Patent Publication Number: US-2022211859-A1

Title: Conjugate production

Description:
TECHNICAL FIELD 
     This invention relates to methods of producing and purifying conjugates of polysaccharides and protein carriers. 
     BACKGROUND 
     Bacterial capsular polysaccharides (CPS) are T-independent antigens and are generally poorly immunogenic. Covalent conjugation of bacterial CPS to a carrier molecule (such as a protein carrier) can convert T-independent antigens into T-dependent antigens, thereby enhancing memory responses and the development of protective immunity. Bacterial capsular polysaccharides conjugated to protein carriers are known for use in vaccines, including  Haemophilus influenzae  type b (Hib) conjugates (Peltola 2000),  Streptococcus pneumoniae  conjugates (Wuorimaa &amp; Kayhty 2002) and serogroup C  Neisseria meningitidis  (MenC) conjugates (Balmer et al. 2002). Carrier proteins used in licensed vaccines include tetanus toxoid (TT), diphtheria toxoid (DT), the nontoxic CRM197 mutant of diptheria toxin, and the outer membrane protein complex from group B  N. meningitidis.    
       Streptococcus agalactiae  (also known as ‘Group B  Streptococcus ’ or ‘GBS’) is a β-hemolytic, encapsulated Gram-positive microorganism that colonizes the anogenital tract of 25-30% of healthy women. It is a major cause of neonatal sepsis and meningitis, particularly in infants born to women carrying the bacteria. The capsular polysaccharides (also referred to as capsular saccharides) of GBS serotypes are being investigated for use in vaccines, including in the form of saccharide-protein conjugates. 
     A need exists for efficient methods of producing and purifying conjugates of polysaccharides and protein carriers, such as conjugates of GBS capsular polysaccharide antigens and carrier proteins. 
     SUMMARY OF THE INVENTION 
     The present invention provides methods of producing conjugates of polysaccharides and carrier proteins, where sized polysaccharides are provided and conjugated with carrier proteins to provide a mixture comprising polysaccharide-protein conjugates and free polysaccharides; and carrying out a chromatography step by contacting the mixture with hydroxyapatite under conditions wherein the conjugate binds to the hydroxyapatite but free polysaccharide does not; removing free polysaccharide from the mixture; and recovering conjugate bound to the hydroxyapatite. 
     In an embodiment of the invention, the mixture further comprises free carrier protein, and the chromatography step is carried out under conditions wherein the conjugate and the free carrier protein bind to the hydroxyapatite. 
     In an embodiment of the present invention, the polysaccharide is a bacterial capsular polysaccharide. 
     In a further embodiment of the invention, the polysaccharide is a GBS capsular polysaccharide that has been sized to an average molecular weight of between about 50 kilodaltons (kDa) and about 200 kDa. 
     In an embodiment of the invention, the carrier protein is selected from tetanus toxoid, diphtheria toxoid, exotoxin A of pseudomonas (EPA), GBS pilus proteins, and CRM197. 
     In a further embodiment of the invention, the recovered capsular polysaccharide conjugate is mixed with a pharmaceutically acceptable diluent or carrier to provide a pharmaceutical composition. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the difference between the repeating structures in GBS serotypes Ia and III. 
         FIG. 2  shows the repeating structures of capsular saccharides in GBS serotypes Ia, Ib, II, III &amp; V. 
         FIG. 3  shows periodate oxidation of a terminal sialic acid residue. 
         FIG. 4  shows the antibody response in mice, obtained using GBS CPS-CRM197 conjugates obtained by different processes. 
         FIG. 5  indicates total IgG titers from individual sera for groups of mice, expressed as Relative Unit (RU) and Geometric Mean Titers±95% confidence intervals (shown as dot and bars, respectively). 
         FIG. 6 : IgG responses. Single mouse Luminex data (RU/ml) are represented as dots; the bars represent the Geometric Mean Titer±95% Confidence Interval (CI). The bar above the graph reports the obtained Geometric Mean IgG Titers (GEOMEAN). 
     
    
    
     DETAILED DESCRIPTION 
     In producing polysaccharide-protein conjugates for medical uses, such as in vaccine preparations, it is desirable to minimize the amounts of free carrier protein (i.e., carrier protein molecules that are not conjugated to a polysaccharide molecule, also referred to as unconjugated carrier protein) and free polysaccharide (i.e., polysaccharide molecules that are not conjugated to a protein molecule, also referred to as unconjugated polysaccharide) in the bulk conjugate product. 
     Various methods for the removal of free carrier protein are known in the art, including hydrophobic chromatography, tangential ultrafiltration, diafiltration etc. See e.g., Lei et al. (2000), WO00/38711, U.S. Pat. No. 6,146,902. The use of gel filtration to purify GBS conjugates (as described in Anonymous (Jan 2002)  Research Disclosure,  453077) requires a large volume of gel filtration matrix and is difficult to apply at a manufacturing scale. An alternative method that involves ultrafiltration, for example using tangential flow diafiltration with a 100 kDa membrane, is not effective unless the saccharide antigen is of a suitably high molecular weight and is thus not suitable for GBS serotype III conjugates or other conjugates where the saccharide antigen is &lt;100kDa. WO2009/010877 (U.S. Pat. No. 9,463,250) provides a method of removing unconjugated carrier protein using hydroxyapatite, where free carrier protein binds to hydroxyapatite. 
     As demonstrated in WO2009/010877 (U.S. Pat. No. 9,463,250) both Type I and Type II hydroxyapatite resin can be used in chromatography to separate conjugates of GBS polysaccharide-CRM197, from unconjugated CRM197 carrier proteins. Both the conjugates and unconjugated CRM197 will bind to the resin under appropriate conditions, and are then separately recoverable. The majority of free polysaccharide flows through without binding to the resin and is thus removed from the starting mixture. The method of hydroxyapatite chromatography described in WO2009/010877 allowed recovery of more than 80% of desialylated GBS type V capsular polysaccharide conjugated to CRM197 from a starting mixture containing both conjugates and unconjugated CRM197. 
     The present inventors determined that, when preparing conjugates of GBS serotype Ia or Ib polysaccharides and CRM197 according to the above process, where native GBS Ia or Ib PS was used as the starting purified saccharide, the resulting bulk conjugate product contained a high free polysaccharide (PS) content (greater than 10% of the total weight of polysaccharide in the resulting bulk product). In contrast, it was found that the amount of free PS was less in GBS serotype III bulk conjugates produced using the same process, where GBS III native PS was used as the starting purified saccharide. 
     Methods of separating free polysaccharide from mixtures are known in the art, including hydrophobic chromatography, tangential ultrafiltration, and diafiltration. See, e.g., Lei et al., (2000); WO 2000/038711. 
     The presently disclosed methods allow efficient removal of free polysaccharide from a starting mixture, where the starting mixture contains both (a) conjugates of sized bacterial polysaccharide and carrier protein, and (b) free sized polysaccharide. The starting mixture may also contain (c) free carrier protein. 
     GBS polysaccharide-CRM197 conjugates for GBS serotypes Ia, Ib, II, III, IV and V have been prepared in bulk using the following process: 
                                            Purified Polysaccharide (PS)               Polysaccharide Oxidation               Ultra Filtration               Conjugation with CRM197               Hydroxyapatite Chromatography               Quenching Reaction               Filtration                                 Bulk PS-CRM197 Conjugate                    
The conjugation step provides a mixture that contains the polysaccharide-protein conjugate, as well as free polysaccharide and free carrier protein. Hydroxyapaptide chromatography is then used to separate free polysaccharide and free carrier protein from the conjugate, providing a final bulk product comprising the polysaccharide-protein conjugate.
 
