RU2791468C2 - Immunogenic polysaccharide-protein conjugates containing a polysaccharide derived from group b streptococcus - Google Patents

Abstract

FIELD: pharmacy.

SUBSTANCE: invention can be used to treat or prevent an infection caused by group B streptococcus. Immunogenic composition containing conjugates of polysaccharide-carrier protein are proposed, in which each of the conjugates includes a capsular polysaccharide from a different serotype of group B streptococcus (GBS) conjugated with a carrier protein, and serotypes include serotypes Ia, Ib, II, III, IV and V, the capsular polysaccharide in each conjugate has a sialic acid level of more than 60%, and the carrier protein contains CRM₁₉₇. While the immunogenic composition additionally contains sodium chloride at a concentration of 150 mm or higher, polysorbate-80, histidine, and optionally an adjuvant, which is aluminum phosphate. The pH of the immunogenic composition is between 6.5 and 7.5 and the immunogenic composition does not elicit a reduced immune response to the III serotype.

EFFECT: stable hexavalent immunogenic formulation.

13 cl, 47 dwg, 51 tab, 26 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The invention relates to immunogenic compositions containing a capsular polysaccharide (CP) from Streptococcus agalactiae , commonly referred to as group B streptococcus (GBS), conjugated to a carrier protein, and optionally containing an aluminum-based adjuvant. The invention also relates to methods for inducing an immune response against GBS in patients and/or alleviating or preventing invasive disease caused by GBS in patients using the compositions disclosed herein. The resulting antibodies can be used to treat or prevent GBS infection by passive immunotherapy.

BACKGROUND OF THE INVENTION

Streptococcus agalactiae are gram-positive, polysaccharide-encapsulated microorganisms, also known as group B streptococci (GBS). They are common commensals in the human gastrointestinal and genital tract and also cause serious disease in infants and the elderly (Baker, CJ, Vaccine, 31(Suppl. 4):D3-D6 (2013)). The main risk factor for GBS infection in infants is maternal colonization (Dillon, HC, et al., J. Pediatr., 110(1):31-36 (1987)). It is estimated that one in four women have GBS rectovaginally, which can infect the amniotic fluid or infant before or during labor, causing sepsis, pneumonia, and meningitis (Baker 2013; Heath, PT, et al., BMJ Clin. Evid (Online), pii:0323 (2014)). Twenty-five percent of infants who survive GBS-induced meningitis suffer from neurological impairment, and 19% have cognitive delay, cerebral palsy, blindness, and hearing loss (Libster, R., et al., Pediatrics, 130(1):e8 -152012 (2012)). GBS can also cause miscarriage and preterm birth and have been associated with stillbirths (McDonald, HM, et al., Infect. Dis. Obstetr. Gynecol., 8(5-6):220-227 (2000); Randis , TM, et al., J. Infect.Dis., 210(2):265-273 (2014): Kessous, R., et al., J. Matern.Fetal Neonatal Med., 25(10):1983 -1986 (2012)). Very low birth weight infants have a much higher risk of infection, with up to 3% of them infected, and mortality as high as 30% even with immediate antibiotic treatment (Heath 2014).

The introduction of GBS screening and intranatal antibiotic prophylaxis (IAP) in the United States in the late 1990s resulted in a reduction in neonatal morbidity in the first week of life (early onset [RND]), but had no measurable effect on rates of late onset disease ( PNZ), which occurs later, within the first 3 months of life. The current rates of RND and PND in the United States are 0.25 and 0.27 per 1000 births, respectively (Centers for Disease Control and Prevention (CDC), Active Bacterial Core (ABC) Surveillance Report (2013) available at http:/ /www.cdc.gov/abcs/reports-findings/survreports/gbs13.pdf). Since the introduction of pneumococcal conjugate vaccines to prevent invasive pneumococcal disease, including bacteremia and meningitis, and despite PAIs to prevent GBS-related disease, GBS has become the single most common cause of neonatal sepsis (RNS) and meningitis (<2 months) in infants in the USA (Verani, JR, et al., MMWR, 59(RR10):1-32 (2010); Thigpen, MC, et al., N. Engl. J. Med., 364(21):2016-2025 (2011)). Contrary to the United States, the introduction of interventions to prevent invasive disease caused by GBS and PAI did not lead to a reduction in the incidence of RNC in the Netherlands or in the United Kingdom (Bekker, V., et al., The Lancet Infectious Dis., 14( 11):1083-1089 (2014); Lamagni, TL, et al., Clin. Infect. Dis., 57(5):682-688 (2013)). This lack of effect may be due to lack of widespread screening and limitation of PAI to mothers at highest risk (eg, fever, prolonged rupture of membranes). RNR rates are significantly higher in countries that do not use PAIs, with a reported median incidence of 0.75 per 1000 live births (95% CI 0.58–0.89) (Edmond, KM, et al., Lancet, 379(9815):547-556 (2012)).

Another population at risk for GBS-induced disease is the elderly. Risk factors include chronic medical problems such as diabetes, cancer, heart failure, and neurological and urological disorders. According to the CDC ABC in the US, the annual incidence of GBS invasion in 2013 was 0.28/1000 adults or 12400 cases/year in adults ≥65 years of age. This rate approaches the incidence of invasive pneumococcal disease in the elderly (versus 0.30/1000 for people over 65). These rates are expected to continue to rise in both the US and Europe (CDC 2013; Lamagni 2013).

One approach to prevent GBS-induced disease in infants and the elderly is the use of a polysaccharide vaccine. Maternal prophylactic vaccination against GBS has the potential to prevent GBS-associated disease in infants in the US, independent of PAI use. Although polysaccharides may be immunogenic per se, conjugation of polysaccharides with protein carriers has been used to increase immunogenicity, especially in children and the elderly. Polysaccharide-protein conjugate vaccines are prepared using polysaccharides, usually from the surface envelope of bacteria associated with protein carriers. The polysaccharide-protein conjugate induces an immune response against bacteria exposed on the surface of the polysaccharide contained in the vaccine, thus preventing disease. Accordingly, vaccination using polysaccharide conjugates from pathogenic bacteria is a potential strategy for stimulating host immunity.

The polysaccharides that coat bacteria vary greatly, even among bacteria of the same species. For example, GBS has ten different serotypes due to variations in the bacterial polysaccharide capsule. Because of this, it is desirable that polysaccharide vaccines contain a panel of polysaccharides to ensure breadth of coverage against different circulating serotypes.

The carrier protein can be either an appropriate protein antigen from the target pathogen that stimulates a specific immune response to that pathogen, or a conventional immunogenic protein that serves more as an adjuvant or a general stimulator of the immune response.

Separate monovalent polysaccharide-protein conjugates for GBS serotypes Ia, Ib, II, III and V have been evaluated in phase 1 and 2 clinical trials in non-pregnant adults (Brigtsen, AK, et al., J. Infect. Dis., 185 (9):1277-1284 (2002); Baker, CJ, et al., J. Infect. Dis., 188(1):66-73 (2003); Baker, CJ, et al., J. Infect. Dis., 189(6):1103-1112 (2004); Baker, CJ, et al., Vaccine, 25(1):55-63 (2007)). Bivalent glycoconjugate vaccines II-TT and III-TT and a trivalent vaccine containing Ia-CRM ₁₉₇ , Ib-CRM ₁₉₇ and III-CRM ₁₉₇ glycoconjugates have also been studied (Baker 2003; Clicaltrials.gov NCT01193920, NCT01412801 and NCT01446289). However, GBS vaccines have not yet been approved for use.

Furthermore, although the trivalent vaccine covers >90% of the invasive strains causing neonatal disease in South Africa (Madzivhandila, M., et al., PloS One, 6(3):e17861 (2011)), these same serotypes represent only 62% and 66% of invasive isolates in North America and Europe, respectively, based on a review of the latest neonatal isolates from a global collection of 901 specimens collected from 2004-2013 in the Tigecycline Evaluation and Control Clinical Trial (TEST, http://www. .testsurveillance.com/).

Analysis of the strains obtained from the samples collected in TEST showed that 95% of the collected strains belonged to one of the five main serotypes reported (Ia, Ib, II, III and V) and another 3% had serotype IV. A series of publications has also confirmed the emergence of serotype IV during the last decade in the Americas and Europe (Diedrick, MJ, et al., J. Clin. Microbiol., 48(9):3100-3104 (2010); Teatero (2014 ); Meehan, M. et al., Eur. J. Clin. Microbiol. Infect. Dis., 33(7):1155-1162 (2014); Florindo, C., et al., Euro Surveillance: Bulletin European sur les Maladies Transmissibles (European Communicable Disease Bulletin), 19(23) (2014); Palmiero, JK, et al., J. Clin. Microbiol., 48(12):4397-4403 (2010)). In a study investigating adult rectovaginal carriage, a risk factor for transmission of GBS to infants, it was also found that 97% of isolates belonged to one of these six serotypes, with serotype IV occurring at a frequency of ~4%. The study was designed to monitor the carriage of beta-hemolytic streptococci (which include GBS), Clostridium difficile , and Staphylococcus aureus in healthy US adults (see Matson, MA, et al., ICAAC, Abstract I-306 (Washington, DC, Sep. 5 -9, 2014)).

Similarly, analysis of samples from T.E.S.T. showed that 98% of isolates from the blood of elderly people aged ≥65 years living in the United States belong to the same six predominant serogroups. The most notable difference between isolates from the elderly and other populations is the distribution of serogroups. In isolates from elderly patients, serotype V strains make up the largest group (34% versus 18% in neonates or 18% in adult strain carriers).

Other studies have found that there is geographic variation in the prevalence of serotypes. For example, isolates of serotypes VI and VIII have been shown to be the predominant colonizing organisms in healthy pregnant women in Japan (Lachenauer, CS, et al., J. Infect. Dis., 179(4):1030-1033 (1999).

Accordingly, there is a need for polysaccharide-protein conjugate vaccines or monoclonal antibodies to confer passive immunity as a means to prevent or treat diseases caused by GBS, including those caused by the new serotype IV, in the general population throughout the world. In addition, such vaccines or immunogenic compositions need to be stable and readily resuspendable in any container known in the art.

SUMMARY OF THE INVENTION

The present invention relates to novel immunogenic compositions containing GBS polysaccharide-protein conjugates and optionally containing an adjuvant. The following paragraphs describe some aspects and embodiments of the invention.

In one aspect, the invention relates to an immunogenic composition comprising a group B streptococcus (GBS) capsular polysaccharide conjugated to a carrier protein, and optionally, an aluminum-based adjuvant. In one embodiment, the aluminum-based adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxyphosphate, and aluminum hydroxide. In one such embodiment, the aluminum-based adjuvant is at a concentration of from about 0.25 mg/mL to about 0.75 mg/mL.

In another embodiment, the capsular polysaccharide is selected from the group consisting of polysaccharides of serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII and IX. In another embodiment, the immunogenic composition is a hexavalent GBS conjugate composition. In one such embodiment, the immunogenic composition comprises capsular polysaccharides from GBS serotypes Ia, Ib, II, III, IV and V.

In another embodiment, the invention provides an immunogenic composition comprising polysaccharide-protein conjugates, wherein the conjugates comprise capsular polysaccharides from group B streptococci (GBS) selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII and IX; and further comprising one or more of a pharmaceutically acceptable excipient, buffer, stabilizer, adjuvant, cryoprotectant, salt, divalent cation, nonionic detergent, free radical oxidation inhibitor, carrier, or a mixture thereof.

In another embodiment, the invention provides an immunogenic composition as described herein wherein the capsular polysaccharides are individually conjugated to the same carrier protein.

In another aspect of the invention, the immunogenic composition is placed in a syringe. In one embodiment, the syringe is glass. In another embodiment, the immunogenic composition is placed in a syringe with a headspace of about 1 mm to about 15 mm above the product.

In a further aspect of the invention, the immunogenic composition is resuspended with about 50 shakes or less.

In one embodiment, the immunogenic composition further comprises a phosphate. In one such embodiment, the phosphate is at a concentration of from about 15 mM to about 25 mM.

In another embodiment, the immunogenic composition further comprises sodium chloride. In one such embodiment, sodium chloride is at a concentration of from about 10 mM to about 350 mM.

In a further embodiment, the immunogenic composition contains a total dose of conjugate from about 60 µg/ml to about 360 µg/ml.

In an additional embodiment, the immunogenic composition has a pH of from about 6.5 to about 7.5.

In one aspect of the invention, the immunogenic composition comprises about 0.25 mg/mL to about 0.75 mg/mL aluminum phosphate, about 15 mM to about 25 mM phosphate, about 225 mM to about 265 mM sodium chloride, and a total dose of conjugate from about 60 μg/ml to about 360 μg/ml, and the immunogenic composition has a pH of from about 6.5 to about 7.5.

In another aspect of the invention, the immunogenic composition comprises about 0.25 mg/ml to about 0.75 mg/ml aluminum hydroxide, about 15 mM to about 25 mM phosphate, about 120 mM to about 170 mM sodium chloride, and a total dose of conjugate from about 60 μg/ml to about 360 μg/ml, and the immunogenic composition has a pH of from about 6.5 to about 7.5.

Another aspect of the invention relates to a method for inducing an immune response against GBS, comprising administering to a patient an effective amount of an immunogenic composition as described herein. In one embodiment, the invention provides a method for preventing, or alleviating, a GBS-associated disease or condition in a patient, comprising administering to the patient an effective amount of an immunogenic composition described herein. In a specific embodiment, the patient is a woman planning a pregnancy or a pregnant woman. In one such embodiment, the pregnant woman is in the second half of her pregnancy, for example, at least 20 weeks pregnant or at least 27 weeks pregnant. In a preferred embodiment, the pregnant woman is between 27 weeks and 36 weeks pregnant. In another embodiment, the patient is an elderly person, such as 50 years of age or older, 65 years of age or older, and 85 years of age or older. In a further embodiment, the patient is immunocompromised. In one aspect, the patient may have a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease, or liver disease. In a preferred embodiment, the group B streptococcus is Streptococcus agalactiae .

In a further aspect, the invention relates to antibodies that bind a capsular polysaccharide in an immunogenic composition of the present invention. In some embodiments, antibodies are obtained by administering an immunogenic composition to a patient. In another aspect, the invention relates to a composition containing the antibodies of the present invention.

The next aspect of the invention relates to a method for creating passive immunity in a patient, comprising the steps of (a) obtaining an antibody preparation using an immunogenic composition described in the present description; and (b) administering the antibody preparation to the patient to confer passive immunity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Comparison of opsonic activity of serum and isolated IgG from mice immunized with monovalent vaccines GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ .

FIG. 2: Stability of GBS Ia-CRM ₁₉₇ (expressed as % change in molecular weight determined by SEC-MALLS) after storage under accelerated aging conditions (4 weeks) at 50°C.

FIG. 3: Stability of GBS Ib-CRM ₁₉₇ (given as % change in molecular weight determined by SEC-MALLS) after storage under accelerated aging conditions (4 weeks) at 50°C.

FIG. 4: Stability of GBS II-CRM ₁₉₇ (given as % change in molecular weight determined by SEC-MALLS) after storage under accelerated aging conditions (4 weeks) at 50°C.

FIG. 5: Stability of GBS III-CRM ₁₉₇ (given as % change in molecular weight determined by SEC-MALLS) after storage under accelerated aging conditions (4 weeks) at 50°C.

FIG. 6: Stability of GBS IV-CRM ₁₉₇ (given as % change in molecular weight determined by SEC-MALLS) after storage under accelerated aging conditions (4 weeks) at 50°C.

FIG. 7: Stability of GBS V-CRM ₁₉₇ (given as % change in molecular weight determined by SEC-MALLS) after storage under accelerated aging conditions (4 weeks) at 50°C.

FIG. 8: Stability of GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS III-CRM ₁₉₇ and GBS IV-CRM ₁₉₇ (represented by the presence of free sialic acid) after storage at 37°C.

FIG. 9: pH stability in the hexavalent GBS vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) using succinate in as a buffer.

FIG. 10: pH stability in the hexavalent GBS vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) using histidine in as a buffer.

FIG. 11: Effect of the concentration of histidine buffer in the hexavalent GBS vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) on binding conjugates of GBS with aluminum at a dose of 10 µg/ml.

FIG. 12: Effect of the concentration of histidine buffer in the hexavalent GBS vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) on binding conjugates of GBS with aluminum at a dose of 40 µg/ml.

FIG. 13: Effect of polysorbate-80 concentration in hexavalent GBS vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) on loss, in percent, of total antigenicity under shaking stress.

FIG. 14: Effect of aluminum concentration in hexavalent GBS vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) on conjugate binding GBS with aluminium.

FIG. 15: Effect of 5.5% (w/v) sucrose at 10 μg/ml of lyophilized hexavalent GBS vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) to restore antigenicity for each serotype.

FIG. 16: Effect of 7.0% (w/v) sucrose at 10 μg/ml of lyophilized hexavalent GBS vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) to restore antigenicity for each serotype.

FIG. 17: Effect of 8.5% (w/v) sucrose at 10 μg/ml of a lyophilized hexavalent GBS vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) to restore antigenicity for each serotype.

FIG. 18: Effect of 5.5% (w/v) sucrose at 50 μg/ml of a lyophilized hexavalent GBS vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) to restore antigenicity for each serotype.

FIG. 19: Effect of 7.0% (w/v) sucrose at 50 μg/ml of lyophilized hexavalent GBS vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) to restore antigenicity for each serotype.

FIG. 20: Effect of 8.5% (w/v) sucrose at 50 μg/ml of lyophilized hexavalent GBS vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) to restore antigenicity for each serotype.

FIG. 21: Effect of 7.0% (w/v) sucrose at 40 μg/ml of lyophilized hexavalent GBS vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) to restore antigenicity for each serotype.

FIG. 22: Effect of 2.0% (w/v) sucrose and 4.0% (w/v) mannitol at 40 μg/ml of lyophilized hexavalent GBS vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) to restore antigenicity for each serotype.

FIG. 23: Effect of 3.0% (w/v) sucrose and 3.0% (w/v) mannitol at 40 μg/ml of lyophilized hexavalent GBS vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) to restore antigenicity for each serotype.

FIG. 24: Effect of 2.0% (w/v) sucrose and 4.0% (w/v) glycine at 40 μg/ml of a lyophilized hexavalent GBS vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) to restore antigenicity for each serotype.

FIG. 25: Effect of 3.0% (w/v) sucrose and 3.0% (w/v) glycine at 40 μg/ml of lyophilized hexavalent GBS vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) to restore antigenicity for each serotype.

FIG. 26: Comparison of GBS6 reconstituted suspension and high and low dose monovalent preparations stored tip down in PFS at time points 0, 2 weeks and 3 months.

FIG. 27: Comparison of GBS6 reconstituted suspension and high and low dose monovalent preparations stored in PFS tip-down and horizontal.

FIG. 28: Zeta potential of AlPO ₄ as a function of pH.

FIG. 29: Zeta potential of GBS conjugates of serotypes Ia, Ib, II, III, IV and V as a function of pH.

FIG. 30: Comparison of sedimentation rates of different GBS6 and high and low dose monovalent preparations stored tip down in PFS at time points 0, 2 weeks and 3 months.

FIG. 31: Sedimentation rate of GBS6 preparations versus dose levels (concentration for each serotype).

FIG. 32: Sedimentation rate of GBS6 preparations as a function of pH at low dose (60 µg/mL) and high dose (240 µg/mL).

FIG. 33: Effect of NaCl dose and concentration on sedimentation rate.

FIG. 34: Comparison of the settling characteristics of different antigen preparations after 45 minutes.

FIG. 35A: Detailed particle size distribution in different antigen preparations.

FIG. 35B: Particle size distribution in different antigen preparations, categorized at ≥10 µm or ≥25 µm.

FIG. 36: GBS6 preparations in PFS with different headspaces.

FIG. 37: Number of shakes required to resuspend high dose GBS6 preparations with different concentrations of AlPO ₄ after 2 weeks of storage in a horizontal position.

FIG. 38: Binding of GBS serotypes Ia, Ib, II, III, IV and V conjugates to AlPO ₄ at various concentrations after 2 weeks of storage in a horizontal position.

FIG. 39: Effect of various flocculants and aluminum salts on resuspension.

FIG. 40: Effect of different flocculants and aluminum salts on settling rate.

FIG. 41: Effect of NaCl concentrations on resuspension time of high dose GBS6 preparations.

FIG. 42: Effect of combination of NaCl and phosphate ions on resuspension time.

FIG. 43A: Effect of NaCl and potassium phosphate concentrations on precipitation after 2 hours.

FIG. 43B: Effect of NaCl and potassium phosphate concentrations on precipitation after 30 minutes.

FIG. 44: Associated antigenicity, in percent, of the GBS6 preparation containing 20 mM phosphate and 240 mM NaCl, compared to the control GBS6 preparation.

FIG. 45: Profiling system recommendations for predicting optimal and reliable drug conditions for easier resuspension (≤10 shakes).

FIG. 46: Associated antigenicity, in percent, of GBS6 preparations based on the recommendations of the profiling system for predictions.

FIG. 47: Binding of GBS serotypes Ia, Ib, II, III, IV and V to Al(OH) ₃ at various concentrations.

DETAILED DESCRIPTION OF THE INVENTION

It should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used in the present description is only for the purpose of describing specific embodiments and is not intended to be limiting.

While any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the invention, preferred methods and materials are described below. All publications mentioned in this specification are incorporated by reference in their entirety.

Terms used in this specification have the meanings recognized and known to those skilled in the art, however, for convenience and completeness of description, specific terms and their meanings are given below and throughout the text of the specification.

When used in this specification and the appended claims, terms in the singular include the corresponding terms in the plural, unless the context clearly indicates otherwise. Thus, for example, the term "method" includes one or more methods and/or steps such as those described herein and/or which will become apparent to those skilled in the art upon reading this description, and so on.

The term "about" or "approximately" means "within a statistically significant range of values." Such a range may be within an order of magnitude, typically within 20%, more specifically within 10%, and even more specifically within 5% of the specified value or range. The allowable variations encompassed by the term "approximately" or "approximately" depend on the particular system under study, and can be readily determined by those skilled in the art. If a range is specified in the text of this application, each integer within this range is also provided as an embodiment of the invention.

As used herein, terms such as "comprises," "comprising," "comprising," "comprises," "comprising," and the like may have the meaning given to them in US patent law; for example, they can mean "includes", "included", "including" and the like. Such terms mean the inclusion of specific ingredients or a set of ingredients, without excluding any other ingredients. Terms such as "consisting principally of" and "consisting principally of" have the meaning given to them in US patent law, for example, they allow the inclusion of other ingredients or steps that do not detract from the novel or essential characteristics of the invention, i.e., they exclude additional ingredients or steps not listed that detract from novel or essential features of the invention, and they exclude prior art ingredients or steps such as documents relating to the art that are cited herein or incorporated herein by reference, in in particular, since the purpose of this document is to identify embodiments that are patentable, for example, new, non-obvious, inventive relative to the prior art, for example, with respect to documents that are cited in the present description or incorporated herein by reference. Also, the terms "consists of" and "consisting of" have the meaning given to them in US patent law; namely, that these terms are closed. Accordingly, these terms mean the inclusion of a particular ingredient or set of ingredients and the exclusion of all other ingredients.

The term "antigen" generally means a biological molecule, typically a protein, peptide, polysaccharide, lipid, or conjugate, containing at least one epitope to which an antibody that recognizes it can selectively bind; or, in some cases, an immunogenic substance that is capable of stimulating antibody production or T cell responses, or both, in an animal, including compositions that are injected into or ingested by the animal. The immune response may be induced on the whole molecule, or on one or more different parts of the molecule (eg, an epitope or a hapten). The term can be used in relation to a single molecule, or to a homogeneous or heterogeneous population of antigenic molecules. The antigen is recognized by antibodies, T-cell receptors, or other elements of specific humoral and/or cellular immunity. The term "antigen" includes all relevant antigenic epitopes. Epitopes of a particular antigen can be identified using various epitope mapping methods well known in the art (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa , NJ). For example, linear epitopes can be determined, for example, by simultaneously synthesizing a large number of peptides corresponding to fragments of a protein molecule on solid supports and reacting the peptides with antibodies while the peptides are still bound to the supports. Such methods are known in the art and are described, for example, in US patent No. 4708871; Geysen, HM, et al., Proc. Natl. Acad. sci. USA, 81:3998-4002 (1984); Geysen, HM, et al., Molec. Immunol., 23(7):709-715 (1986), all of which are incorporated herein by reference in their entirety. Similarly, conformational epitopes can be identified by determining the spatial conformation of amino acids, for example, by X-ray crystallography and 2D nuclear magnetic resonance (see, for example, Epitope Mapping Protocols, supra). In addition, for the purposes of the present invention, the term "antigen" can also be used in relation to a protein that has modifications such as deletions, additions and substitutions (generally conservative in nature, but they may not be conservative), in the natural sequence, provided that the protein retains the ability to elicit an immune response. Such modifications may be intentional, eg, as a result of site-directed mutagenesis, or due to the use of a particular synthetic method or genetic engineering approach, or may be accidental, eg, due to mutations in hosts that produce antigens. In addition, the antigen may be produced, obtained or isolated from a microorganism, such as a bacterium, or may be an entire microorganism. Likewise, an oligonucleotide or polynucleotide that expresses an antigen, such as in nucleic acid immunization protocols, is also covered by this definition. Synthetic antigens are also included, for example, polyepitopes, flanking epitopes, and other recombinant or synthetic antigens (Bergmann, C., et al., Eur. J. Immunol., 23(11):2777-2781(1993); Bergmann, CC, et al., J. Immunol., 157(8):3242-3249 (1996); Suhrbier, A., Immunol. and Cell Biol., 75(4):402-408 (1997)).

The terms "vaccine" or "vaccine composition", which are used interchangeably, means pharmaceutical compositions containing at least one immunogenic composition that induces an immune response in an animal.

Capsular polysaccharides

As used herein, the term "saccharide" means a single sugar moiety or monosaccharide unit, as well as a combination of two or more single sugar moieties or monosaccharide units covalently linked to form disaccharides, oligosaccharides and polysaccharides. The term "saccharide" can be used interchangeably with the term "carbohydrate". The polysaccharide may be linear or branched.

Used in the present description, the term "monosaccharide" means one sugar residue in the oligosaccharide. Used in the present description, the term "disaccharide" means a polysaccharide consisting of two monosaccharide units, or fragments linked together by a glycosidic bond.

In one embodiment, the polysaccharide is an oligosaccharide (OS). Used in the present description, the term "oligosaccharide" means a compound containing two or more monosaccharide units, or fragments. In the context of an oligosaccharide, a single monomeric unit, or moiety, is a monosaccharide that is, or can be linked, via a hydroxyl group, to another monosaccharide unit, or moiety. Oligosaccharides can be obtained either by chemical synthesis from a protective group of single sugar residues, or by chemical degradation of biologically obtained polysaccharides. Alternatively, the oligosaccharides can be produced in vitro by enzymatic methods.

In a preferred embodiment, the PS polysaccharide is a linear or branched polymer of at least 5 monosaccharide units, or fragments. For clarity, a polymer with a large number of repeating units, where n is greater than 5, for example, greater than about 10, is referred to herein as a polysaccharide.

In one embodiment, the polysaccharide is a cell surface polysaccharide. The term "cell surface polysaccharide" means a polysaccharide, at least part of which is located on the outermost membrane of a bacterial cell, or the surface of a bacterial cell, including the peptidoglycan layer, the cell wall and the capsule. Typically, the cell surface polysaccharide is associated with the induction of an immune response in vivo . The cell surface polysaccharide may be a "cell wall polysaccharide" or "capsular polysaccharide". The cell wall polysaccharide typically forms a discontinuous layer on the surface of bacteria.

