vials, reusable vials or pre-filled syringes.

In another embodiment, the compositions of the present invention are administered orally and are thus formulated to be suitable for oral administration, ie. in the form of a solid or liquid preparation. Solid oral formulations include tablets, capsules, pills, granules, pellets, and the like. Liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils, and the like.

Pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are oils of animal, vegetable or synthetic origin, such as peanut oil, soybean oil, olive oil, sunflower oil, fish oil, oil from other marine animals or lipid from milk or eggs.

The pharmaceutical composition may be isotonic, hypotonic or hypertonic. However, it is often preferred that the pharmaceutical composition for infusion or injection be substantially isotonic when administered. Thus, in case of storage, the pharmaceutical composition may preferably be isotonic or hypertonic. If the pharmaceutical composition is hypertonic on storage, it may be diluted prior to administration so that it becomes an isotonic solution.

The isotonic agent may be an ionic isotonic agent such as a salt or a non-ionic isotonic agent such as a carbohydrate. Non-limiting examples of ionic isotonic agents include NaCl, CaCl ₂ , KCl and MgCl ₂ . Non-limiting examples of non-ionic isotonic agents include mannitol, sorbitol, and glycerin.

Also preferably, at least one pharmaceutically acceptable additive is a buffer. For some purposes, for example, if the pharmaceutical composition is intended for infusion or injection, it is often desirable that the composition contains a buffer with which the solution can be buffered to a pH in the range of 4 to 10, such as 5 to 9, such as 6 to eight.

The buffer, for example, can be selected from the group consisting of Tris, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate and triethanolamine buffer.

In addition, the buffer, for example, can be selected from USP-compatible buffers for parenteral use, in particular if the pharmaceutical composition is intended for parenteral use. For example, the buffer may be selected from the group consisting of monobasic acids such as acetic, benzoic, gluconic, glyceric and lactic acids; dibasic acids such as aconitic, adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric acids; polybasic acids such as citric and phosphoric acids; and bases such as ammonia, diethanolamine, glycine, triethanolamine and Tris.

Parenteral vehicles (for subcutaneous, intravenous, intra-arterial, or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, and fixed oils. Intravenous vehicles include fluids and nutrient supplements, electrolyte supplements such as Ringer's dextrose supplements, and the like. Examples are sterile liquids such as water and oils with or without the addition of a surfactant and other pharmaceutically acceptable excipients. In general, the preferred liquid carriers, particularly for injectables, are water, saline, aqueous dextrose and related sugar solutions, glycols such as propylene glycol or polyethylene glycol, polysorbate 80 (PS-80), polysorbate 20 (PS-20 ) and poloxamer 188 (P188). Examples of oils are oils of animal, vegetable or synthetic origin, such as peanut oil, soybean oil, olive oil, sunflower oil, fish oil, oil from other marine animals or lipid from milk or eggs.

The compositions of the invention may also contain a surfactant. Surfactants include, as non-limiting examples: surfactants based on esters of polyoxyethylene and sorbitan (generally referred to as Tween), in particular, PS-20 and PS-80; ethylene oxide (EO), propylene oxide (PO) and/or butylene oxide (BO) copolymers sold under the trade name DOWFAX™, such as EO/PO linear block copolymers; octoxynols, the number of repeating ethoxy groups (oxy-1,2-ethanediyl) in which may vary, while octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) is of particular interest; (octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin); nonylphenol ethoxylates such as the Tergitol™ NP series; polyoxyethylene fatty acid ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as triethylene glycol monolauryl ether (Brij 30); and esters of sorbitan (known as SPAN) such as sorbitan trioleate (Span 85) and sorbitan monolaurate. The preferred surfactant for inclusion in the emulsion is PS-80.

Mixtures of surfactants can be used, such as PS-80/Span 85 mixtures. Also suitable is a combination of a polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan monooleate (PS-80) and an octoxynol such as t-octylphenoxypolyethoxyethanol (Triton X-100). Another combination that can be used contains an ester of polyoxyethylene and laureth-9 plus sorbitan and/or octoxynol.

Preferred amounts of surfactants (% by weight) are: esters of polyoxyethylene and sorbitan (such as PS-80) - from 0.01 to 1%, in particular about 0.1%; octyl- or nonylphenoxypolyoxyethanols (such as Triton X-100 or other detergents of the Triton series) from 0.001 to 0.1%, in particular from 0.005 to 0.02%; polyoxyethylene ethers (such as laureth-9) 0.1 to 20%, preferably 0.1 to 10%, and in particular 0.1 to 1% or about 0.5%.

In some embodiments, the composition consists essentially of histidine (20 mM), saline (150 mM) and 0.02% PS-20 or 0.04% PS-80 at pH 5.8 with 250 μg/ml APA (Aluminum Phosphate Adjuvant). The amount of PS-20 can range from 0.005% to 0.1% (w/v), while the presence of PS-20 or PS-80 in the composition allows you to control aggregation during simulated production and transportation using primary packaging. The method consists of combining a mixture of up to 24 serotypes in histidine, saline, and PS-20 or PS-80, and then combining this mixed material with APA and saline with or without antimicrobial preservatives.

For various drug products and drug substances, optimization of the choice of surfactant may be required. In the case of multivalent vaccines containing 15 or more serotypes, PS-20 and P188 are preferred. The choice of chemical reaction used to produce the conjugate can also play an important role in stabilizing the composition. In particular, it has been found that when the conjugation reactions used to prepare various polysaccharide-protein conjugates in a multivalent composition involve the use of an aqueous solvent and a DMSO solvent, specific surfactant systems result in significant differences in stability. Improved stability of polysaccharide-protein conjugates was observed when using polysorbate 20 alone or poloxamer 188 in combination with a polyol.

The specific mechanism of protection by a particular detergent of a biotherapeutic agent is not understood and cannot be predicted a priori. Possible stabilization mechanisms include preferential hydration, preferential exclusion, competition at the air/liquid interface between the biotherapeutic agent and the surface, surface tension, and/or direct binding of the detergent to the biotherapeutic agent to mask hydrophobic sites serving as a seed for aggregation.