     The present inventors determined that the size of the starting polysaccharide molecules affected the free PS content of the final bulk product, and further determined that using sized GBS Ia polysaccharide (i.e., GBS Ia polysaccharide molecules with reduced MW as compared to native or unsized GBS Ia) as the starting material (the Purified Polysaccharide) in the above process, resulted in a bulk conjugate product with decreased levels of free GBS Ia polysaccharide (compared to the level of free PS when using native or unsized GBS Ia polysaccharide as the Purified Polysaccharide in the same process). The resulting GBS serotype Ia-CRM197 conjugate was further shown to retain immunogenicity, using an animal model. 
     The present inventors additionally determined that using sized GBS Ib polysaccharide (i.e., GBS Ib polysaccharide molecules with reduced MW as compared to native or unsized GBS Ib) as the starting material (the Purified Polysaccharide) in the above process, resulted in a bulk conjugate product with decreased levels of free GBS Ib polysaccharide (compared to the level of free PS when using native or unsized GBS Ib polysaccharide as the Purified Polysaccharide in the same process). The resulting GBS serotype Ib-CRM197 conjugate was further shown to retain immunogenicity, using an animal model. 
     The present inventors additionally determined that using sized GBS II polysaccharide (i.e., GBS II polysaccharide molecules with reduced MW as compared to native or unsized GBS II) as the starting material (the Purified Polysaccharide) in the above process increased the yield of conjugate (compared to the yield when using native or unsized GBS II polysaccharide as the Purified Polysaccharide in the same process). The resulting GBS serotype II-CRM197 conjugate was further shown to retain immunogenicity, using an animal model. 
     Each of native GBS polysaccharide serotypes Ia, Ib and II have a higher molecular weight than native GBS polysaccharide serotype III. The MW of native GBS III is approximately 100 kDa; the MW of native GBS Ia is approximately 270 kDa; and the MW of native GBS Ib is approximately 185 kDa; the MW of native GBS II is approximately 419 kDa. The MW of CRM197 is about 60 kDa. 
     Thus, the invention provides a method of producing polysaccharide antigen-carrier protein conjugates, where the amount of free PS in the Bulk Conjugate Product is reduced due to the use of a sized polysaccharide as the starting purified polysaccharide (compared to amount of free PS in Bulk Conjugate Product when native or unsized polysaccharide is used). During the process, the mixture of saccharide-protein conjugate, free protein, and free polysaccharide (obtained after the conjugation step) is contacted with hydroxyapatite. Under appropriate chromatographic conditions, both the conjugate and the free protein carrier bind to the hydroxyapatite while the free polysaccharide does not; free PS can thus be separated, e.g., in chromatography flow-through. The conjugate is then recovered (eluted) from the hydroxyapatite. The free carrier protein may then also be recovered (eluted) from the hydroxyapatite and re-used in a subsequent conjugation reaction, assayed, or discarded. 
     Carrier Protein 
     Any carrier protein may be used in the methods of the present invention, provided that the carrier protein binds to hydroxyapatite under the conditions used in the present methods. Diphtheria toxoid (DT), tetanus toxoid (TT) and CRM197 (detoxified DT variant) are currently in use in commercial pediatric vaccines as carrier proteins for polysaccharide antigens. (CRM stands for Cross Reacting Material). Thus the carrier protein may be selected from those known in the art, such as, tetanus toxoid (TT), diphtheria toxoid (DT), or derivatives thereof such as CRM197 or other detoxified variants of DT (see e.g., Anderson (1983) and Anderson et al. (1985)). Other suitable carrier proteins include EPA (exotoxin A of pseudomonas, see e.g., Broker et al. (2017)), and GBS pilus proteins (see e.g. WO02/34771, WO2002/012294), the  N. meningitidis  outer membrane protein (EP-A-0372501), synthetic peptides (EP-A-0378881, EP-A-0427347), heat shock proteins (WO93/17712, WO94/03208), pertussis proteins (WO98/58668, EP-A-0471177), cytokines (WO91/01146), lymphokines (WO91/01146), hormones (WO91/01146), growth factors (WO91/01146), artificial proteins comprising multiple human CD4 +  T cell epitopes from various pathogen-derived antigens (Falugi et al. (2001) such as N19 (Baraldo et al. 2004), protein D from  H. influenzae  (Ruan et al. 1990; EP-A-0594610, WO00/56360), pneumolysin (Kuo et al. 1995), pneumococcal surface protein PspA (WO02/091998), iron-uptake proteins, toxin A or B from  C. difficile  (WO00/61761), etc. 
     In one embodiment of the invention the carrier protein is CRM197. CRM197 is a non-toxic form of the diphtheria toxin but is immunologically indistinguishable from the diphtheria toxin. (Uchida et al., Nature New Biology (1971) 233; 8-11). The CRM197 protein has the same molecular weight as the diphtheria toxin but differs from it by a single base change in the structural gene, a glycine to glutamic acid change at position 52 which makes fragment A unable to bind NAD and therefore non-toxic (Pappenheimer 1977; Rappuoli et al., 1983). The non-toxic DT carrier protein may have the amino acid sequence of SEQ ID NO:1 (CRM197); or the non-toxic DT carrier protein may have an amino acid sequence at least 90%, 95% or 99% identical to SEQ ID NO: 1, provided it is non-toxic and contains an amino acid substitution at the amino acid position corresponding to position 52 in SEQ ID NO:1, such that fragment A is unable to bind NAD (e.g., a glutamic acid residue at the amino acid position corresponding to position 52 in SEQ ID NO:1). 
     
       
         
           
               
               
            
               
                   
                   C. diphtheriae  mutant CRM197 
               
               
                   
                 SEQ ID NO: 1 
               
               
                   
                 Gly Ala Asp Asp Val Val Asp Ser 
               
               
                   
                   
               
               
                   
                 Ser Lys Ser Phe Val Met Glu Asn 
               
               
                   
                   
               
               
                   
                 Phe Ser Ser Tyr His Gly Thr Lys 
               
               
                   
                   
               
               
                   
                 Pro Gly Tyr Val Asp Ser Ile Gln 
               
               
                   
                   
               
               
                   
                 Lys Gly Ile Gln Lys Pro Lys Ser 
               
               
                   
                   
               
               
                   
                 Gly Thr Gln Gly Asn Tyr Asp Asp 
               
               
                   
                   
               
               
                   
                 Asp Trp Lys Glu Phe Tyr Ser Thr 
               
               
                   
                   
               
               
                   
                 Asp Asn Lys Tyr Asp Ala Ala Gly 
               
               
                   
                   
               
               
                   
                 Tyr Ser Val Asp Asn Glu Asn Pro 
               
               
                   
                   
               
               
                   
                 Leu Ser Gly Lys Ala Gly Gly Val 
               
               
                   
                   
               
               
                   
                 Val Lys Val Thr Tyr Pro Gly Leu 
               
               
                   
                   
               
               
                   
                 Thr Lys Val Leu Ala Leu Lys Val 
               
               
                   
                   
               
               
                   
                 Asp Asn Ala Glu Thr Ile Lys Lys 
               
               
                   
                   
               
               
                   
                 Glu Leu Gly Leu Ser Leu Thr Glu 
               
               
                   
                   
               
               
                   
                 Pro Leu Met Glu Gln Val Gly Thr 
               
               
                   
                   
               
               
                   
                 Glu Glu Phe Ile Lys Arg Phe Gly 
               
               
                   
                   
               
               
                   
                 Asp Gly Ala Ser Arg Val Val Leu 
               
               
                   
                   
               
               
                   
                 Ser Leu Pro Phe Ala Glu Gly Ser 
               
               
                   
                   
               
               
                   
                 Ser Ser Val Glu Tyr Ile Asn Asn 
               
               
                   
                   
               
               
                   
                 Trp Glu Gln Ala Lys Ala Leu Ser 
               
               
                   
                   
               
               
                   
                 Val Glu Leu Glu Ile Asn Phe Glu 
               
               
                   
                   
               
               
                   
                 Thr Arg Gly Lys Arg Gly Gln Asp 
               
               
                   
                   
               
               
                   
                 Ala Met Tyr Glu Tyr Met Ala Gln 
               
               
                   
                   
               
               
                   
                 Ala Cys Ala Gly Asn Arg Val Arg 
               
               
                   
                   
               
               
                   
                 Arg Ser Val Gly Ser Ser Leu Ser 
               
               
                   
                   
               
               
                   
                 Cys Ile Asn Leu Asp Trp Asp Val 
               
               
                   
                   
               
               
                   
                 Ile Arg Asp Lys Thr Lys Thr Lys 
               
               
                   
                   
               
               
                   
                 Ile Glu Ser Leu Lys Glu His Gly 
               
               
                   
                   
               
               
                   
                 Pro Ile Lys Asn Lys Met Ser Glu 
               
               
                   
                   
               
               
                   
                 Ser Pro Asn Lys Thr Val Ser Glu 
               
               
                   
                   
               
               
                   
                 Glu Lys Ala Lys Gln Tyr Leu Glu 
               
               
                   
                   
               
               
                   
                 Glu Phe His Gln Thr Ala Leu Glu 
               
               
                   
                   
               
               
                   
                 His Pro Glu Leu Ser Glu Leu Lys 
               
               
                   
                   
               
               
                   
                 Thr Val Thr Gly Thr Asn Pro Val 
               
               
                   
                   
               
               
                   
                 Phe Ala Gly Ala Asn Tyr Ala Ala 
               
               
                   
                   
               
               
                   
                 Trp Ala Val Asn Val Ala Gln Val 
               
               
                   
                   
               
               
                   