In one embodiment, the polysaccharide is a capsular polysaccharide. A capsular polysaccharide is a glycopolymer containing repeating units of one or more monosaccharides linked by glycosidic bonds. The capsular polysaccharide typically forms a capsule-like layer around the bacterial cell. "Capsular polysaccharide" or "capsule polysaccharide" means the polysaccharide capsule that is external to the cell wall in most streptococcal isolates. For example, the capsular polysaccharides of all GBS have a branched repeat structure with a terminal α2-3-linked sialic acid, which is essential for bacterial virulence. Capsule bound sialic acid (quantified by HPLC) was detected in >94% of in vitro cultured invasive neonatal isolates from the TEST study.

The present inventors have found that the level of sialic acid in GBS capsular polysaccharides is an important factor in inducing an immune response. Previous applications have only provided conflicting information regarding sialic acid levels for serotype V, the conclusion that desialized serotype V was preferred (International Patent Publication No. WO 2012/035519), and that a sialic acid content of >50% for serotype V could be used. (International Patent Application Publication No. WO 2014/053612). However, nothing in these documents indicates the importance of sialic acid levels for the immunogenicity of at least most of the GBS polysaccharides. The present inventors surprisingly found that GBS capsular polysaccharides should contain approximately 60% or more sialic acid prior to conjugation to provide an immune response comparable to such polysaccharides having natural levels of sialic acid (i.e., 100% or greater than approximately 95%) . Even sialic acid levels of 58%, which are within the ranges previously described for serotype V, adversely affect immunogenicity.

Accordingly, in one embodiment of the invention, the capsular polysaccharides have their natural level of sialic acid, for example, about 100% or greater than about 95%. In another embodiment, the desialylation of the capsular polysaccharides can be up to about 40% (sialylation level above about 60%), for example up to about 35% (sialylation level above about 65%), up to about 30% (sialylation level above about 70%) , up to about 25% (sialylation level above about 75%), up to about 20% (sialylation level above about 80%), up to about 15% (sialylation level above about 85%), up to about 10% (sialylation level above about 90%). %) and up to about 5% (sialylation level above about 95%).

It should be noted that a 100% sialic acid level corresponds to approximately 1.0 mM sialic acid per mM polysaccharide. Thus, capsular polysaccharides may have about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide. In a further embodiment, the capsular polysaccharide may have at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0, 9 mM sialic acid per mM polysaccharide or at least about 0.95 mM sialic acid per mM polysaccharide.

The terminal sialyl residues of capsular polysaccharides (CP) of some serotypes are partially O-acetylated (OAc) (Lewis, AL, et al., Proceedings of the National Academy of Sciences USA, 101(30):11123-8 (2004)). The polysaccharides of serotypes Ib, III, IV, V, VI, and IX have partial O-acetylation (up to ~40%), while serotypes Ia, II, and VII have little or no O-acetylation (less than about 5%) ( Lewis 2004). In one embodiment, the capsular polysaccharides have their natural level of O-acetylation (about 0% to about 40%). In another embodiment, the capsular polysaccharides may be de-O-acetylated (less than about 5% level). The degree of O-acetylation of a polysaccharide or oligosaccharide can be determined by any method known in the art, such as proton NMR (Lemercinier, X., et al., Carbohydrate Research, 296:83-96 (1996); Jones, C., et al., Journal of Pharmaceutical and Biomedical Analysis, 30:1233-1247 (2002); International Patent Application Publication Nos. WO 2005/033148 and WO 00/56357). Another commonly used method is that described in Hestrin, S., J. Biol. Chem. 180:249-261 (1949).

In addition, 100% O-acetate corresponds to approximately 1.0 mM O-acetate per mM repeating saccharide unit. Suitably, partially O-acetylated polysaccharides contain at least about 0.1, 0.2, 0.3, 0.35, or about 0.4 mM O-acetate per mM repeating saccharide unit. The de-O-acetylated polysaccharide contains less than about 0.01, 0.02, 0.03, 0.04, or 0.05 mM O-acetate per mM repeating saccharide unit.

Streptococcal microorganisms capable of causing invasive disease are also generally capable of producing CP, which encapsulates the bacterium and increases its resistance to removal by the host's innate immune system. CP encloses the bacterial cell with a protective capsule that makes the bacterium resistant to phagocytosis and intracellular destruction. Bacteria lacking a capsule are more susceptible to phagocytosis. Capsular polysaccharides are often an important virulence factor for many bacterial pathogens, including Haemophilus influenzae , Streptococcus pneumoniae , Neisseria meningitidis , and Staphylococcus aureus .

The capsular polysaccharide can be used to serotype specific bacterial species. Typing is typically performed by reacting with a specific antisera or monoclonal antibodies raised against a particular structure or unique epitope characteristic of the capsular polysaccharide. There are ten GBS serotypes: Ia, Ib and II-IX (Ferrieri, P., et al., Emerg. Infect. Dis. [Internet], 19(4) (2013), available at http://wwwnc. cdc.gov/eid/article/19/4/12-1572_article.

In one embodiment, the polysaccharide is isolated from Streptococcus agalactiae . The polysaccharide can be isolated from any encapsulated strain of S. agalactiae such as 090, A909 (ATCC Registry BAA-1138), 515 (ATCC Registry BAA-1177), B523, CJB524, MB 4052 (ATCC Registry 31574), H36B (ATCC P/N 12401), S40, S42, MB 4053 (ATCC P/N 31575), M709, 133, 7357, PFEGBST0267, MB 4055 (ATCC P/N 31576), 18RS21 (ATCC P/N BAA-1175), S16, S20 , V8 (ATCC P/N 12973), DK21, DK23, UAB, 5401, PFEGBST0708, MB 4082 (ATCC P/N 31577), M132, 110, M781 (ATCC P/N BAA-22), D136C(3) (ATCC P/N 12403), M782, S23, 120, MB 4316 (M-732; ATCC P/N 31475), M132, K79, COH1 (ATCC P/N BAA-1176), PFEGBST0563, 3139 (ATCC P/N 49446), CZ-NI- 016, PFEGBST0961, 1169-NT1, CJB111 (ATCC P/N BAA-23), CJB112, 2603 V/R (ATCC P/N BAA-611), NCTC 10/81, CJ11, PFEGBST0837, 118754, 114852, 114862, 114866, 118775 , B 4589, B 4645, SS1214, CZ-PW-119, 7271, CZ-PW-045, JM9130013, JM9130672, IT-NI-016, IT-PW-62 and IT-PW-64.

The polysaccharides described herein can be isolated by methods known in the art, including, for example, the methods described herein. Used in the present description, the term "isolated" means obtained from, and separated from, a particular source. The term "isolated" also means not in the appropriate natural form, condition and/or environment. For example, "isolated from streptococcus" means a substance that has been obtained from, and separated from, streptococcal cells. The isolated polysaccharide is not natural. The term "isolated" means that the material is removed from its natural environment (eg, the natural environment if it is natural, or from the host organism if it is a recombinant molecule) or transferred from one environment to another environment. For example, an "isolated" capsular polysaccharide, protein, or peptide is substantially free of cellular material or other protein contaminants of the cellular or tissue source from which the protein is derived, or substantially free of chemical precursors or other reagents if chemically synthesized or otherwise present in the mixture as a component of a chemical reaction. In the present invention, proteins or polysaccharides can be isolated from a bacterial cell or from cell debris in order to obtain them in a form useful for the production of an immunogenic composition. The term "isolated" or "isolation" may encompass purification, or purification, including methods for purifying an isolated polysaccharide known in the art and/or methods described herein. The phrase "substantially free of cellular material" refers to polypeptide/protein preparations in which the polypeptide/protein is separated from the cellular components of the cells from which it is isolated or recombinantly produced. Thus, a capsular polysaccharide, protein, or peptide that is substantially free of cellular material includes preparations of a capsular polysaccharide, protein, or peptide containing less than about 30%, 20%, 10%, 5%, 2.5%, or 1% ( based on dry weight) of contaminating protein or polysaccharide, or other cellular material. If the polypeptide/protein is recombinantly produced, it is also preferably substantially free of the culture medium, ie, the culture medium is less than about 20%, 10%, or 5% of the volume of the protein preparation. If the polypeptide/protein or polysaccharide is obtained by chemical synthesis, it is preferably substantially free of chemical precursors or other reagents, that is, it is separated from the chemical precursors or other reagents that are used in protein or polysaccharide synthesis. Accordingly, such polypeptide/protein or polysaccharide preparations contain less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide/protein or polysaccharide fragment of interest.

In one embodiment of the invention, the polysaccharide is isolated from a bacterium. In another embodiment of the invention, the polysaccharide is obtained by recombinant methods. In a further embodiment, the polysaccharide is synthetic or chemically synthesized by conventional methods. In another embodiment of the invention, the polysaccharide is obtained by expression in a surrogate host after cloning and expression of the biosynthetic pathway to produce the polysaccharide. In one embodiment, the polysaccharide is immunogenic. For example, the inventors have found that each polysaccharide described herein is capable of inducing, or causing, an immune response. The term "immunogenic" means having the ability to initiate, trigger, induce, enhance, improve and/or increase the humoral and/or cellular immune response in a mammal. In one embodiment, the mammal is a human, a primate, a rabbit, a pig, a mouse, and so on.

The molecular weight of the capsular polysaccharide is a factor to consider when using it in immunogenic compositions. High molecular weight capsular polysaccharides are able to induce certain immune responses in the form of antibody production due to the higher valency of the epitopes present on the surface of the antigen. The isolation and purification of high molecular weight capsular polysaccharides is intended to be used in the preparation of conjugates, compositions, and also in the methods of the present invention.

However, in one embodiment, the polysaccharide may have a size in the molecular weight (MW) range that is lower than the molecular weight of the natural capsular polysaccharide prior to conjugation to the carrier protein. The size of the purified capsular polysaccharide is reduced to obtain conjugates with more suitable filterability and/or yield characteristics.

In one such embodiment, the size of the purified capsular polysaccharide is reduced by high pressure homogenization. In high pressure homogenization, a high shear rate is achieved by pumping the process stream through a relatively small flow channel. The shear rate is increased by using a high applied homogenizing pressure and the exposure time can be increased by recycling the feed stream through the homogenizer.

In one embodiment, the polysaccharide described herein is capable of inducing opsonic activity. In another embodiment, the polysaccharide described herein is capable of inducing opsonic and phagocytic activity (eg, opsonophagocytic activity).

The term "opsonic activity" or "opsonization" means the process by which an opsonin (eg, an antibody or complement factor) binds to an antigen (eg, an isolated polysaccharide described herein) that facilitates attachment of the antigen to a phagocyte, or phagocytic cell. (e.g., macrophage, dendritic cell, and polymorphonuclear leukocyte (PMNL). Some bacteria, such as encapsulated bacteria, which are usually not phagocytized due to the presence of a capsule, are more likely to be recognized by phagocytes if they are coated with an opsonic antibody In one embodiment, the polysaccharide induces an immune response, such as, for example, the production of an antibody that is opsonic.In one embodiment, the opsonic activity is directed against Gram-positive cocci, preferably against a species of Streptococcus , more preferably against at least one strain of S. agalactiae .

In another embodiment, the polysaccharide described herein is capable of inducing a bactericidal immune response. In one embodiment, the bactericidal activity is directed against Gram-positive cocci, preferably against a species of Streptococcus , more preferably against at least one strain of S. agalactiae .

Methods for quantifying opsonization, phagocytosis, and/or bactericidal activity are known in the art, for example, methods such as measuring reduction in bacterial load in vivo (for example, by measuring levels of bacteremia in mammals injected with Streptococcus spp.) and/or measuring bacterial kill. cells in vitro (for example, in vitro analysis of opsonophagocytic activity). In one embodiment, the polysaccharide is capable of inducing opsonic, phagocytic, and/or bactericidal activity when compared to an appropriate control, for example, compared to an antiserum prepared against heat-killed Gram-positive cocci.

Serotype Ia

One embodiment relates to GBS serotype Ia capsular polysaccharide. The structure of a serotype Ia polysaccharide can be represented as follows:

a)

or

b)

The molecular weight of the serotype Ia capsular polysaccharides before conjugation is from about 5 kDa to about 1000 kDa, for example, from about 25 kDa to about 750 kDa, from about 25 kDa to about 500 kDa, from about 25 kDa to about 450 kDa, from about 25 about 25 kDa to about 350 kDa, about 25 kDa to about 300 kDa, about 25 kDa to about 250 kDa, about 25 kDa to about 200 kDa, about 50 kDa to about 750 kDa , from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa up to about 250 kDa, from about 50 kDa to about 200 kDa, from about 75 kDa up to about 750 kDa, from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa up to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 3 00 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is from about 25 kDa to about 200 kDa. In another preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is from about 100 kDa to about 400 kDa. Any integer in any of the above ranges is provided as an embodiment of the invention.

In a particular embodiment, a high pressure homogenization process is used to reduce the size of the naturally occurring GBS serotype Ia capsular polysaccharide while retaining structural features of the polysaccharide, such as sialic acid.

In one embodiment, the serotype Ia capsular polysaccharide has its natural sialic acid level, eg, about 100% or greater than about 95%. In another embodiment, the desialylation of the capsular polysaccharides can be up to about 40% (sialylation level above about 60%), for example up to about 35% (sialylation level above about 65%), up to about 30% (sialylation level above about 70%) , up to about 25% (sialylation level above about 75%), up to about 20% (sialylation level above about 80%), up to about 15% (sialylation level above about 85%), up to about 10% (sialylation level above about 90%). %) and up to about 5% (sialylation level above about 95%) before conjugation.

In another embodiment, the serotype Ia capsular polysaccharide has about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation. In a further embodiment, the capsular polysaccharide may have at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0, 9 mM sialic acid per mM polysaccharide or at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation.

Serotype Ia capsular polysaccharides have an O-acetylation level of less than about 5%. Some exemplary strains having serotype Ia capsular polysaccharides of the invention include 090, A909 (ATCC Accession No. BAA-1138), 515 (ATCC Accession No. BAA-1177), B523, CJB524, and MB 4052 (ATCC Accession No. 31574).

Serotype Ib

One embodiment relates to GBS serotype Ib capsular polysaccharide. The structure of a serotype Ib polysaccharide can be represented as follows:

a)

or

b)

The molecular weight of the serotype Ib capsular polysaccharides before conjugation is from about 5 kDa to about 1000 kDa, for example, from about 25 kDa to about 750 kDa, from about 25 kDa to about 500 kDa, from about 25 kDa to about 450 kDa, from about 25 about 25 kDa to about 350 kDa, about 25 kDa to about 300 kDa, about 25 kDa to about 250 kDa, about 25 kDa to about 200 kDa, about 50 kDa to about 750 kDa , from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa up to about 250 kDa, from about 50 kDa to about 200 kDa, from about 75 kDa up to about 750 kDa, from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa up to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 3 00 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is from about 25 kDa to about 400 kDa. Any integer in any of the above ranges is provided as an embodiment of the invention.

In one embodiment, the serotype Ib capsular polysaccharide has its natural sialic acid level, eg, about 100% or greater than about 95%. In another embodiment, the desialylation of the capsular polysaccharides can be up to about 40% (sialylation level above about 60%), for example up to about 35% (sialylation level above about 65%), up to about 30% (sialylation level above about 70%) , up to about 25% (sialylation level above about 75%), up to about 20% (sialylation level above about 80%), up to about 15% (sialylation level above about 85%), up to about 10% (sialylation level above about 90%). %) and up to about 5% (sialylation level above about 95%) before conjugation.

In another embodiment, the serotype Ib capsular polysaccharide has about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation. In a further embodiment, the capsular polysaccharide may have at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0, 9 mM sialic acid per mM polysaccharide or at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation.

Serotype Ib capsular polysaccharides have an O-acetylation level of from about 0% to about 40%. In one embodiment, the polysaccharide is de-O-acetylated (ie, the level of O-acetylation is less than about 5%). Some exemplary strains having serotype Ib capsular polysaccharides of the invention include H36B (ATCC Accession No. 12401), S40, S42, MB 4053 (ATCC Accession No. 31575), M709, 133, 7357, and PFEGBST0267.

Serotype II

One embodiment relates to GBS serotype II capsular polysaccharide. The structure of a serotype II polysaccharide can be represented as follows:

a)

or

b)

The molecular weight of the serotype II capsular polysaccharides before conjugation is from about 5 kDa to about 1000 kDa, for example, from about 25 kDa to about 750 kDa, from about 25 kDa to about 500 kDa, from about 25 kDa to about 450 kDa, from about 25 about 25 kDa to about 350 kDa, about 25 kDa to about 300 kDa, about 25 kDa to about 250 kDa, about 25 kDa to about 200 kDa, about 50 kDa to about 750 kDa , from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa up to about 250 kDa, from about 50 kDa to about 200 kDa, from about 75 kDa up to about 750 kDa, from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa up to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 3 00 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is from about 25 kDa to about 400 kDa. Any integer in any of the above ranges is provided as an embodiment of the invention.

In one embodiment, the serotype II capsular polysaccharide has its natural sialic acid level, eg, about 100% or greater than about 95%. In another embodiment, the desialylation of the capsular polysaccharides can be up to about 40% (sialylation level above about 60%), for example up to about 35% (sialylation level above about 65%), up to about 30% (sialylation level above about 70%) , up to about 25% (sialylation level above about 75%), up to about 20% (sialylation level above about 80%), up to about 15% (sialylation level above about 85%), up to about 10% (sialylation level above about 90%). %) and up to about 5% (sialylation level above about 95%) before conjugation.

In another embodiment, the serotype II capsular polysaccharide has about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation. In a further embodiment, the capsular polysaccharide may have at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0, 9 mM sialic acid per mM polysaccharide or at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation.

Serotype II capsular polysaccharides have an O-acetylation level of less than about 5%. Some exemplary strains having serotype II capsular polysaccharides of the invention include MB 4055 (ATCC Accession No. 31576), 18RS21 (ATCC Accession No. BAA-1175), S16, S20, V8 (ATCC Accession No. 12973), DK21, DK23, UAB, 5401 and PFEGBST0708.

Serotype III

One embodiment relates to GBS serotype III capsular polysaccharide. The structure of a serotype III polysaccharide can be represented as follows:

a)

or

b)

The molecular weight of the serotype III capsular polysaccharides before conjugation is from about 5 kDa to about 1000 kDa, for example, from about 25 kDa to about 750 kDa, from about 25 kDa to about 500 kDa, from about 25 kDa to about 450 kDa, from about 25 about 25 kDa to about 350 kDa, about 25 kDa to about 300 kDa, about 25 kDa to about 250 kDa, about 25 kDa to about 200 kDa, about 50 kDa to about 750 kDa , from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa up to about 250 kDa, from about 50 kDa to about 200 kDa, from about 75 kDa up to about 750 kDa, from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa up to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 300 kD to about 550 kD, or about 300 kD to about 500 kD. In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is from about 25 kDa to about 200 kDa. In another preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is from about 100 kDa to about 400 kDa. Any integer in any of the above ranges is provided as an embodiment of the invention.

In a particular embodiment, a high pressure homogenization process is used to reduce the size of the naturally occurring GBS serotype III capsular polysaccharide while retaining structural features of the polysaccharide, such as sialic acid.

In one embodiment, the serotype III capsular polysaccharide has its natural sialic acid level, eg, about 100% or greater than about 95%. In another embodiment, the desialylation of the capsular polysaccharides can be up to about 40% (sialylation level above about 60%), for example up to about 35% (sialylation level above about 65%), up to about 30% (sialylation level above about 70%) , up to about 25% (sialylation level above about 75%), up to about 20% (sialylation level above about 80%), up to about 15% (sialylation level above about 85%), up to about 10% (sialylation level above about 90%). %) and up to about 5% (sialylation level above about 95%) before conjugation.

In another embodiment, the serotype III capsular polysaccharide has about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation. In a further embodiment, the capsular polysaccharide may have at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0, 9 mM sialic acid per mM polysaccharide or at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation.

Serotype III capsular polysaccharides have an O-acetylation level of from about 0% to about 40%. In one embodiment, the polysaccharide is de-O-acetylated (ie, the level of O-acetylation is less than about 5%). Some exemplary strains having serotype III capsular polysaccharides of the invention include MB 4082 (ATCC Accession No. 31577), M132, 110, M781 (ATCC Accession No. BAA-22), D136C(3) (ATCC Accession No. 12403), M782, S23 , 120, MB 4316 (M-732; ATCC Registry 31475), M132, K79, COH1 (ATCC Registry BAA-1176), and PFEGBST0563.

Serotype IV

One embodiment relates to GBS serotype IV capsular polysaccharide. The structure of a serotype IV polysaccharide can be represented as follows:

a)

or

b)

The molecular weight of the serotype IV capsular polysaccharides before conjugation is from about 5 kDa to about 1000 kDa, for example, from about 25 kDa to about 750 kDa, from about 25 kDa to about 500 kDa, from about 25 kDa to about 450 kDa, from about 25 about 25 kDa to about 350 kDa, about 25 kDa to about 300 kDa, about 25 kDa to about 250 kDa, about 25 kDa to about 200 kDa, about 50 kDa to about 750 kDa , from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa up to about 250 kDa, from about 50 kDa to about 200 kDa, from about 75 kDa up to about 750 kDa, from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa up to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 3 00 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is from about 25 kDa to about 400 kDa. Any integer in any of the above ranges is provided as an embodiment of the invention.

In one embodiment, the serotype IV capsular polysaccharide has its natural sialic acid level, eg, about 100% or greater than about 95%. In another embodiment, the desialylation of the capsular polysaccharides can be up to about 40% (sialylation level above about 60%), for example up to about 35% (sialylation level above about 65%), up to about 30% (sialylation level above about 70%) , up to about 25% (sialylation level above about 75%), up to about 20% (sialylation level above about 80%), up to about 15% (sialylation level above about 85%), up to about 10% (sialylation level above about 90%). %) and up to about 5% (sialylation level above about 95%) before conjugation.

In another embodiment, the serotype IV capsular polysaccharide has about 1.0 mM sialic acid per mM polysaccharide, eg, at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation. In a further embodiment, the capsular polysaccharide may have at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0, 9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation.

Serotype IV capsular polysaccharides have an O-acetylation level of from about 0% to about 40%. In one embodiment of the invention, the polysaccharide is de-O-acetylated (ie, the level of O-acetylation is less than about 5%). Some exemplary strains having serotype IV capsular polysaccharides of the invention include 3139 (ATCC Accession No. 49446), CZ-NI-016, and PFEGBST0961.

Serotype V

One embodiment relates to GBS serotype V capsular polysaccharide. The structure of a serotype V polysaccharide can be represented as follows:

a)

or

b)

The molecular weight of the serotype V capsular polysaccharides before conjugation is from about 5 kDa to about 1000 kDa, for example, from about 25 kDa to about 750 kDa, from about 25 kDa to about 500 kDa, from about 25 kDa to about 450 kDa, from about 25 about 25 kDa to about 350 kDa, about 25 kDa to about 300 kDa, about 25 kDa to about 250 kDa, about 25 kDa to about 200 kDa, about 50 kDa to about 750 kDa , from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa up to about 250 kDa, from about 50 kDa to about 200 kDa, from about 75 kDa about 750 kDa, from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 30 0 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. In one preferred embodiment, the molecular weight of the capsular polysaccharide prior to conjugation is from about 25 kDa to about 400 kDa. Any integer in any of the above ranges is provided as an embodiment of the invention.

In one embodiment, the serotype V capsular polysaccharide has its natural sialic acid level, eg, about 100% or greater than about 95%. In another embodiment, the desialylation of the capsular polysaccharides can be up to about 40% (sialylation level above about 60%), for example up to about 35% (sialylation level above about 65%), up to about 30% (sialylation level above about 70%) , up to about 25% (sialylation level above about 75%), up to about 20% (sialylation level above about 80%), up to about 15% (sialylation level above about 85%), up to about 10% (sialylation level above about 90%). %) and up to about 5% (sialylation level above about 95%) before conjugation.

In another embodiment, the serotype V capsular polysaccharide has about 1.0 mM sialic acid per mM polysaccharide, eg, at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation. In a further embodiment, the capsular polysaccharide may have at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0, 9 mM sialic acid per mM polysaccharide or at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation.

Serotype V capsular polysaccharides have an O-acetylation level of from about 0% to about 40%. In one embodiment, the polysaccharide is de-O-acetylated (ie, the level of O-acetylation is less than about 5%). Some exemplary strains having serotype V capsular polysaccharides of the invention include 1169-NT1, CJB111 (ATCC Accession No. BAA-23), CJB112, 2603 V/R (ATCC Accession No. BAA-611), NCTC 10/81, CJ11, and PFEGBST0837 .

Serotype VI

GBS serotype VI capsular polysaccharides are described in von Hunolstein, C., et al., Infection and Immunity, 6194):1272-1280 (1993), the contents of which are incorporated herein by reference in their entirety. The structure of a serotype VI polysaccharide can be represented as follows:

a)

or

b)

The molecular weight of the serotype VI capsular polysaccharides before conjugation is from about 5 kDa to about 1000 kDa, for example, from about 50 kDa to about 750 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 about 50 kDa to about 350 kDa, about 50 kDa to about 300 kDa, about 50 kDa to about 250 kDa, about 50 kDa to about 200 kDa, about 75 kDa to about 750 kDa , from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa up to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa up to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa to approximately 700 kDa, approximately 200 kDa to approximately 650 kDa, approximately 200 kDa to approximately 600 kDa, approximately 200 kDa to approximately 550 kDa, approximately 200 kDa to approximately 500 kDa, approximately 200 kDa to approximately 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kD to about 700 kD, from about 250 kD to about 650 kD, from about 250 kD to about 600 kD, from about 250 kD to about 550 kD, from about 250 kD to about 500 kD, from about 250 kD to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 300 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. Any integer in any of the above ranges is provided as an embodiment of the invention.

In one embodiment, the serotype VI capsular polysaccharide has its natural sialic acid level, eg, about 100% or greater than about 95%. In another embodiment, the desialylation of the capsular polysaccharides can be up to about 40% (sialylation level above about 60%), for example up to about 35% (sialylation level above about 65%), up to about 30% (sialylation level above about 70%) , up to about 25% (sialylation level above about 75%), up to about 20% (sialylation level above about 80%), up to about 15% (sialylation level above about 85%), up to about 10% (sialylation level above about 90%). %) and up to about 5% (sialylation level above about 95%) before conjugation.

In another embodiment, the serotype VI capsular polysaccharide has about 1.0 mM sialic acid per mM polysaccharide, eg, at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation. In a further embodiment, the capsular polysaccharide may have at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0, 9 mM sialic acid per mM polysaccharide or at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation.

Serotype VI capsular polysaccharides have an O-acetylation level of from about 0% to about 40%. In one embodiment, the polysaccharide is de-O-acetylated (ie, the level of O-acetylation is less than about 5%). Some exemplary strains having serotype VI capsular polysaccharides of the invention include 118754, 114852, 114862, 114866, 118775, B 4589, B 4645, SS1214, and CZ-PW-119.

Serotype VII

GBS serotype VII capsular polysaccharides are described in Kogan, G., et al., Carbohydrate Research, 277(1):1-9 (1995), the contents of which are incorporated herein by reference in their entirety. The serotype VII repeat unit is as follows:

The molecular weight of the serotype VII capsular polysaccharides before conjugation is from about 5 kDa to about 1000 kDa, for example, from about 50 kDa to about 750 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 about 50 kDa to about 350 kDa, about 50 kDa to about 300 kDa, about 50 kDa to about 250 kDa, about 50 kDa to about 200 kDa, about 75 kDa to about 750 kDa , from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa up to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa a to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa , from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa up to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kD to about 700 kD, from about 250 kD to about 650 kD, from about 250 kD to about 600 kD, from about 250 kD to about 550 kD, from about 250 kD to about 500 kD, from about 250 kD to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 300 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. Any integer in any of the above ranges is provided as an embodiment of the invention.