Poloxamer can also be used in the compositions of the invention. Poloxamer is a non-ionic triblock copolymer consisting of a central hydrophobic polyoxypropylene (poly(propylene oxide)) chain flanked by two hydrophilic polyoxyethylene (poly(ethylene oxide)) chains. Poloxamers are also known under the trade name Pluronic ^® . Because the length of the polymer blocks can be adapted, there are many different poloxamers with slightly different properties. Because the generic term is "poloxamer", these copolymers are commonly referred to by the letter "P" (for "poloxamer") and three digits, the first two digits times 100 is the approximate molecular weight of the polyoxypropylene core, and the last digit times 10 is percentage of polyoxyethylene content (eg P407=Poloxamer with a polyoxypropylene molecular weight of 4000 g/mol and a polyoxyethylene content of 70%). In the case of the trade name Pluronic ^® , the coding of these copolymers begins with a letter defining its physical form at room temperature (L=liquid, P=paste, F=crumb (solid)), followed by two or three digits. The first digit (two digits in three-digit format) in the numerical designation, multiplied by 300, indicates the approximate molecular weight of the hydrophobic substance; and the last figure multiplied by 10 is the percentage of polyoxyethylene content (eg ^L61 =Pluronic® with a polyoxypropylene molecular weight of 1800 g/mol and a polyoxyethylene content of 10%). See US Pat. No. 3,740,421.

Examples of poloxamers having the general formula:

HO(C ₂ H ₄ O) _a (C ₃ H ₆ O) _b (C ₂ H ₄ O) _a H,

where blocks a and b have the following meanings:

Pluronic ^® Poloxamer a b Molecular mass L31 2 16 1100 (medium) L35 1900 (middle) L44NF 124 12 twenty 2090-2360 L64 2900 (medium) L81 2800 (medium) L121 4400 (medium) P123 twenty 70 5750 (medium) F68NF 188 80 27 7680-9510 F87NF 237 64 37 6840-8830 F108NF 338 141 44 12700-17400 F127NF 407 101 56 9840-14600

Within the scope of the invention, the units of molecular weight are daltons (Da) or g/mol.

Preferably, the poloxamer typically has a molecular weight in the range of 1100 to 17400 Da, 7500 to 15000 Da, or 7500 to 10000 Da. The poloxamer can be selected from poloxamer 188 or poloxamer 407. The final concentration of poloxamer in the formulations ranges from 0.001% to 5% w/v. or from 0.025% to 1% w/v. In some aspects, the polyol is propylene glycol, and its final concentration is from 1% to 20% wt./about. In some aspects, the polyol is polyethylene glycol 400, and its final concentration is from 1% to 20% wt./about.

Suitable polyols for the compositions of the invention are polymer polyols, in particular polyester diols, including, but not limited to, propylene glycol and polyethylene glycol, polyethylene glycol monomethyl ethers. Available propylene glycol with a range of monomer molecular weights from ~425 to ~2700. Also available are polyethylene glycol and polyethylene glycol monomethyl ether with a molecular weight range of ~200 to ~35,000, including, but not limited to, PEG200, PEG300, PEG400, PEG1000, PEG MME 550, PEG MME 600, PEG MME 2000, PEG MME 3350, and PEG MME 4000. The preferred polyethylene glycol is polyethylene glycol 400. The final concentration of the polyol in the compositions according to the invention can be from 1% to 20% wt./about. or 6% to 20% wt./about.

The composition also contains pH-buffered saline. The buffer, for example, can be selected from the group consisting of Tris, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate buffer, HEPES buffer (4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid) and triethanolamine buffer. The buffer can buffer the solution to a pH in the range of 4 to 10, 5.2 to 7.5, or 5.8 to 7.0. In a certain aspect of the invention, the buffer is selected from the group consisting of phosphate, succinate, histidine, MES, MOPS, HEPES, acetate, or citrate. In addition, the buffer, for example, can be selected from USP-compatible buffers for parenteral use, in particular if the pharmaceutical composition is intended for parenteral use. Buffer concentrations will range from 1 mM to 50 mM, or from 5 mM to 50 mM. In some aspects, the buffer is histidine at a final concentration of 5 mM to 50 mM or succinate at a final concentration of 1 mM to 10 mM. In some aspects, the histidine is at a final concentration of 20 mM ± 2 mM.

Although physiological saline (ie, a solution containing NaCl) is preferred, other salts suitable for formulation include, but are not limited to, CaCl ₂ , KCl, MgCl ₂ and combinations thereof. Instead of salt, non-ionic isotonic agents can be used, including, but not limited to, sucrose, trehalose, mannitol, sorbitol, and glycerin. Suitable salt concentration ranges include, but are not limited to, 25 mM to 500 mM or 40 mM to 170 mM. In one aspect, the saline solution is a NaCl solution, optionally at a concentration of 20 mM to 170 mM.

In an embodiment, the formulations contain L-histidine buffer with sodium chloride.

In some embodiments of the formulations provided herein, the polysaccharide-protein conjugates comprise one or more pneumococcal polysaccharides conjugated to a carrier protein. The carrier protein can be selected from CRM ₁₉₇ , diphtheria toxin B fragment (DTFB), DTFBC8, diphtheria toxoid (DT), tetanus toxoid (TT), TT fragment C, pertussis toxoid, cholera toxoid, E. coli LT, E. coli ST , Pseudomonas aeruginosa exotoxin A, and combinations thereof. In one aspect, all of the polysaccharide-protein conjugates are prepared using an aqueous chemical reaction. In another aspect, one or more of the polysaccharide-protein conjugates are prepared using a DMSO solvent. For example, the polysaccharide-protein conjugate composition may be a 15-valent formulation of pneumococcal conjugate (15vPnC), where polysaccharide-protein conjugates of serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F are prepared using a DMSO solvent, and polysaccharide-protein conjugates of serotypes 1, 3, 4, 5, 9V, 14, 22F and 33F were prepared using an aqueous solvent.

In another embodiment, the pharmaceutical composition is administered in a controlled release system. For example, the agent can be administered using intravenous infusion, a transdermal patch, liposomes, or other routes of administration. In another embodiment, polymeric materials are used, for example in microspheres or an implant.

Compositions of the present invention may also include one or more S. pneumoniae proteins. Examples of S. pneumoniae proteins suitable for inclusion include those defined in International Patent Application Publication Nos. WO 02/083855 and WO 02/053761.

Although various embodiments of the invention are described with reference to the accompanying description and drawings, it should be understood that the invention is not limited to these specific embodiments, and that a person skilled in the art can make various changes and modifications to the invention without deviating from its scope or spirit as defined in the claims inventions.