                 Ile Asp Ser Glu Thr Ala Asp Asn 
               
               
                   
                   
               
               
                   
                 Leu Glu Lys Thr Thr Ala Ala Leu 
               
               
                   
                   
               
               
                   
                 Ser Ile Leu Pro Gly Ile Gly Ser 
               
               
                   
                   
               
               
                   
                 Val Met Gly Ile Ala Asp Gly Ala 
               
               
                   
                   
               
               
                   
                 Val His His Asn Thr Glu Glu Ile 
               
               
                   
                   
               
               
                   
                 Val Ala Gln Ser Ile Ala Leu Ser 
               
               
                   
                   
               
               
                   
                 Ser Leu Met Val Ala Gln Ala Ile 
               
               
                   
                   
               
               
                   
                 Pro Leu Val Gly Glu Leu Val Asp 
               
               
                   
                   
               
               
                   
                 Ile Gly Phe Ala Ala Tyr Asn Phe 
               
               
                   
                   
               
               
                   
                 Val Glu Ser Ile Ile Asn Leu Phe 
               
               
                   
                   
               
               
                   
                 Gln Val Val His Asn Ser Tyr Asn 
               
               
                   
                   
               
               
                   
                 Arg Pro Ala Tyr Ser Pro Gly His 
               
               
                   
                   
               
               
                   
                 Lys Thr Gln Pro Phe Leu His Asp 
               
               
                   
                   
               
               
                   
                 Gly Tyr Ala Val Ser Trp Asn Thr 
               
               
                   
                   
               
               
                   
                 Val Glu Asp Ser Ile Ile Arg Thr 
               
               
                   
                   
               
               
                   
                 Gly Phe Gln Gly Glu Ser Gly His 
               
               
                   
                   
               
               
                   
                 Asp Ile Lys Ile Thr Ala Glu Asn 
               
               
                   
                   
               
               
                   
                 Thr Pro Leu Pro Ile Ala Gly Val 
               
               
                   
                   
               
               
                   
                 Leu Leu Pro Thr Ile Pro Gly Lys 
               
               
                   
                   
               
               
                   
                 Leu Asp Val Asn Lys Ser Lys Thr 
               
               
                   
                   
               
               
                   
                 His Ile Ser Val Asn Gly Arg Lys 
               
               
                   
                   
               
               
                   
                 Ile Arg Met Arg Cys Arg Ala Ile 
               
               
                   
                   
               
               
                   
                 Asp Gly Asp Val Thr Phe Cys Arg 
               
               
                   
                   
               
               
                   
                 Pro Lys Ser Pro Val Tyr Val Gly 
               
               
                   
                   
               
               
                   
                 Asn Gly Val His Ala Asn Leu His 
               
               
                   
                   
               
               
                   
                 Val Ala Phe His Arg Ser Ser Ser 
               
               
                   
                   
               
               
                   
                 Glu Lys Ile His Ser Asn Glu Ile 
               
               
                   
                   
               
               
                   
                 Ser Ser Asp Ser Ile Gly Val Leu 
               
               
                   
                   
               
               
                   
                 Gly Tyr Gln Lys Thr Val Asp His 
               
               
                   
                   
               
               
                   
                 Thr Lys Val Asn Ser Lys Leu Ser 
               
               
                   
                   
               
               
                   
                 Leu Phe Phe Glu Ile Lys Ser 
               
            
           
         
       
     
     Group B  Streptococcus  (GBS) 
     The  Streptococcus agalactiae  (Group B  Streptococcus  or GBS) capsule is a major virulence factor that assists the bacterium in evading human innate immune defences. The GBS capsule consists of high molecular weight polymers made of multiple identical repeating units of four to seven monosaccharides. GBS can be classified into ten serotypes (Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX) based on the chemical composition and the pattern of glycosidic linkages of the capsular polysaccharide repeating units. Non-typeable strains of GBS are also known to exist. Description of the structure of GBS CPS may be found in the published literature (see e.g., WO2012/035519). 
     It is estimated that 65-95% of global disease-causing isolates are either serotype Ia, Ib or III. These three serotypes of GBS (Ia, Ib and III) are estimated to cause from 65% to 75% of early onset GBS disease and from 80-90% of late onset GBS disease in Europe and the US. 
     Compositions comprising GBS capsular polysaccharides conjugated to a carrier protein (CPS from GBS serotypes Ia, Ib, II, III, IV and V) have been shown, in clinical testing, to be immunogenic in humans. Conjugates suitable for use in such immunogenic compositions include those where a single CPS molecule is conjugated to a single carrier protein molecule, or where a single carrier protein molecule is conjugated to multiple molecules of saccharide antigen (e.g., multiple GBS Ia saccharide molecules conjugated to the same CRM197 molecule). 
     GBS capsular polysaccharides used according to the present invention may be chemically modified or depolymerized. See e.g., WO2006/050341. 
     Saccharide &amp; Sizing Definition 
     The term “saccharide” throughout this specification refers to polysaccharides (PS) and oligosaccharides. A GBS serotype polysaccharide, as used herein, refers to the GBS bacterial capsular polysaccharide of that serotype. Isolated GBS serotype polysaccharides may be “sized,” i.e., their size reduced, by known methods (see for example EP497524 and EP497525). Methods of sizing polysaccharides include acid hydrolysis treatment, hydrogen peroxide treatment, sizing by EMULSIFLEX followed by a hydrogen peroxide treatment to generate saccharide fragments, and microfluidization. 
     Preferably the sized polysaccharide used in the methods of the present invention has an average molecular weight of between about 50 kilodaltons (kDa) and about 200 kDa, between about 70 kDa and about 170 kDa, between about 80 kDa and about 170 kDa, between about 90 kDa and about 170 kDa, between about 100 kDa and about 170 kDa, between about 100 kDa and about 160 kDa, or about 70 kDa, about 80 kDa, about 90 kDa, about 100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, about 150 kDa, about 160 kDa, about 170 kDa, about 180 kDa, about 190 kDa, or about 200 kDa. 
     Preferably GBS capsular polysaccharide used in the methods of the present invention is selected from: sized GBS serotype Ia capsular polysaccharide having an average molecular weight of between about 80 kDa to about 110 kDa, between about 90 kDa and about 110 kDa, or about 100 kDa; sized GBS serotype Ib capsular polysaccharide having an average molecular weight of between about 80 kDa to about 110 kDa, between about 90 kDa and about 110 kDa, or about 100 kDa; and sized GBS serotype II capsular polysaccharide having an average molecular weight of between about 100 kDa to about 170 kDa, between about 110 kDa and about 170 kDa, between about 120 kDa to about 170 kDa, between about 130 kDa and about 170 kDa, between about 140 kDa to about 170 kDa, between about 150 kDa to about 170 kDa, or about 150 kDa, about 160 kDa, or about 170 kDa. 
     As used herein, a ‘sized’ polysaccharide is one that has undergone a process to reduce its molecular weight, compared to the molecular weight of the starting polysaccharide prior to the sizing process. The starting polysaccharide may be a wild-type (native) polysaccharide. 
     In an embodiment of the present invention, the bacterial polysaccharides are sized by mechanical cleavage, for instance by microfluidisation or sonication. 
     Sizing may be by a factor of no more than x20, x10, x8, x 6, x5, x4, x3 or x2. 
     For the purposes of the invention, “sized by a factor of x2” means the saccharide is subject to a process that reduces the size of the saccharide to one-half that of the starting polysaccharide (the polysaccharide before the sizing process, which may be a native polysaccharide). “Sized by a factor of x3” means the saccharide is subject to a process that reduces the size of the saccharide to one-third that of the starting polysaccharide. 
     For the purposes of the invention, “sized by a factor no more than x2 (or up to x2)” means the saccharide is subject to a process that reduces the size of the saccharide, but wherein the saccharide retains a size that is at least one-half the starting polysaccharide (i.e., one-half or more). “Sized by a factor no more than x3 (or up to x3)” means that the saccharide is subject to a process that reduces the size of the saccharide, but wherein the saccharide retains a size of at least one-third the starting polysaccharide (i.e., one-third or more). 
     For the purposes of the invention, “sized by a factor of at least x2” refers to a sizing process wherein the polysaccharide is reduced to a size that is one half or less of the starting saccharide. “Sized by a factor of at least x3” refers to a sizing process wherein the polysaccharide is reduced to one-third or less of the starting saccharide. 
     For the purposes of the invention, “sized by a factor of at least x2 and up to x3” refers to a sizing process wherein the polysaccharide is reduced to a size that is one half or less of the starting saccharide, but is no less than one third, the size of the starting polysaccharide, inclusive of the end points (e.g., a starting saccharide of 300 kDa that is sized by a factor of at least x2 and up to x3 is reduced to a size between 150-100 kDa, inclusive). 
     The terms x4, x5, x6, x7, x8, x9, x10, x20 are to be interpreted in the same way as described above for x2 and x3. 
     For the purposes of the invention, “native” or “wildtype” polysaccharide refers to a saccharide that has not been subjected to a process, the purpose of which is to reduce the size of the saccharide. 
     Molecular Weight 
     The molecular weight or average molecular weight of a saccharide herein refers to the weight-average molecular weight (Mw) of the saccharide as measured by MALLS (Multi-Angle Laser Light Scattering). The MALLS technique is well known in the art. The molecular weight of a free or unconjugated saccharide is measured prior to conjugation to any carrier protein. 
     Saccharide Antigens 
     In one embodiment, the saccharide antigen used in the methods of the present invention is a bacterial saccharide, in particular a bacterial capsular saccharide, more particularly a GBS capsular polysaccharide. In one embodiment of the present invention, the saccharide is GBS serotype Ia capsular polysaccharide. In a further embodiment of the present invention, the saccharide is GBS serotype Ib capsular polysaccharide. In a further embodiment of the present invention, the saccharide is GBS serotype II capsular polysaccharide. 
     In one embodiment of the invention, the wild-type or native saccharide antigen has a molecular weight of more than 100 kDa; in a further embodiment, the wild-type or native saccharide antigen has a molecular weight of more than about 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480 or 500 kDa; between about 160-about 300, or between any of the provided molecular weights. 
     In one embodiment of the invention, the saccharide antigen is a capsular saccharide selected from GBS serotype Ia, Ib and II. In particular, GBS capsular saccharides from these serotypes may be covalently linked to GlcNAc residues in the bacterium&#39;s peptidoglycan backbone. The capsular polysaccharides of different GBS serotypes are chemically related but are antigenically very different. All GBS capsular polysaccharides share the following trisaccharide core: 
       β-D-GlcpNAc(1→3)β-D-Galp(1→4)β-D-Glcp
 