In one embodiment, the serotype VII capsular polysaccharide has its natural sialic acid level, eg, about 100% or greater than about 95%. In another embodiment, the desialylation of the capsular polysaccharides can be up to about 40% (sialylation level above about 60%), for example up to about 35% (sialylation level above about 65%), up to about 30% (sialylation level above about 70%) , up to about 25% (sialylation level above about 75%), up to about 20% (sialylation level above about 80%), up to about 15% (sialylation level above about 85%), up to about 10% (sialylation level above about 90%). %) and up to about 5% (sialylation level above about 95%) before conjugation.

In another embodiment, the serotype VII capsular polysaccharide has about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation. In a further embodiment, the capsular polysaccharide may have at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0, 9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation.

Serotype VII capsular polysaccharides have an O-acetylation level of less than about 5%. Some exemplary strains having serotype VII capsular polysaccharides of the invention include 7271 and CZ-PW-045.

Serotype VIII

GBS serotype VIII capsular polysaccharides are described in Kogan, G., et al., The Journal of Biological Chemistry, 271(15):8786-8790 (1996), the contents of which are incorporated herein by reference in their entirety. The serotype VIII repeat unit is as follows:

The molecular weight of the serotype VIII capsular polysaccharides before conjugation is from about 5 kDa to about 1000 kDa, for example, from about 50 kDa to about 750 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 about 50 kDa to about 350 kDa, about 50 kDa to about 300 kDa, about 50 kDa to about 250 kDa, about 50 kDa to about 200 kDa, about 75 kDa to about 750 kDa , from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa up to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa Yes to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa , from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa up to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from about 200 kDa to about 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about about 250 kDa to about 700 kDa, about 250 kDa to about 650 kDa, about 250 kDa to about 600 kDa, about 250 kDa to about 550 kDa, about 250 kDa to about 500 kDa, from about 250 kDa to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 300 kD to about 550 kD, or about 300 kD to about 500 kD. Any integer in any of the above ranges is provided as an embodiment of the invention.

In one embodiment, the serotype VIII capsular polysaccharide has its natural sialic acid level, eg, about 100% or greater than about 95%. In another embodiment, the desialylation of the capsular polysaccharides can be up to about 40% (sialylation level above about 60%), for example up to about 35% (sialylation level above about 65%), up to about 30% (sialylation level above about 70%) , up to about 25% (sialylation level above about 75%), up to about 20% (sialylation level above about 80%), up to about 15% (sialylation level above about 85%), up to about 10% (sialylation level above about 90%). %) and up to about 5% (sialylation level above about 95%) before conjugation.

In another embodiment, the serotype VIII capsular polysaccharide has about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation. In a further embodiment, the capsular polysaccharide may have at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0, 9 mM sialic acid per mM polysaccharide, or at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation.

Serotype VIII capsular polysaccharides have an O-acetylation level of from about 0% to about 40%. In one embodiment, the polysaccharide is de-O-acetylated (ie, the level of O-acetylation is less than about 5%). Some exemplary strains having serotype VIII capsular polysaccharides of the invention include JM9130013 and JM9130672.

Serotype IX

GBS serotype IX capsular polysaccharides are described in Berti, F., et al., The Journal of Biological Chemistry, 289(34):23437-2348 (2014), the contents of which are incorporated herein by reference in their entirety. The structure of a serotype IX polysaccharide can be represented as follows:

The molecular weight of the serotype IX capsular polysaccharides before conjugation is from about 5 kDa to about 1000 kDa, for example, from about 50 kDa to about 750 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 about 50 kDa to about 350 kDa, about 50 kDa to about 300 kDa, about 50 kDa to about 250 kDa, about 50 kDa to about 200 kDa, about 75 kDa to about 750 kDa , from about 75 kDa to about 500 kDa, from about 75 kDa to about 450 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa up to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa up to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa, from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa to about 750 kDa, from about 200 kDa to approximately 700 kDa, approximately 200 kDa to approximately 650 kDa, approximately 200 kDa to approximately 600 kDa, approximately 200 kDa to approximately 550 kDa, approximately 200 kDa to approximately 500 kDa, approximately 200 kDa to approximately 450 kDa, from about 200 kDa to about 400 kDa, from about 250 kDa to about 750 kDa, from about 250 kD to about 700 kD, from about 250 kD to about 650 kD, from about 250 kD to about 600 kD, from about 250 kD to about 550 kD, from about 250 kD to about 500 kD, from about 250 kD to about 450 kDa, from about 250 kDa to about 400 kDa, from about 300 kDa to about 750 kDa, from about 300 kDa to about 700 kDa, from about 300 kDa to about 650 kDa, from about 300 kDa to about 600 kDa, from about 300 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. Any integer in any of the above ranges is provided as an embodiment of the invention.

In one embodiment, the serotype IX capsular polysaccharide has its natural sialic acid level, eg, about 100% or greater than about 95%. In another embodiment, the desialylation of the capsular polysaccharides can be up to about 40% (sialylation level above about 60%), for example up to about 35% (sialylation level above about 65%), up to about 30% (sialylation level above about 70%) , up to about 25% (sialylation level above about 75%), up to about 20% (sialylation level above about 80%), up to about 15% (sialylation level above about 85%), up to about 10% (sialylation level above about 90%). %) and up to about 5% (sialylation level above about 95%) before conjugation.

In another embodiment, the serotype IX capsular polysaccharide has about 1.0 mM sialic acid per mM polysaccharide, such as at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation. In a further embodiment, the capsular polysaccharide may have at least about 0.6 mM sialic acid per mM polysaccharide, such as at least about 0.65 mM sialic acid per mM polysaccharide, at least about 0.7 mM sialic acid per mM polysaccharide, at least about 0.75 mM sialic acid per mM polysaccharide, at least about 0.8 mM sialic acid per mM polysaccharide, at least about 0.85 mM sialic acid per mM polysaccharide, at least about 0, 9 mM sialic acid per mM polysaccharide or at least about 0.95 mM sialic acid per mM polysaccharide prior to conjugation.

Serotype IX capsular polysaccharides have an O-acetylation level of from about 0% to about 40%. In one embodiment, the polysaccharide is de-O-acetylated (ie, the level of O-acetylation is less than about 5%). Some exemplary strains having serotype IX capsular polysaccharides of the invention include IT-NI-016, IT-PW-62, and IT-PW-64.

Polysaccharide-protein conjugates

As used herein, the term "conjugates" encompasses a capsular polysaccharide, typically in the desired molecular weight range, and a carrier protein, wherein the capsular polysaccharide is conjugated to the carrier protein. The conjugates may or may not contain some free capsular polysaccharide. As used herein, the term "free capsular polysaccharide" means a capsular polysaccharide that is non-covalently associated with (ie, non-covalently attached to, adsorbed on, or enclosed in or together with) a capsular polysaccharide-carrier protein conjugate. The terms "free capsular polysaccharide", "free polysaccharide" and "free saccharide" may be used interchangeably and should have the same meaning. Regardless of the nature of the carrier molecule, it can be conjugated to the capsular polysaccharide either directly or via a linker. As used herein, the terms "conjugate", "conjugated", and "conjugation" refer to the process by which a bacterial capsular polysaccharide is covalently attached to a carrier molecule. Conjugation results in an increase in the immunogenicity of the bacterial capsular polysaccharide. Conjugation can be performed by the methods described below or by methods known in the art.

As used herein, the term "conjugate immunogenic composition" means an immunogenic composition in which the immunogenic material comprises an antigenic polysaccharide covalently linked to a carrier protein to form a polysaccharide-protein conjugate. In one embodiment, the polysaccharide-protein conjugate of the invention may be formulated as a polyvalent immunogenic composition.

As used herein, the term "molecular weight" of a polysaccharide or carrier protein-polysaccharide conjugate means the molecular weight determined by size exclusion chromatography (SEC) in combination with a multi-angle laser light scattering (MALLS) detector.

As used herein, the term "polysaccharide-protein conjugate" refers to a polysaccharide molecule conjugated to a carrier protein molecule through one or more covalent bonds. It may be desirable to conjugate the polysaccharide to a protein from another species known to be immunogenic in the target host. Accordingly, in one embodiment, the carrier molecule is a carrier protein. In the present description, such a foreign protein is called a "carrier protein". Carrier proteins serve to enhance the antigenicity and immunogenicity of the polysaccharide. As used herein, the term "carrier effect" refers to the phenomenon where the antigenicity and immunogenicity of a weakly immunogenic or non-immunogenic molecule is enhanced by attachment to a more immunogenic carrier molecule (eg, a heterologous protein). In this case, the polysaccharide in the combined polysaccharide-protein conjugate becomes more immunogenic than in the free state. Carrier proteins contain T-cell epitopes to stimulate T-cells to help elicit immune responses in the form of antibody production.

As used herein, the term "carrier protein" or "carrier protein" means any protein molecule that can be conjugated to an antigen (such as capsular polysaccharides) against which an immune response is desired. Conjugation of an antigen, such as a polysaccharide, with a carrier protein can render the antigen immunogenic. Carrier proteins are preferably proteins which are non-toxic, non-reactive, and which can be obtained in sufficient quantity and in a sufficiently pure state. Examples of carrier proteins are toxins, toxoids, or any mutant cross-reactive material (CRM ₁₉₇ ) of tetanus, diphtheria, pertussis toxin, Pseudomonas spp ., E. coli spp ., Staphylococcus spp., and Streptococcus spp. toxin. Carrier proteins should be amenable to standard conjugation procedures. In a specific embodiment of the present invention, CRM ₁₉₇ is used as the carrier protein.

Cross-reacting materials, or CRMs, are particularly useful in some embodiments of the present invention. Genetically modified proteins can be produced that are similar in antigenicity to some bacterial toxins, but are not toxic. They are called "cross-reacting materials", or CRM. Mention should be made of CRM ₁₉₇ (Wyeth/Pfizer Inc., Sanford, NC) as it has one amino acid alteration compared to natural diphtheria toxin and is immunologically indistinguishable from it. See Pappenheimer, AM, et al., Immunochem., 9(9):891-906 (1972); US patent No. 5614382, the contents of which are incorporated herein by reference in their entirety. CRM ₁₉₇ is a non-toxic variant (ie, toxoid) of diphtheria toxin isolated from cultures of Corynebacterium diphtheriae strain C7 (β197) grown in a medium based on cas amino acids and yeast extract. CRM ₁₉₇ is purified by ultrafiltration, ammonium sulfate precipitation and ion exchange chromatography. A culture of C. diphtheriae strain C7 (β197) producing the CRM ₁₉₇ protein has been deposited with the American Type Culture Collection, Rockville, Maryland and assigned accession number ATCC 53281. Other diphtheria toxoids are also suitable for use as carrier proteins. CRM3201 is a genetically modified variant of the pertussis toxin. See Black, WJ, et al., Science, 240(4852):656-659 (1988), the contents of which are incorporated herein by reference in their entirety.

In addition to diphtheria toxoid (DT), CRM₁₉₇ and pertussis toxoid, further examples of carrier proteins include tetanus toxoid (TT), cholera toxoid (e.g. described in International Patent Application Publication No. WO 2004/083251), heat labile toxoid (LT)E. coli, thermostable toxoid (ST)E. coli, pneumolysin fromS. pneumonia(wild type or reduced toxicity mutant), pneumococcal surface protein A (PspA), pneumococcal adhesin protein A (PsaA), C5a peptidase fromStreptococcus, hemolysin fromStaphylococcal aureus,non-typeable strain proteinshaemophilus influenzae(NTHi), protein Dhaemophilus influenzae, exotoxins/toxoidsClostridium perfringens, hepatitis B surface antigen, hepatitis B core antigen, rotavirus VP7 protein and respiratory syncytial virus F and G proteins, ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), purified protein derived from tuberculin (PPD) ), as well as exotoxinPseudomonas or derivatives thereof, including recombinantly produced non-toxic mutant exotoxin APseudomonas aeruginosa. Bacterial outer membrane proteins such as outer membrane protein complex c (OMPC), porins, transferrin-binding proteins or enterotoxin (toxin A) and cytotoxin (toxin B) can also be used.C. difficile. Other proteins such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified tuberculin derived protein (PPD) can also be used as carrier proteins. In a preferred embodiment, the carrier protein is a diphtheria toxoid. More preferably, the carrier protein is CRM₁₉₇. In another embodiment, the carrier protein is a tetanus toxoid.

To create a polyvalent conjugate immunogenic composition, polysaccharide-protein conjugates can be obtained by conjugating a mixture of polysaccharides purified from two different species of bacteria with a carrier protein. Alternatively, a polyvalent conjugate immunogenic composition can be prepared by combining polysaccharides purified from bacteria of two or more different serotypes of the same bacterial species and conjugating them as a mixture with a carrier protein. Alternatively, polysaccharide-protein conjugates obtained from separate reactions of several types of polysaccharides with a carrier protein can be mixed. Thus, a polyvalent immunogenic composition may comprise a carrier protein carrying a homogeneous or heterogeneous population of associated polysaccharides.

After the capsular polysaccharide has been conjugated to the carrier protein, the polysaccharide-protein conjugates are purified (enriched in terms of the amount of polysaccharide-protein conjugate) by various methods. These methods include, for example, concentration/diafiltration, precipitation/elution, column chromatography and depth filtration procedures.

As described above, the present invention relates to conjugates containing GBS capsular polysaccharides conjugated to carrier proteins. One embodiment of the invention relates to conjugates comprising a GBS serotype IV capsular polysaccharide conjugated to a carrier protein and at least one additional conjugate comprising a GBS serotype Ia capsular polysaccharide conjugated to a carrier protein, a GBS serotype Ib capsular polysaccharide conjugated to carrier protein, GBS serotype II capsular polysaccharide conjugated to a carrier protein, GBS serotype III capsular polysaccharide conjugated to a carrier protein, GBS serotype V capsular polysaccharide conjugated to a carrier protein, GBS serotype VI capsular polysaccharide conjugated to a carrier protein a carrier, a GBS serotype VII capsular polysaccharide conjugated to a carrier protein, a GBS serotype VIII capsular polysaccharide conjugated to a carrier protein, or a GBS serotype IX capsular polysaccharide conjugated to a carrier protein. In one aspect of the invention, the polysaccharides have a molecular weight of from about 5 kDa to 1000 kDa; the conjugates have a molecular weight of from about 300 kDa to about 20,000 kDa, and the conjugates contain less than about 40% free polysaccharide relative to the total polysaccharide. In one embodiment, the conjugates contain less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% free polysaccharide relative to the total polysaccharide.

In one embodiment, the serotype Ia, Ib, II, III, IV, V, VI, VII, VIII, and/or IX polysaccharide has a molecular weight before conjugation from about 5 kDa to about 1000 kDa, for example, from about 50 kDa to about 750 kDa, from about 50 kDa to about 500 kDa, from about 50 kDa to about 450 kDa, from about 50 kDa to about 400 kDa, from about 50 kDa to about 350 kDa, from about 50 kDa to about 300 kDa, from about 50 kDa to about 250 kDa, about 50 kDa to about 200 kDa, about 75 kDa to about 750 kDa, about 75 kDa to about 500 kDa, about 75 kDa to about 450 kDa, about 75 kDa to about 400 kDa , from about 75 kDa to about 350 kDa, from about 75 kDa to about 300 kDa, from about 75 kDa to about 250 kDa, from about 75 kDa to about 200 kDa, from about 100 kDa to about 750 kDa, from about 100 kDa to about 700 kDa, from about 100 kDa to about 650 kDa, from about 100 kDa to about 600 kDa, from about 100 kDa to about 550 kDa , from about 100 kDa to about 500 kDa, from about 100 kDa to about 450 kDa, from about 100 kDa to about 400 kDa, from about 100 kDa to about 350 kDa, from about 100 kDa to about 300 kDa, from about 200 kDa and 750 kDa, from about 200 kDa to about 700 kDa, from about 200 kDa to about 650 kDa, from about 200 kDa to about 600 kDa, from about 200 kDa to about 550 kDa, from about 200 kDa to about 500 kDa, from approximately 200 kDa to approximately 450 kDa, approximately 200 kDa to approximately 400 kDa, approximately 250 kDa to n about 750 kDa, from about 250 kDa to about 700 kDa, from about 250 kDa to about 650 kDa, from about 250 kDa to about 600 kDa, from about 250 kDa to about 550 kDa, from about 250 kDa to about 500 kDa, from approximately 250 kDa to approximately 450 kDa, approximately 250 kDa to approximately 400 kDa, approximately 300 kDa and 750 kDa, approximately 300 kDa to approximately 700 kDa, approximately 300 kDa to approximately 650 kDa, approximately 300 kDa to approximately 600 kDa kDa, from about 300 kDa to about 550 kDa, or from about 300 kDa to about 500 kDa. Any integer in any of the above ranges is provided as an embodiment of the invention.

In one embodiment, the conjugate has a molecular weight of from about 300 kDa to about 20,000 kDa, for example, from about 300 kDa to about 15,000 kDa, from about 300 kDa to about 10,000 kDa, from about 300 kDa to about 9000 kDa, from about 300 kDa up to about 8000 kDa, from about 300 kDa to about 7000 kDa, from about 300 kDa to about 6000 kDa, from about 300 kDa to about 5000 kDa, from about 300 kDa to about 4000 kDa, from about 300 kDa to about 3000 kDa, from about 300 kDa to about 2000 kDa, from about 300 kDa to about 1000 kDa, from about 500 kDa to about 20,000 kDa, from about 500 kDa to about 15,000 kDa, from about 500 kDa to about 10,000 kDa, from about 500 kDa to about 9000 kDa, from about 500 kDa to about 8000 kDa, about t about 500 kDa to about 7000 kDa, from about 500 kDa to about 6000 kDa, from about 500 kDa to about 5000 kDa, from about 500 kDa to about 4000 kDa, from about 500 kDa to about 3000 kDa, from about 500 kDa to about 2000 kDa, from about 500 kDa to about 1000 kDa, from about 1000 kDa to about 20000 kDa, from about 1000 kDa to about 15000 kDa, from about 1000 kDa to about 10000 kDa, from about 1000 kDa to about 8000 kDa, from about 1000 kDa to about 7000 kDa, from about 1000 kDa to about 6000 kDa, from about 1000 kDa to about 5000 kDa, from about 1500 kDa to about 20000 kDa, from about 1500 kDa to about 15000 kDa, from about 1500 kDa to about 10000 kDa, from approx. 1500 kDa to approximately 9000 kDa, approximately 1500 kDa to approximately 8000 kDa, approximately 1500 kDa to approximately 7000 kDa, approximately 1500 kDa to approximately 6000 kDa, approximately 1500 kDa to approximately 5000 kDa, approximately 2000 kDa to approximately 20000 kDa, from about 2000 kDa to about 15000 kDa, from about 2000 kDa to about 10000 kDa, from about 2000 kDa to about 9000 kDa, from about 2000 kDa to about 8000 kDa, from about 2000 kDa to about 7000 kDa, from about 2000 kDa to approximately 6000 kDa, approximately 2500 kDa to approximately 20000 kDa, approximately 2500 kDa to approximately 15000 kDa, approximately 2500 kDa to approximately 10000 kDa, approximately 2500 kDa to approximately 9000 kDa, approximately 2500 kDa to approximately 8000 kDa, from about 2500 kDa to about 7000 kDa, from about about 2500 kDa to about 6000 kDa, from about 3000 kDa to about 20000 kDa, from about 3000 kDa to about 15000 kDa, from about 3000 kDa to about 10000 kDa, from about 3000 kDa to about 9000 kDa, from about 3000 kDa to about 8000 kDa, from about 3000 kDa to about 7000 kDa, or from about 3000 kDa to about 6000 kDa.

In one embodiment, the GBS serotype IV capsular polysaccharide conjugate has a molecular weight in any of the above ranges.

In one embodiment, the GBS serotype Ia capsular polysaccharide conjugate has a molecular weight in any of the above ranges.

In one embodiment, the GBS serotype Ib capsular polysaccharide conjugate has a molecular weight in any of the above ranges.

In one embodiment, the GBS serotype II capsular polysaccharide conjugate has a molecular weight in any of the above ranges.

In one embodiment, the GBS serotype III capsular polysaccharide conjugate has a molecular weight in any of the above ranges.

In one embodiment, the GBS serotype V capsular polysaccharide conjugate has a molecular weight in any of the above ranges.

In one embodiment, the GBS serotype VI capsular polysaccharide conjugate has a molecular weight in any of the above ranges.

In one embodiment, the GBS serotype VII capsular polysaccharide conjugate has a molecular weight in any of the above ranges.

In one embodiment, the GBS serotype VIII capsular polysaccharide conjugate has a molecular weight in any of the above ranges.

In one embodiment, the GBS serotype IX capsular polysaccharide conjugate has a molecular weight in any of the above ranges.

In one embodiment, the conjugates of the invention have at least about 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 0.97, or 0. 98 mM sialic acid per mM polysaccharide. In a preferred embodiment, the conjugates have at least about 0.9 or 0.95 mM sialic acid per mM polysaccharide.

In one embodiment, the GBS serotype IV capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

In one embodiment, the GBS serotype Ia capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

In one embodiment, the GBS serotype Ib capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

In one embodiment, the GBS serotype II capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

In one embodiment, the GBS serotype III capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

In one embodiment, the GBS serotype V capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

In one embodiment, the GBS serotype VI capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

In one embodiment, the GBS serotype VII capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

In one embodiment, the GBS serotype VIII capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

In one embodiment, the GBS serotype IX capsular polysaccharide conjugate has a sialic acid content of at least any of the above values.

In one embodiment, the conjugate of the invention contains less than about 0.01, 0.02, 0.03, 0.04, or 0.05 mM O-acetate per mM saccharide repeat unit. In another embodiment, the conjugate contains at least about 0.1, 0.2, 0.3, 0.35, or about 0.4 mM O-acetate per mM saccharide repeating unit.

In one embodiment, the GBS serotype IV capsular polysaccharide conjugate has an O-acetate content of any of the above values.

In one embodiment, the GBS serotype Ia capsular polysaccharide conjugate has an O-acetate content of any of the above values.

In one embodiment, the GBS serotype Ib capsular polysaccharide conjugate has an O-acetate content of any of the above values.

In one embodiment, the GBS serotype II capsular polysaccharide conjugate has an O-acetate content of any of the above values.

In one embodiment, the GBS serotype III capsular polysaccharide conjugate has an O-acetate content of any of the above values.

In one embodiment, the GBS serotype V capsular polysaccharide conjugate has an O-acetate content of any of the above values.

In one embodiment, the GBS serotype VI capsular polysaccharide conjugate has an O-acetate content of any of the above values.

In one embodiment, the GBS serotype VII capsular polysaccharide conjugate has an O-acetate content of any of the above values.

In one embodiment, the GBS serotype VIII capsular polysaccharide conjugate has an O-acetate content of any of the above values.

In one embodiment, the GBS serotype IX capsular polysaccharide conjugate has an O-acetate content of any of the above values.

In a further embodiment, the immunogenic conjugate contains less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% free GBS capsular polysaccharide compared to total GBS capsular polysaccharide. In a preferred embodiment, the immunogenic conjugate contains less than about 5% unreacted free saccharide compared to the total GBS capsular polysaccharide.

In another embodiment, the ratio (by weight) of GBS capsular polysaccharide to carrier protein in the conjugate is from about 0.5 to about 3.0. In one aspect, the ratio of GBS capsular polysaccharide to carrier protein in the conjugate is from about 0.5 to about 2.0, from about 0.5 to about 1.5, from about 0.5 to about 1.0, from about 1 0 to about 1.5, or from about 1.0 to about 2.0. In a preferred embodiment, the ratio of GBS capsular polysaccharide to carrier protein in the conjugate is from about 0.8 to about 1.0.

In another embodiment, the degree of conjugation of the conjugate is 2 to 15, 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 15, 5 to 10, 8 to 15, 8 to 12, 10 up to 15 or 10 to 12. In a preferred embodiment, the degree of conjugation of the conjugate is from 2 to 5.

Conjugation

Conjugation can be direct when the atoms of the polysaccharide are covalently linked to the surface atoms of the protein. Alternatively, conjugation can occur via a linker molecule that reacts with both the polysaccharide and the protein and joins them to link the carbohydrate to the protein.

When conjugating (ie, covalently linking) the carrier and one or more antigens, such as a polysaccharide, the conjugation can be performed by any chemical method, method, or genetic method known in the art. For example, a carrier polypeptide and one or more antigens selected from the group consisting of carbohydrate, oligosaccharide, lipid, lipooligosaccharide, polysaccharide, oligosaccharide-protein conjugate, polysaccharide-protein conjugate, peptide-protein conjugate, oligosaccharide-peptide conjugate, polysaccharide-peptide conjugate, protein-protein conjugate, lipooligosaccharide-protein conjugate, polysaccharide-protein conjugate, or any combination thereof, can be conjugated by methods including, but not limited to: carboxyl bond, amine-aldehyde bond; enzymatic direct linkage); (2) homobifunctional binding of amines (eg using bis-aldehydes); (3) homobifunctional binding of thiols (eg using bis-maleimides); (4) homobifunctional binding using photoactivated reagents; (5) heterobifunctional coupling of amines to thiols (eg using maleimides); (6) heterobifunctional coupling with photoactivated reagents (eg, the β-carbonyldiazo family); (7) introducing amino-reactive groups into the poly- or oligosaccharide by cyanogen bromide activation or carboxymethylation; (8) introducing thiol-reactive groups into the poly- or oligosaccharide using a heterobifunctional compound such as maleimido-hydrazide; (9) protein-lipid conjugation by introducing a hydrophobic group into the protein; and (10) protein-lipid conjugation by introducing a reactive group into the lipid. In addition, methods for heterobifunctional "non-covalent binding" are also contemplated, for example through biotin-avidin interactions. Other methods well known in the art for conjugating oligosaccharides and polysaccharides with immunogenic carrier proteins are also within the scope of some embodiments of the invention.

In one embodiment, GBS capsular polysaccharide-protein conjugates are prepared by activating the polysaccharide with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The activated polysaccharide can be linked directly or through a spacer (linker) group to the amino group on the carrier protein. For example, the spacer may be a cystamine or cysteamine to form a thiolated polysaccharide that may be linked to the carrier by a thioether bond formed after reaction with a maleimide-activated carrier protein (e.g., using GMBS) or a haloacetylated carrier protein (e.g., with using iodoacetimide, SIB, SIAB, sulfo-SIAB, SIA, or SBAP).

In one aspect, a cyanate ester (optionally prepared using CDAP chemistry) is coupled to hexanediamine or adipic acid dihydrazide (ADH) and the amino-derivatized saccharide is conjugated to a carrier protein using carbodiimide (e.g., EDAC or EDC) chemistry through the carboxyl group. on the carrier protein. Such conjugates are described, for example, in International Patent Application Publication Nos. WO 93/15760, WO 95/08348 and WO 96/29094.

Other suitable methods use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S--NHS, EDC and TSTU. Many of them are described in International Patent Application Publication No. WO 98/42721. The conjugation may use a carbonyl linker, which may be formed by reacting the free hydroxyl group of the saccharide with 1,1-carbonyldiimidazole (CDI) or 1,1-carbonyldi-1,2,4-triazole (CDT) (see Bethell, et al . al., J. Biol. Chem., 254:2572-2574 (1979); Hearn, et al., J. Chromatogr., 218:509-518 (1981)), followed by reaction with the protein to form a carbamate bond. This may include reducing the anomeric end to the primary hydroxyl group, optionally protecting/deprotecting the primary hydroxyl group, reacting the primary hydroxyl group with the CDI/CDT to form the CDI/CDT carbamate intermediate, and linking the CDI/CDT carbamate intermediate to the amino group. on protein.