The following examples are presented for illustrative purposes and not to limit the invention.

EXAMPLES

EXAMPLE 1 Preparation of capsular polysaccharides S. pneumoniae

Methods for cultivating pneumococci are well known in the art. See, for example, Chase, 1967, Methods of Immunology and Immunochemistry 1:52. Methods for preparing pneumococcal capsular polysaccharides are also well known in the art. See, for example, European Patent No. EP0497524. Pneumococcal subtype isolates are available from the American Type Culture Collection (Manassas, VA). The bacteria are identified as encapsulated, non-motile, Gram-positive, lanceolate diplococci that are alpha-hemolytic on blood agar. Subtypes can be differentiated by the capsule swelling test using specific antisera. See, for example, US Pat. No. 5,847,112.

Cell banks representing each of the available S. pneumococcus serotypes were obtained from Merck Culture Collection (Rahway, NJ) in frozen vials. The thawed seed culture was transferred to an seed fermenter containing pre-sterilized growth media suitable for S. pneumoniae . The culture was grown in a seed fermenter with temperature and pH control. The entire volume of the seed fermenter was transferred to the production fermenter containing pre-sterilized growth media. Production fermentation was the final stage of cell cultivation. The temperature, pH and stirring speed were controlled.

The fermentation was stopped by adding an inactivating agent. After inactivation, the batch was transferred to an inactivation tank where it was kept at a controlled temperature and agitation speed. Cellular debris was removed using a combination of centrifugation and filtration. The batch was subjected to ultrafiltration and diafiltration. The batch was then subjected to fractionation with a solvent, which removes impurities and isolation of the polysaccharide.

EXAMPLE 2 Conjugation of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F with CRM ₁₉₇ via reductive amination in aqueous solution

Different polysaccharide serotypes were separately conjugated to purified CRM ₁₉₇ carrier protein using a common workflow. The polysaccharide was dissolved, ground, chemically activated and subjected to a buffer exchange by ultrafiltration. The purified CRM ₁₉₇ was then conjugated to the activated polysaccharide using NiCl ₂ (2 mM) in the reaction mixture and the resulting conjugate was purified by ultrafiltration before final filtration with a 0.2 µm filter. Several process parameters at each stage, such as pH, temperature, concentration, and time, were controlled to maintain the serotype-specific values defined in the section below.

Grinding and oxidation of the polysaccharide

Purified pneumococcal capsular polysaccharide powder was dissolved in water and all serotypes except serotype 19A were filtered with a 0.45 µm filter. All serotypes, with the exception of serotype 19A, were homogenized to reduce the molecular weight of the polysaccharide. Serotype 19A was not minced due to its relatively small initial size. The homogenization pressure and the number of passes through the homogenizer were controlled at the level of serotype-specific target values (150-1000 bar; 4-7 passes) to achieve serotype-specific molecular weight. The milled polysaccharide was filtered with a 0.2 μm filter and then concentrated and diafiltered against water using a 10 kDa NMWCO tangential filtration membrane.

The polysaccharide solution is then adjusted to serotype-specific temperature (4-22° C.) and pH (4-5) with sodium acetate buffer to minimize polysaccharide size reduction due to activation. For all serotypes (except serotype 4), polysaccharide activation was initiated by adding a 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was serotype specific and ranged from about 0.1 to 0.5 moles of sodium metaperiodate per mole of polysaccharide repeating unit. The serotype-specific charge of sodium metaperiodate is designed to achieve a target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeating unit). For serotype 4, the batch was incubated at approximately 50° C. and pH 4.1 prior to addition of sodium metaperiodate to partially deketalize the polysaccharide.

For all serotypes except serotypes 5 and 7F, the activated product was diafiltered against 10 mM potassium phosphate, pH 6.4, using a 10 kDa NMWCO tangential filtration membrane. Serotypes 5 and 7F were diafiltered against 10 mM sodium acetate. Ultrafiltration of all serotypes was carried out at 2-8°C.

Conjugation of polysaccharide with CRM ₁₉₇

The oxidized polysaccharide solution was mixed with water and 1.5 M potassium phosphate, pH 6.0 or pH 7.0, depending on the serotype. The selected buffer pH is designed to improve the stability of the activated polysaccharide during the conjugation reaction. Purified CRM ₁₉₇ obtained by expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1) was filtered with a 0.2 μm filter and combined with a buffered polysaccharide solution at a weight ratio of polysaccharide and CRM ₁₉₇ ranging from 0.4 to 1.0 w/w depending on the serotype. The weight ratio was chosen to control the ratio of polysaccharide and CRM ₁₉₇ in the resulting conjugate. The concentrations of polysaccharide and phosphate were serotype-specific and ranged from 3.6 to 10.0 g/l and from 100 to 150 mm, respectively, depending on the serotype. The serotype-specific concentration of the polysaccharide was chosen to control the size of the resulting conjugate. The solution was then filtered with a 0.2 µm filter. Nickel chloride was added to a concentration of approximately 2 mM using a 100 mM nickel chloride solution. Sodium cyanoborohydride (2 moles per mole repeating unit of polysaccharide) was added. Conjugation was performed over a serotype-specific time period (72 to 120 hours) to maximize polysaccharide and protein consumption.

Recovery with sodium borohydride

After the conjugation reaction, the batch was diluted to a polysaccharide concentration of approximately 3.5 g/l, cooled to 2-8°C and filtered using a 1.2 μm filter. All serotypes (except serotype 5) were diafiltered against 100 mM potassium phosphate, pH 7.0 at 2-8° C. using a 100 kDa cutoff NMWCO tangential filtration membrane. The batch isolated in the retentate was then diluted to approximately 2.0 g polysaccharide/l and the pH was adjusted by adding 1.2 M sodium bicarbonate, pH 9.4. Sodium borohydride (1 mol per mol polysaccharide repeating unit) was added. Later, 1.5 M potassium phosphate, pH 6.0, was added. Serotype 5 was diafiltered against 300 mM potassium phosphate using a 100 kDa NMWCO tangential filtration membrane.

Final filtration and product storage

The batch was then concentrated and diafiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0 at 4° C. using a 300 kDa cutoff NMWCO tangential filtration membrane. The batch of retentate was filtered with a 0.2 µm filter.

Serotype 19F conjugate was incubated for approximately 7 days at 22° C., diafiltered against 10 mM L-histidine in 150 mM sodium chloride, pH 7.0, at 4° C. using a 100 kDa cutoff NMWCO tangential filtration membrane, and filtered with a 0.2 µm filter.