     The GBS serotypes differ by the way in which this core is modified. The difference between serotypes Ia and III, for instance, arises from the use of either the GlcNAc (Ia) or the Gal (III) in this core for linking consecutive trisaccharide cores ( FIG. 1 ). Serotypes Ia and Ib both have a [α-D-NeupNAc(2→3)β-D-Galp-(1→] disaccharide linked to the GlcNAc in the core, but the linkage is either 1→4 (Ia) or 1→3 (Ib). 
     As shown in  FIG. 2 , the capsular saccharides of five GBS serotypes include: (a) a terminal N-acetyl-neuraminic acid (NeuNAc) residue (commonly referred to as neuaminic acid or sialic acid), which in all cases is linked 2→3 to a galactose residue; and (b) a N-acetyl-glucosamine residue (GlcNAc) within the trisaccharide core. 
     Examples of additional bacterial capsular saccharides which may be used in the methods of the present invention include capsular saccharides from  Neisseria meningitidis  (serogroups A, B, C, W135 and/or Y),  Streptococcus pneumoniae  (serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, particularly 4, 6B, 9V, 14, 18C, 19F and/or 23F),  Streptococcus agalactiae  (types Ia, Ib, II, III, IV, V, VI, VII, and/or VIII),  Haemophilus influenzae  (typeable strains: a, b, c, d, e and/or f),  Pseudomonas aeruginosa, Staphylococcus aureus  (from, for example, serotypes 5 and 8),  Enterococcus faecalis  or  E. faecium  (trisaccharide repeats), Y ersinia enterocolitica, Vibrio cholerae, Salmonella typhi, Klebsiella  spp., etc. Another saccharide which may be included is the  Streptococcus pyogenes  group-specific antigen (GAS carbohydrate). 
     Preparation of Capsular Saccharide Antigens 
     Methods for the preparation of bacterial capsular saccharide antigens are known in the art. For example, WO03/007985 describes the preparation of saccharide antigens from  N. meningitidis . The preparation of saccharide antigens from  H. influenzae  is described in chapter 14 of  Vaccine  (ed Plotkin et al). The preparation of saccharide antigens and conjugates from  S. pneumoniae  is also described in the art. Processes for the preparation of saccharide antigens from  S. agalactiae  are described in detail in Wessels et al. (1990), Wessels et al. (1989), WO2006/082527. 
     The saccharide may be chemically modified relative to the capsular saccharide as found in nature. For example, the saccharide may be de-O-acetylated (partially or fully), de-N-acetylated (partially or fully), N-propionylated (partially or fully), etc. De-acetylation may occur before, during or after conjugation, but preferably occurs before conjugation. Depending on the particular saccharide, chemical modification such as de-acetylation may or may not affect immunogenicity; the effect of chemical modification on antigen immunogenicity can be assessed by routine assays. 
     Saccharide antigens from GBS may be modified. For example, when the saccharide antigen is the  Streptococcus agalactiae  serotype V capsular saccharide, then the saccharide antigen may be modified as described in WO2006/050341. In particular, the  Streptococcus agalactiae  serotype V capsular saccharide may be desialylated ( FIG. 3 ). Desialylated GBS serotype V capsular saccharide may be prepared by treating purified GBS serotype V capsular saccharide under mildly acidic conditions (e.g. 0.1M sulphuric acid at 80° C. for 60 minutes) or by treatment with neuraminidase, as described in WO2006/050341. A method for preparaing desialylated GBS serotype V capsular saccharide is by treating the purified saccharide with 1M acetic acid at 81° C. +/−3° C. for 2 h. 
     Saccharide-Carrier Conjugates 
     Bulk Conjugate product produced by the method of the present invention may include small amounts of free (i.e. unconjugated) protein carrier. When a given carrier protein is present in both free and conjugated form in a composition produced by the method of the invention, the unconjugated form is preferably present at no more than 5% of the total amount of the carrier protein in the composition as a whole (by weight), and more preferably present at less than 2%, less than 1.5%, or less than 1.0% of the total amount of the carrier protein in the composition as a whole (by weight). 
     Bulk Conjugate product produced by the method of the present invention may include small amounts of free (i.e. unconjugated) polysaccharide. When a given polysaccharide is present in both free and conjugated form in a composition produced by the method of the invention, the unconjugated form is preferably present at no more than 5% of the total amount of the total polysaccharide in the composition as a whole (by weight), and more preferably present at less than 2%, less than 1%, or less than 0.5% of the total amount of the carrier protein in the composition as a whole (by weight). 
     Any suitable conjugation reaction can be used in the methods of the present invention, with any suitable linker where necessary. Attachment of the saccharide antigen to the carrier is preferably via a —NH 2  group e.g. in the side chain of a lysine residue in a carrier protein, or of an arginine residue. Where a saccharide has a free aldehyde group then this can react with an amine in the carrier to form a conjugate by reductive amination. Attachment may also be via a —SH group e.g. in the side chain of a cysteine residue. 
     The saccharide will typically be activated or functionalised prior to conjugation. Activation may involve, for example, cyanylating reagents such as CDAP (e.g. 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (see e.g., Lees et al. (1996); WO95/08348). Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU (see e.g., WO98/42721). 
     When the saccharide antigen is a  Streptococcus agalactiae  capsular saccharide, conjugation typically involves reductive amination of the saccharide to a carrier protein, as described in Wessels et al. (1990). The reductive amination involves an amine group on the side chain of an amino acid in the carrier and an aldehyde group in the saccharide. Where GBS capsular saccharides do not include an aldehyde group in their natural form, one is generated before conjugation by periodate oxidation of a portion of the saccharide&#39;s sialic acid residues, as shown in  FIG. 3  (Wessels et al. (1990), U.S. Pat. No. 4,356,170. Conjugation of a  Streptococcus agalactiae  capsular saccharide to a carrier protein may also be carried using the methods described in WO2006/082530. 
     Hydroxyapatite 
     The hydroxyapatite ((Ca 5 (PO 4 ) 3 OH) 2 ) used in the invention may be in one of number of forms known in the art. The hydroxyapatite (HA) may be in the form of crystals, a gel or a resin. The normal crystalline form may alternatively be sintered at high temperatures to modify it to a ceramic form. Ceramic HA is available from Bio-Rad as CHT™ Ceramic Hydroxyapatite, which hydroxyapatite that has been sintered at high temperatures to modify it from a crystalline to a ceramic form, providing spherical particles. Such ceramic HA may be obtained in different particle sizes, such as about 20 μm in diameter, about 40 μm in diameter, or about 80 μm in diameter. 
     The hydroxyapatite may be in the form of a gelpacked into a column, as commonly used in chromatography purification. 
     If the hydroxyapatite is in particulate form, preferably the particles have a diameter of about 20 μm, about 40 μm, about 80 μm, between about 20 μm and about 80 μm, or between about 40 μm and about 80 μm. 
     pH 
     Preferably the present method is carried out at a pH of between 6 and 8, more preferably 6.5 to 7.5, more preferably 7.2. A pH of 7.2 is preferred as this helps stabilize the saccharide. The pH may be adjusted using acids/bases known in the art. 
     Phosphate Concentration 
     As will be known to one of skill in the art of chromatography, conditions such as the pH and salt concentration of buffers affects (a) the binding of components in the mobile phase to the stationary phase of the chromatographic column, and (b) subsequent elution (recovery) of bound components from the stationary phase. In the chromatographic step of the present methods, the chromatographic conditions are such that both free and conjugated protein initially binds to the HA solid phase while free polysaccharide does not; conditions can then be modified to separately recover the conjugate and the free protein. 
     In one embodiment of the present method, buffers comprising different phosphate concentrations, but at essentially the same pH, are used in the chromatography step such that both free protein and conjugated protein initially bind to HA, while free polysaccharide does not. Subsequently, chromatographic conditions are varied to elute (recover) the conjugate. Additionally, the conditions may be varied to separately recover the free protein. 
     Preferably the phosphate concentration of the starting material (loading buffer) is less than about 10 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, from about 1 mM to about 2 mM, or about 2mM. Elution of the conjugate (carrier protein+polysaccharide) is then carried out at a phosphate concentration of from about 5 mM to about 50 mM, from about 10 mM to about 50 mM, from about 10 mM to about 40 mM, from about 10 mM to about 35 mM. The conjugate may be eluted using two different elution buffers, for example, a first elution using a buffer of about 10 mM phosphate, and a second buffer of about 35 mM phosphate. 
     The free protein may subsequently be stripped from the chromatography column using a buffer with a phosphate concentration higher than that used for eluting the conjugate, e.g., from about 50 mM to about 500 mM, from about 100 mM to about 500 mM, from about 200 mM to about 500 mM, or about 400 mM. 
     Typically a sodium phosphate buffer is used. Alternative buffers include MES (2-(N-morpholino) ethanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), tris(hydroxymethyl)aminomethane, imidazole, or acetate. 
     Formulations, Medical Methods and Uses 
     The invention further provides a method of preparing a pharmaceutical composition, comprising the steps of producing polysaccharide antigen-protein conjugates according to the present methods using sized polysaccharides as the starting purified polysaccharide, and then mixing said polysaccharide antigen-carrier protein conjugates with a pharmaceutically acceptable diluent or carrier. The invention also provides the compositions prepared by said method. 
     Thus, conjugates (or bulk conjugate) obtained using the methods of the invention may be formulated as a pharmaceutical composition by the addition of a pharmaceutically acceptable diluent or carrier. Such formulations may further include immunological adjuvants, additional antigens, or both an immunological adjuvant and additional antigen(s). 
     Once formulated in a final medicinal form, such as a vaccine, the compositions of the invention can be administered to a subject in order to induce (or raise) an immune response against the polysaccharide component of the conjugate. The subjects can be non-human mammals such as mice, rats, rabbits, non-human primates, etc., or may be human subjects. The compositions may be formulated as vaccines. They may be delivered by any suitable route. The compositions of the invention may be used in prophylactic measures (to prevent disease or infection) or in therapeutic measures (to treat disease or infection). Where the polysaccharide antigen is a bacterial polysaccharide, the compositions of the invention may be used in preventin or treating infection or disease caused by that bacteria. 
     The invention provides a conjugate obtained using the method of the invention, for use in medicine (in therapeutic purposes, for treating an infection, for raising an immune response, and/or as a vaccine). 
     The invention also provides the use of a conjugate of the invention in the manufacture of a medicament for raising an immune response in a patient or treating a bacterian infection in a patient. 
     Conjugates of the invention may be administered in conjunction with other immunoregulatory agents. In particular, immunogenic or vaccine compositions may include an adjuvant. Adjuvants which may be used in compositions of the invention include, but are not limited to: mineral containing compositions such as aluminum salts and calcium salts; oil emulsions such as oil-in-water or squalene-water emulsions; saponin formulations; virosomes and Virus-Like Particles (VLPs); bacterial or microbial derivatives such as lipopolysaccharides and non-toxic derivatives of lipopolysaccharides such as 3-0-deacylated monophosphoryl lipid A (3dMPL); immunostimulatory oligonucleotides; liposomes; imidazoquinolone compounds; and TLR4 antagonists. 
     Definitions 
     The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X+Y. 
     The term “about” or “approximately” in relation to a numerical value x means, for example, x±10%. All numerical values herein can be considered to be qualified by “about” or “approximately”, unless the context indicates otherwise. 
     The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention. 
     As used herein, a “bulk” product is one that has completed all processing stages but has not been formulated into a final medicinal product. 
     As used herein, “flow through” in chromatography is the fraction which contains primarily protein which does not bind to the chromatographic column under the chromatographic conditions being used. 
     EXAMPLES 
     Example 1—Production of Un-Sized GBS Conjugates 
     Conjugates of native (un-sized) GBS CPS were produced as follows. Purified capsular polysaccharides were obtained from a  Streptococcus agalactiae  serotype (serotype Ia, Ib, II and III). The purified GBS CPS was conjugated to a carrier protein by periodate oxidation followed by reductive amination. The carrier protein was CRM197, providing conjugates of GBS serotype Ia capsular polysaccharide-CRM197 (serotype Ia-CRM197), GBS serotype Ib capsular polysaccharide-CRM197 (serotype Ib-CRM197), GBS serotype II capsular polysaccharide-CRM197 (serotype II-CRM197) and GBS serotype III capsular polysaccharide-CRM197 (serotype III-CRM197). 
     As described in WO2009/010877, conjugates of GBS CPS and protein carriers can be separated from free carrier protein using hydroxyapatite (HA) chromatography. Free polysaccharide is not bound. WO2009/010877 describes using BIO-RAD HA resin of Type I and Type II, 80 μm particle size. Type I HA resin is reported to have a higher protein binding capacity and better capacity for acidic proteins, while Type II is reported to have a lower protein binding capacity but better resolution of certain proteins. Both Type I and Type II hydroxyapatite resins were reported in WO2009/010877 to be useful in separating conjugated from unconjugated CRM197, using hydroxyapatite chromatography conducted at a pH of 7.2 to ensure the stability of the saccharide (a pH of 6.8 is more typically used for hydroxyapatite chromatography; this change in pH did not affect the efficiency of the chromatography as reported in WO2009/010877). WO2009/010877 also described the use of different phosphate concentrations; CRM197 was reported to essentially completely bind to the hydroxyapatite column at a phosphate concentration of less than or equal to 35 mM in the starting material, with the equilibration/post loading wash buffer pH 7.2. Conjugate and free carrier protein could then be eluted separately. 
     WO2009/010877 describes a pilot process using the following parameters:
         Column volume (CV)=4 Liters (L) (acceptable range: &gt;3 L). Column selected had 20 cm diameter (corresponding height about 11 centimeters plus/minus 2.5 cm).   Column rinsing and equilibration: 5 CV of 400 mM sodium phosphate pH 6.8 buffer at 80 cm/hour (419 ml/min) plus 5 CV of 35 mM sodium phosphate pH 7.2 buffer at 80 cm/h.   Column loading and product (conjugate) collection: load product (80 cm/h) and then wash with equilibration buffer; waste flow-through up to 0.4 CV, collect conjugate product up to 2.2 CV, then waste up to 2.9 CV.   To then collect unconjugated CRM bound to the column, Column eluted with 2 CV of 400 mM sodium phosphate pH 6.8 buffer at 80 cm/h.       