In preferred embodiments, the GBS capsular polysaccharide-protein conjugates of the invention are prepared using reductive amination. Reductive amination involves two steps: (1) oxidation of the polysaccharide to form aldehyde functional groups from adjacent diols in a separate hexasaccharide unit and (2) reduction of the activated polysaccharide and carrier protein to form a conjugate.

In one embodiment, the GBS capsular polysaccharide is activated (oxidized) by a method comprising the steps of:

(a) reacting the isolated GBS capsular polysaccharide with an oxidizing agent; And

(b) quenching the oxidation reaction by adding an quencher to obtain an activated GBS capsular polysaccharide.

In one aspect of the invention, the concentration of the isolated capsular polysaccharide is from about 0.1 mg/ml to about 10.0 mg/ml, for example, from about 0.5 mg/ml to about 5.0 mg/ml, from about 1, 0 mg/ml to about 3.0 mg/ml or about 2.0 mg/ml.

In a specific embodiment, the oxidizing agent is a periodate. Periodate oxidizes hydroxyl groups attached to neighboring atoms to form carbonyl or aldehyde groups and causes cleavage of the CC bond. The term "periodate" includes both periodate and iodic acid. The term also includes both metaperiodate (IO ₄ ^- ) and orthoperiodate (IO ₆ ^5- ). The term "periodate" also includes various periodate salts, including sodium periodate and potassium periodate. In a preferred embodiment, the oxidizing agent is sodium periodate. In a preferred embodiment, the periodate used to oxidize the GBS capsular polysaccharides is metaperiodate. In a preferred embodiment, the periodate used to oxidize the serotype capsular polysaccharide is sodium metaperiodate.

In another embodiment, the polysaccharide reacts with 0.01-10.0, 0.05-5.0, 0.1-1.0, 0.5-1.0, 0.7-0.8, 0 05-0.5 or 0.1-0.3 molar equivalents of the oxidizing agent. In a particular embodiment, the polysaccharide reacts with about 0.05, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, or about 0.95 molar equivalents of oxidizing agent. In a further embodiment, the polysaccharide is reacted with about 0.1 molar equivalents of an oxidizing agent. In a further embodiment, the polysaccharide is reacted with about 0.15 molar equivalents of an oxidizing agent. In a further embodiment, the polysaccharide is reacted with about 0.25 molar equivalents of an oxidizing agent. In another embodiment, the polysaccharide is reacted with about 0.5 molar equivalents of an oxidizing agent. In an alternative embodiment, the polysaccharide is reacted with about 0.6 molar equivalents of an oxidizing agent. In a further embodiment, the polysaccharide is reacted with about 0.7 molar equivalents of an oxidizing agent.

In one aspect of the invention, the duration of the oxidation reaction is from about 1 hour to about 50 hours, from about 10 hours to about 30 hours, from about 15 hours to about 20 hours, from about 15 hours to about 17 hours, or about 16 hours.

In another aspect of the invention, the oxidation reaction temperature is maintained at about 2°C to about 25°C, from about 2°C to about 8°C, or from about 20°C to about 25°C. In one preferred embodiment, the oxidation reaction temperature is maintained at about 23°C. In another preferred embodiment, the oxidation reaction temperature is maintained at about 5°C.

In a further aspect, the oxidation reaction is carried out in a buffer selected from the group consisting of sodium phosphate, potassium phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), and Bis-Tris. In a preferred embodiment, the buffer is potassium phosphate.

In a further aspect, the buffer has a concentration of about 1 mM to about 500 mM, about 1 mM to about 300 mM, or about 50 mM to about 200 mM. In a preferred embodiment, the buffer has a concentration of approximately 100 mm.

In one aspect, the oxidation reaction is carried out at a pH of about 4.0 to about 8.0, about 5.0 to about 7.0, or about 5.5 to about 6.5. In a preferred embodiment, the pH is about 6.0.

In one embodiment, an activated GBS capsular polysaccharide is prepared by reacting an isolated capsular polysaccharide at a concentration of about 0.5 mg/l to about 5.0 mg/ml with about 0.05-0.3 molar periodate equivalents at a temperature of about 20 °C to 25°C.

In another embodiment, an activated GBS capsular polysaccharide is prepared by reacting an isolated capsular polysaccharide at a concentration of about 0.5 mg/l to about 5.0 mg/ml with about 0.05-0.3 molar periodate equivalents at a temperature of about 2 °C to approximately 8°C.

In another embodiment, the activated GBS capsular polysaccharide is purified by methods known to the person skilled in the art, such as gel permeation chromatography (GPC), dialysis, or ultrafiltration/diafiltration. For example, the activated capsular polysaccharide is purified by concentration and diafiltration using an ultrafiltration device.

In one embodiment, the oxidation state of the activated GBS capsular polysaccharide is 5 to 25, e.g., 5 to 15, 5 to 10, 10 to 25, 10 to 20, 10 to 15. In a preferred embodiment, the oxidation state of the activated capsular polysaccharide GBS is 10 to 20, 11 to 19, 12 to 18, 13 to 17, or 14 to 16.

In another embodiment, the activated GBS capsular polysaccharide has a molecular weight of from about 5 kDa to about 1000 kDa, for example, from about 50 kDa to about 300 kDa, from about 75 kDa to about 400 kDa, from about 75 kDa to about 200 kDa, from approximately 100 kDa to approximately 700 kDa, approximately 100 kDa to approximately 500 kDa, approximately 100 kDa to approximately 400 kDa, approximately 100 kDa to approximately 300 kDa, approximately 200 kDa to approximately 400 kDa, approximately 300 kDa to approximately 700 kDa. In a preferred embodiment, the activated GBS capsular polysaccharide has a molecular weight of from about 75 kDa to about 400 kDa.

In one embodiment, the activated GBS capsular polysaccharide is lyophilized, optionally in the presence of a saccharide. In a preferred embodiment, the saccharide is selected from sucrose, trehalose, raffinose, stachyose, melisitose, dextran, mannitol, lactitol and palatinite. In a preferred embodiment, the saccharide is sucrose. The lyophilized activated capsular polysaccharide can then be combined with a solution containing a carrier protein.

In another embodiment, the activated GBS capsular polysaccharide is combined with a carrier protein and lyophilized, optionally in the presence of the saccharide. In one aspect, the saccharide is selected from sucrose, trehalose, raffinose, stachyose, melisitose, dextran, mannitol, lactitol, and palatinite. In a preferred embodiment, the saccharide is sucrose. The co-lyophilized polysaccharide and carrier protein can then be resuspended in solution and reacted with a reducing agent.

The activated GBS capsular polysaccharide can be conjugated to a carrier protein by a method comprising the steps of:

(a) combining the activated GBS capsular polysaccharide with a carrier protein, and

(b) reacting the combined activated GBS capsular polysaccharide and carrier protein with a reducing agent to produce a GBS capsular polysaccharide-carrier protein conjugate.

Conjugation of the activated GBS capsular polysaccharide to a carrier protein by reductive amination in a polar aprotic solvent is suitable for maintaining low levels of free polysaccharide, as compared to, for example, reductive amination in aqueous solution where the level of unreacted (free) polysaccharide is significantly increased. In a preferred embodiment, step (a) and step (b) are performed in a polar aprotic solvent.

In one embodiment, step (a) comprises dissolving the lyophilized GBS capsular polysaccharide in a solution containing a carrier protein and a polar aprotic solvent. In another embodiment, step (a) comprises dissolving the co-lyophilized GBS capsular polysaccharide and carrier protein in a polar aprotic solvent.

In one embodiment, the polar aprotic solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), sulfolane, dimethylformamide (DMF), and hexamethylphosphoramide (HMPA). In a preferred embodiment, the polar aprotic solvent is DMSO.

If steps (a) and (b) are performed in an aqueous solution, steps (a) and (b) are performed in a buffer in an aqueous medium, preferably selected from PBS, MES, HEPES, Bis-tris, ADA, PIPES, MOPSO, BES, MOPS, DIPSO, MOBS, HEPPSO, POPSO, TEA, EPPS, bicine, or HEPB with a pH of about 6.0 to about 8.5, about 7.0 to about 8.0, or about 7.0 to about 7 ,5. In a preferred embodiment, the buffer is PBS. In a preferred embodiment, the pH is about 7.3.

In one embodiment, the concentration of the activated GBS capsular polysaccharide in step (b) is from about 0.1 mg/mL to about 10.0 mg/mL, from about 0.5 mg/mL to about 5.0 mg/mL, or from about 0.5 mg/ml to about 2.0 mg/ml. In a preferred embodiment, the concentration of activated GBS serotype capsular polysaccharide in step (b) is about 0.1 mg/ml, about 0.2 mg/ml, about 0.3 mg/ml, about 0.4 mg/ml, about 0 .5 mg/mL, approximately 0.6 mg/mL, approximately 0.7 mg/mL, approximately 0.8 mg/mL, approximately 0.9 mg/mL, approximately 1.0 mg/mL, approximately 1.1 mg/mL, approximately 1.2 mg/mL, approximately 1.3 mg/mL, approximately 1.4 mg/mL, approximately 1.5 mg/mL, approximately 1.6 mg/mL, approximately 1.7 mg/mL ml, about 1.8 mg/ml, about 1.9 mg/ml, about 2.0 mg/ml, about 2.1 mg/ml, about 2.2, about 2.3 mg/ml, about 2, 4 mg/mL, approximately 2.5 mg/mL, approximately 2.6 mg/mL, approximately 2.7 mg/mL, approximately 2.8 mg/mL, approximately 2.9 mg/mL, or approximately 3.0 mg /ml

In a preferred embodiment, the starting ratio (by weight) of activated GBS serotype capsular polysaccharide to carrier protein is 5:1 to 0.1:1, 2:1 to 0.1:1, 2:1 to 1:1 , 1.5:1 to 1:1, 0.1:1 to 1:1, 0.3:1 to 1:1, 0.6:1 to 1:1. In a preferred embodiment, the starting ratio of activated GBS serotype capsular polysaccharide to carrier protein is approximately 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9 :1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1 .8:1, 1.9:1, 2:1.

In one embodiment, the reducing agent is sodium cyanoborohydride, sodium triacetoxyborohydride, sodium or zinc borohydride in the presence of Bronsted or Lewis acids, amine boranes such as pyridine borane, 2-picoline borane, 2,6-diborane-methanol, dimethylamine -borane, tert-BuMe ⁱ PrN-BH ₃ , benzylamine-BH ₃ or 5-ethyl-2-methylpyridine-borane (PEMB). In a preferred embodiment, the reducing agent is sodium cyanoborohydride.

In another embodiment, the amount of reducing agent used in step (b) is from about 0.1 to about 10.0 molar equivalents, from about 0.5 to about 5.0 molar equivalents, or from about 1.0 to about 2, 0 molar equivalents. In a preferred embodiment, the amount of reducing agent used in step (b) is approximately 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1, 8, 1.9 or 2.0 molar equivalents.

In a preferred embodiment, the duration of step (b) is 1 hour to 60 hours, 10 hours to 50 hours, 40 hours to 50 hours, or 42 hours to 46 hours. In a preferred embodiment, the duration of step (b) is approximately 44 hours.

In a further embodiment, the reaction temperature in step (b) is maintained at 10°C to 40°C, 15°C to 30°C, or 20°C to 26°C. In a preferred embodiment, the reaction temperature in step (b) is maintained at approximately 23°C.

In a further embodiment, the method for preparing an immunogenic conjugate comprising a GBS capsular polysaccharide covalently linked to a carrier protein further comprises the step (step (c)) of capping unreacted aldehydes (quenching) by adding a borohydride.

In one embodiment, the capping agent is a borohydride selected from the group consisting of sodium borohydride (NaBH ₄ ), sodium cyanoborohydride, lithium borohydride, potassium borohydride, tetrabutylammonium borohydride, calcium borohydride, and magnesium borohydride. In a preferred embodiment, the capping agent is sodium borohydride.

In another embodiment, the amount of borohydride used in step (c) is from about 0.1 to about 10.0 molar equivalents, from about 0.5 to about 5.0 molar equivalents, or from about 1.0 to 3.0 molar equivalents. In a preferred embodiment, the amount of borohydride used in step (c) is approximately 2.0 molar equivalents.

In one preferred embodiment, the borohydride used in step (c) is NaBH ₄ at a concentration of approximately 2.0 molar equivalents.

In one embodiment, the duration of step (c) is from 0.1 hour to 10 hours, from 0.5 hour to 5 hours, from 2 hours to 4 hours. In a preferred embodiment, the duration of step (c) is approximately 3 hours.

In another embodiment, the reaction temperature in step (c) is maintained at about 15°C to about 45°C, from about 15°C to about 30°C, or from about 20°C to about 26°C. In a preferred embodiment, the reaction temperature in step (c) is maintained at approximately 23°C.

After conjugation of the GBS capsular polysaccharide with the carrier protein and capping, the polysaccharide-protein conjugate can be purified (enriched in terms of the amount of polysaccharide-protein conjugate) by various methods known to the skilled person. These methods include dialysis, concentration/diafiltration procedures, flow-through filtration, precipitation/elution, column chromatography (DEAE chromatography or hydrophobic interaction chromatography), and depth filtration.

In a further embodiment, the immunogenic conjugate contains less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% free GBS capsular polysaccharide compared to total GBS capsular polysaccharide. In a preferred embodiment, the immunogenic conjugate contains less than about 5% unreacted free saccharide compared to the total GBS capsular polysaccharide.

In a preferred embodiment, the GBS polysaccharide-protein conjugate has a molecular weight of from about 300 kD to about 20,000 kD, for example, from about 1000 kD to about 15,000 kD or from about 1000 kD to about 10,000 kD.

In another embodiment, the ratio (by weight) of GBS capsular polysaccharide to carrier protein in the conjugate is from about 0.5 to about 3.0. In one aspect, the ratio of GBS capsular polysaccharide to carrier protein in the conjugate is from about 0.5 to about 2.0, from about 0.5 to about 1.5, from about 0.5 to about 1.0, from about 1 0 to about 1.5, or from about 1.0 to about 2.0. In a preferred embodiment, the ratio of GBS capsular polysaccharide to carrier protein in the conjugate is from about 0.8 to about 1.0.

In another embodiment, the degree of conjugation of the conjugate is 2 to 15, 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 15, 5 to 10, 8 to 15, 8 to 12, 10 up to 15 or 10 to 12. In a preferred embodiment, the degree of conjugation of the conjugate is from 2 to 5.

In one aspect of the invention, GBS capsular polysaccharide-protein conjugates are prepared by the reductive amination method described above. For example, in one aspect, the present invention relates to a GBS capsular polysaccharide-protein conjugate containing a polysaccharide conjugated to a carrier protein, which is made, or can be made, by a method comprising the steps of:

(a) reacting the isolated GBS capsular polysaccharide with an oxidizing agent;

(b) quenching the oxidation reaction by adding an quencher to obtain an activated GBS capsular polysaccharide;

(c) combining the activated GBS capsular polysaccharide with a carrier protein,

(d) reacting the combined activated GBS capsular polysaccharide and carrier protein with a reducing agent to produce a GBS capsular polysaccharide and carrier protein conjugate, and optionally

(e) capping unreacted aldehyde by adding sodium borohydride (NaBH ₄ ).

In a preferred embodiment, steps (c) and (d) are performed in DMSO.

In another aspect of the invention, the GBS capsular polysaccharide-protein conjugates of the invention are prepared by the reductive amination method described above, but with the free radical 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) as co-oxidant. at the activation/oxidation stage. See International Patent Application Publication No. WO 2014/097099, the contents of which are incorporated herein by reference in their entirety. In such an embodiment, glycoconjugates from GBS capsular polysaccharides are prepared using the free radical TEMPO to oxidize primary saccharide alcohols to aldehydes using NCS as a co-oxidant (hereinafter "TEMPO/NCS oxidation"), for example, as described in Example 7 and in International Patent Application Publication No. WO 2014/097099. Thus, in one aspect, GBS capsular polysaccharide conjugates can be prepared by a process comprising the steps of: a) reacting the GBS capsular polysaccharide with TEMPO and NCS in a solvent to form an activated saccharide; and b) reacting the activated saccharide with a carrier protein containing one or more amino groups (hereinafter "TEMPO/NCS reductive amination"). In one embodiment, the solvent may be an aqueous solvent or DMSO.

In one aspect, GBS capsular polysaccharide-protein conjugates can be obtained by the specified method. For example, in one aspect, the present invention relates to a GBS capsular polysaccharide-protein conjugate containing a polysaccharide conjugated to a carrier protein, which is, or can be obtained, by a method comprising the steps of: a) reacting a saccharide with 2,2,6, 6-tetramethyl-1-piperidinyloxy (TEMPO) and N-chlorosuccinimide (NCS) in a solvent to give an activated saccharide; and b) reacting the activated saccharide with a carrier protein containing one or more amino groups. In one embodiment, the solvent may be an aqueous solvent or DMSO.

Immunogenic compositions

Once the individual conjugates have been purified, they can be combined to formulate an immunogenic composition of the present invention, which can be used, for example, in a vaccine. The formulation of the immunogenic composition of the present invention can be performed using methods recognized in the art.

An "immune response" to an immunogenic composition is the development in a patient of a humoral and/or cellular immune response to molecules present in the composition of interest (eg, an antigen, such as a protein or polysaccharide). For the purposes of the present invention, a "humoral immune response" is an antibody-mediated immune response and includes the production of antibodies with affinity for antigens present in the immunogenic compositions of the invention, while a "cellular immune response" is a T- lymphocytes and/or other white blood cells. A "cellular immune response" is initiated by the presentation of antigenic epitopes in association with major histocompatibility complex (MHC) class I or class II molecules. This leads to the activation of antigen-specific CD4+ T helpers or CD8+ cytotoxic T lymphocytes (CTL). CTLs have specificity for peptide or lipid antigens that are presented in association with proteins encoded by MHC or CD1 and expressed on the cell surface. CTL contribute to the induction and stimulation of intracellular destruction of intracellular microbes, or lysis of cells infected with such microbes. Another aspect of cellular immunity involves the antigen-specific response of T helpers. T-helpers contribute to the stimulation of the function, and the direction of activity, of non-specific effector cells against cells on the surface of which peptide antigens are exposed in association with classical or non-classical MHC molecules. "Cellular immune response" also means the production of cytokines, chemokines, and other such molecules produced by activated T cells and/or other white blood cells, including those produced by CD4+ and CD8+ T cells. The ability of a particular antigen or composition to stimulate a cellular immune response can be determined in a number of assays, for example, in lymphoproliferation (lymphocyte activation) assays, cytotoxic CTL assays, antigen-specific T-lymphocyte assays in a sensitized patient, or by measuring cytokine T -cells in response to re-stimulation with antigen. Such assays are well known in the art. See, for example, Erickson, AL, et al., J. Immunol., 151(8):4189-4199 (1993); Doe, B., et al., Eur. J. Immunol. 24(10):2369-2376 (1994).

The term "immunogenic" means the ability of an antigen or vaccine to elicit an immune response, either humoral or cellular, or both.

The terms "immunogenic amount" or "immunologically effective amount" or "dose", which are used interchangeably herein, generally means an amount of an antigen or immunogenic composition sufficient to elicit an immune response, either cellular (T-cell) or humoral. (B-cell or antibody production) response, or both, when measured in standard assays known to those skilled in the art.

As used herein, the term "immune interference" or "significant immune interference" means a statistically significant decrease in the immune response to a single antigen in a polyvalent or multicomponent vaccine, compared to the immune response to the same antigen when administered in a monovalent vaccine.

"Protective immune response" means the ability of an immunogenic composition to elicit an immune response, either humoral or cellular, that protects the patient from infection. The protection provided need not be absolute, i.e., infection need not be completely prevented or eliminated if there is a statistically significant improvement compared to a control patient population, such as infected animals that have not received a vaccine or immunogenic composition. Protection may be limited by reducing the severity or rate of infection symptoms. Several assays are known in the art for determining whether an immune response is a "protective immune response". For example, an increase in antibody levels can be determined in a binding assay, such as a cell-based ELISA, described below. Other assays include measuring responses in terms of production of functional antibodies, eg, bacterial killers, which can be tested in the opsonophagocytosis assay (OPA) described below. In specific situations, a "protective immune response" may include the induction of a two-fold increase in antibody levels or a four-fold increase in antibody levels specific for a particular antigen in at least 50% of patients. In another situation, a "protective immune response" may include a reduction in the number of bacteria by at least 10%, 25%, 50%, 65%, 75%, 80%, 85%, 90%, 95% or more.

The amount of a particular conjugate in a composition is generally calculated based on the total amount of polysaccharide, conjugated and unconjugated, for that conjugate. For example, a GBS capsular polysaccharide conjugate with 20% free polysaccharide will contain approximately 80 μg/ml conjugated GBS capsular polysaccharide and approximately 20 μg/ml unconjugated GBS capsular polysaccharide at a dose of 100 μg/ml GBS capsular polysaccharide. The contribution of the protein carrier to the conjugate is generally not taken into account when calculating the dose of the conjugate. The amount of conjugate may vary depending on the serotype of streptococci. Typically, each dose will contain from about 0.01 mg/ml to about 100 µg/ml of each polysaccharide, in particular from about 1 µg/ml to about 70 µg/ml, and more specifically from about 5 µg/ml up to approximately 50 µg/ml. The "immunogenic amount" of the different polysaccharide components in an immunogenic composition may vary, and in each case may be about 0.01 µg/ml, about 0.1 µg/ml, about 0.25 µg/ml, about 0.5 µg/ml , approximately 1 μg/mL, approximately 2 μg/mL, approximately 3 μg/mL, approximately 4 μg/mL, approximately 5 μg/mL, approximately 6 μg/mL, approximately 7 μg/mL, approximately 8 μg/mL, approximately 9 mcg/mL, approximately 10 mcg/mL, approximately 15 mcg/mL, approximately 20 mcg/mL, approximately 25 mcg/mL, approximately 30 mcg/mL, approximately 40 mcg/mL, approximately 50 mcg/mL, approximately 60 mcg /ml, approximately 70 μg/ml, approximately 80 μg/ml, approximately 90 μg/ml, or approximately 100 μg/ml of a particular polysaccharide antigen. The dose, or immunogenic amount, of a polyvalent immunogenic composition will be indicative of the dose of each polysaccharide, unless otherwise indicated. For example, a 10 μg/ml dose for a hexavalent immunogenic composition would include 10 μg/ml of each of the six polysaccharides, for a total dose of 60 μg/ml of antigen or conjugate.

In one aspect of the invention, the total dose of the conjugate is at least about 60 μg/ml, such as at least about 120 μg/ml or at least about 240 μg/ml. In one embodiment, the total dose of the conjugate is from about 60 µg/ml to about 600 µg/ml, for example, from about 60 µg/ml to about 540 µg/ml, from about 60 µg/ml to about 480 µg/ml, from about 60 µg/mL to about 420 µg/mL, about 60 µg/mL to about 360 µg/mL, about 60 µg/mL to about 300 µg/mL, about 60 µg/mL to about 240 µg/mL , from about 60 µg/ml to about 180 µg/ml, from about 60 µg/ml to about 120 µg/ml, from about 120 µg/ml to about 600 µg/ml, from about 120 µg/ml to about 540 µg /ml, from about 120 µg/ml to about 480 µg/ml, from about 120 µg/ml to about 420 µg/ml, from about 120 µg/ml to about 360 µg/ml, from about 120 µg/ml to about 300 µg/ml, from about 120 µg/ml to about 240 µg/mL, from about 120 µg/mL to about 180 µg/mL, from about 180 µg/mL to about 600 µg/mL, from about 180 µg/mL to about 540 µg/mL, from about 180 µg/mL to about 480 µg/mL, from about 180 µg/mL to about 420 µg/mL, from about 180 µg/mL to about 360 µg/mL, from about 180 µg/mL to about 300 µg/mL, from about 180 µg/mL ml to about 240 µg/ml, from about 240 µg/ml to about 600 µg/ml, from about 240 µg/ml to about 540 µg/ml, from about 240 µg/ml to about 480 µg/ml, from about 240 µg/ml to about 420 µg/ml, from about 240 µg/ml to about 360 µg/ml, or from about 240 µg/ml to about 300 µg/ml. In a preferred embodiment, the total dose of the conjugate is from about 60 µg/ml to about 360 µg/ml, and most preferably from about 120 mg/ml to about 240 µg/ml.

The effectiveness of an antigen as an immunogen can be determined by measuring levels of B-cell activity by measuring levels of circulating antibodies specific for the antigen in serum using immunoassays, immunoprecipitation assays, functional antibody assays such as an in vitro opsonic activity assay, and using a variety of other assays known in the art. Another measure of the effectiveness of an antigen as a T cell immunogen can be determined in proliferation assays, in cytolytic activity assays such as chromium release assays, to measure the ability of T cells to lyse their specific target cells. In addition, in the present invention, the "immunogenic amount" can also be determined by measuring serum levels of antigen-specific antibodies induced after antigen challenge, or by measuring the ability of antibodies thus induced to enhance the opsonophagocytic activity of specific white blood cells, as described herein. The level of protection resulting from an immune response can be determined by challenging the immunized host with the antigen that has been injected. For example, if the antigen to which an immune response is desired is a bacterium, the level of protection induced by the "immunogenic amount" of the antigen can be measured by determining percentage survival or percentage mortality after challenge of animals with bacterial cells. In one embodiment, the degree of protection can be determined by quantifying at least one symptom associated with a bacterial infection, such as fever associated with the infection. The amount of each of the antigens in the polyantigenic or multicomponent vaccine or immunogenic compositions will vary relative to each of the other components and can be determined by methods known to the skilled artisan. Such methods will include, for example, the measurement of immunogenicity and/or in vivo efficacy.

The term "immunogenic composition" means any pharmaceutical composition containing an antigen, such as a microorganism, or a component thereof, which can be used to elicit an immune response in a patient. The immunogenic compositions of the present invention can be used to treat a human susceptible to GBS infection by administering the immunogenic compositions by systemic, transdermal or mucosal route of administration. Such routes of administration may include intramuscular (IM), intraperitoneal (IP), intradermal (IC), or subcutaneous injection; application with a patch or other transdermal delivery device; or mucosal administration to the oral cavity/digestive system, respiratory or genitourinary tract. In one embodiment, the immunogenic composition can be used to produce a vaccine or induce polyclonal or monoclonal antibodies that can be used to passively protect or treat an animal.

In one aspect, the present invention relates to immunogenic compositions containing an effective amount of at least one polysaccharide, oligosaccharide, polysaccharide-protein conjugate, or their biological equivalent described in the present description. For example, in one embodiment, the immunogenic composition comprises polysaccharide-protein conjugates, wherein the capsular polysaccharide is selected from the group consisting of capsular polysaccharides of group B streptococci serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX, and wherein the capsular polysaccharide has a sialic acid level greater than about 60%. In another example, the immunogenic composition comprises polysaccharide-protein conjugates, wherein the conjugates comprise capsular polysaccharides from group B streptococci serotype IV and at least one additional serotype selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In another embodiment, the immunogenic composition comprises polysaccharide-protein conjugates, wherein the conjugates comprise capsular polysaccharides from group B streptococci serotype IV and at least two additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In another embodiment, the immunogenic composition comprises polysaccharide-protein conjugates, wherein the conjugates comprise capsular polysaccharides from group B streptococci serotype IV and at least three additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In a further embodiment, the immunogenic composition comprises polysaccharide-protein conjugates, wherein the conjugates comprise capsular polysaccharides from group B streptococci serotype IV and at least four additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In a specific embodiment, the immunogenic composition comprises polysaccharide-protein conjugates, wherein the conjugates comprise capsular polysaccharides from Group B Streptococcus serotypes Ia, Ib, II, III, and V. In another embodiment, the immunogenic composition comprises polysaccharide-protein conjugates, wherein the conjugates comprise capsular polysaccharides from group B streptococci serotypes Ia, Ib, II, III and IV. In another embodiment, the immunogenic composition comprises polysaccharide-protein conjugates, wherein the conjugates comprise capsular polysaccharides from group B streptococci serotype IV and at least five additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII and IX. In one such embodiment, the immunogenic composition comprises six polysaccharide-protein conjugates, wherein the conjugates comprise a capsular polysaccharide from group B streptococci serotypes Ia, Ib, II, III, IV, and V.