The batch was adjusted to a polysaccharide concentration of 1.0 g/l with additional 10 mM L-histidine in 150 mM sodium chloride, pH 7.0. The batch was divided into aliquots and frozen at ≤-60°C.

EXAMPLE 3 Methods for conjugating serotypes 3, 4, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F to CRM ₁₉₇ via reductive amination in dimethyl sulfoxide

Various polysaccharide serotypes 3, 4, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F were separately conjugated to purified CRM carrier protein₁₉₇ using a common technological scheme. The polysaccharide was dissolved, sized to the target molecular weight, chemically activated, and subjected to buffer exchange via ultrafiltration. Activated polysaccharide and purified CRM₁₉₇ separately lyophilized and redissolved in dimethyl sulfoxide (DMSO). Then the solutions of the re-dissolved polysaccharide and CRM₁₉₇ combined and conjugated as described below. The resulting conjugate was purified by ultrafiltration before final filtration with a 0.2 µm filter. Several process parameters at each stage, such as pH, temperature, concentration and time, were controlled to maintain the serotype-specific values defined in the section below.

Grinding and oxidation of the polysaccharide

Purified pneumococcal capsular polysaccharide powder was dissolved in water and all serotypes except serotype 19A were filtered with a 0.45 µm filter. All serotypes, except for serotypes 18C and 19A, were homogenized to reduce the molecular weight of the polysaccharide. The homogenization pressure and the number of passes through the homogenizer were controlled at the level of serotype-specific target values (150-1000 bar; 4-7 passes). Serotype 18C was ground by acid hydrolysis at ≥90°C .

The milled polysaccharide was filtered with a 0.2 μm filter and then concentrated and diafiltered against water using a 10 kDa NMWCO tangential filtration membrane. For serotype 18C, a 5 kD cutoff NMWCO membrane was used.

The polysaccharide solution was then adjusted to serotype-specific temperature (4-22° C.) and pH (4-5) using sodium acetate buffer to minimize polysaccharide grinding due to activation. For all serotypes (except serotype 4), polysaccharide activation was initiated by adding a 100 mM sodium metaperiodate solution. The amount of sodium metaperiodate added was serotype specific and ranged from about 0.1 to 0.5 moles of sodium metaperiodate per mole of polysaccharide repeating unit. The serotype-specific charge of sodium metaperiodate is designed to achieve a target level of polysaccharide activation (moles of aldehyde per mole of polysaccharide repeating unit). For serotype 4, the batch was incubated at approximately 50° C. and pH 4.1 prior to addition of sodium metaperiodate to partially deketalize the polysaccharide.

The activated product was diafiltered against 10 mM potassium phosphate, pH 6.4 using a 10 kDa NMWCO tangential filtration membrane, then diafiltered or dialyzed against water using a 10 kDa NMWCO membrane. For serotype 18C, a 5 kD cutoff NMWCO membrane was used. Ultrafiltration or dialysis of all serotypes was carried out at 2-8°C.

Conjugation of polysaccharide with CRM ₁₉₇

Purified CRM ₁₉₇ obtained by expression in Pseudomonas fluorescens as described previously (WO 2012/173876 A1) was diafiltered against 2-5 mM phosphate buffer, pH 7.0 using an NMWCO tangential filtration membrane with a cutoff of 5 kDa and filtered using a 0.2 µm filter.

In the case of all serotypes, with the exception of serotype 3, oxidized polysaccharides were at a concentration of 6 mg polysaccharide/ml and 5% wt./about. sucrose (50 mg sucrose/ml) in water. In the case of serotype 3, the oxidized polysaccharide was at a concentration of 2 mg polysaccharide/ml and 10% w/v. sucrose (100 mg sucrose/ml) in water. The protein solution was at a concentration of 6 mg protein/ml using 1% wt./about. sucrose (10 mg sucrose/ml) in phosphate buffer.

The formulated solutions of polysaccharide and CRM _{197 were} separately lyophilized. The lyophilized polysaccharide and CRM ₁₉₇ materials were redissolved in DMSO and combined using a T-shaped stirrer. Sodium cyanoborohydride (1 mol per mol polysaccharide repeating unit) was added and conjugated for a serotype-specific time period (1 to 48 hours) to achieve the target conjugate size.

Reduction of sodium borohydride

After the conjugation reaction, sodium borohydride (2 moles per mole repeating unit of polysaccharide) was added. The batch was diluted with 150 mM sodium chloride with or without about 0.025% (w/v) polysorbate 20 at about 4°C. Potassium phosphate buffer was then added to neutralize the pH. For serotypes 3, 6A, 6B, 7F, 9V, 18C, 19A, 19F, 22F, 23F and 33F, the batch was concentrated and diafiltered at approximately 4°C against 150 mM sodium chloride with 25 mM potassium phosphate, pH 7, or without it, using a NMWCO tangential filtration membrane with a cutoff of 30 kDa.

Final filtration and product storage

Serotypes 3, 6A, 6B, 7F, 9V, 18C, 19A, 22F, 23F, and 33F were concentrated and diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, with 0.015% (w/v) polysorbate 20 or no at 4°C using a NMWCO tangential filtration membrane with a cutoff of 300 kDa. The batch of retentate was filtered with a 0.2 µm filter.

Serotype 19F was incubated for approximately 5 days, diafiltered against 10 mM histidine in 150 mM sodium chloride, pH 7.0, at approximately 4° C. using a 300 kDa cutoff NMWCO tangential filtration membrane, and filtered with a 0.0 filter. 2 µm.

Serotypes 3, 6A, 6B, 7F, 9V, 18C, 19A, 19F, 22F, 23F and 33F were diluted with additional 10 mM histidine in 150 mM sodium chloride, pH 7.0, aliquoted and frozen at ≤-60°C.

Serotypes 4 and 14 were dialyzed against 150 mM sodium chloride at approximately 4° C. using a 300 kDa NMWCO membrane, filtered through a 0.2 μm filter, aliquoted and frozen at ≤-60° C.