     Example 2—Sized GBS Serotype Ia and Ib 
     Sizing of GBS Serotype Ia and Ib Capsular Polysaccharide 
     Microfluidisation technology was used to reduce the size of GBS Ia and Ib polysaccharides, to an average MW of about 100 kDa. The size reduction was done using a high-pressure homogenizer (EMULSIFLEX C50 from Avestin) equipped with a fixed geometry homogenizing cell (MICROFLUIDICS F20Y-75 μm). The size of the polysaccharide is reduced due to the high pressure in the system and shock between molecules due the configuration of homogenizing cell. The sizing is a continuous process and is followed by in-process testing to stop the process when the target size is reached. 
     Native GBS Ia polysaccharide at a concentration of 7.25 mg/ml, was sized via microfluidization, using a pressure of between 10000 and 15000 psi (pounds per square inch). The size reduction was followed by Size Exclusion-High Performance Liquid Chromatography (SEC-HPLC) to stop the microfluidisation. After 65 cycles, the polysaccharide was analyzed by MALLS, which indicated that the sized GBS Ia polysaccharide was about 80 kDa. 
     Further trials using microfluidization to size native GBS Ia polysaccharide (saccharide concentration of 7.25 mg/ml, pressure of between 10000 and 15000 psi) determined that 25 cycles of SEC-HPLC provided sized GBS Ia polysaccharide of about 100 kDa (confirmed by MALLS analysis). Integrity of sialic acid (important for immunogenicity) after sizing was demonstrated by nuclear magnetic resonance (NMR). 
     For the size reduction of native GBS Ib polysaccharide, 23 cycles were needed to reach the target size of about 100 kDa (saccharide concentration of 7.25 mg/ml, pressure of between 10000 and 15000 psi). MALLS analysis confirmed a size of about 105.6 kDa. Integrity of sialic acid (important for immunogenicity) after sizing was demonstrated by NMR. 
     Conjugation and HA Purification of Sized GBS Ia and Ib 
     Conjugation of the sized GBS Ia (about 100 kDa) polysaccharide, obtained as described above, was conducted as follows: purified sized polysaccharide underwent oxidation and subsequent ultrafiltration, followed by conjugation with CRM197. 
     Oxidation: The oxidation step was done using the same conditions as those used with a native polysaccharide. The same oxidation rate was observed for the sized GBS Ia and Ib polysaccharides, as that observed with native polysaccharides (±20%). 
     Coupling times: Four different coupling times were evaluated using sized GBS Ia and Ib polysaccharide (24 hours, 48 hours, 72 hours, and 96 hours). 
     Oxidation and conjugation of sized GBS Ib (about 105 kDa) polysaccharide (obtained as described above) was carried out using the same process as used for sized GBS Ia. 
     The resulting mixtures (containing conjugate, unconjugated CRM197, and unconjugated sized GBS Ia or Ib polysaccharide) underwent hydroxyapatite (HA) chromatography (CHT™ ceramic hydroxyapatite type I, 80 μm particle size, Bio-Rad Laboratories, Inc.) to separate the polysaccharide-CRM197 conjugate from free polysaccharide and free CRM197 carrier protein. The chromatography step (purification) was carried out as follows: 
     Load 0.6 mg CRM/ml resin; 
     Equilibration: 2 mM Na/Na 2 PO 4 , pH 7.2, 550 mM NaCl, 4 CV; 
     Load/rinsing: 2 mM Na/Na 2 PO 4 , pH 7.2, 550 mM NaCl, 5 CV; 
     Elution 1: 10 mM Na/Na 2 PO 4 , pH 7.2, 4 CV; 
     Elution 2: 35 mM Na/Na 2 PO 4 , pH 7.2, 4 CV; 
     Pool (conjugate)=from 0.5 CV of elution 1 to 3.75 CV of elution 2. 
     Stripping (collection of unconjugated carrier protein): 400 mM Na/Na 2 PO 4 , pH 7.2, 3 CV. Free polysaccharide did not bind to the HA and was removed in the chromatography flow-through. Conjugate was collected by elution with phosphate buffer, as described. The conjugate obtained from the HA chromatography then underwent a quenching reaction, and subsequent filtration to provide the purified polysaccharide conjugate. 
     Characterization of Conjugates Produced Using Sized GBS Ia and Sized GBS Ib 
     As described above, four lots of CPS-CRM197 conjugate were produced using sized GBS Ia (MW about 100 kDa); each lot used a different coupling time as shown in Table 1. The lots were then treated with hydroxyapatite chromatography as described above to provide final product. For each lot, the free polysaccharide in the final product was &lt;1% (of total PS by weight), showing that free (uncoupled) sized GBS Ia polysaccharide is removed during hydroxyapatite purification. Similar results were achieved using sized GBS Ib (mw about 105 kDa) polysaccharide. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Sized GBS Ia -- characterization of final conjugate bulk 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 PS 
                   