In one embodiment, the immunogenic composition of the invention comprises polysaccharides from 2-10 different S. agalactiae serotypes. Thus, in one embodiment, the immunogenic composition of the invention is a 2, 3, 4, 5, 6, 7, 8, 9, or 10-valent GBS conjugate composition. In one such embodiment, the immunogenic composition is a 5-valent GBS conjugate composition. In another embodiment, the immunogenic composition is a 6-valent GBS conjugate composition. In another embodiment, the immunogenic composition is a 7-valent GBS conjugate composition. In a further embodiment, the immunogenic composition is an 8-valent GBS conjugate composition.

Despite previously obtained results using less than six, less than five, or less than four GBS antigens in a formulation (see U.S. International Patent Applications Nos. WO 2006/082527 and WO 2006/082530), which indicated immune interference, in particular in connection with the use of serotype V in multivalent compositions (see International Patent Publication No. WO 2012/035519), the present invention is not associated with any significant immune interference when four or more GBS antigens are used, or when serotype V is used in a multivalent composition. Accordingly, the present invention relates to polyvalent immunogenic compositions containing polysaccharide-protein conjugates containing capsular polysaccharides of at least four GBS serotypes, for example, capsular polysaccharides of at least five GBS serotypes, capsular polysaccharides of at least six GBS serotypes, at least seven GBS serotypes, capsular polysaccharides of at least eight GBS serotypes, or capsular polysaccharides of at least nine GBS serotypes, and the composition is free from significant immune interference. In a particular embodiment, the immunogenic composition comprises a GBS serotype V capsular polysaccharide.

Polysaccharide-protein conjugates may contain the same or different protein carriers. In one embodiment, the conjugates contain the same carrier protein and the saccharides are conjugated to the same carrier protein molecule (2 or more different polysaccharides are conjugated to the carrier molecules) [see, for example, International Patent Application Publication No. WO 2004/083251] . In another embodiment, all polysaccharides are individually conjugated to different carrier protein molecules (only one type of polysaccharide is conjugated to each carrier protein molecule). In this embodiment, the capsular saccharides are said to be individually conjugated to a carrier protein.

The optimal amounts of components for a particular immunogenic composition can be established using standard studies, including the observation of appropriate immune responses in patients. Following the initial vaccination, patients may receive one or more booster immunizations at appropriate intervals.

The immunogenic compositions of the invention may additionally contain one or more preservatives in addition to several capsular polysaccharide-protein conjugates. Biologics in multi-dose (multidose) vials are required by the FDA to contain a preservative, with only a few exceptions. The present invention contemplates the use of such multi-dose vials. Vaccine preparations containing preservatives include those containing benzethonium chloride (anthrax), 2-phenoxyethanol (DPT, hepatitis A, Lyme disease, polio (parenteral)) and phenol (pneumococcal infection, typhoid (parenteral)). Preservatives approved for use in injectables include, for example, chlorobutanol, m-cresol, methylparaben, propylparaben, 2-phenoxyethanol, benzethonium chloride, benzalkonium chloride, benzoic acid, benzyl alcohol, phenol, and phenylmercury nitrate.

In another aspect, the invention relates to a composition comprising at least one of any of the polysaccharides described herein and a pharmaceutically acceptable excipient, buffer, stabilizer, adjuvant, cryoprotectant, salt, divalent cation, non-ionic detergent, inhibitor of free radical-induced oxidation, diluent or a carrier, or a mixture thereof.

The immunogenic composition may optionally contain one or more physiologically acceptable buffers selected from, but not limited to, HEPES, PIPES, MES, Tris (trimethamine), phosphate, acetate, borate, citrate, glycine, histidine, and succinate. In a preferred embodiment, the buffer is histidine or phosphate.

In one embodiment, the immunogenic composition comprises a buffer at a concentration of from about 5 mM to about 50 mM, from about 5 mM to about 40 mM, from about 5 mM to about 30 mM, from about 5 mM to about 20 mM, from about 5 mM up to about 10 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, from about 10 mM to about 30 mM, from about 10 mM to about 25 mM, from about 10 mM to about 20 mM, from about 10 mM to about 15 mM, from about 15 mM to about 50 mM, from about 15 mM to about 40 mM, from about 15 mM to about 35 mM, from about 15 mM to about 30 mM, from about 15 mM to about 25 mM, or from about 15 mM to about 20 mM. In a preferred embodiment, the immunogenic composition contains a buffer at a concentration of from about 10 mM to about 25 mM, such as from about 15 mM to about 25 mM, and most preferably about 20 mM.

In one preferred embodiment, the immunogenic composition contains histidine at a concentration of approximately 20 mM. In another preferred embodiment, the immunogenic composition contains phosphate at a concentration of approximately 20 mM.

In specific embodiments, the formulation buffer pH is in the range of about 5.0 to about 7.5, for example, about 5.3 to about 7.5, about 5.5 to about 7.5, about 6 0 to about 7.5, about 6.5 to about 7.5, about 5.3 to about 7.1, about 5.5 to about 7.0, about 6.0 to about 7, 0, about 6.0 to about 6.5, about 6.3 to about 7.0, or about 6.5 to about 7.0. In another embodiment, the pH of the formulation buffer is about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5. In a preferred embodiment, the pH of the formulation buffer is in the range of about 6.0 to about 7.5, such as about 6.5 to about 7.5, and most preferably about 6.5 or about 7.0.

The immunogenic composition may optionally contain one or more non-ionic surfactants, including, but not limited to, polyoxyethylene sorbitan fatty acid esters, polysorbate-80 (TWEEN 80), polysorbate-60 (TWEEN 60), polysorbate-40 (TWEEN 40), polysorbate-20 (TWEEN 20) and polyoxyethylene alkyl ethers including but not limited to BRIJ 58, BRIJ 35, and others such as TRITON X-100; TRITON X-114, NP40, SPAN 85 and Pluronic series non-ionic surfactants (e.g. Pluronic 121). In one embodiment, the immunogenic composition comprises polysorbate-80 or polysorbate-40, preferably polysorbate-80 (PS80).

In one embodiment, the immunogenic composition comprises a surfactant at a concentration of from about 0.001% to about 2% (v/w), from about 0.001% to about 1%, from about 0.001% to about 0.5%, from about 0.001% to about 0.1%, from about 0.001% to about 0.05%, from about 0.001% to about 0.01%, from about 0.001% to about 0.005%, from about 0.005% to about 2%, from about 0.005% to about 1%, from about 0.005% to about 0.5%, from about 0.005% to about 0.1%, from about 0.005% to about 0.05%, from about 0.005% to about 0.01%, from about 0.01% to about 2%, about 0.01% to about 1%, about 0.01% to about 0.5%, about 0.01% to about 0.1%, about 0, 01% to about 0.05%, from about 0.01% to n about 0.04%, about 0.01% to about 0.03%, about 0.015% to about 2%, about 0.015% to about 1%, about 0.015% to about 0.5%, about 0.015% to about 0.1%, about 0.015% to about 0.05%, about 0.015% to about 0.04%, about 0.015% to about 0.03%, about 0.02% to about 2%, about 0.02% to about 1%, about 0.02% to about 0.5%, about 0.02% to about 0.1%, about 0.02% to about 0, 05%, about 0.02% to about 0.04%, about 0.02% to about 0.03%, about 0.05% to about 2%, about 0.05% to about 1% , from about 0.05% to about 0.5%, from about 0.05% to about 0.1%, from about 0.1% to about 2%, from about 0, 1% to about 1%, about 0.1% to about 0.5%, or about 0.1% to about 0.25%. In a preferred embodiment, the immunogenic composition contains a surfactant at a concentration of from about 0.01% to about 0.03%, and most preferably about 0.02%.

In another embodiment, the immunogenic composition comprises polysorbate-80 at a concentration of from about 0.001% to about 2% (preferably up to about 0.25%) or polysorbate-40 at a concentration of from about 0.001% to 1% (while preferably to about 0.5%).

In a preferred embodiment, the immunogenic composition contains PS80 at a concentration of approximately 0.02%.

Pharmaceutically acceptable carriers should not be confused with "carrier proteins" which are used to attach a carbohydrate of the invention to a protein and modify the immune response to that carbohydrate. To avoid confusion with the protein carriers described herein, the term "pharmaceutically acceptable diluent" will be preferred over "pharmaceutically acceptable carriers", however, these terms can sometimes be used interchangeably. The term "pharmaceutically acceptable carrier" means a carrier approved by a federal, state, or other regulatory agency, or listed in the USP or other recognized pharmacopoeias, for use in animals, including humans, as well as non-human mammals. The term "carrier" means a diluent, adjuvant, excipient, or medium with which a pharmaceutical composition is administered. Suitable pharmaceutically acceptable diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Such pharmaceutically acceptable diluents may be sterile liquids such as water and oils, including petroleum, animal, vegetable or synthetic oils. As liquid carriers, in particular for injection solutions, water, water for injection (WFI), sterile isotonic saline solutions, phosphate-buffered saline, adjuvant suspensions, aqueous solutions of dextrose and glycerol, and combinations thereof can be used. Pharmaceutically acceptable diluents may also contain minor amounts of excipients such as wetting agents or emulsifiers, preservatives or buffers that enhance shelf life or potency in the body. The preparation and use of pharmaceutically acceptable diluents is well known in the art. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, edited by EW Martin. In one embodiment, the diluent is water, water for injection (WFI), adjuvant suspension, or saline. In a particular embodiment, the diluent is a suspension of any adjuvant described herein. In a preferred embodiment, the diluent is an aluminum based adjuvant suspension, such as aluminum phosphate suspension .

Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melisitose, dextran, mannitol, lactitol, palatinite, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerin, propylene glycol, water, ethanol, and the like. In a preferred embodiment, the excipient is NaCl.

In one embodiment, the immunogenic composition contains the excipient at a concentration of from about 10 mM to about 500 mM, from about 10 mM to about 450 mM, from about 10 mM to about 400 mM, from about 10 mM to about 350 mM, from about 10 mM up to about 300 mM, from about 10 mM to about 250 mM, from about 10 mM to about 200 mM, from about 10 mM to about 150 mM, from about 10 mM to about 100 mM, from about 10 mM to about 50 mM, from about 10 mM to about 30 mM, from about 10 mM to about 20 mM, from about 20 mM to about 500 mM, from about 20 mM to about 450 mM, from about 20 mM to about 400 mM, from about 20 mM to about 350 mM, from about 20 mM to about 300 mM, from about 20 mM to about 250 mM, from approx. 20 mM to about 200 mM, about 20 mM to about 150 mM, about 20 mM to about 100 mM, about 20 mM to about 50 mM, about 20 mM to about 30 mM, about 50 mM to about 500 mM, about 50 mM to about 450 mM, about 50 mM to about 400 mM, about 50 mM to about 350 mM, about 50 mM to about 300 mM, about 50 mM to about 250 mM, from about 50 mM to about 200 mM, about 50 mM to about 150 mM, about 50 mM to about 100 mM, about 100 mM to about 500 mM, about 100 mM to about 450 mM, about 100 mM to about 400 mM, from about 100 mM to about 350 mM, from about 100 mM to about 300 mM, from about 100 mM to about 250 mM, from approx. 100 mM to about 200 mM, about 100 mM to about 150 mM, about 120 mM to about 470 mM, about 120 mM to about 420 mM, about 120 mM to about 370 mM, about 120 mM to about 320 mM, about 120 mM to about 270 mM, about 120 mM to about 220 mM, about 120 mM to about 170 mM, about 150 mM to about 500 mM, about 150 mM to about 450 mM, from about 150 mM to about 400 mM, about 150 mM to about 350 mM, about 150 mM to about 300 mM, about 150 mM to about 250 mM, about 150 mM to about 200 mM, about 200 mM to about 500 mM, from about 200 mM to about 450 mM, from about 200 mM to about 400 mM, from about 200 mM to approx. but 350 mM, from about 200 mM to about 300 mM, from about 200 mM to about 250 mM, from about 225 mM to about 465 mM, from about 225 mM to about 425 mM, from about 225 mM to about 365 mM, from about 225 mM to about 325 mM, from about 225 mM to about 265 mM, from about 250 mM to about 500 mM, from about 250 mM to about 450 mM, from about 250 mM to about 400 mM, from about 250 mM to about 350 mM, from about 250 mM to about 300 mM, from about 300 mM to about 500 mM, from about 300 mM to about 450 mM, from about 300 mM to about 400 mM, from about 300 mM to about 350 mM, from about 350 mM to about 500 mM, from about 350 mM to about 450 mM, from about 350 mM to about 400 mM, from about 400 mM to about 500 mM, about 400 mM to about 450 mM, or about 450 mM to about 500 mM. In a preferred embodiment, the immunogenic composition contains the excipient at a concentration of about 10 mM to about 350 mM, such as about 130 mM to about 170 mM and about 225 mM to about 265 mM, and most preferably about 150 mM or about 245 mM.

In one preferred embodiment, the excipient is NaCl at a concentration of approximately 150 mM. In another preferred embodiment, the excipient is NaCl at a concentration of approximately 245 mM.

The composition, if necessary, may also contain minor amounts of wetting agents, bulking agents, emulsifiers or pH adjusters. Such compositions can be taken in the form of solutions, suspensions, emulsions, lyophilized powder or lozenges, and the like. The drug must correspond to the route of administration. Unless any of the conventional media or agents are incompatible with the active ingredient, their use in the immunogenic compositions of the present invention is intended.

In one embodiment, the immunogenic composition is lyophilized, optionally in the presence of at least one excipient. In a preferred embodiment, at least one excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melisitose, dextran, mannitol, lactitol, palatinite, gelatin, malt, rice, flour, chalk, silica gel , sodium stearate, glycerin monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerin, propylene glycol, water and ethanol. In a preferred embodiment, at least one excipient is selected from the group consisting of sucrose, mannitol and glycine. In a specific embodiment, at least one excipient is sucrose. In another embodiment, the lyophilized composition contains an additional excipient. In one such embodiment, the additional excipient is mannitol or glycine.

In another embodiment, the lyophilized composition contains from about 1% (w/v) to about 10% (w/v) of at least one saccharide, e.g., about 1.5%, 2.0%, 2, 5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7, 5%, 8.0%, 8.5%, 9.0%, 9.5% or 10.0%. In a preferred embodiment, the lyophilized composition contains greater than about 5.5% (w/v) of at least one excipient, such as greater than about 7.0% (w/v). In the following embodiment, the lyophilized composition contains from about 1% (w/v) to about 10% (w/v) of additional excipient, for example, about 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5% or 10.0%. In a preferred embodiment, the lyophilized composition contains from about 1% (w/v) to about 10% (w/v) of at least one excipient and from about 1% (w/v) to about 10% (w/v) additional excipient.

In another embodiment, the lyophilized composition is reconstituted with water, water for injection (WFI), adjuvant suspension, or saline. In a preferred embodiment, the diluent is an aluminum adjuvant suspension, such as aluminum phosphate suspension .

In one embodiment, the composition contains an isolated polysaccharide as described herein and a carrier molecule. Suitable carrier molecules may include proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactivated viral particles. Examples of particulate carriers include those derived from poly(methyl methacrylate) polymers as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides) known as PLGs.

The immunogenic compositions of the present invention may also contain one or more additional "immunomodulators", which are agents that cause disturbances or changes in the immune system, resulting in either stimulation or suppression of humoral and/or cellular immunity. In one particular embodiment, stimulation of the humoral and/or cellular components of the immune system is preferred. Examples of specific immunomodulators include, for example, an adjuvant or cytokine, or ISCOMATRIX (CSL Limited, Parkville, Australia) described in US Pat. No. 5,254,339, among others. The term "adjuvant" means a compound or mixture that elicits an enhanced immune response to an antigen, as further described herein.

Non-limiting examples of adjuvants that can be used in the composition of the present invention include the RIBI adjuvant system (Ribi Inc., Hamilton, Mont.); mineral gels such as aluminum hydroxide gel; water-in-oil emulsions such as complete and incomplete Freund's adjuvants; block copolymer (CytRx, Atlanta Ga.); SAF-M (Chiron, Emeryville, Calif.); adjuvant AMPHIGEN ^® ; saponin; Quil A or another saponin fraction; monophosphoryl lipid A and the lipid-amine adjuvant avridine. Non-limiting examples of oil-in-water emulsions useful as an adjuvant in a vaccine of the invention include MF59 (US Pat. No. 6,299,884) (containing 5% squalene, 0.5% polysorbate-80 and 0.5% Span 85 (optional, containing varying amounts of MTP-PE) formulated as submicron particles using a microfluidizer such as the Model 110Y Microfluidizer (Microfluidics, Newton, MA)), and SAF (containing 10% squalene, 0.4% polysorbate-80, 5% pluronic -block polymer L121 and thr-MDP, either microfluidized into a submicron emulsion or vortexed to form an emulsion with larger particles); modified SEAM62 (containing 5% (v/v) squalene (Sigma), 1% (v/v) ^SPAN® 85 detergent (ICI Surfactants), 0.7% (v/v) polysorbate-80 detergent (ICI Surfactants), 2.5 % (v/v) ethanol, 200 μg/ml Quil A, 100 μg/ml cholesterol and 0.5% (v/v) lecithin); and modified SEAM 1/2 (containing 5% (v/v) squalene, 1% (v/v) ^SPAN® 85 detergent, 0.7% (v/v) polysorbate-80 detergent, 2.5% (v/v) ethanol, 100 µg/ml Quil A and 50 µg/ml cholesterol).

Suitable adjuvants used to enhance the immune response further include, without limitation, MPL™ (3-O-deacylated monophosphoryl lipid A, Corixa, Hamilton, MT), which is described in US patent No. 4912094. Synthetic analogs are also suitable for use as adjuvants. lipid A or an aminoalkylglucosamine phosphate (AGP) compound, or derivatives or analogs thereof, available from Corixa (Hamilton, MT) 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 which is also known as 529 (formerly RC529 ). This adjuvant 529 is formulated in aqueous (AF) or stable emulsion (SE) form.

Other adjuvants include a cyclodextrin derivative (US Pat. No. 6,165,995); polyanionic polymer (US patent No. 6610310); muramyl peptides such as N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP) and N-acetylnormuramyl-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamine ( MTP-PE); amphigen; avridine; L121/squalene; D-lactide-polylactide/glycoside; pluronic polyols; killed by Bordetella ; saponins such as Stimulon™ QS-21 (Antigenics, Framingham, MA.) described in US Pat. No. 5,057,540; Mycobacterium tuberculosis ; bacterial lipopolysaccharides; synthetic polynucleotides such as oligonucleotides containing a CpG motif (eg US Pat. No. 6,207,646); IC-31 (Intercell AG, Vienna, Austria), described in European patents Nos. 1296713 and 1326634; pertussis toxin (PT) or its mutant, cholera toxin or its mutant (for example, US patent No. 7285281, 7332174, 7361355 and 7384640); or heat-labile toxin E. coli (LT) or its mutant, in particular, LT-K63, LT-R72 (for example, US patents No. 6149919, 7115730 and 7291588).

Other "immunomodulators" that may be included in a vaccine include, for example, one or more of the interleukins 1-α, 1-β, 2, 4, 5, 6, 7, 8, 10, 12 (see, for example, US Pat. No. 5723127), 13, 14, 15, 16, 17 and 18 (and its mutants); interferons-α, β and γ; granulocyte-macrophage colony stimulating factor (GM-CSF) (see, for example, US patent No. 5078996 and registration number ATCC 39900); macrophage colony stimulating factor (M-CSF); granulocyte colony stimulating factor (G-CSF); or tumor necrosis factors α and β. Other adjuvants that may be used in the immunogenic compositions described herein include chemokines, including but not limited to MCP-1, MIP-1α, MIP-1β, and RANTES; adhesion molecules such as selectin, eg L-selectin, P-selectin and E-selectin; mucin-like molecules such as CD34, GlyCAM-1 and MadCAM-1; members of the integrin family such as LFA-1, VLA-1, Mac-1 and p150.95; members of the immunoglobulin superfamily such as PECAM, ICAM, eg ICAM-1, ICAM-2 and ICAM-3, CD2 and LFA-3; costimulatory molecules such as B7-1, B7-2, CD40 and CD40L; growth factors including vascular growth factor, nerve growth factor, fibroblast growth factor, epidermal growth factor, PDGF, BL-1 and vascular endothelial growth factor; receptor molecules including Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2 and DR6; and caspase (ICE).

It should be understood that the decision to use an immunomodulator and/or adjuvant, or the choice of immunomodulator and/or adjuvant used, will depend on the patient to whom the vaccine or immunogenic composition will be administered, the route of administration and the number of injections. For example, if a patient has been naturally exposed to a pathogen, an adjuvant may not be needed because vaccine antigens can effectively induce a memory cell response. In specific embodiments, the implementation of the immunogenic composition will contain one or more adjuvants. In one embodiment, the immunogenic composition contains an aluminum-based adjuvant. In one such embodiment, the aluminum-based adjuvant is aluminum hydroxide, aluminum phosphate, or aluminum hydroxyphosphate. In a particular embodiment, the adjuvant is aluminum phosphate. In another embodiment of the invention, the immunogenic composition contains QS-21 as an adjuvant.

In one embodiment, the immunogenic composition contains an adjuvant at a concentration of from about 0.1 mg/ml to about 1.0 mg/ml, from about 0.1 mg/ml to about 0.9 mg/ml, from about 0.1 mg /ml to about 0.8 mg/ml, from about 0.1 mg/ml to about 0.7 mg/ml, from about 0.1 mg/ml to about 0.6 mg/ml, from about 0.1 mg/mL to about 0.5 mg/mL, from about 0.1 mg/mL to about 0.4 mg/mL, from about 0.1 mg/mL to about 0.3 mg/mL, from about 0, 1 mg/mL to about 0.2 mg/mL, from about 0.25 mg/mL to about 0.95 mg/mL, from about 0.25 mg/mL to about 0.85 mg/mL, from about 0 .25 mg/mL to about 0.75 mg/mL, about 0.25 mg/mL to about 0.65 mg/mL, about 0.25 mg/mL to about 0.55 mg/mL, from about 0.25 mg/mL to about 0.45 mg/mL, from about 0.25 mg/mL to about o 0.35 mg/ml, from about 0.5 mg/ml to about 1.0 mg/ml, from about 0.5 mg/ml to about 0.9 mg/ml, from about 0.5 mg/ml to about 0.8 mg/ml, from about 0.5 mg/ml to about 0.75 mg/ml, from about 0.5 mg/ml to about 0.7 mg/ml, from about 0.5 mg/ml ml to about 0.65 mg/ml, from about 0.5 mg/ml to about 0.6 mg/ml, from about 0.75 mg/ml to about 1.0 mg/ml, from about 0.75 mg /ml to about 0.95 mg/ml, from about 0.75 mg/ml to about 0.9 mg/ml, and from about 0.75 mg/ml to about 0.85 mg/ml. In a preferred embodiment, the immunogenic composition contains an adjuvant at a concentration of from about 0.25 mg/ml to about 0.75 mg/ml, and most preferably about 0.5 mg/ml.

In a preferred embodiment, the adjuvant is an aluminum based adjuvant at a concentration of approximately 0.5 mg/mL. In one such embodiment, the aluminum-based adjuvant is aluminum phosphate or aluminum hydroxyphosphate. In another embodiment, the aluminum-based adjuvant is aluminum hydroxide.

It should be noted that immunogenic compositions containing an aluminum-based adjuvant tend to phase separate over time; aluminum particles are separated from the liquid, precipitating during storage. To ensure successful administration, the immunogenic composition must be visually uniform and resuspended prior to administration. Accordingly, it is important that the composition can be easily resuspended to a uniform state. In one aspect of the invention, the immunogenic composition is resuspended using about 50 shakes or less, for example, about 40 shakes or less, about 30 shakes or less, about 25 shakes or less, about 20 shakes or less, about 15 shakes or less, about 10 or less shakes. less shakes, or approximately 5 shakes or less. In a preferred embodiment, the immunogenic composition is resuspended using about 10 shakes or less. In another aspect of the invention, the immunogenic composition is resuspended using from about 1 to about 50 shakes, for example, from about 1 to about 40 shakes, from about 1 to about 30 shakes, from about 1 to about 25 shakes, from about 1 to about 20 shakes , about 1 to about 15 shakes, about 1 to about 10 shakes, or about 1 to about 5 shakes. In a preferred embodiment, the immunogenic composition is resuspended using from about 1 to about 10 shakes.

In one aspect of the invention, the immunogenic composition is placed in a container. In one embodiment, the container is selected from the group consisting of ampoule, vial, bag and syringe. In one embodiment, the container is made of glass, metal (eg, steel, stainless steel, and aluminum), and polymers (eg, thermoplastics, elastomers, thermoplastic elastomers). In one embodiment, the container is siliconized. In a preferred embodiment, the container is a syringe, and most preferably a glass syringe.

In one particular embodiment, the immunogenic composition is placed in a syringe with a headspace of about 1 mm to about 15 mm, e.g., about 1 mm to about 11 mm, about 1 mm to about 10 mm, about 1 mm up to about 9 mm, from about 1 mm to about 8 mm, from about 1 mm to about 7 mm, from about 1 mm to about 6 mm, from about 1 mm to about 5 mm, from about 1 mm to about 4 mm, from about 1 mm to about 3 mm, from about 2 mm to about 15 mm, from about 2 mm to about 11 mm, from about 2 mm to about 10 mm, from about 2 mm to about 9 mm, from about 2 mm to about 8 mm, from about 2 mm to about 7 mm, from about 2 mm to about 6 mm, from about 2 mm to about 5 mm, from about 2 mm to about 4 mm, from about 3 mm to about 15 mm, from about 3 mm to about 11 mm, from about 3 mm to about 10 mm, from about 3 mm to about 9 mm, from about 3 mm to about 8 mm, from about 3 mm to about 7 mm, from about 3 mm to about 6 mm, from about 3 mm to about 5 mm, from about 4 mm to about 15 mm, from about 4 mm to about 11 mm, from approximately 4 mm to approximately 10 mm, approximately 4 mm to approximately 9 mm, approximately 4 mm to approximately 8 mm, approximately 4 mm to approximately 7 mm, approximately 4 mm to approximately 6 mm, approximately 5 mm to approximately 15 mm, about 5 mm to about 11 mm, about 5 mm to about 10 mm, about 5 mm to about 9 mm, about 5 mm to about 8 mm, or about t approximately 5 mm to approximately 7 mm. In a preferred embodiment, the headspace above the product is from about 2 mm to about 6 mm, and most preferably about 4 mm.

In one embodiment, the immunogenic composition comprises a polysaccharide-protein conjugate as described herein, a buffer, a surfactant, an excipient, and optionally an adjuvant, wherein the pH of the composition buffer is from about 6.0 to about 7.5.

In one such embodiment, the immunogenic composition comprises a GBS polysaccharide-protein conjugate, a buffer, a surfactant, an excipient, and optionally an adjuvant, wherein the pH of the composition buffer is from about 6.0 to about 7.5, and wherein the capsular polysaccharide has a sialic acid level. acid more than about 60%.

In one embodiment, the immunogenic composition comprises a GBS polysaccharide-protein conjugate, histidine, polysorbate-80, sodium chloride, and optionally aluminum phosphate, wherein the pH of the composition buffer is from about 6.0 to about 7.0.

In one specific embodiment, the immunogenic composition comprises a GBS polysaccharide-protein conjugate, histidine, polysorbate-80, sodium chloride, and optionally aluminum phosphate, wherein the pH of the composition buffer is from about 6.0 to about 7.0, and the capsular the polysaccharide has a sialic acid level greater than about 60%.