EXAMPLE 4: Analysis of conjugates

Molecular weight and concentration analysis of conjugates using HPSEC/UV/MALS/RI analysis

Conjugate samples were injected and separated by high performance size exclusion chromatography (HPSEC). Detection was performed using an ultraviolet (UV) detector, a multi-angle light scattering (MALS) detector and a refractive index detector (RI) arranged in series. The protein concentration was calculated from the UV absorbance at 280 nm using the extinction coefficient. The polysaccharide concentration was obtained by deconvolution of the RI signal (contributed by both the protein and the polysaccharide) using the dn/dc coefficients, which are the change in the refractive index of the solution with the change in solute concentration, given in ml/g. The average molecular weight of the samples was calculated using Astra software (Wyatt Technology Corporation, Santa Barbara, CA) using measured concentration and light scattering information across the sample peak.

Analysis of the degree of activation of the polysaccharide

The conjugation is carried out by reductive amination between the activated aldehydes and especially the lysine residues on the carrier protein. The level of activation, expressed as moles of aldehyde per mole of polysaccharide repeating unit, is important in controlling conjugation reactions. An assay for measuring the degree of activation is described in US Patent Application Publication No. 2017/0021006.

We developed our own assay to measure the degree of activation based on the reaction of aldehyde groups (formed from the oxidation of a polysaccharide with periodate) with thiosemicarbazide (commercially available).

Quantitative analysis can be carried out using NMR (Nuclear Magnetic Resonance), or by comparing the derivatized polysaccharide with suitable standards, and/or using the extinction coefficients of the derivative. The extinction coefficients for this assay are used similarly to those used in the HPSEC/UV/MALS/RI method.

Typically, the assay can be performed under the following reaction conditions:

Time: 0.5-35 hours (this time is serotype-specific, but the reaction is carried out to completion, i.e. reaching a time dependence plateau)

Temperature: 15-37°C, preferably around 21-27°C

TSC concentration : 1-5 mg/mL

reaction pH: pH 3-5.5, preferably 4.0

In Example 4, the polysaccharide was derivatized using 1.25 to 2.5 mg/mL thiosemicarbazide (TSC) at pH 4.0 to incorporate the chromophore (1.25 mg/mL TSC is used when derivatizing activated polysaccharide serotypes 1, 5, and 9V) . The derivatization reactions were allowed to proceed until a plateau was reached. The exact time varied depending on the reaction rate for each serotype. TSC-Ps were then separated from TSC and other low molecular weight components by high performance size exclusion chromatography. The signal was determined by UV absorption at 266 nm. Calculated the level of activated aldehyde relative to injections of mono-TSC on the standard curve or directly using predetermined extinction coefficients. Mono-TSC is a synthesized thiosemicarbazone monosaccharide derivative. The level of aldehydes was then converted to moles of aldehyde per mole of repeating unit (Ald/RU) using the polysaccharide concentration measured by HPSEC/UV/MALS/RI analysis.

A similar derivatization can be carried out using structural analogs of thiosemicarbazide, hydrazides, hydrazine, semicarbazide, structural analogs of semicarbazide, aminooxy compounds or aromatic amines, provided that the derivatives exhibit significant UV absorption. UV absorption can be due to the chromophore attached to the derivatizing agents, or the chromophore resulting from aldehyde derivatization, as in the case of thiosemicarbazide.

Determination of the consumption of lysine in a conjugated protein as a measure of the amount of covalent bonds between the polysaccharide and the carrier protein

Waters AccQ-Tag (AAA) amino acid analysis was used to measure the degree of conjugation in the conjugate samples. Samples were hydrolyzed using gas phase acid hydrolysis using an Eldex workstation to break down carrier proteins to their amino acid components. Free amino acids were derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). The derivatized samples were then analyzed using UPLC with UV detection on a C18 column. The average protein concentration was obtained using characteristic amino acids other than lysine. The consumption of lysine during conjugation (ie loss of lysine) was determined by the difference between the average measured amount of lysine in the conjugate and the expected amount of lysine in the original protein.

Properties of conjugates obtained by reductive amination in aqueous solution and DMSO

The results of analyzes of polysaccharide activation and consumption of lysine (i.e. loss of lysine) for the conjugates obtained by the methods described in examples 2 and 3 are shown in the table. 1. There is a distinct difference that the conjugates prepared in DMSO (Example 3) have a higher consumption of lysine with lower polysaccharide activation than the conjugates prepared in aqueous solution (Example 2). This suggests that making conjugates in DMSO solution allows the polysaccharide to attach to more conjugation sites on the carrier protein with less activation or degradation to native polysaccharide structures. As a result, the conjugates, on average, contain more glycopeptide per polysaccharide repeat unit due to higher cross-linking in conjugates prepared in DMSO solution than in aqueous solution. The glycopeptide is believed to be an antigenic domain that elicits an immune response. Thus, conjugates prepared in DMSO are expected to be more immunogenic than conjugates prepared in aqueous solution.

Average molecular weight (Mw) of the conjugates in the table 1 was measured by HPSEC UV-MALS-RI assay. The conjugates obtained by reductive amination in aqueous solution had a molecular weight ranging from 990 to 3410 kDa. Conjugates prepared in DMSO were generally larger, ranging in size from 1300 to 5822 kDa.

Table 1: Loss of lysine for conjugates of pneumococcal serotypes 3, 4, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F and CRM ₁₉₇ obtained by reductive amination in aqueous solution or DMSO.

conjugate conjugate batch number Conjugation reaction in aqueous solution or DMSO Polysaccharide activation (mol aldehyde/mol repeating unit) Loss of lysine (mol/mol protein) Serotype 3-CRM ₁₉₇ one Water solution 0.10 3.1 2 0.10 2.5 3 0.10 3.1 four DMSO 0.092 16.3 5 0.053 9.6 Serotype 4-CRM ₁₉₇ one Water solution 0.43 2.7 2 DMSO 0.25 3.0 Serotype 6A-CRM ₁₉₇ one Water solution 0.19 4.5 2 DMSO 0.11 9.1 Serotype 6B-CRM ₁₉₇ one Water solution 0.18 4.6 2 DMSO 0.11 9.6 Serotype 7F-CRM ₁₉₇ one Water solution 0.26 2.0 2 DMSO 0.22 10.6 Serotype 9V-CRM ₁₉₇ one Water solution 0.30 4.7 2 DMSO 0.15 7.9 Serotype 14-CRM ₁₉₇ one Water solution 0.22 6.4 2 DMSO 0.22 12.7 Serotype 18C-CRM ₁₉₇ one Water solution 0.12 3.5 2 DMSO 0.11 9.2 Serotype 19A-CRM ₁₉₇ one Water solution 0.38 4.9 2 DMSO 0.14 9.5 Serotype 19F-CRM ₁₉₇ one Water solution 0.13 2.7 2 DMSO 0.15 9.6 Serotype 22F-CRM ₁₉₇ one Water solution 0.12 1.7 2 DMSO 0.15 7.2 3 0.15 7.0 Serotype 23F-CRM ₁₉₇ one Water solution 0.39 3.2 2 DMSO 0.19 10.8 Serotype 33F-CRM ₁₉₇ one Water solution 0.23 4.5 2 DMSO 0.14 7.0