                 Glycosylation 
                   
               
               
                   
                 Coupling 
                 Content 
                 Protein 
                 Degree 
                 Free 
               
               
                   
                 Time 
                 μg/ml 
                 μg/ml 
                 (w/w) 
                 PS % 
               
               
                   
               
               
                 1 
                 24 H 
                 478 
                 518 
                 0.92 
                 &lt;1.0 
               
               
                 2 
                 48 H 
                 424 
                 463 
                 0.91 
                 &lt;1.1 
               
               
                 3 
                 72 H 
                 511 
                 507 
                 1.00 
                 &lt;0.9 
               
               
                 4 
                 96 H 
                 469 
                 491 
                 0.96 
                 &lt;1.0 
               
               
                   
               
            
           
         
       
     
     Example 3—Sized GBS Ia Conjugation Process without Quenching and Filtration 
     Sized GBS Ia conjugation: The oxidation step was done using the same conditions as those used with a native polysaccharide. Three different oxidized batches were used, with 8 runs per batch. Conjugation temperature and reaction volume were respectively fixed to 22° C. and 4 ml. Conjugation reaction conditions were: CRM197 9 mg/ml, GBS Polysaccharide 11 mg/ml, NaCNBH3 9 mg/ml, 48 hours of reaction. This present example did not include quenching and filtration steps following hydroxyapatite chromatography. 
     Each conjugation reaction run was sampled and monitored at 24 h, 48 h, 72 h and 96 hours. 
     For each batch, an additional run at GBS Ia native PS set points (PS: 6.35 mg/ml, CRM197: 8.47 mg/ml, NaCNBH3: 6.35 mg/ml) was included. 
                     TABLE 2                  GBS Ia                                 Reaction conditions   Conjugation Efficacy                                                     Conjugation               PS   CRM197   Final Bulk           Time   CRM197   PS   NaCNBH3   conjugated   Conversion   Free PS           (Hours)   (mg/ml)   (mg/ml)   (mg/ml)   (%)   (%)   (%)                                                     GBS Ia Existing   IPC* to stop   8.47   6.35   6.35   −80%   −35%   −17-29%       process (Native PS)   reaction                               GBS Ia sized   48 H   9   11   9   &gt;70%   &gt;80%   &lt;1%       Polysaccharide                   (predicted)   (predicted)               *IPC: in-process characterization            
The sized parameters selected were then evaluated on a process including HA chromatography and filtration, to confirm the above results.
 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Sized GBS IA 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 T Predicted range 
               
               
                   
                   
                   
                   
                 (95% confidence 
               
               
                   
                 023 
                 024 
                 026 
                 interval) 
               
               
                   
               
               
                 Conjugation scale (mg) 
                 100 mg 
                 400 mg 
                 400 mg 
                   
               
               
                 CRM197 Conversion (%) 
                   73% 
                 82.5% 
                 80.2% 
                 82.2-85.4 
               
               
                 PS Conjugated (%) 
                 72.1% 
                 74.0% 
                 73.3% 
                 71.7-75.2 
               
               
                 % Polydispersity &gt; 180 Å 
                 39.3 
                 44.1 
                 41.9 
                   34-40.8 
               
               
                 Radius (Å) 
                 183 
                 195.1 
                 192 
                 193.6-198.6 
               
               
                 Glycosylation Degree 
                 1.1 
                 1.1 
                 1.1 
                 1.0-1.1 
               
               
                   
               
            
           
         
       
     
                     TABLE 4                  Sized GBS Ia                                                 Free    Free                Lot   Glycosylation   PS   Carrier   Hydrodynamic           number   Degree (w/w)   (%)*   (%)**   Radius (Å)̊                                             Existing    007   1.7   20.1   &lt;1.7   387 (372)       (Native   008   1.5   19.7   &lt;2.3   407       PS) Process   009   1.5   18.4   &lt;1.5   407 (390)           010   1.5   17   &lt;2.2   430       Sized PS   023   1.0   &lt;1.3   &lt;1.0   238       process   024   1.0   &lt;1.2   &lt;1.0   249           026   1.1   &lt;1.2   &lt;1.0   248               Free PS by HPAEC-PAD Gal I; Free protein by μBCA (IPC)       *percentage of PS in the final product that is unconjugated       **percentage of carrier protein in final product that is unconjugated            
The present example using sized GBS Ia polysaccharide provided, after hydroxyapatite chromatography, a product with reduced free polysaccharide content (compared to the process using native GBS Ia polysaccharide). As expected, the size of the GBS Ia-CRM197 conjugate prepared using sized GBS Ia polysaccharide was smaller (compared to GBS Ia-CRM197 conjugate prepared using native GBS Ia polysaccharide) but was similar to the size of native GBS serotype III-CRM197 conjugate. The glycosylation degree was lower.
 