In a preferred embodiment, the immunogenic composition contains from about 5 μg/ml to about 50 μg/ml of GBS polysaccharide-protein conjugate, from about 10 mM to about 25 mM histidine, from about 0.01% to about 0.03% (v / mass) polysorbate-80, from about 10 mm to about 250 mm sodium chloride and, optionally, from about 0.25 mg/ml to about 0.75 mg/ml of aluminum in the form of aluminum phosphate, and the capsular polysaccharide has a sialic acid level of more approximately 60%.

In another embodiment, the immunogenic composition comprises a GBS polysaccharide-protein conjugate, phosphate, polysorbate-80, sodium chloride, and aluminum phosphate, wherein the pH of the composition buffer is from about 6.0 to about 7.5.

In a further embodiment, the immunogenic composition comprises a GBS polysaccharide-protein conjugate, phosphate, polysorbate-80, sodium chloride, and aluminum phosphate, wherein the pH of the composition buffer is from about 6.0 to about 7.5, and wherein the capsular polysaccharide has a sialic acid level. acid more than about 60%.

In a preferred embodiment, the immunogenic composition contains from about 5 µg/ml to about 50 µg/ml GBS polysaccharide-protein conjugate, from about 10 mM to about 25 mM phosphate, from about 0.01% to about 0.03% (vol. /wt.) polysorbate-80, from about 10 mm to about 350 mm sodium chloride and from about 0.25 mg/ml to about 0.75 mg/ml aluminum in the form of aluminum phosphate, and the capsular polysaccharide has a level of sialic acid of more than about 60%.

In one such embodiment, the immunogenic composition comprises at least two GBS polysaccharide-protein conjugates, a buffer, a surfactant, an excipient, and optionally an adjuvant, wherein the pH of the composition buffer is from about 6.0 to about 7.5, and wherein the conjugates contain capsular polysaccharides from group B streptococcus (GBS) serotype IV and at least one additional serotype selected from the group consisting of Ia, Ib, II, III, V, VI, VII, VIII and IX.

In one specific embodiment, the immunogenic composition comprises at least two GBS polysaccharide-protein conjugates, histidine, polysorbate-80, sodium chloride, and optionally aluminum phosphate, wherein the pH of the composition buffer is from about 6.0 to about 7.0, and wherein the conjugates comprise capsular polysaccharides from Group B Streptococcus (GBS) serotype IV and at least one additional serotype selected from the group consisting of Ia, Ib, II, III, V, VI, VII, VIII and IX.

In a preferred embodiment, the immunogenic composition contains from about 5 μg/ml to about 50 μg/ml of each of at least two GBS polysaccharide-protein conjugates, from about 10 mM to about 25 mM histidine, from about 0.01% to about 0 03% (v/w) polysorbate-80, about 10 mM to about 250 mM sodium chloride, and optionally about 0.25 mg/ml to about 0.75 mg/ml aluminum as aluminum phosphate, moreover, the conjugates contain capsular polysaccharides from group B streptococcus (GBS) serotype IV and at least one additional serotype selected from the group consisting of Ia, Ib, II, III, V, VI, VII, VIII and IX.

In another specific embodiment, the immunogenic composition comprises at least two GBS polysaccharide-protein conjugates, phosphate, polysorbate-80, sodium chloride, and aluminum phosphate, wherein the pH of the composition buffer is from about 6.0 to about 7.5, and the conjugates contain capsular polysaccharides from group B streptococcus (GBS) serotype IV and at least one additional serotype selected from the group consisting of Ia, Ib, II, III, V, VI, VII, VIII and IX.

In one preferred embodiment, the immunogenic composition contains from about 5 μg/ml to about 50 μg/ml of each of at least two GBS polysaccharide-protein conjugates, from about 10 mM to about 25 mM phosphate, from about 0.01% to about 0.03% (v/w) polysorbate-80, about 10 mM to about 350 mM sodium chloride, and about 0.25 mg/mL to about 0.75 mg/mL aluminum as aluminum phosphate, with the conjugates contain capsular polysaccharides from group B streptococcus (GBS) serotype IV and at least one additional serotype selected from the group consisting of Ia, Ib, II, III, V, VI, VII, VIII and IX.

Evaluation of Immunogenic Compositions

Various in vitro tests are used to evaluate the immunogenicity of the immunogenic compositions of the invention. For example, in vitro analysis of opsonic activity is carried out by co-incubation of a mixture of streptococcal cells, heat inactivated serum containing specific antibodies to antigens of interest, and complement from an exogenous source. Onsonophagocytosis occurs during the incubation of freshly isolated polymorphonuclear cells (PMN) or differentiated effector cells such as HL60 and an antibody/complement/streptococcal mixture. Bacterial cells that are coated with antibody and complement are destroyed by opsonophagocytosis. The number of colony-forming units (CFU) of bacteria that survived opsonophagocytosis is determined by seeding the analytical mixture. Titers are defined as the reciprocal of the highest dilution at which 50% bacteria are killed, as determined by comparison with analytical controls.

For in vitro assessment of immunogenicity and antigen surface exposure, a cellular ELISA assay can also be used, in which cells of a bacterial strain of interest ( S. agalactiae ) are introduced into the wells of a plate, such as a 96-well plate, and the test serum from the immunized animal reacts with the bacterial cells. If an antibody specific for the antigen under test reacts with a surface-exposed epitope of the antigen, this can be detected by standard methods known to one of skill in the art. Alternatively, flow cytometry can be used to measure the surface exposure of capsular polysaccharide antigens and the specificity of antibodies, including monoclonal antibodies.

An antigen showing the desired in vitro activity can then be tested in an in vivo animal challenge model. In specific embodiments, the immunogenic compositions are used to immunize an animal (e.g., a mouse) using immunization methods and routes known to those skilled in the art (e.g., intranasal, parenteral, oral, rectal, vaginal, transdermal, intraperitoneal, intravenous, subcutaneous, etc.). ). After immunizing the animal with the GBS immunogenic composition, the animal is injected with Streptococcus agalactiae cells and tested for resistance to streptococcal infection.

In one embodiment, pathogen-free mice are immunized and challenged with S. agalactiae . For example, mice are immunized with one or more doses of the desired antigen in an immunogenic composition. Mice are then challenged with S. agalactiae and survival is monitored over time after challenge.

Application methods

Used in the present description, the term "having immune deficiency" refers to a patient suffering from insufficiency of the cellular and/or humoral link(s) of the immune system. Accordingly, imply a degree of deficiency of immune function, ranging from mild impairment of immune processes to complete immunosuppression.

The term "patient" refers to a mammal, bird, fish, reptile, or any other animal. The term "patient" also covers people. The term "patient" also covers pets. Non-limiting examples of pets include: dogs, cats, pigs, rabbits, rats, mice, gerbils, hamsters, guinea pigs, ferrets, birds, snakes, lizards, fish, turtles, and frogs. The term "patient" also includes pets. Non-limiting examples of domestic animals include: alpacas, bison, camels, cattle, deer, pigs, horses, llamas, mules, donkeys, sheep, goats, rabbits, reindeer, yaks, chickens, geese, and turkeys.

As used herein, the term "treatment" (including variations thereof, such as "treat" or "treated") refers to any one or more of the following: (i) prevention of infection or re-infection, as in the case of a conventional vaccine, (ii) reduction in severity or elimination of symptoms; and (iii) substantial or complete elimination of a particular pathogen or disease. Thus, treatment can be applied prophylactically (before infection) or therapeutically (after infection). In accordance with the present invention, prophylactic or therapeutic treatment can be used. In accordance with a particular embodiment of the present invention, compositions and methods are provided that treat, including through prophylactic and/or therapeutic immunization, a host animal against an infection caused by a microbe (eg, a bacterium such as S. agalactiae ). The methods of the present invention are useful for prophylactically and/or therapeutically inducing immunity in a patient. The methods of the present invention can also be applied to patients for the purpose of biomedical research.

In another aspect, the invention relates to a method of inducing an immune response against GBS in a patient by administering to the patient an effective amount of the immunogenic composition described herein. In one embodiment, the invention relates to a method of preventing, or alleviating, a disease or condition associated with group B streptococcus in a patient by administering to the patient an effective amount of the immunogenic composition described herein. In one aspect, the invention provides an immunogenic composition as described herein for use as a medicament. In one aspect, the invention provides an immunogenic composition as described herein for use in a method for inducing an immune response against GBS in a patient. In a specific embodiment, the patient is a woman planning a pregnancy or a pregnant woman. In one such embodiment, the pregnant woman is in the second half of her pregnancy, for example, at least 20 weeks pregnant or at least 27 weeks pregnant. In a preferred embodiment, the pregnant woman is between 27 weeks and 36 weeks pregnant. In another embodiment, the patient is an elderly person, such as 50 years of age or older, 65 years of age or older, and 85 years of age or older. In a further embodiment, the patient is immunocompromised. In one aspect, the patient may have a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease, or liver disease. In a preferred embodiment, group B streptococcus is S. agalactiae .

In one embodiment, the immunogenic composition comprises polysaccharide-protein conjugates comprising a GBS serotype IV polysaccharide and at least one additional serotype selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII, and IX. In another embodiment, the conjugates comprise a serotype IV GBS polysaccharide and at least two additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII, and IX. In a further embodiment, the conjugates comprise a serotype IV GBS polysaccharide and at least three additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII, and IX. In another embodiment, the conjugates comprise a serotype IV GBS polysaccharide and at least four additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII, and IX. In a particular embodiment, the conjugates comprise GBS serotypes Ia, Ib, II, III and IV polysaccharides. In a further embodiment, the conjugates comprise a GBS serotype IV polysaccharide and at least five additional serotypes selected from the group consisting of serotypes Ia, Ib, II, III, V, VI, VII, VIII, and IX. In a further embodiment, the composition comprises a GBS serotype V polysaccharide. In a specific embodiment, the conjugates comprise GBS serotypes Ia, Ib, II, III, and V polysaccharides. In a preferred embodiment, the immunogenic composition comprises six polysaccharide-protein conjugates from GBS serotypes Ia, Ib, II , III, IV and V. One aspect of the invention relates to an immunogenic composition in which there is no immune interference.

An immunogenic, or effective, amount of an immunogenic composition can be determined in a dose-response study in which patients are immunized with incremental amounts of the immunogenic composition and the immune response is analyzed to determine the optimal dose. Initial doses in the study may be determined based on immunization data in animal models. The amount of dose may vary depending on the specific conditions for the individual. The amount can be determined in conventional tests by methods known to those skilled in the art.

An immunologically effective amount of the immunogenic composition in an appropriate number of doses is administered to a patient to initiate an immune response. The amount of dose may vary depending on the specific conditions for the individual, such as age and body weight. This amount can be determined in conventional tests by methods known to those skilled in the art.

In one embodiment, patients receiving the immunogenic compositions of the invention experience a decrease in S. agalactiae carriage. Such a reduction in carriage, or a longer period of time without carriage after administration of the immunogenic composition, is of medical importance. For example, the reduction in the overall degree of carriage of S. agalactiae in carriers can be assessed after administration of a single dose of an immunogenic composition of the invention. For example, 1 day prior to administration of the immunogenic composition, a group of adults aged 18-50 years can be screened for carriage by obtaining nasal, throat, axillary, rectal, perineal, and vaginal swabs, followed by culture to determine their carrier status. A group of people can then be administered the immunogenic composition of the invention, with the other group receiving control. Nasal, throat, axillary, rectal, perineal, and vaginal swabs are collected weekly for a 12-week period, and monthly up to 6 months after administration of the immunogenic composition, and compared with placebo results. One main measure of effectiveness is the results of comparing the levels of carriage in patients after administration of the immunogenic composition and placebo at 3-month intervals after immunization.

Antibodies

An "antibody" is an immunoglobulin molecule capable of specifically binding to a target such as a carbohydrate, polynucleotide, lipid, polypeptide, and so on, through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, unless the context dictates otherwise, the term should include not only intact polyclonal or monoclonal antibodies, but also recombinant antibodies (e.g., chimeric, humanized, and/or derivatized to alter effector functions, stability, and other biological activities) and antibody fragments (such as Fab, Fab', F(ab') ₂ , Fv), single chain (ScFv) and domain antibodies, including shark and camelid antibodies, as well as antibody fragment containing fusion proteins, multivalent antibodies, multispecific antibodies (eg, bispecific antibodies, provided they have the desired biological activity), and antibody fragments described herein, as well as any other modified configuration of the immunoglobulin molecule that contains an antigen recognition site. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or a subclass thereof), and the antibody need not be of any particular class. Depending on the amino acid sequence of the constant domain of the heavy chains of antibodies, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 in humans. The heavy chain constant domains that define the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structure and three-dimensional configurations of immunoglobulins of different classes are well known.

"Antibody fragments" comprise only a portion of an intact antibody, the portion preferably retaining at least one, preferably most or all, of the functions normally associated with the portion when present in an intact antibody.

As used herein, the terms "functional activity" of an antibody or "functional antibody" refer to an antibody that, at a minimum, is capable of specifically binding an antigen. Additional functions are known in the art and may include additional components of the immune system that effect clearance or destruction of the pathogen, such as through opsonization, ADCC, or complement-mediated cytotoxicity. After antigen binding, any subsequent functions of the antibody may be mediated by the Fc region of the antibody. The Antibody-Mediated Opsonophagocytosis Assay (OPA) is an in vitro assay designed to measure in vitro Ig- and complement-mediated bacterial killing by effector cells (white blood cells), thus mimicking the biological process. The binding of an antibody can also directly inhibit the biological function of the antigen it binds. In some embodiments, the term "functional antibody" means an antibody that is functional in a measure of bacterial killing in an animal model of efficacy or in an opsonophagocytic killing assay indicating that the antibodies kill bacteria.

In one aspect, the invention provides an isolated antibody, or fragment thereof, that specifically binds the polysaccharide described herein. As used herein, the term "isolated" antibody means an antibody that has been identified and separated and/or recovered from components of its natural environment. Impurity components of its natural environment are materials that would interfere with the diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In illustrative embodiments, the antibody will be purified (1) to greater than 95% by weight of the antibody as determined by the Lowry method, and most preferably greater than 99% by weight, (2) to a degree sufficient to detect at least 15 N- terminal or internal amino acid sequence using a rotating beaker sequencer, or (3) homogenous as determined by SDS-PAGE under reducing and non-reducing conditions by staining with Coomassie blue or preferably silver. An isolated antibody includes the antibody in situ in recombinant cells, since at least one component of the antibody's natural environment will not be present. Typically, however, an isolated antibody is obtained using at least one purification step.

An antibody that "specifically binds" or is "specific for" a particular polysaccharide or epitope on a particular polysaccharide is an antibody that binds a particular polysaccharide or epitope on a particular polysaccharide without substantially binding to any other polysaccharide or epitope of the polysaccharide.

As used herein, the term “label” means a detectable compound or composition that is directly or indirectly conjugated to an antibody to produce a “labeled” antibody. The label may be itself detectable (eg, radioactive isotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze a chemical change in the substrate compound or composition that can be detected.

The invention also relates to antibodies and antibody compositions that specifically and selectively bind one or more antigens of the immunogenic composition of the present invention. In some embodiments, antibodies are obtained after administration of an immunogenic composition of the present invention to a patient. In some embodiments, the invention provides purified or isolated antibodies directed against one or more antigens of the immunogenic composition of the present invention. In some embodiments, the antibodies of the present invention are functional in measuring bacterial kill in either an animal efficacy model or an opsonophagocytic kill assay. In some embodiments, the antibodies of the invention confer passive immunity in a patient. In addition, the present invention relates to polynucleotide molecules encoding an antibody or antibody fragment of the invention, as well as to a cell or cell line (for example, hybridoma cells or other genetically modified cell lines for recombinant production of antibodies) and a transgenic animal producing an antibody or antibody composition. according to the invention using methods well known to those skilled in the art.

The antibodies or antibody compositions of the invention can be used in a method for treating or preventing a streptococcal infection, disease or condition associated with S. agalactiae in a patient, comprising obtaining a polyclonal or monoclonal antibody preparation and using said antibodies or antibody composition to confer passive immunity in the patient . The antibodies of the invention may also be useful in diagnostic methods, for example, to detect the presence or quantify the levels of one or more antigens of an immunogenic composition of the invention.

The production of antibodies to repeat structures, such as the polysaccharide of the present invention, may have some unique features. For example, the regularity of repeating units may mean that antigen molecules that differ greatly in molecular weight can bind to antibodies specific for the polysaccharide. Second, the repeating structures of longer polysaccharides are able to induce T-cell independent antibody production. Therefore, by using polysaccharides conjugated to protein carriers having epitopes for T helpers, both T cell independent and T cell dependent antibody production can be stimulated. Thus, the immune response can be modified by appropriate selection of the size of the polysaccharide, as well as by whether or not a carrier protein is used.

Polyclonal antibodies

In specific embodiments, the anti-polysaccharide antibody is a polyclonal antibody. As used herein, the term "polyclonal antibodies" means a mixture of antibodies with different specificities derived from a serum preparation and derived from different B-cell clones. Methods for obtaining and characterizing polyclonal antibodies are known in the art.

Polyclonal antibodies are generated in a patient, eg a mammal, upon administration of one or more injections of an immunogen or immunogenic composition as described herein and, if necessary, an adjuvant, buffer and/or diluent. Animals of different species can be used to obtain a specific antiserum. Typically, the animal used to prepare the antisaccharide polyclonal antiserum is a non-human primate, goat, sheep, rabbit, mouse, rat, hamster, or guinea pig. Typically, the immunogen or immunogenic composition, with or without an adjuvant, is administered to a mammal in multiple injections. The immunogenic material may include a polysaccharide, an oligosaccharide, a polysaccharide, a polysaccharide-protein conjugate as described herein, or a plurality of immunogens. Typically, starting 2-6 weeks after the first immunization, blood is taken from the immunized animal, allowed to clot, and serum is collected. The serum contains polyclonal antibodies against the saccharide from the immunized animal and is often referred to as an antiserum.

Monoclonal antibodies

An anti-saccharide monoclonal antibody can be prepared by the known hybridoma method. Typically, the production of monoclonal antibodies involves first immunizing the appropriate target animal host with a selected immunogen comprising the polysaccharide, oligosaccharide, polysaccharide, or polysaccharide-protein conjugate of the present invention. If necessary, an adjuvant, buffer and/or diluents may be included. Immunization is performed in such a way as to stimulate B-lymphocytes to produce or express antibodies that specifically bind the polysaccharide or its conjugate. Alternatively, lymphocytes are immunized in vitro .

The lymphocytes are then fused with the immortalized lineage cells using a suitable fusion agent such as polyethylene glycol to obtain a hybridoma cell. The source of lymphocytes determines whether the monoclonal antibodies will be of human or animal origin. Generally, peripheral blood lymphocytes ("PBL") are used when antibodies and human cells are needed, and spleen or lymph node cells are used when non-human mammals are needed as a source.

Immortalized lineage cells are generally transformed mammalian cells, in particular rodent, bovine and human myeloma cells. Typically, rat or mouse myeloma cell lines are used. The hybridoma cells are cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of non-fused immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), hybridoma culture media will typically contain hypoxanthine, aminopterine, and thymidine ("HAT media"), substances that prevent the growth of HGPRT-deficient cells.

Immortalized lineage cells are selected based on practical considerations such as original species, fusion and growth characteristics. For example, suitable immortalized lineage cells are those that efficiently participate in fusion, maintain stable high levels of antibody expression by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Examples of immortalized cell lines include mouse myeloma cell lines. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies.

The monoclonal antibody is secreted into the culture medium by hybridoma cells. The culture medium is then analyzed for the presence of monoclonal antibodies that recognize and bind the polysaccharide. The polysaccharide binding specificity of particular monoclonal antibodies produced by hybridoma cells is determined by one of the many methods that are well known in the art. For example, antibody binding specificity can be determined by immunoprecipitation, radioimmunoassay (RIA), Western blot, ELISA, or surface plasmon resonance (eg, Biacore). The specific epitope recognized by a monoclonal antibody is determined by epitope mapping. Such methods and assays are well known in the art.

After identification of hybridoma cells producing antibodies with the desired specificity, the clones are subcloned by serial dilution and cultured by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, hybridoma cells grow in vivo as ascites in a mammal. The monoclonal antibodies secreted by the subclones are isolated or purified from the culture medium or ascitic fluid by conventional immunoglobulin purification methods such as, for example, protein A sepharose, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography.

Alternatively, antibodies having the desired specificity and obtained from animals of the desired species can be obtained using phage display libraries. In addition, examples of methods and reagents particularly suitable for generating and screening an antibody display library can be found in the relevant literature.

Application of antibodies

In one aspect, the invention relates to the use of the immunogenic composition described herein for the preparation of an anti-GBS antibody and/or antibody fragment. The polysaccharide-protein conjugates described herein and/or antibodies generated using them may find use in a variety of immunological diagnostic methods known to those skilled in the art, including ELISA and microarray technologies. In addition, these reagents can be used to assess antibody production, including serum levels of antibodies against, for example, immunogenic polysaccharide conjugates. The analytical methods of the invention may include the use of labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules and/or secondary immunological reagents to directly or indirectly detect a complex between an antigen or antibody in a biological sample and the corresponding antibody or antigen. associated with the solid substrate.

The resulting antibody, or antibody fragment, may also be useful for passive immunotherapy or for the prevention of streptococcal infection.

Method for obtaining a polysaccharide

In another aspect, the invention relates to a method for producing polysaccharides described in the present description. The method includes culturing GBS and harvesting polysaccharides produced by the bacterium. In one embodiment, the GBS includes S. agalactiae . The bacterium may refer to any strain of S. agalactiae . In a preferred embodiment, the bacterium is an encapsulated S. agalactiae bacterium strain. S. agalactiae strains for use in the present invention include 090, A909 (ATCC Registry BAA-1138), 515 (ATCC Registry BAA-1177), B523, CJB524, MB 4052 (ATCC Registry 31574), H36B (ATCC Registry 31574), 12401), S40, S42, MB 4053 (ATCC reg. no. 31575), M709, 133, 7357, PFEGBST0267, MB 4055 (ATCC reg. no. 31576), 18RS21 (ATCC reg. ATCC 12973), DK21, DK23, UAB, 5401, PFEGBST0708, MB 4082 (ATCC P/N 31577), M132, 110, M781 (ATCC P/N BAA-22), D136C(3) (ATCC P/N 12403), M782 , S23, 120, MB 4316 (M-732; ATCC P/N 31475), M132, K79, COH1 (ATCC P/N BAA-1176), PFEGBST0563, 3139 (ATCC P/N 49446), CZ-NI-016, PFEGBST0961, 1169-NT1, CJB111 (ATCC P/N BAA-23), CJB112, 2603 V/R (ATCC P/N BAA-611), NCTC 10/81, CJ11, PFEGBST0837, 118754, 114852, 114862, 114866, 118775, B 4589 , B4645, SS 1214, CZ-PW-119, 7271, CZ-PW-045, JM9130013, JM9130672, IT-NI-016, IT-PW-62 and IT-PW-64.

The polysaccharide described herein can be obtained by culturing GBS in an appropriate medium. An appropriate medium may include Colombian broth. The medium may include dextrose, hemin and/or glucose. Preferably, the medium includes Colombian broth and dextrose. If S. agalactiae is cultured using Columbia broth and dextrose, preferably the culture temperature is 20-40°C, preferably 37°C. In a preferred embodiment, the bacterium is cultured under aerobic conditions. In another preferred embodiment, the bacterium is cultured for 12-60 hours.

The polysaccharide can be harvested from the resulting culture by methods known in the art to harvest the target substance from the culture, such as, for example, heating, enzymatic treatment, centrifugation, precipitation, activated carbon treatment, and/or filtration. (See, for example, US Patent Application Publication Nos. 2006/0228380, 2006/0228381, 2007/0184071, 2007/0184072, 2007/0231340 and 2008/0102498; International Patent Application Publication No. WO 2008/118752). In one embodiment, a culture containing a bacterium and a polysaccharide is centrifuged and treated with an enzyme such as, for example, lysozyme, RNase, DNase, pronase, mutanolysin, or a combination thereof. For example, in one embodiment, an appropriate organic solvent is added to the resulting supernatant to precipitate proteins, and the precipitate is removed by centrifugation. The polysaccharide can then be precipitated by adding an appropriate organic solvent to the supernatant, and the polysaccharide can be collected by centrifugation. More specifically, the polysaccharide described herein can be obtained by adding ethanol at a final concentration of approximately 25% by volume to the supernatant from which bacteria have been removed, removing the pellet containing the protein, centrifuging, then adding ethanol to the supernatant to a final concentration of approximately 75% by volume. %, followed by collection of sediment by centrifugation. The resulting precipitate can be dried with nitrogen. The resulting pellet can be resuspended in Tris with 0.05% Na azide.

A further aspect of the invention relates to a novel method for isolating substantially intact high molecular weight CPs retaining N- and O-acetyl groups using organic reagents such as derivatized hydroxylamine compounds. Since this method does not lead to cell lysis, CP isolated by centrifugation have minimal impurities of intracellular components and their overall yield can be higher. In addition, these reagents cleave the Group B antigen contaminant to very small fragments due to multiple phosphodiester linkages, and the fragments can be easily removed by diafiltration.

In one embodiment, CP is isolated by reacting hydroxylamine with a cell paste containing a capsular polysaccharide producing bacterium. In a particular embodiment, the method further includes a centrifugation step. In another embodiment, the method further includes a filtering step. In another embodiment, the capsular polysaccharide producing bacterium is selected from the group consisting of Streptococcus agalactiae , Streptococcus pneumoniae , Staphylococcus aureus , Neisseria meningitidis, Escherichia coli, Salmonella typhi, Haemophilus influenzae, Klebsiella pneumoniae, Enterococcus faecium, and Enterococcus faecalis.

In one aspect of the invention, the hydroxylamine may be one of those listed in Table 2 of Example 2. In a preferred embodiment, the hydroxylamine is selected from the group consisting of dibenzylhydroxylamine; diethylhydroxylamine; hydroxylamine; ethylenediamine; triethylenetetramine; 1,1,4,7,10,10-hexamethyltriethylenetetramine and 2,6,10-trimethyl-2,6,10-triazoundecane.

In one aspect of the invention, the concentration of hydroxylamine is from about 5 mM to about 200 mM, for example, from about 5 mM to about 150 mM, from about 5 mM to about 100 mM, from about 5 mM to about 75 mM, from about 5 mM to about 50 mM, from about 5 mM to about 25 mM, from about 5 mM to about 10 mM, from about 10 mM to about 200 mM, for example, from about 10 mM to about 150 mM, from about 10 mM to about 100 mM , from about 10 mM to about 75 mM, from about 10 mM to about 50 mM, from about 10 mM to about 25 mM, from about 25 mM to about 200 mM, from about 25 mM to about 150 mM, from about 25 mM up to about 100 mM, from about 25 mM to about 75 mM, from about 25 mM to about 50 mM, from about 50 mM to about 50 mM and about 200 mM, about 50 mM to about 150 mM, about 50 mM to about 100 mM, and about 50 mM to about 75 mM.

In another aspect, the pH of the reaction mixture is maintained at about 5.5 to about 9.5, for example, from about 5.5 to about 9.0, from about 5.5 to about 8.5, from about 5.5 to about 8.0, from about 5.5 to about 7.5, from about 5.5 to about 7.0, from about 5.5 to about 6.5, from about 6.0 to about 9.5, about 6.0 to about 9.0, about 6.0 to about 8.5, about 6.0 to about 8.0, about 6.0 to about 7.5, about 6.0 to about about 7.0, about 6.5 to about 9.5, about 6.5 to about 8.5, about 6.5 to about 8.0, about 6.5 to about 7.5, from about 7.0 to about 9.5, about 7.0 to about 9.0, about 7.0 to about 8.5, and about 7 .0 to about 8.0.