Quantitative analysis of the degree of conjugation at different sites on CRM ₁₉₇

The polysaccharide can be conjugated to the amino group at the N-terminus of the carrier protein or any of the 39 lysine side chains in the CRM₁₉₇. Amino acid sequence of CRM₁₉₇ shown in the table 2, where the lysines (abbreviated as K) are underlined and in bold. To localize and quantify the degree of polysaccharide conjugation at different sites on the CRM protein₁₉₇ used the method of peptide mapping LC/UV/MS. Typical conjugate samples (prepared in DMSO or aqueous solution) were digested in duplicate using trypsin to give tryptic peptides. The mixtures were then separated on a reversed-phase column C_eighteen and analyzed with UV and mass spectrometer. CRM protein sample₁₉₇ (non-conjugated to the polysaccharide) was also treated in triplicate simultaneously with the control. Because trypsin cleaves the protein at the C-terminal side of the lysine and arginine residues; conjugation at the lysine residue makes this site resistant to the action of the protease. The degree of conjugation at a particular site was determined by calculating the decrease in the intensity of the tryptic peptide peak compared to the control CRM.₁₉₇. Depending on the cleavage sites and sequences, a decrease in the signal of a particular peptide may be the result of incorrect cleavage of lysins in the preceding peptide, or incorrect cleavage of a lysine at the end of the peptide, or conjugation in the middle of the peptide sequence.

The relative percentage reduction in peptide signal for serotype 19A conjugates compared to CRM ₁₉₇ control relative to possible conjugation sites was _plotted in FIG. are possible sites of conjugation of the analyzed peptides. For example, the designation "33" means that the decrease in peptide signal was the result of conjugation on the 33rd lysine, and the designation "6, 7" means that the decrease in peptide signal was the result of conjugation on the 6th, or 7th, or both lysines . The data presented in Figure 1 suggests that not only was the degree of conjugation at each site generally higher for conjugates prepared in DMSO compared to aqueous solution, but there were more conjugation sites when conjugated in DMSO. These additional conjugation sites include the 29th, 30th, 31st, and 32nd lysins, which are the only lysins located in previously identified common human T-cell peptide epitopes (see Raju et al ., 1995, Eur. J Immunol 25:3207-3214 located at peptide 411-430 and peptide 431-450 of the CRM ₁₉₇ sequence). Similar results were observed for the other serotypes tested.

Table 2: Amino acid sequence of CRM ₁₉₇

Amino acids Amino acid sequence 1-535 GADDVVDSS K SFVMENFSSY HGT K PGYVDS IQ K GIQ K P K S GTQGNYDDDW
K EFYSTDN K Y DAAGYSVDNE NPLSG K AGGV V K VTYPGLT K VLAL K VDNAE
TI KK ELGLSL TEPLMEQVGT EEFI K RFGDG ASRVVLSLPF AEGSSSVEYI
NNWEQA K ALS VELEINFETR G K RGQDAMYE YMAQACAGNR VRRSVGSSLS
CINLDWDVIR D K T K T K IESL K EHGPI K N K M SESPN K TVSE E K A K QYLEEF
HQTALEHPEL SEL K TVTGTN PVFAGANYAA WAVNVAQVID SETADNLE K T
TAALSILPGI GSVMGIADGA VHHNTEEIVA QSIALSSLMV AQAIPLVGEL
VDIGFAAYNF VESIINLFQV VHNSYNRPAY SPGH K TQPFL HDGYAVSWNT
VEDSIIRTGF QGESGHDI K I TAENTPLPIA GVLLPTIPG K LDVN K S K THI
SVNGR K IRMR CRAIDGDVTF CRP K SPVYVG NGVHANLHVA FHRSSSE K IH
SNEISSDSIG VLGYQ K TVDH T K VNS K LSLF FEI K S (SEQ ID NO: 1)

EXAMPLE 5 Immunogenicity studies in mice comparing serotype 3-CRM polysaccharide conjugates ₁₉₇ obtained in aqueous solution and DMSO

All animal experiments were performed in strict accordance with the recommendations given in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Laboratory Animal Care and Use Committee (IACUC), MRL, West Point, PA.

Eight-week-old female CD1 mice were housed in microisolator cages (n=10/cage) at the MRL Vivarium, West Point, PA. Food and water were available without restrictions. Mice (n=10/group) were immunized intramuscularly (IM) with ST3-CRM ₁₉₇ conjugates (0.4 μg of ST3 polysaccharide) formulated with aluminum phosphate adjuvant (APA) as described in Table 3. Negative control animals received only APA. Immunizations were performed on days 0, 14 and 28. Blood was collected in serum separation tubes (BD, Franklin Lakes, NJ) via the tail vein on days 6 and 34.

Table 3: Groups of a mouse study comparing 3-CRM ₁₉₇ serotype polysaccharide conjugates prepared in aqueous solution and DMSO solution.

group number Group Description of the conjugate Description of composition one APA Control without the use of conjugate 250 μg/ml APA, 20 mM L-histidine, pH 5.8, and 150 mM NaCl with 0.2% w/v. PS-20 2 ST3-CRM ₁₉₇ (water solution)/APA Monovalent conjugate ST3-CRM₁₉₇ (party No. 1 in the table 1) obtained by reductive amination in aqueous solution as described in example 2 0.4 μg ST3-CRM ₁₉₇ , 250 μg/ml APA, 20 mM L-histidine, pH 5.8, and 150 mM NaCl with 0.2% w/v. PS-20 3 ST3-CRM ₁₉₇ (DMSO)/APA Monovalent conjugate ST3-CRM₁₉₇ (batch number 4 in the table 1) obtained by reductive amination in DMSO as described in example 3 0.4 μg ST3-CRM ₁₉₇ , 250 μg/ml APA, 20 mM L-histidine, pH 5.8, and 150 mM NaCl with 0.2% w/v. PS-20

Electrochemiluminescent assays (ECL) immunogenicity

Mouse humoral responses were measured in multiplex electrochemiluminescent assays in a 96-well format as previously described with minor modifications. See Marchese et al ., 2009, Clin Vaccine Immunol 16(3):387-96; Skinner et al ., 2011, Vaccine 29(48):8870-6; and Caro-Aguilar et al ., 2017 Vaccine 35(6):865-72. Briefly, after incubating test sera for 1 hour on Meso-Scale Discovery plates (Meso Scale Diagnostics, Rockville, MD) and washing, 25 μl of 2 μg/ml labeled Sulfo-tag (Meso Scale Diagnostics, Rockville, MD) was added to each well. MD) Goat anti-mouse IgG. The plates were incubated for 1 hour at room temperature with shaking, and then processed as described previously and analyzed using MESO Sector S600.