     Example 4—Use of Sized GBS Serotype Ib Polysaccharide 
     For GBS Ib, a similar series of experiments was done as above for GBS Ia. Sized Lots were produced using the following conjugation parameters: CRM197 8 mg/ml, PS 10 mg/ml, NaCNBH3 9 mg/ml and 48 hours of reaction 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 GBS Ib 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Free 
                 Free 
               
               
                   
                 Lot  
                 Glycosylation 
                 PS 
                 Carrier** 
               
               
                   
                 number 
                 Degree (w/w) 
                 (%)* 
                 (%) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Existing  
                 001 
                 1.3 
                 17.4 
                 &lt;1.2 
               
               
                 (Native 
                 002 
                 1.1 
                 18 
                 &lt;1.8 
               
               
                 PS) Process 
                 003 
                 1.2 
                 16.4 
                 &lt;1.2 
               
               
                   
                 004 
                 1.1 
                 17.2 
                 &lt;1.8 
               
               
                 Sized PS  
                 008A 
                 1.1 
                 &lt;0.9* 
                 &lt;0.6 
               
               
                 process 
                 008B 
                 1.2 
                 &lt;0.7* 
                 &lt;0.6 
               
               
                   
               
               
                 *percentage of PS in the final product that is unconjugated 
               
               
                 **percentage of carrier protein in final product that is unconjugated 
               
            
           
         
       
     
     Example 5—Use of Sized GBS Serotype II Polysaccharide Increases Yield of Conjugate 
     The present inventors investigated the use of sized GBS serotype II polysaccharides in the production of polysaccharide-protein conjugates. Native GBS II polysaccharide has a mean molecular weight of about 419 kDa. Microfluidisation technology as described above was used for the size reduction of native GBS II polysaccharide. Six cycles at 8000 psi produced GBS serotype II capsular polysaccharide having a mean molecular weight of 159 kDa. GBS PS are in liquid form and may vary slightly in PS concentration. Here, a first microfluidization of GBS II was done with PS at 5.42 mg/ml; the next microfluidization used GBS II with a PS concentration of 5.47 mg/ml. 
     Oxidation was performed using the same conditions as with a native GBS serotype II polysaccharide. Oxidation level of sized GBS II was near to that observed with a native GBS II PS (18.2% vs. 19.5-20.1%, respectively). 
     Conjugation and purification were performed according to existing process conditions for native GBS II PS. The CRM197 conversion (percentage of CRM197 that becomes conjugated) appeared slightly lower using sized GBS II PS than that obtained with a native GBS II PS (52.5% vs. 58-60%). For sized GBS serotype II PS, the free polysaccharide content before hydroxyapatite chromatography was estimated at 23.6%, compared to 6-7% with native GBS serotype II PS. 
     Using sized GBS serotype II polysaccharide rather than native polysaccharide, the yield of GBS serotype II PS was increased from 40% to 53%. To further improve the yield, conjugation conditions were assessed to increase % of conjugated PS. 
     To improve the conjugation step (increase the % of conjugated sized GBS II PS before the hydroxyapatite purification step), three lots at 700 mg PS scale were produced using the following conjugation reaction conditions: CRM197 6 mg/ml; Polysaccharide 11 mg/ml; NaCNBH3 9 mg/ml; 48 hours of reaction at 22° C. 
     The three lots demonstrated the same behavior in conjugation, as assessed by SEC-UPLC monitoring. 
     In purification, no significant loss of sized GBSII-CRM197 conjugate was observed in the flow through from the HA chromatography column. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 6 
               
               
                   
                   
               
               
                   
                   
                 Hydrodynamic 
                 %  
                 %  
               
               
                   
                   
                 Radius (Rh) 
                 CRM197 
                 conjugated 
               
               
                   
                 Study 
                 (Å) 
                 conversion 
                 PS 
               
               
                   
                   
               
             
            
               
                   
                 003 
                 154 
                 79 
                 76.4 
               
               
                   
                 004 
                 155 
                 75 
                 77.3 
               
               
                   
                 005 
                 154 
                   76.5 
                 76.8 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                   
                   
                   
                   
                 CRM197 
                   
               
               
                   
                   
                 PS (μg/ml) 
                 PS content 
                 concentration 
                   
               
               
                   
                 Time 
                 RP-SEC- 
                 (HPAEC-PAD) 
                 (μBCA assay) 
                 Glycosylation 
               
               
                 Study 
                 (hour) 
                 HPLC(1) 
                 (2) μg/ml 
                 (3) μg/ml 
                 degree 
               
               
                   
               
             
            
               
                 003 
                 48 
                 1931  
                 1712  
                 1170  
                 1.46 
               
               
                 004 
                 48 
                 904 
                 865 
                 598 
                 1.45 
               
               
                 005 
                 48 
                 898 
                 842 
                 571 
                 1.47 
               
               
                   
               
            
           
         
       
     
     (1) Reversed Phase (RP) Size Exclusion Chromatography (SEC) High Performance Liquid Chromatography (HPLC); 
     (2) High Performance Anion Exchange Chromatography (HPAEC) with Pulsed Amperometric Detection (PAD);
 
(3) Micro-bicinchoninic acid (μBCA) assay.
 
     Example 6—GBS Ia-CRM197 Antibody Response in Mice 
     Mouse studies were carried out using GBS Ia-CRM197 conjugates produced using either native or sized GBS Ia PS (produced as described above), and using one of two conjugation processes, as listed in Table 8. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 GBS Ia - CRM197 conjugates 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Conjugation 
                   
               
               
                   
                 Group 
                 Process 
                 PS source 
               
               
                   
                   
               
               
                   
                 1 
                 existing 
                 native 
               
               
                   
                 (RS2505) 
                   
                   
               
               
                   
                 2 
                 existing 
                 native 
               
               
                   
                 (009) 
                   
                   
               
               
                   
                 3 
                 existing 
                 Sized (80 kDa) 
               
               
                   
                 (014) 
                   
                   
               
               
                   
                 4 
                 existing 
                 native 
               
               
                   
                 (021) 
                   
                   
               
               
                   
                 5 
                 improved 
                 Sized (100 kDa) 
               
               
                   
                 (024) 
                   
                   
               
               
                   
                 6 
                 existing 
                 Sized (100 kDa) 
               
               
                   
                 (025) 
               
               
                   
                   
               
               
                   
                 The “Existing” conjugation process: PS conc = 6.35 mg/ml; initial PS/CRM ratio = 0.75/1 w/w; NaBH3C conc = 6.35 mg/ml. 
               
               
                   
                 “Improved” conjugation process: PS conc = 11 mg/ml; initial PS/CR ratio = 1.22/1 w/w; NaBH3CN conc = 9 mg/ml. 
               
            
           
         
       
     
     The study as set forth in Table 8 was repeated three times (with 32 mice per group, 16 mice per group, and 16 mice per group, for a total of 64 mice per group). Five-week old female CD1 mice were used in all groups. Conjugated polysaccharides were provided in aluminium hydroxide formulation 2 mg/ml, 1 μg immunization PS dose, administered at days 1, 21 and 35 via an intraperitoneal route (IP). With a 0.6 Standard Deviation, 64 mice was determined as the sample size per group to appreciate a two- to three-fold difference in IgG titers with at least 80% power. 
     Sera was collected from each mouse two weeks after the third immunization and tested for IgG titers using Luminex in monoplex PS Ia-Biotin (Relative Unit/ml or RU/ml). Streptavidin beads were coupled with 1 ug/ml of GBS PS Ia-biotin, and the bead set was incubated (1 hour) with individual mouse sera. Analysis confirmed that Group 5 (sized GBS Ia PS of 100 kDa, using improved conjugation process as described above) elicited a significantly higher antibody response in the tested mice (between 1.3- and 5.2-fold difference). Both Groups 3 and 5 showed higher titers compared to other groups. See  FIG. 4 , showing total results on the 64 sera/group. No differences within groups were detected among the three independent experiments. ANOVA P value adjusted for multiple comparison &gt;0.1. 
     Example 7—GBS Ib-CRM197 Antibody Response in Mice 
     GBS Ib-CRM197 conjugates were also assessed in preclinical mouse studies. Five-week old female CD1 mice (64 mice per group) were immunized with the following: 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 GBS Ib - CRM197 conjugates 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Conjugation  
                   
               
               
                   
                 Group 
                 process* 
                 PS source 
               
               
                   
                   
               
               
                   