In a further aspect of the invention, the reaction proceeds at a temperature of from about 20°C to about 85°C, for example, from about 20°C to about 80°C, from about 20°C to about 75°C, from about 20°C to about 70°C, about 20°C to about 65°C, about 20°C to about 60°C, about 20°C to about 55°C, about 20°C to about 50°C, from about 25°C to approximately 85°C, approximately 25°C to approximately 80°C, approximately 25°C to approximately 75°C, approximately 25°C to approximately 70°C, approximately 25°C to approximately 65 °C, approximately 25°C to approximately 60°C, approximately 25°C to approximately 55°C, approximately 25°C to approximately 50°C, approximately 30°C to approximately 85°C, approximately 30 °C to approx. 80°C, approx. 30°C to approx. 75°C, approx. 30°C to approx. 70°C, approximately 30°C to approximately 65°C, approximately 30°C to approximately 60°C, approximately 30°C to approximately 55°C, approximately 30°C to approximately 50°C, from approximately 35°C to approximately 85°C, approximately 35°C to approximately 80°C, approximately 35°C to approximately 75°C, approximately 35°C to approximately 70°C, approximately 35°C to approximately 65°C, approximately 35°C to approximately 60°C, approximately 35°C to approximately 55°C, approximately 40°C to approximately 85°C, approximately 40°C to approximately 80°C, approximately 40°C to approximately 75°C, approximately 40°C to approximately 70°C, approximately 40°C to approximately 65°C, approximately 40°C to approximately 60°C, approximately 45°C to approximately 85 °C, approximately 45°C to approximately 80°C, approximately 45°C to approximately 75°C, approximately 45°C to approximately 70° C, approximately 45°C to approximately 65°C, approximately 50°C to approximately 85°C, approximately 50°C to approximately 80°C, approximately 50°C to approximately 75°C, approximately 50°C C to approximately 70°C, approximately 55°C to approximately 85°C, approximately 55°C to approximately 80°C, approximately 55°C to approximately 75°C, approximately 60°C to approximately 85°C and from about 65°C to about 85°C.

In another aspect, the reaction time is from about 10 hours to about 90 hours, for example, from about 10 hours to about 85 hours, from about 10 hours to about 80 hours, from about 10 hours to about 75 hours, from about 10 hours to about 70 hours, from about 10 hours to about 60 hours, from about 10 hours to about 50 hours, from about 10 hours to about 40 hours, from about 10 hours to about 30 hours, from about 10 hours to about 25 hours, from about 10 hours to about 20 hours, about 10 hours to about 15 hours, from about 15 hours to about 90 hours, from about 15 hours to about 85 hours, from about 15 hours to about 80 hours, from about 15 hours to about 75 hours hours, from about 15 hours to about 70 hours, about t about 15 hours to about 60 hours, from about 15 hours to about 50 hours, from about 15 hours to about 40 hours, from about 15 hours to about 30 hours, from about 15 hours to about 20 hours, for example, from about 20 hours to about 90 hours, from about 20 hours to about 85 hours, from about 20 hours to about 80 hours, from about 20 hours to about 75 hours, from about 20 hours to about 70 hours, from about 20 hours to about 60 hours , from about 20 hours to about 50 hours, from about 20 hours to about 40 hours, from about 20 hours to about 30 hours, and from about 20 hours to about 25 hours.

Alternatively, in another embodiment of the invention, the polysaccharide is synthesized by chemical means. The polysaccharide can be synthesized by conventional chemical methods.

In another embodiment, the polysaccharide is produced by expression in a surrogate host after cloning and expression of the biosynthetic pathway to produce the polysaccharide. For example, a host cell can be modified to produce a polysaccharide having structural similarity to the polysaccharide described herein, wherein the polysaccharide repeat unit produced in the host cell is partially identical to the polysaccharide repeat unit described herein. A polysaccharide has structural similarity to a polysaccharide described herein if, for example, the polysaccharide repeating unit is devoid of a branch, differs in size, and/or differs in branch arrangement from the polysaccharide repeat unit described herein. Preferably, the host cell is a bacterial host cell.

EXAMPLES

The following examples illustrate some embodiments of the present invention. However, it should be understood that these examples are for illustrative purposes only and do not purport to be definitive as to the terms and scope of the present invention. It should be understood that where typical reaction conditions (eg, temperature, reaction time, etc.) are given, conditions outside or below the ranges indicated may also be used, although less conveniently. All parts and percentages given in this specification are by weight and all temperatures are in degrees Celsius unless otherwise noted.

In addition, in the following examples, standard methods have been used that are well known and routine to those skilled in the art, except where described in detail. As noted above, the following examples are provided for the purpose of illustration and should not be taken as limiting the scope of the present invention in any way.

Example 1 Preparation of Polysaccharide-Protein Conjugates with De-O-Acetylated Polysaccharides

The fermentation of S. agalactiae strains for the respective serotypes was carried out in a pH-controlled flooded culture in the indicated medium. The methods and media used have been optimized through experimentation and are modifications of the basic methods previously described by von Hunolstein, C. et al., Appl. micro. Biotech. 38(4):458-462 (1993). The capsular polysaccharide was removed from the cells by treatment with NaOH. After the steps of clarification, a series of UV/DF, precipitation and filtration through a carbon filter, the purified polysaccharide was obtained. See, for example, US Pat. No. 8,652,480. Reductive amination chemistry was used to conjugate the activated polysaccharide to CRM ₁₉₇ . See, for example, US Pat. No. 5,360,897.

Example 2 Isolation of O-acetylated polysaccharides

A paste of GBS cells with capsular polysaccharide (CP) serotype Ia, obtained after killing and centrifuging the fermentation broth (1.2 L), was resuspended in 175 ml of 25 mM potassium phosphate buffer (25 mM, pH 6.9). The suspension was mixed with an aqueous solution of hydroxylamine-O-sulfonic acid at a final concentration of 10 mM. The pH value of the suspension when determined was approximately 5.8. The suspension was stirred at 55°C for 72 hours. The suspension was then centrifuged at approximately 10,000 rpm and the supernatant was collected. The supernatant containing crude digested CP was analyzed for molecular weight and yield. The remainder was purified by diafiltration using a 30 kDa HOMM membrane using water for injection (WFI). An additional analysis of the molecular weight of the purified polysaccharide was carried out by size exclusion chromatography in combination with a detector of multi-angle laser light scattering (SEC-MALS) (Table 1).

Table 1 Purification of serotype Ia GBS polysaccharide by diafiltration

Sample MM (kDa) Polydispersity (PD) Raw 340 1.2 Purified polysaccharide 320 1.3

Several hydroxylamines, both nitrogen- and oxygen-substituted compounds, were screened for their activity using the method described above. Yields were calculated using gel permeation chromatography coupled with multi-angle laser light scattering (GPC-MALS) detection of raw supernatants using refractive index response (RI) and specific refractive index increment squared (dn/dc) of 0.135. The yield depended on the type of hydroxylamines and the optimization of conditions such as concentration, temperature and reaction time (see Table 2). As a rule, increasing the concentration of hydroxylamine, increasing the temperature and increasing the duration of the reaction led to a higher yield.

Table 2. Screening of various hydroxylamines and optimization of conditions for capsular polysaccharide GBS serotype Ia

The ex.
No. Reagent Conc. (mm) Pace. (°C) Time (hour) Yield/1l fermentation broth MM polysaccharide (kDa) Polydispersity (PD) 1

O-бензилгидроксиламин гидрохлорид

O-benzylhydroxylamine hydrochloride 10 55 72 42 2240 1.2 2

benzaldehyde oxime 10 55 72 68 1400 1.2 3

N,N-dibenzylhydroxylamine 10 55 72 101 980 1.3 4

N,N-dibenzylhydroxylamine 50 55 17 320 590 1.3 5

O-phenylhydroxylamine hydrochloride 10 55 72 109 850 1.3 6

4-(dimethylamino)benzaldehyde oxime 10 55 72 90 1400 1.2 7

benzylhydroxyl carbamate 10 55 84 323 1835 1.5 8

O-(tetrahydro-2H-pyran-2-yl)hydroxylamine 10 55 84 304 1270 1.4 9

O-((perfluorophenyl)methyl)hydroxylamine 10 55 84 185 1330 1.6 10

O-tritylhydroxylamine 10 55 84 371 1275 1.7 eleven

O-(4-nitrobenzyl)hydroxylamine hydrochloride 10 55 84 190 3500 1.3 12

2-(aminooxy)acetic acid hydrochloride 10 55 84 252 2600 1.2 13

(aminooxy)sulfonic acid 10 55 84 463 490 1.4 14

(aminooxy)sulfonic acid 50 55 17 460 270 1.2 15

(aminooxy)sulfonic acid 100 23 21 240 500 1.2 16

N,N-dioctadecylhydroxylamine 10 55 84 380 4700 1.7

The substituted and unsubstituted hydroxylamines have been found to be very effective in releasing the GBS capsular polysaccharide from the cell wall. This approach leads to the isolation of the high molecular weight polysaccharide GBS with preserved N- and O-acetyl groups. Of the several compounds screened, dibenzylhydroxylamine was found to be the most effective. The data are shown in Table 3 ([dibenzylhydroxylamine] -50 mm; pH - 7-8; temperature - 50°C; time - 24 hours).

Table 3. CP GBS release data using dibenzylhydroxylamine

GBS type Ia Ib III Output upon release of the polysaccharide 86% 81% 46% Total Cleaning Yield 63% 54% thirty% Molecular weight (MM) 330 kDa 212 kDa 171 kDa O-acetylation (NMR) on 31% 37% N-acetylation (NMR) 106% 104% 87%

n/a - serotype Ia polysaccharide is not O-acetylated.

Since dibenzylhydroxylamine is poorly soluble in water, an alternative hydroxylamine derivative is needed that is readily soluble in water and has a similar or higher activity than dibenzylhydroxylamine. After screening several compounds, diethylhydroxylamine was found to be a good alternative. The data are shown in Table 4 ([diethylhydroxylamine] -100 mm;, pH - 7-8; temperature - 60°C; time - 19 hours).

Table 4. CP GBS release data using diethylhydroxylamine

GBS type Ia Ib III Yield, %, upon release of the polysaccharide 100 94 59 Molecular weight (MM) 890 kDa 560 kDa 309 kDa

It has been found that hydroxylamine (NH ₂ -OH) is also effective in cleaving the CPS polysaccharide from the cell wall. The data are shown in Table 5 ([hydroxylamine] -100 mm; pH - 7-7.5; temperature - 65°C; time - 17 hours). For serotype III, the yield was 54% after 17 hours; however, the yield increased to 70% after three and a half days.

Table 5. CP GBS release data using hydroxylamine

GBS type Ia III Yield, %, upon release of the polysaccharide 100 54 Molecular weight (MM) 1160 kDa 500 kDa

Screening of oligoamines for release of GBS capsular polysaccharide from cells

Hydroxylamine and its substituted compounds have been found to be very effective in cleaving capsular polysaccharides from the GBS cell wall. However, they were found to be less effective for serotypes II and V. Thus, oligoamines were tested due to the suggestion that they may be more active due to the presence of multiple amino functional groups.

Ethylenediamine has been found to be effective in releasing capsular polysaccharides of all serotypes. The data are shown in Table 6 ([ethylenediamine] -50 or 100 mm; pH 8.0; 16 hours; 80°C; 25 mm EDTA).

Table 6: CP GBS release data using ethylenediamine

Serotype GBS Recovery (%) MM (kDa) Ia 96% 242 Ib 83% 225 II thirty% 76 III 68% 94 V thirty% 235

Other representative oligoamines were tested for their activity using a paste of serotypes Ia and V cells, but showed less efficacy against serotype V. The data are shown in Tables 7 ([triethylenetetramine] -100 mm; pH - 8.9; temperature - 60°C ; time - 15 hours), 8 ([1,1,4,7,10,10-hexamethyltriethylenetetramine] - 10 mm; pH - 6.3; temperature - 60°C; time - 20 hours) and 9 ([2 ,6,10-trimethyl-2,6,10-triazoundecane] -10 mM; pH - 7-8; temperature - 60°C; time - 19 hours).

Table 7. CP GBS release data using triethylenetetramine

GBS type Ia V Yield, %, upon release of the polysaccharide 100 ~10 in 2.5 days Molecular weight (MM) 1280 But

n/a - not defined.

Table 8. CP GBS release data using 1,1,4,7,10,10-hexamethyltriethylenetetramine

GBS type Ia V Yield, %, upon release of the polysaccharide 100 ~1% Molecular weight (MM) 980 But

n/a - not defined.

Table 9. CP GBS release data using 2,6,10-trimethyl-2,6,10-triazoundecane

GBS type Ia V Yield, %, upon release of the polysaccharide 100 ~1% Molecular weight (MM) 1100 But

n/a - not defined.

Example 3 Conjugation of GBS Capsular Polysaccharides by Reductive Amination

Polysaccharide activation

Polysaccharide oxidation was performed in 100 mM potassium phosphate buffer (pH 6.0 ± 0.5) by successively adding the calculated amount of 500 mM potassium phosphate buffer (pH 6.0) and water for injection (WFI) until the final concentration of polysaccharide 2 was reached. .0 g/l. If necessary, the pH of the reaction mixture was adjusted to approximately pH 6.0. After adjusting the pH, the temperature of the reaction mixture was brought to 23°C. The oxidation was started by adding approximately 0.25 molar equivalents of sodium periodate. The oxidation reaction was carried out at 5±3°C for about 16 hours.

Concentration and diafiltration of the activated polysaccharide was performed using ultrafiltration cassettes with 5K HOMM. Diafiltration was performed against 20 times the diavolume of WFI. The purified activated polysaccharide was then stored at 5±3°C. The purified activated saccharide was characterized, in particular, by determining (i) the concentration of saccharide by colorimetric method; (ii) aldehyde concentration by colorimetric method; (iii) oxidation states; and (iv) molecular weight by SEC-MALLS.

The oxidation state (CO=moles of sugar repeating unit/moles of aldehyde) of the activated polysaccharide was determined as follows.

Moles of sugar repeating unit were determined by various colorimetric methods, for example the anthrone method. In the anthrone method, the polysaccharide is first broken down into monosaccharides with sulfuric acid and heat. The anthrone reagent reacts with hexoses to form a yellow-green complex, the absorption of which is determined spectrophotometrically at 625 nm. Within the sensitivity of the method, the absorbance is directly proportional to the amount of hexose present.

At the same time, moles of aldehyde are also determined by a colorimetric method using MBTH. The MBTH assay involves the formation of an azine compound by reacting aldehyde groups (from a specific sample) with 3-methyl-2-benzothiazolonehydrazone (MBTH analytical reagent). Excess 3-methyl-2-benzothiazolonehydrazone is oxidized to form a reactive cation. The reactive cation and azine react to form a blue chromophore. The resulting chromophore is then determined spectrophotometrically at 650 nm.

Combination of activated polysaccharide with sucrose excipient and lyophilization

The activated polysaccharide was combined with sucrose at a ratio of 25 grams of sucrose per gram of activated polysaccharide. The mixture in the vial was then lyophilized. After lyophilization, the vials containing the lyophilized activated polysaccharide were stored at -20±5°C. The calculated amount of CRM ₁₉₇ protein was frozen in a container and lyophilized separately. Lyophilized CRM ₁₉₇ was stored at -20±5°C.

Recovery of lyophilized activated polysaccharide and carrier protein

The lyophilized activated polysaccharide was reconstituted in anhydrous dimethyl sulfoxide (DMSO). After complete dissolution of the polysaccharide, an equal amount of anhydrous DMSO was added to the lyophilized CRM ₁₉₇ for reconstitution.

Conjugation and capping

The reduced activated polysaccharide was combined with the reduced CRM ₁₉₇ in a reaction vessel, then thoroughly mixed to give a clear solution prior to conjugation using sodium cyanoborohydride. The final concentration of the polysaccharide in the reaction solution was approximately 1 g/L. Conjugation was started by adding 1.0-1.5 mEq of sodium cyanoborohydride to the reaction mixture and incubating at 23 ± 2°C for 20-48 hours. The conjugation reaction was stopped by adding 2 mEq of sodium borohydride (NaBH ₄ ) to cap the unreacted aldehydes. This capping reaction was continued at 23±2° C. for 3±1 hours.

Purification of the conjugate

The conjugate solution was diluted 1:10 with a chilled mixture of 5 mM succinate-0.9% saline (pH 6.0) in preparation for purification by downstream flow filtration using 100-300K HOMM membranes.

The diluted conjugate solution was passed through a 5 μm filter and diafiltered using a mixture of 5 mM succinate/0.9% saline (pH 6.0) as medium. After completion of diafiltration, the conjugate concentrate was passed through a 0.22 μm filter. The conjugate was further diluted with 5 mM succinate/0.9% saline (pH 6) to bring the saccharide concentration to approximately 0.5 mg/mL. Alternatively, the conjugate was purified using 20 mM histidine/0.9% saline (pH 6.5) by a downstream flow filtration method using 100-300K HOMM membranes. A final filtration step was performed through a 0.22 µm filter to obtain an immunogenic conjugate.

Example 4 Effect of Different Conjugation Conditions on GBS Polysaccharide-CRM Conjugates ₁₉₇

GBS polysaccharide conjugates of serotypes Ia, Ib, II, III, IV and V were prepared by deliberately varying the conditions of periodate oxidation/reductive amination (PO/RA) chemical reactions, including the solvent for the reagent (aqueous medium against DMSO), varying the levels of sialic acid in the initial polysaccharide and the degree of oxidation/modification of the saccharide epitope. Generally, conjugates prepared using DMSO as solvent have been found to have lower levels of unreacted (free) polysaccharide, higher conjugate molecular weight, and higher saccharide/protein ratios than conjugates prepared using an aqueous medium.

A conjugation method that results in conjugates with lower levels of unreacted (free) polysaccharide is advantageous and is preferred. It is well known that high levels of unreacted (free) polysaccharide can elicit a T-cell independent immune response that has the potential to "dilute" the T-cell dependent response elicited by the polysaccharide-protein conjugate, thus reducing the immune response elicited by the conjugate.

Selected GBS polysaccharides were chemically desiallated using methods known in the art (see Chaffin, DO, et al., J Bacteriol 187(13):4615-4626 (2005)), to generate conjugate variants to determine the effect of % desialization on immunogenicity. Desialylation at levels greater than about 40% (ie, sialic acid levels less than about 60%) adversely affects immunogenicity.

Similarly, in most cases, an oxidation state of less than about 5 or a saccharide epitope modification of more than about 20% adversely affects immunogenicity. Since oxidation occurs via sialic acid on the capsular polysaccharide, the results seem to indicate that modification of the saccharide epitope at a level greater than about 20% results in a decrease in sialic acid, leading to a decrease in immunogenicity.

In contrast, conjugates having different saccharide/protein ratios or polysaccharide molecular weights elicited an immune response in mice, indicating a relatively wide range of acceptable criteria for these parameters.

Additional conjugate variants have also been generated using alternative chemical routes. One alternative chemistry involved preparing conjugates by reacting the polysaccharide with carbonyl ditriazole (CDT) and performing the conjugation reaction in DMSO. In another alternative chemistry route, conjugates were prepared by oxidizing the polysaccharide with the TEMPO reagent [(2,2,6,6-tetramethylpiperidin-1-yl)oxyl] (instead of sodium periodate), followed by conjugation via a reductive amination reaction (TEMPO/BA ) in DMSO as detailed in Example 3 above. All conjugates made using these alternative chemistries were shown to be immunogenic in mice, suggesting that alternative chemistries besides PO/VA could be used. However, some variants of conjugation reactions for some serotypes were more effective than for others.

OPA analysis was performed according to Nanra, JS, et al., Hum. Vaccin. Immunother., 9(3):480-487 (2013), with group B streptococcal isolates replacing Staphylococcus aureus isolates and skipping the preopsonization step. After dose 3 (PD3), titers in OPA were presented as the geometric mean of a group of 10-20 mice immunized with the respective conjugate at a dose of 1 μg/ml.

Polysaccharide conjugates GBS serotype Ia-CRM ₁₉₇

The PO/VA conjugates and activated polysaccharides having CO 16-17 (approximately 6% saccharide epitope modification) were shown to be immunogenic (conjugates 1 and 3). However, the use of activated polysaccharides with CO 5.4 (approximately 19% saccharide epitope modification) adversely affected immunogenicity (conjugate 2). Similarly, at a sialic acid level of 50%, the immune response was almost completely absent (conjugate 4). The results are shown in Table 10.

Table 10. Effect of different conditions of the periodate oxidation/reductive amination method on GBS serotype Ia-CRM polysaccharide conjugates ₁₉₇

conjugate 1 2 3 4 Solvent DMSO DMSO Water DMSO MM polysaccharide (kDa) 190 190 190 190 % Sialic acid in the original polysaccharide >95 >95 >95 50 % Modifications 6 19 6 6 Oxidation state (CO) 16.8 5.4 16.2 16.4 Saccharide/Protein Ratio 0.8 0.8 2.0 1.1 % Free Saccharide <5 <5 26 <5 MM conjugate by SEC-MALLS method, kDa 6040 15390 806 3763 Titer in OPA assay 300 172 406 68

Additional variants of the conjugates were obtained using alternative methods of conjugation and molecular indicators of the conjugates (the results are shown in Table 11). Conjugate 5 was obtained by reacting the polysaccharide with carbonyl ditriazole (CDT) and the conjugation reaction was carried out in DMSO. Conjugate 6 was prepared by oxidation of the polysaccharide using the TEMPO reagent (instead of sodium periodate), followed by reductive amination conjugation in DMSO as detailed in Example 3 above. with a high saccharide/protein (S/P) ratio. Conjugate 8 was prepared by the PO/VA method using a low MW (40 kDa) polysaccharide. All of these conjugates were shown to be immunogenic in mice, suggesting alternative conjugation methods besides periodate oxidation/reductive amination reactions, as well as alternative conjugate measures such as C/B ratio and low molecular weight of the parent polysaccharide.

Table 11. Effect of various conditions of the periodate oxidation/reductive amination method and alternative chemistries on serotype Ia-CRM GBS polysaccharide conjugates ₁₉₇

Conjugate 5 6 7 8 conjugation reaction CDT TEMPO/VA PO/VA PO/VA MM polysaccharide (kDa) 383 220 383 40 % Sialic acid in the original polysaccharide >95 >95 >95 >95 % Modifications on eleven 6 8 Oxidation state (CO) on 9.2 17.5 12 Saccharide/Protein Ratio 1.2 0.9 2.5 1 % Free Saccharide 12.5 11.2 13.3 24.3 MM by SEC-MALLS method, kDa 7128 1678 4347 2000 Titer in OPA assay 1028 371 303 1484

Polysaccharide conjugates GBS serotype Ib-CRM ₁₉₇

Conjugates prepared using the PO/VA method and activated polysaccharides with SD 15.8 (approximately 6% saccharide epitope modification) in DMSO were immunogenic in mice (conjugates 9 and 11). The conjugates prepared by the PO/VA method in DMSO were slightly more immunogenic than the conjugate prepared by the PO/VA method in an aqueous medium, while all other molecular parameters of the conjugates were similar (conjugates 9 and 11, respectively). However, the use of activated polysaccharides with CO 4.7 (approximately 21% saccharide epitope modification) adversely affected immunogenicity (conjugate 10). Immunogenicity was almost completely absent, with very few responses, in the case of the conjugate obtained using the PO/VA method and 95% desialized (5% sialic acid level) polysaccharide (conjugate 12). The results are shown in Table 12.

Table 12. Effect of different conditions of the periodate oxidation/reductive amination method on serotype Ib-CRM GBS polysaccharide conjugates ₁₉₇

conjugate 9 10 eleven 12 Solvent DMSO DMSO Water DMSO MM polysaccharide (kDa) 120 120 120 120 % Sialic acid in the original polysaccharide >95 >95 >95 5 % Modifications 6 21 6 9 Oxidation state (CO) 15.8 4.7 15.8 11.7 Saccharide/Protein Ratio 1.1 1 2 1.1 % Free Saccharide eleven <5 33 7 MM by SEC-MALLS method, kDa 2608 7302 381 8418 Titer in OPA assay 417 159 278 62

Additional variants of the conjugates were obtained using alternative methods of conjugation and molecular indicators of the conjugates (the results are shown in Table 13). Conjugate 13 was obtained by the PO/VA method using a polysaccharide initially having a low level of sialylation (65%). Conjugate 14 was prepared by the PO/VA method with intentional variation of the conjugation parameters to obtain a conjugate with a high saccharide/protein (S/P) ratio. Conjugate 15 was prepared by reacting the polysaccharide with carbonyl ditriazole (CDT) and the conjugation reaction was carried out in DMSO. Conjugate 16 was prepared by oxidizing the polysaccharide using the TEMPO reagent (instead of sodium periodate), followed by reductive amination conjugation in DMSO as detailed in Example 3 above. alternative conjugation methods other than periodate oxidation/reductive amination reactions; and alternative conjugate parameters such as C/B ratio and low molecular weight of the parent polysaccharide.

Table 13. Effect of different conditions of the periodate oxidation/reductive amination method and alternative chemistries on serotype Ib-CRM GBS polysaccharide conjugates ₁₉₇

conjugate 13 14 15 16 conjugation reaction PO/VA PO/VA CDT TEMPO/VA MM polysaccharide (kDa) 141 141 141 150 % Sialic acid in the original polysaccharide 65 >95 >95 >95 % Modifications 8 7 on 13 Oxidation state (CO) 12 14.7 on 7.8 Saccharide/Protein Ratio 1.06 2.1 1.29 0.85 % Free Saccharide <5% 16% 21 7 MM by SEC-MALLS method, kDa 5345 1594 2760 1400 Titer in OPA assay 246 118 287 548

Polysaccharide conjugates GBS serotype II-CRM ₁₉₇

Conjugates prepared using the PO/VA method and activated polysaccharides with CO 4-15 (approximately 7-23% saccharide epitope modification) were immunogenic in mice (conjugates 17-20). The conjugate obtained using the PO/VA method and a polysaccharide with a sialylation level of 74% (desialization level of 26%) was also immunogenic (conjugate 20). The results are shown in Table 14.

Table 14. Effect of different conditions of the periodate oxidation/reductive amination method on GBS serotype II-CRM polysaccharide conjugates ₁₉₇

conjugate 17 18 19 20 Solvent DMSO DMSO Water DMSO MM polysaccharide (kDa) 95 95 109 109 % Sialic acid in the original polysaccharide >95 >95 >95 74 % Modifications 8 23 10 7 Oxidation state (CO) 12.6 4.3 9.8 15.2 Saccharide/Protein Ratio 0.84 0.90 1.13 0.63 % Free Saccharide 16 <5 6 <5 MM by SEC-MALLS method, kDa 3600 4650 1611 6140 Titer in OPA assay 610 967 2149 684

Additional variants of the conjugates were obtained using alternative methods of conjugation and molecular indicators of the conjugates (the results are shown in Table 15). Conjugates 21 and 22 were prepared by the PO/VA method with intentional variation in conjugation parameters to produce low and high saccharide/protein (S/P) conjugates, respectively. Conjugate 23 was prepared by oxidizing the polysaccharide using a TEMPO reagent (instead of sodium periodate), followed by reductive amination conjugation in DMSO as detailed in Example 3 above. Conjugate 24 was prepared by reacting the polysaccharide with carbonylditriazole (CDT), and conjugating carried out in DMSO. All of these conjugates were shown to be immunogenic in mice, suggesting the possibility of using alternative conjugation methods other than periodate oxidation/reductive amination reactions, as well as alternative conjugate measures such as C/B ratio.