The ECL titer was calculated as the reciprocal of the linearly interpolated dilution corresponding to the cut-off value (geometric mean pneumococcal polysaccharide ECL signal in predetermined pooled sera from positive control mice). Interpolation was performed by ECL logarithmic scaling and dilution. The titer was then obtained by inversely transforming the linearly interpolated dilution. Titers were extrapolated for samples falling outside the study range from 100 to 1562500 by linear extrapolation (at double logarithmic scaling) using the intercept on the axis and the slope for the last 3 ECL data points on the sampling curve, located completely above the threshold line, or using the intercept on the axis and the slope for the first 2 ECL data points on the sampling curve that are completely below the threshold line. The titer was then obtained by inverse transformation of the linearly extrapolated dilution.

Opsonophagocytic cytolysis assay (OPA)

Opsonophagocytic cytolysis assay (OPA) of pneumococcal serotype 3 was performed as previously described with minor modifications (Caro-Aguilar et al ., 2017 Vaccine 35(6):865-72; and Burton et al ., 2006, Clin Vaccine Immunol 13( 9):1004-9). After incubation of the sera, bacteria, complement, HL-60 cells, 10 μl of the opsonophagocytic reaction mixture was transferred to a separate well on a 96-well Millipore filter plate containing 200 μl/well of sterile water. The plate was vacuum filtered and 100 μl of Todd-Hewitt broth (THYE, Teknova) was added. The medium was filtered and the wet plate was placed in a sealed plastic bag overnight at 27°C. Plate filters were then stained with 100 μl/well 0.1% Coomassie blue (Bio-Rad, Hercules, CA). The dye was filtered through the plate, the colonies were decolorized with Coomassie decolorization solution (Bio-Rad) and again subjected to vacuum filtration until dry. Stained bacterial colonies were counted using a CTL Immunospot reader (Shaker Heights, OH). The OPK titer was determined as the reciprocal of a dilution of serum with at least 50% cytolysis compared to the mean growth in wells with control complement (control without serum), and was calculated by linear interpolation between serial dilutions, the signals of which covered 50% - ny cytolysis.

The results obtained before immunization and after dose 3 are shown in figure 2 and in table 4 for ECL immunogenicity analysis and on the figure 3 for OPA. Both conjugates, obtained by methods using an aqueous solution and DMSO, are immunogenic and provide functional cytolysis of bacteria. Notably, the conjugate prepared using the DMSO solution method provided both higher immunogenicity in the ECL assay and stronger responses in the OPA assay than the conjugate prepared using the aqueous solution. Differences in immunogenicity in the ECL assay are statistically significant. The GMT ratio in group 3 and group 2 is 3.41 (with lower and upper limits of the 95% confidence interval of 1.26 and 9.26).

Table 4: Results of ECL immunogenicity analysis after dose 3 in groups of a study in mice comparing serotype 3-CRM ₁₉₇ polysaccharide conjugates prepared in aqueous solution and DMSO solution.

group number Group Geometric mean titer, GMT Lower limit of 95% confidence interval Upper limit of 95% confidence interval one APA 368 227 596 2 ST3-CRM ₁₉₇ (water solution)/APA 355.207 187.905 671.466 3 ST3-CRM ₁₉₇ (DMSO)/APA 1211654 719.297 2041028

EXAMPLE 6 Immunogenicity Studies in Adult Humans Comparing Pneumococcal Polysaccharide-Protein Conjugates Produced by Aqueous Reductive Amination and DMSO

This example describes the immunogenicity and safety of two 15-valent pneumococcal conjugate vaccines (PCV15) in a study in healthy, pneumococcal vaccine-naive adults 50 years of age or older.

Test design

A randomized, multicenter, double-blind trial was performed to compare the safety, tolerability, and immunogenicity of a single dose of 2 different formulations of PCV15 (PCV15-A and PCV15-B) and Prevnar 13™ (pneumococcal 13-valent conjugate vaccine [CRM ₁₉₇ diphtheria protein], Wyeth Pharmaceuticals Inc., a subsidiary of Pfizer Inc., Philadelphia, PA, USA) on healthy (any chronic comorbidity must be documented to be stable) adults 50 years of age or older, conducted in accordance with Good Clinical Practice.

A total of 690 healthy, pneumococcal vaccine-naïve individuals aged 50 years or older were included in the study and randomly assigned to three different vaccination groups: Prevnar 13™, PCV15-A and PCV15-B in a 1:1:1 ratio. Randomization was stratified by age at study entry (50 to 64 years, 65 to 74 years, and ≥75 years).

PCV15 contained a dose of 2 μg/0.5 ml of each of the following CRM ₁₉₇ -conjugated pneumococcal polysaccharide serotypes (1, 3, 4, 5, 6A, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, 33F) , a dose of 4 μg/0.5 ml of pneumococcal polysaccharide serotype 6B conjugated with CRM ₁₉₇ , a dose of 125 μg/0.5 ml of aluminum phosphate adjuvant, 20 mm L-histidine, 150 mm sodium chloride, pH 5.8. PCV15-A was with 0.2% wt./about. P188. PCV15-B was with 0.1% wt./about. PS-20.

In the case of PCV15-A, all fifteen polysaccharide serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F) were conjugated to CRM₁₉₇ by reductive amination in aqueous solution as described in Example 2. The properties of some of these conjugates (Conjugate Lot #1 materials) are shown in Table one.