                 1 
                 Existing 
                 Native 
               
               
                   
                 (RS2606) 
                   
                   
               
               
                   
                 2 
                 Existing 
                 Sized (100 kDa) 
               
               
                   
                 (006) 
                   
                   
               
               
                   
                 3 
                 Improved 
                 Sized (100 kDa) 
               
               
                   
                 (008A) 
               
               
                   
                   
               
               
                   
                 *as described above 
               
            
           
         
       
     
     Conjugated polysaccharides were provided in aluminium hydroxide (2 mg/ml) formulation, 1 μg immunization PS dose, administered at days 1, 21 and 35 via an intraperitoneal route (IP). Sera was collected from each mouse two weeks after the third immunization and tested for IgG titers by Luminex in monoplex PS Ib-Biotin assay; antibody functionality was tested by Opsonophagocytosis Killing Assay (OPKA) using pooled sera from each group. 
       FIG. 5  indicates total IgG titers from individual sera for each group, expressed as Relative Unit (RU) and Geometric Mean Titers ±95% confidence intervals (shown as dot and bars, respectively). No significant differences in IgG response were found among the three GBS Ib groups. 
     OPKA Titers, expressed as reciprocal of the serum dilution resulting in 50% GBS killing, showed no major differences among the three GBS Ib groups (data not shown). 
     Example 8—GBS II-CRM197 Antibody Response in Mice 
     Three different preparations of GBS II-CRM197 (1 ug CPS, formulation with 2 mg/ml Alum, 200 ul administered by IP route at days 1, 21 and 35). Five-week old CD1 female mice, 64 total mice per formulation. Sera from immunized mice were collected two weeks after the third immunization and tested for IgG titers by Luminex assay, and for antibody functional activity by OPKA titers. 
     Three studies were conducted as shown in Tables 10-12: 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Study 1/GBS II-CRM197 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Conjugation 
                   
               
               
                 Group 
                 # Mice 
                 Bacterial Seed 
                 Process* 
                 PS Source 
               
               
                   
               
               
                 1 
                 8 
                 Old seed, Lot EB26 
                 Existing 
                 Native 
               
               
                 2 
                 8 
                 Old seed, Lot EB26 
                 Existing 
                 Native 
               
               
                 3 
                 8 
                 Old seed, Lot EB26 
                 Existing 
                 Native 
               
               
                 4 
                 8 
                 New Seed, Lot 002 
                 Existing 
                 Sized (160 kDa) 
               
               
                 5 
                 8 
                 New Seed, Lot 002 
                 Existing 
                 Sized (160 kDa) 
               
               
                 6 
                 8 
                 New Seed, Lot 002 
                 Existing 
                 Sized (160 kDa) 
               
               
                 7 
                 8 
                 New seed, Lot 003 
                 Improved 
                 Sized (160 kDa) 
               
               
                 8 
                 8 
                 New seed, Lot 003 
                 Improved 
                 Sized (160 kDa) 
               
               
                 9 
                 8 
                 New seed, Lot 003 
                 Improved 
                 Sized (160 kDa) 
               
               
                   
               
               
                 *as described above 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Study 2/GBS II-CRM197 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Conjugation 
                   
               
               
                 Group 
                 # Mice 
                 Bacterial Seed 
                 Process* 
                 PS Source 
               
               
                   
               
               
                 1 
                 8 
                 Old seed, Lot EB26 
                 Existing 
                 Native 
               
               
                 2 
                 8 
                 Old seed, Lot EB26 
                 Existing 
                 Native 
               
               
                 3 
                 8 
                 Old seed, Lot EB26 
                 Existing 
                 Native 
               
               
                 4 
                 8 
                 New Seed, Lot 002 
                 Existing 
                 Sized (160 kDa) 
               
               
                 5 
                 8 
                 New Seed, Lot 002 
                 Existing 
                 Sized (160 kDa) 
               
               
                 6 
                 8 
                 New Seed, Lot 002 
                 Existing 
                 Sized (160 kDa) 
               
               
                 7 
                 8 
                 New Seed, Lot 003 
                 Improved 
                 Sized (160 kDa) 
               
               
                 8 
                 8 
                 New Seed, Lot 003 
                 Improved 
                 Sized (160 kDa) 
               
               
                 9 
                 8 
                 New Seed, Lot 003 
                 Improved 
                 Sized (160 kDa) 
               
               
                   
               
               
                 *as described above 
               
            
           
         
       
     
                     TABLE 12                  Study 3/GBS II-CRM197                                             Conjugation           Group   # Mice   Bacterial seed   Process*   PS Source               1   8   Old seed, Lot EB26   Existing   Native       2   8   Old seed, Lot EB26   Existing   Native       3   8   New Seed, Lot 002   Existing   Sized (160 kDa)       4   8   New Seed, Lot 002   Existing   Sized (160 kDa)       5   8   New Seed, Lot 003   Improved   Sized (160 kDa)       6   8   New Seed, Lot 003   Improved   Sized (160 kDa)               *as described above            
Results from each of the three GBS II studies, and total results of merged studies:
 
     In each of the three studies, all groups of mice immunized with a GBS II-CRM197 conjugate responded to vaccination, compared to control mice. In the exploratory statistical analysis (P value Adjusted for multiple comparison by ANOVA), significantly lower and more dispersed IgG responses were observed in Study 1 for Lot 002, with respect to the other two preparations (EB26 and 003); however, no significant differences in IgG responses were observed among the three (EB26, 002, and 003) in Studies 2 and 3. Overall, consistent data among the three studies were observed. Therefore, as primary objective, the data from all three studies (including a total of 64 mice) were merged in a final analysis, as shown in  FIG. 6 . The the exploratory statistical analysis (P value Adjusted for multiple comparison by ANOVA) indicated a statistically significant difference in IgG responses between preparation EB26 and 002. 
       FIG. 6 : IgG responses—single mouse Luminex data (RU/ml) are represented as dots; the bars represent the Geometric Mean Titer ±95% Confidence Interval (CI). The bar above the graph reports the obtained Geometric Mean IgG Titers (GEOMEAN). 
     OPKA Titers, expressed as reciprocal of the serum dilution resulting in 50% GBS killing, showed no major differences among the GBS II groups in the three studies (data not shown), though in accordance with IgG response measured by Luminex, in Study 1, groups 5 and 6 (002) showed lower OPKA titers with respect to the other conjugate preparations. 
     REFERENCES 
     Anderson (1983)  Infect Immun  39(1):233-238. 
     Anderson et al. (1985)  J Clin Invest  76(1):52-59. 
     Anonymous (Jan 2002)  Research Disclosure,  453077. 
     Balmer et al. (2002)  J Med Microbiol  51:717-722. 
     Baraldo et al. (2004)  Infect Immun  72(8):4884-7. 
     Broker et al. (2017)  Vaccine  35(25): 3286-3294. 
     Falugi et al. (2001)  Eur J Immunol  31:3816-3824. 
     Kuo et al. (1995)  Infect Immun  63:2706-13. 
     Lees et al. (1996)  Vaccine  14:190-198. 
     Lei et al. (2000)  Dev Biol  ( Basel ) 103:259-264. 
     Pappenheimer (1977)  Ann Rev, Biochem.  46: 69-94. 
     Peltola (2000)  Clin Microbiol Rev  13:302-317 
     Rappuoli et al. (1983)  Applied and Environ Microbiol  46(3):p560-564 
     Ruan et al. (1990)  J Immunol  145:3379-3384. 
     Uchida et al., (1971)  Nature New Biology  233: 8-11. 
       Vaccine  (ed Plotkin et al) Fourth Edition ISBN 0-7216-9688-0 
     Wessels et al. (1989)  Infect Immun  57:1089-94. 
     Wessels et al. (1990)  J Clin Invest  86:1428-33. 
     Wuorimaa &amp; Kayhty (2002)  Scand J Immunol  56:111-129. 
     EP-A-0372501 
     EP-A-0378881 
     EP-A-0427347 
     EP-A-0471177 
     EP-A-0594610 
     EP0497524 
     EP0497525 
     WO1991/001146 
     WO1993/017712 
     WO1994/003208 
     WO1995/008348 
     WO1998/042721 
     WO1998/058668 
     WO2000/038711 
     WO2000/056360 
     WO2000/061761 
     WO2001/072337 
     WO2002/091998 
     WO2002/034771 
     WO2002/012294 
     WO2003/007985 
     WO2006/050341 
     WO2006/082527 
     WO2006/082530 
     WO2009/010877 
     WO2012/035519 
     U.S. Pat. No. 4,356,170
 
U.S. Pat. No. 6,146,902
 
U.S. Pat. No. 9,463,250