In the case of PCV15-B, serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F were conjugated to CRM₁₉₇ by reductive amination in DMSO as described in Example 3. The properties of these conjugates (Conjugate Batch #2 materials) are shown in Table one. The conjugates of the remaining serotypes (1, 3, 4, 5, 9V, 14, 22F and 33F) are the same conjugates used in PCV15-A.

Both formulations of PCV15 generally had comparable safety profiles to Prevnar 13™ based on the overall safety score (data not shown). Serotype-specific GMC IgG and GMT in the OPA assay were measured on day 30 (OPA results not included).

results

Geometric mean concentrations (GMC) of IgG and confidence intervals (CI) are shown in Table 6. Conjugates of serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F in PCV15-A and PCV15-B were obtained by different conjugation methods as described above. According to the results in the table 4, immunogenicity responses for each of the serotypes presented in the table 6 improved if polysaccharide serotypes were conjugated to CRM₁₉₇ in DMSO. GMCs for serotypes 18C, 19A, 19F and 23F in PCV15-B were significantly higher than for PCV15-A (two-sided significance level 0.05). These data strongly suggest the benefit of DMSO conjugation for improved immunogenicity. This is a discovery that has not previously been demonstrated for either pneumococcal or other conjugate vaccines.

Table 6: Humoral IgG responses to PCV15-A and PCV15-B formulations of serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F. Conjugates of these serotypes were obtained by reductive amination in aqueous solution (PCV15-A) or reductive amination in DMSO (PCV15-B).

Serotypes PCV15-A (N=231), GMC ^† (day 30) PCV15-B (N=231), GMC ^† (day 30) Approximate GMC Ratio ^† [PCV15-B/PCV15-A] n Approximate answer n Approximate answer (95% CI) ^† 6A 217 3.74 217 4.93 1.32 (0.96, 1.81) 6B 217 3.69 217 4.95 1.34 (0.98, 1.85) 7F 217 4.09 217 4.53 1.11 (0.86, 1.43) 18C 217 6.61 217 10.99 1.66 (1.27, 2.18) 19A 217 8.77 217 13.83 1.58 (1.23, 2.02) 19F 217 4.11 217 6.80 1.66 (1.26, 2.17) 23F 217 3.92 217 5.53 1.41 (1.04, 1.91)

†Approximate GMC, ratio of GMC and 95% CI obtained using cLDA model

N=Number of randomized and vaccinated individuals

n=Number of individuals with post-vaccination serology on day 30 included in the analysis

GMC=Geometric Mean Concentration

CI=Confidence interval

--->

SEQUENCE LIST

<110> He Jian

McNair, John, E.

SMITH, William J.

WINTERS, Michael A.

Joyce, Joseph, J.

ABEIGUNAVARDANA, Chitrananda

MUSEY, Luvi

PUJAR, Hari

SKINNER, Julie, M.

WEN, Emily

McHugh, Patrick

WILLIAMS, John Michael

LANCASTER, Katherine

<120> INCREASING THE IMMUNOGENICITY OF STREPTOCOCCUS POLYSACCHARIDE CONJUGATES

PNEUMONIAE-PROTEIN

<130> 24424

<150> 62/555.444

<151> 2017-09-07

<150> 62/463.216

<151> 2017-02-24

<160> 1

<170>PatentIn version 3.5

<210> 1

<211> 535

<212> protein

<213> Artificial sequence

<220>

<223> CRM197 detoxified diphtheria toxin variant)

<400> 1

Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn

1.5 10 15

Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln

20 25 30

Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp Asp

35 40 45

Asp Trp Lys Glu Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala Gly

50 55 60

Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly Val

65 70 75 80

Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys Val

85 90 95

Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu

100 105 110

Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe Gly

115 120 125

Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly Ser

130 135 140

Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser

145 150 155 160

Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp

165 170 175

Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg

180 185 190

Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val

195 200 205

Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His Gly

210 215 220

Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu

225 230 235 240

Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu

245 250 255

His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val

260 265 270

Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val

275 280 285

Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu

290 295 300

Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala

305 310 315 320

Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser

325 330 335

Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp

340 345 350

Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe

355 360 365

Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His

370 375 380

Lys Thr Gln Pro Phe Leu His Asp Gly Tyr Ala Val Ser Trp Asn Thr

385 390 395 400

Val Glu Asp Ser Ile Ile Arg Thr Gly Phe Gln Gly Glu Ser Gly His

405 410 415

Asp Ile Lys Ile Thr Ala Glu Asn Thr Pro Leu Pro Ile Ala Gly Val

420 425 430

Leu Leu Pro Thr Ile Pro Gly Lys Leu Asp Val Asn Lys Ser Lys Thr

435 440 445

His Ile Ser Val Asn Gly Arg Lys Ile Arg Met Arg Cys Arg Ala Ile

450 455 460

Asp Gly Asp Val Thr Phe Cys Arg Pro Lys Ser Pro Val Tyr Val Gly

465 470 475 480

Asn Gly Val His Ala Asn Leu His Val Ala Phe His Arg Ser Ser Ser

485 490 495

Glu Lys Ile His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Val Leu

500 505 510

Gly Tyr Gln Lys Thr Val Asp His Thr Lys Val Asn Ser Lys Leu Ser

515 520 525

Leu Phe Phe Glu Ile Lys Ser

530 535

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Claims (4)

1. Immunogenic composition for the prevention of invasive disease caused by S. pneumoniae , consisting essentially of the polysaccharide of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F, each conjugated to CRM ₁₉₇ ; where the conjugation reaction for S. pneumoniae serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F is carried out in an aprotic solvent, where the aprotic solvent is DMSO; and the conjugation reaction of S. pneumoniae serotypes 1, 3, 4, 5, 9V, 14, 22F and 33F is carried out in an aqueous solvent. 2. The immunogenic composition according to claim 1, additionally containing approximately 0.2% wt./about. polysorbate 20. 3. Immunogenic composition for the prevention of an invasive disease caused by S. pneumoniae , consisting of a polysaccharide of S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F , each of which is conjugated with CRM ₁₉₇ ; where the conjugation reaction for S. pneumoniae serotypes 6A, 6B, 7F, 18C, 19A, 19F and 23F is carried out in an aprotic solvent, where the aprotic solvent is DMSO; and the conjugation reaction of S. pneumoniae serotypes 1, 3, 4, 5, 9V, 14, 22F and 33F is carried out in an aqueous solvent. 4. The immunogenic composition according to claim 3, additionally containing approximately 0.2% wt./about. polysorbate 20.

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