Table 15. Effect of various conditions of the periodate oxidation/reductive amination method and alternative chemistries on serotype II-CRM GBS polysaccharide conjugates ₁₉₇

conjugate 21 22 23 24 conjugation reaction PO/VA PO/VA TEMPO/VA CDT MM polysaccharide (kDa) 109 109 109 109 % Sialic acid in the original polysaccharide >95 >95 >95 >95 % Modifications 10 10 eleven on Oxidation state (CO) 10 10 8.8 on Saccharide/Protein Ratio 0.61 2.03 0.66 0.87 % Free Saccharide <5 25 <5% 12 MM by SEC-MALLS method, kDa 8850 1480 5270 603 Titer in OPA assay 3117 891 2167 631

GBS serotype III-CRM polysaccharide conjugates ₁₉₇

Conjugates prepared using the PO/VA method and activated polysaccharides with CO 10-17 (approximately 6-10% saccharide epitope modification in DMSO) were immunogenic in mice (conjugates 25 and 30). Conjugates with CO 2.9 (approximately 34% saccharide epitope modification) or high saccharide/protein ratio (2.1) (conjugates 26 and 27, respectively) were relatively less immunogenic. The conjugate obtained using the PO/VA method and a polysaccharide with a sialylation level of 81% (desialization level of 19%) was immunogenic (conjugate 30). However, the conjugate prepared using the 58% sialylation polysaccharide (42% desialization level) was weakly immunogenic (conjugate 29). The PO/VA conjugate in DMSO was slightly more immunogenic than the aquatic PO/VA conjugate, while all other molecular parameters of the conjugates were similar (conjugates 25 and 28, respectively). The results are shown in Table 16.

Table 16. Effect of different conditions of the periodate oxidation/reductive amination method on serotype III-CRM GBS polysaccharide conjugates ₁₉₇

conjugate 25 26 27 28 29 thirty Solvent DMSO DMSO DMSO Water DMSO DMSO MM polysaccharide (kDa) 263 358 358 358 355 358 % Sialic acid in the original polysaccharide >95 >95 >95 >95 58 81 % Modifications 10 34 6 8 6 6 Oxidation state (CO) 10 2.9 17 13 16 17 Saccharide/Protein Ratio 1.1 1.2 2.1 1.7 1.15 1.19 % Free Saccharide 10 7 24 19 <5 <5 MM by SEC-MALLS method, kDa 2396 14340 3066 1885 5110 4643 Titer in OPA assay 701 57 252 248 137 505

Additional variants of the conjugates were obtained using alternative methods of conjugation and molecular indicators of the conjugates (the results are shown in Table 17). Conjugates 31-35 were obtained by the PO/VA method with deliberate variation of the conjugation parameters to obtain conjugates with different MW values. Conjugate 36 was prepared by reacting the polysaccharide with carbonyl ditriazole (CDT) and the conjugation reaction was carried out in DMSO. Conjugate 37 was prepared by oxidizing the polysaccharide using the TEMPO reagent (instead of sodium periodate), followed by reductive amination conjugation in DMSO as detailed in Example 3 above. alternative conjugation methods other than periodic oxidation/reductive amination reactions; and alternative conjugate indicators such as MM. Conjugates made with CO as little as 5 (approximately 20% saccharide epitope modification) were still immunogenic in mice (conjugate 32 in Table 17) compared to conjugates prepared with CO 2.9 (approximately 34% saccharide epitope modification) (conjugate 26 in Table 16 above).

Table 17. Effect of different conditions of the periodate oxidation/reductive amination method and alternative chemistries on serotype III-CRM GBS polysaccharide conjugates ₁₉₇

conjugate 31 32 33 34 35 36 37 conjugation reaction PO/VA PO/VA PO/VA PO/VA PO/VA CDT TEMPO/VA MM polysaccharide (kDa) 353 350 355 50 349 355 355 % Sialic acid in the original polysaccharide >95 >95 >95 >95 >95 >95 >95 % Modifications 6 5 6 8 20 on 10 Oxidation state (CO) 17 19 17 12 5 on 10 Saccharide/Protein Ratio 0.8 1.1 1.2 0.9 1.0 1.16 0.96 % Free Saccharide <5 <5 <5 20 <5 <5 <5 MM by SEC-MALLS method, kDa 9278 5291 4982 1201 8024 10740 3415 Titer in OPA assay 646 204 176 441 1116 448 336

Polysaccharide conjugates GBS serotype IV-CRM ₁₉₇

Conjugates prepared using the PO/VA method and activated polysaccharides with SD 6.9-14.2 (approximately 7-14% saccharide epitope modification) were immunogenic in mice (conjugates 38-41). The conjugate obtained using the PO/VA method and a polysaccharide with a sialylation level of 60% (desialization level of 40%) was also immunogenic (conjugate 41). The results are shown in Table 18.

Table 18. Effect of different conditions of the periodate oxidation/reductive amination method on serotype IV-CRM GBS polysaccharide conjugates ₁₉₇

conjugate 38 39 40 41 Solvent DMSO DMSO Water DMSO MM polysaccharide (kDa) 143 143 133 121 % Sialic acid in the original polysaccharide >95 >95 >95 60 % Modifications 7 14 7 8 Oxidation state (CO) 14.2 6.9 13.6 13.2 Saccharide/Protein Ratio 0.80 0.91 1.92 1.0 % Free Saccharide <5 <5 33.4 <5 MM by SEC-MALLS method, kDa 8268 10210 657 5691 Titer in OPA assay 3140 2379 3080 6708

Additional conjugate variants were generated using alternative conjugation methods and conjugate molecular weights. The results are shown in Table 19. Conjugates 42 and 45 were prepared by the PO/VA method with intentional variation in conjugation parameters to produce conjugates with high CO (lower oxidation) and high C/B ratio, respectively. Conjugate 43 was prepared by reacting the polysaccharide with carbonylditriazole (CDT) and the conjugation reaction was carried out in DMSO. Conjugate 44 was prepared by oxidizing the polysaccharide using the TEMPO reagent (instead of sodium periodate), followed by reductive amination conjugation in DMSO as detailed in Example 3 above. All serotype IV polysaccharide conjugates were immunogenic in mice, suggesting that alternative methods could be used conjugation, in addition to periodate oxidation/reductive amination reactions, as well as alternative indicators of conjugates, such as the C/B ratio. Conjugates made with CO (lower oxidation) up to at least 20 (approximately 5% saccharide epitope modification) were still immunogenic in mice.

Table 19. Effect of different conditions of the periodate oxidation/reductive amination method and alternative chemistries on serotype IV-CRM GBS polysaccharide conjugates ₁₉₇

conjugate 42 43 44 45 conjugation reaction PO/VA CDT TEMPO PO/VA MM polysaccharide (kDa) 143 133 133 140 % Sialic acid in the original polysaccharide >95 >95 >95 >95 % Modifications 5 on 7 7 Oxidation state (CO) 20 on 13.7 14.6 Saccharide/Protein Ratio 1.0 0.9 0.52 1.96 % Free Saccharide <5 <5 6 <5 MM by SEC-MALLS method, kDa 3580 12390 4580 2710 Titer in OPA assay 8614 1989 7567 3695

Polysaccharide conjugates GBS serotype V-CRM ₁₉₇

Conjugates prepared using the PO/VA method and activated polysaccharides with SD 4.4-14.6 (approximately 7-23% saccharide epitope modification) were immunogenic in mice (conjugates 46 and 47). The desialized (sialylation level 5%) conjugate was not immunogenic (conjugate 49), and the conjugate obtained using the PO/VA method and an aqueous solvent caused a weak immune response (conjugate 48). The results are shown in Table 20.

Table 20. Effect of different conditions of the periodate oxidation/reductive amination method on V-CRM serotype GBS polysaccharide conjugates ₁₉₇

conjugate 46 47 48 49 Solvent DMSO DMSO Water DMSO MM polysaccharide (kDa) 132 132 132 132 % Sialic acid in the original polysaccharide >95% >95% >95% 5% % Modifications 7 23 8 6 Oxidation state (CO) 14.6 4.4 12.1 18 Saccharide/Protein Ratio 1.32 1.43 1.27 0.94 % Free Saccharide eleven <5 25.4 <5 MM by SEC-MALLS method, kDa 4304 14510 573 4847 Titer in OPA assay 335 181 93 60

Additional conjugate variants were generated using alternative conjugation methods and conjugate molecular weights. The results are shown in Table 21. Conjugates 50 and 53 were prepared by the PO/VA method with deliberate variation in conjugation parameters to obtain conjugates with lower sialylation (81% sialylation) and low MW, respectively. Conjugate 51 was prepared by reacting the polysaccharide with carbonyl ditriazole (CDT) and the conjugation reaction was carried out in DMSO. Conjugate 52 was prepared by oxidizing the polysaccharide using the TEMPO reagent (instead of sodium periodate), followed by reductive amination conjugation in DMSO as detailed in Example 3 above. All serotype V polysaccharide conjugates except for the conjugate prepared using CDT were immunogenic in mice, suggesting the possibility of using alternative methods of conjugation, in addition to reactions of periodic oxidation/reductive amination, as well as alternative indicators of conjugates, such as MM. The conjugate obtained using CDT was significantly less immunogenic compared to other conjugates obtained by the BA method. The conjugate with a sialylation level of 81% (conjugate 50) elicited a weaker immune response compared to the conjugate with a sialylation level of >95% (conjugate 53), but stronger than the conjugate with a sialylation level of 5% (conjugate 49 in Table 20). , higher).

Table 21. Effect of various conditions of the periodate oxidation/reductive amination method and alternative chemistries on V-CRM serotype GBS polysaccharide conjugates ₁₉₇

conjugate 50 51 52 53 conjugation reaction PO/VA CDT TEMPO PO/VA MM polysaccharide (kDa) 159 193 193 37 % Sialic acid in the original polysaccharide >81% >95% >95% >95% % Modifications 7 on 5 10 Oxidation state (CO) 13.5 on 18.2 10.3 Saccharide/Protein Ratio 1.2 1.14 0.71 0.53 % Free Saccharide <5 <5 22.3 7.3 MM by SEC-MALLS method, kDa 3037 4756 3501 3044 Titer in OPA assay 160.3 101 320 279

Example 5 Monovalent GBS III-CRM Conjugate Vaccines ₁₉₇ and GBS V-CRM ₁₉₇ elicited an OPA response in mice

Female CD-1 mice were immunized with 1 μg, 0.1 μg, or 0.01 μg of group B Streptococcus (GBS) serotype III polysaccharide conjugated to CRM ₁₉₇ (GBS III-CRM ₁₉₇ ) or GBS serotype V conjugated to CRM ₁₉₇ ( GBS V-CRM ₁₉₇ ), three times subcutaneously at weeks 0, 3 and 6. After the third dose (PD3), serum was assessed in the opsonophagocytosis assay (OPA). OPA assays were performed as described in Example 4. Both conjugates induced OPA responses in mice (Table 22). Samples without a detectable OPA response were assigned a value of 50.

Table 22: GBS III and GBS V conjugates induce OPA responses in mice

Conjugate type Dose (mcg) Geometric Mean Titer in OPA Assay III 1 701 0.1 103 0.01 50 V 1 378 0.1 204

Example 6 Monovalent GBS Ia-CRM Conjugate Vaccines ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ elicited an OPA response in mice

Six groups of female CD-1 mice were immunized three times subcutaneously with a vaccine containing 1 μg of a single capsular polysaccharide (CP) GBS conjugated to CRM ₁₉₇ at weeks 0, 3, and 6. Initial studies showed that the mice were previously titre-free in the OPA assay none of the six serotypes tested. Sera obtained in PD3 were analyzed in the OPA assay against the GBS polysaccharide of the corresponding serotype contained in the vaccine. OPA analyzes were performed as described in Example 4. The results are shown in Tables 23 and 24 below.

Table 23: Geometric mean titer of OPA assays in mice after immunization with individual GBS CP-CRM conjugates ₁₉₇

Serotype Geometric Mean Titer in OPA Assay Ia 300 Ib 417 II 610 III 188 IV 3140 V 378

Table 24: Titer increase in OPA assays in mice after immunization with individual GBS CP-CRM conjugates ₁₉₇

Serotype Geometric Mean Titer in OPA Assay Ia 6 Ib 8 II 12 III 4 IV 63 V 8

Note: The multiplicity of the increase was calculated, assuming that the mice had previously no titer.

Example 7 Opsonic Activity of Serum Versus Isolated IgG from Mice Immunized with Monovalent GBS Ia-CRM Conjugate Vaccines ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇

Female CD-1 or BALB/c mice were immunized with 1 μg of single GBS CP conjugated to CRM ₁₉₇ three times subcutaneously at weeks 0, 3 and 6. Either AlPO ₄ or QS-21 was used as an adjuvant. Serum obtained in PD3 was tested in a serotype-specific OPA assay and then an immunoglobulin G fraction was isolated and tested for OPA activity. OPA activity of purified IgG was normalized to 5 mg/ml (in the range of IgG in normal mouse serum). All six GBS CP conjugates caused the production of IgG antibodies with opsonic activity (Figure 1).

Example 8 Monovalent GBS Ia-TT, GBS Ib-TT, GBS II-TT, GBS III-TT, GBS IV-TT and GBS V-TT Conjugate Vaccines Induced OPA Responses in Rabbits

Rabbits were immunized three times with 50 μg/ml of GBS serotype Ia polysaccharide conjugated to tetanus toxoid, 10 μg/ml serotype Ib GBS polysaccharide tetanus toxoid conjugated 50 µg/ml serotype GBS II polysaccharide tetanus toxoid conjugated 50 µg/ml serotype III GBS polysaccharide tetanus toxoid conjugated 50 µg/ml serotype IV GBS polysaccharide toxoid conjugated tetanus, or 50 μg/ml GBS serotype V polysaccharide conjugated to tetanus toxoid, using complete Freund's adjuvant at the first dose and incomplete Freund's adjuvant at the second and third doses as adjuvant. Conjugates were prepared using polysaccharides having a sialic acid level >95% and chemical reaction with CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate). PD3 day immune responses were determined in the OPA assay as described in Example 4. Serum titers are shown in Table 25 and purified IgG titers are shown in Table 26 below. GBS serotype Ia, Ib, II, III, IV and V polysaccharides conjugated with TT showed high immunogenicity in rabbits.

Table 25: Geometric mean titer in OPA assays of rabbit serum after immunization with individual GBS CP-TT conjugates

Serotype GBS Geometric Mean Titer in OPA Assay Ib 11550 II 36753 IV 34345

Table 26: Geometric mean titer in OPA assays of purified rabbit IgG after immunization with individual GBS CP-TT conjugates

Serotype GBS OPA Geometric Mean Titer (1 mg/ml vp) Ia 7190 Ib 2817 II 41870 III 40146 IV 15565 V 12124

Example 9 Hexavalent GBS Conjugate Vaccine Induced OPA Response in Primates

Three groups of rhesus monkeys were immunized with hexavalent group B streptococcus vaccine (GBS6) intramuscularly three times at weeks 0, 4 and 8. The GBS6 vaccine contained GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III- CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ . Animals in two groups used aluminum phosphate (AlPO ₄ ) as an adjuvant and doses of either 5 μg of each conjugate or 50 μg of each conjugate. Animals in the third group received 5 μg of each conjugate without adjuvant. Table 27 below shows the immunization schedule.

Table 27: Immunization schedule for rhesus monkeys

Group Number of primates Vaccine dose
(each conjugate) Alum as AlPO4
(mg/ml) Volume of vaccine administered Delivery Immunization schedule (weeks) 1 10 5 mcg 0.5 1.0 ml i/m 0, 4, 8 2 10 50 mcg 0.5 1.0 ml i/m 0, 4, 8 3 10 5 mcg No 1.0 ml i/m 0, 4, 8

Pre-immune sera and sera obtained on PD3 day were analyzed in the OPA assay for the polysaccharides of all six GBS serotypes contained in the vaccine. OPA analyzes were performed as described in Example 4. The results are shown in Tables 28 and 29 below. For all six serotypes, AlPO ₄ adjuvanted formulations elicited a detectable OPA response (increase in titers from pre-immunization to PD3 day, or pre/PD3 >1-fold increase). The 5 μg dose of the conjugate without adjuvant elicited detectable OPA responses in five of the six serotypes.

Table 28: Geometric mean titer of OPA assays in rhesus monkeys before and after GBS6 immunization

5 µg+AlPO ₄ 50 µg+AlPO ₄ 5 mcg without adjuvant Serotype Pre PD3 Pre PD3 Pre PD3 Ia 97 2907 118 8106 166 69 Ib 53 3821 53 2957 50 313 II 54 980 62 764 61 362 III 105 7759 204 8448 762 4515 IV 78 2350 89 3331 309 899 V 233 5556 442 16476 6192 4226

Table 29: Titer increase in OPA assays in rhesus monkeys after GBS6 immunization

Serotype 5 µg+AlPO ₄ 50 µg+AlPO ₄ 5 mcg without adjuvant Ia thirty 69 4 Ib 72 56 6 II 18 12 6 III 74 41 6 IV thirty 37 3 V 24 37 1

Example 10 Hexavalent GBS Conjugate Vaccine Induced OPA Response in Rats

Female Sprague-Dawley rats were immunized twice subcutaneously with 5 μg/ml of each conjugate in the GBS6 polysaccharide conjugate vaccine as described in Example 9, formulated with or without aluminum phosphate (AlPO ₄ ). Two sera, pre-immune (baseline) and post-dose, were evaluated for titers in the OPA assay against polysaccharides of all six respective GBS serotypes. The titers in the OPA assay were measured for each GBS serotype in a 384-well assay format and the fold increase in titer was calculated. GBS6-vaccinated rats had a strong immune response, producing functional antibodies against each serotype polysaccharide after the second dose; in the absence of AlPO _4, an increase in titers from 7 to 205 times was observed in the case of different serotypes, and in the presence of adjuvant this increase ranged from 11 to 294 times (Table 30).

Table 30. Post-dose 2 (PD2) titer increase in opsonophagocytic activity (OPA) assay in rats immunized with GBS hexavalent conjugate vaccine

Multiplicity of increase in the geometric mean value of the titer from preimmune to PD2 serum in the OPA analysis Serotype GBS Without AlPO ₄ With AlPO ₄ Ia 36 eleven Ib 7 39 II 205 294 III 107 141 IV 45 33 V 185 195

Example 11: Immunization of pregnant females with monovalent or hexavalent GBS glycoconjugate vaccine demonstrated a protective effect against GBS III or V infection in their offspring after birth

Female CD-1 mice were immunized three times subcutaneously with the GBS6 vaccine as described in Example 9 containing 5 μg/ml of each conjugate and 100 μg/ml of AlPO ₄ , GBS III or V monovalent glycoconjugate vaccine (each containing 10 μg/ml of the conjugate and 100 μg/ml AlPO ₄ ) or control solvent alone. The mice were allowed to mate until the third immunization. The offspring of immunized mice were given a lethal dose of either serotype GBS III or GBS serotype V, according to the vaccine they received, and survival was monitored for 90 hours. Immunization of females with GBS6+AlPO ₄ or GBS III-CRM ₁₉₇ +AlPO ₄ resulted in a significant degree of protection (p<0.0001) of their offspring from a lethal dose of GBS serotype III bacteria. Similarly, immunization of females with GBS V-CRM ₁₉₇ + AlPO ₄ resulted in a significant degree of protection (p<0.0001) of their offspring from a lethal dose of GBS serotype V bacteria. The results are shown in Table 31.

Table 31: Immunization with monovalent and hexavalent GBS vaccine resulted in increased offspring survival

Number of surviving offspring/total offspring (% survival) Serotype used for infection Offspring of females immunized with GBS6 Offspring of females immunized with the appropriate monovalent vaccine Offspring of solvent-immunized females III 20/22 (91%) 28/28 (100%) 9/29 (31%) V 35/40 (85%) 3/27 (11%)

Example 12: Passive immunization with anti-GBS III monoclonal antibodies of neonatal rats showed a protective effect

Monoclonal antibodies (mAbs) against group B streptococcus serotype III (GBS III) were obtained by immunizing mice with a pentavalent vaccine containing polysaccharides of serotypes Ia, Ib, II, III, and V. Clones of GBS III-specific mAbs were selected, and mAbs recognizing CP GBS III , was obtained using standard methods. Anti-GBS serotype III mAbs were passively administered to neonatal rats (n=10 per group; results from 2 independent experiments shown) 16 hours prior to challenge with a clinical GBS III isolate. Four hours after infection, blood was collected and CFU of residual bacteria were counted. The introduction of anti-GBS III mAb resulted in a decrease in CFU for residual bacteria in neonatal rats by 4 log or more (Table 32).

Table 32: Anti-GBS III mAbs resulted in a reduction in CFU of residual bacteria in neonatal rats

Impact CFU of residual bacteria (log) Experiment 1 Anti-GBS III mat 1.8 Control 7.5 Experiment 2 Anti-GBS III mat 2.4 Control 6.4

Example 13: Passive immunization with monoclonal antibodies against GBS Ib, III and V of pregnant mice demonstrated a protective effect for their offspring after birth

Monoclonal antibodies (MAbs) were obtained from mice immunized with pentavalent vaccine (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) using standard methods. Mabs specifically recognizing the capsular polysaccharides of each of the five serotypes have been identified. Doses of 500 μg/ml mAb against GBS serotype Ib (GBS Ib), mAb against GBS serotype III (GBS III), mAb against GBS serotype V (GBS V) or isotype control mAbs were passively administered to pregnant mice approximately 24-48 hours before childbirth. 24-48 hours after birth, the offspring of immunized female mice were challenged with a lethal dose of GBS Ib, GBS III, or GBS V bacteria. Survival was monitored for 96 hours. Significantly higher survival rates were noted in offspring born from females immunized with anti-GBS Ib mAb, anti-GBS III mAb, or anti-GBS V mAb compared to control mAbs after GBS challenge (Table 33).

Table 33: Anti-GBS III and V mAb resulted in increased progeny survival

% Survival of offspring born from females passively immunized: GBS serotype at infection corresponding mat Control mAb Ib 80 0 III 93 0 V 100 12

Example 14 Stability of GBS Conjugates

GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ were separately formulated in 10 mM succinate-phosphate buffer with 155 mM NaCl at different pH values for testing the stability of conjugates under conditions of accelerated drug aging. The percentage change in molecular weight determined by the SEC-MALLS method was determined after 4 weeks of storage at 50°C. The results are presented in Figures 2-7.

GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS III-CRM ₁₉₇ and GBS IV-CRM ₁₉₇ conjugates were tested for sialylation stability under various buffer conditions shown in Table 34. Free sialic acid (N-acetylneuraminic acid; NANA) content measured by HPLC after 1 month storage at 37°C. The results are presented in Figure 8.

Both studies showed that the conjugates were better preserved above pH 6.0 and optionally at pH around 6.5.

Table 34. Buffer conditions for sialylation stability testing

pH Salt Salt concentration (mM) Buffer Buffer concentration (mM) 5.8 No 0 Succinate 10 6.2 No 0 Succinate 10 6.6 No 0 Succinate 10 6.9 No 0 Succinate 10 5.8 NaCl 150 Succinate 10 6.3 NaCl 150 Succinate 10 6.6 NaCl 150 Succinate 10 6.9 NaCl 150 Succinate 10 6.1 No 0 Histidine 10 6.5 No 0 Histidine 10 6.9 No 0 Histidine 10 6.1 NaCl 150 Histidine 10 6.5 NaCl 150 Histidine 10 6.9 NaCl 150 Histidine 10

Example 15 GBS6 Preparation

To determine the preferred buffer, GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ (GBS6) were formulated using the buffer conditions specified in Table 33 above. The actual pH value of the preparations was determined at the following time points: 0 (at the time of creation of the preparation), after 1 month at 5°C, after 1 month at 25°C and after 1 month at 37°C. A pH shift was noted in preparations using succinate as a buffer, while no shift was observed in preparations prepared using histidine as a buffer. The results are presented in Figures 9-10.

Also tested the effect of the concentration of histidine buffer on the binding of GBS conjugates with aluminum. A preparation containing 150 mM NaCl, 0.01% polysorbate-80 at pH 6.5, and 0.5 mg/ml aluminum as AlPO4 was tested with conjugates at two different concentrations (10 µg/ml and 40 mg/ml of each serotype ) and histidine at several different concentrations. The percentage of aluminum-bound conjugates was determined by measuring the total amount of each of the conjugates in the vaccine and the amount of each of the aluminum-bound conjugates. The amount of bound conjugates was determined by centrifugation of the vaccine preparation, resuspension of the aluminum pellet, aluminum solubilization and quantification of bound conjugates by nephelometry with serotype-specific polyclonal antibodies against each of the serotypes. The results are presented in Figures 11-12. It was found that the concentration of histidine buffer affected the amount, in percent, of the conjugate of each serotype associated with aluminum, and the effect was more pronounced at a lower dose than at a higher dose.

Shaking studies were conducted to determine the preferred amount of polysorbate-80 (PS80). GBS6 preparations containing 20 mM histidine, 150 mM NaCl, 0.5 mg/ml AlPO ₄ (if present) and either no PS80 or containing 0.01% PS80, 0.02% PS80, or 0.03% PS80 when pH 6.5, tested for percentage loss of total antigenicity under shaking stress. Syringes prefilled with drugs were shaken at 500 rpm for 72 hours at room temperature. Control samples (without shaking) were stored at room temperature for 72 hours. The results are presented in Figure 13.

The concentration of aluminum in the GBS6 preparations was also tested to determine its effect on the binding of GBS conjugates to aluminum. GBS6 preparations containing 10 mM histidine, 150 mM NaCl, 0.02% PS80 and aluminum phosphate (AlPO ₄ ) at either 0.25 mg/mL, 0.5 mg/mL, 0.75 mg/mL, or 1 0 mg/ml aluminum at pH 6.5 was tested for the amount, in percent, of the conjugate associated with aluminum. The percentage binding to AlPO ₄ increased with increasing concentration of AlPO ₄ . The results are presented in Figure 14.

Example 16 GBS6 Lyophilized Preparation

Various lyophilized preparations of GBS6 (GBS Ia-CRM ₁₉₇ , GBS Ib-CRM ₁₉₇ , GBS II-CRM ₁₉₇ , GBS III-CRM ₁₉₇ , GBS IV-CRM ₁₉₇ and GBS V-CRM ₁₉₇ ) were tested for stability. Low (10 µg/mL) and high (50 µg/mL) dose formulations containing 20 mM histidine at pH 6.5, 0.02% PS80, approximately 28 mM NaCl, and either 5.5%, 7.0% or 8.5% (w/v) sucrose, lyophilized. The stability of lyophilized preparations was tested by measuring pH and moisture content after 4 months at 5°C, 4 months at 37°C and 1 month at 50°C. All formulations were stable with respect to pH and moisture content (data not shown). In addition, percentage recovery of antigenicity for each serotype was tested for all formulations at 1, 4 and 9 months at both 5°C and 37°C, and at 1, 2 and 4 weeks at 50°C. The results are presented in Figures 15-20.

The following variations in excipients were also used and evaluated for the 40 μg/mL dose of GBS6 formulation: 1) 7% (w/v) sucrose, 2) 2% (w/v) sucrose and 4% (w/v) .) mannitol, 3) 3% (w/v) sucrose and 3% (w/v) mannitol, 4) 2% (w/v) sucrose and 4% (w/v) glycine or 5) 3% (w/v) sucrose and 3% (w/v) glycine. The pH and moisture content of all five preparations were stable after 3 months at 5°C, 3 months at 25°C, 3 months at 37°C and 1 month at 50°C (data not shown). In addition, percent recovered antigenicity for each serotype was tested for all preparations after storage for 1, 3, and 7 months at 2-8°C, 25°C, and 37°C, and after 1, 2, and 4 weeks. at 50°C. The results are presented in Figures 21-25.

The amount, in percent, of antigen bound to the aluminum phosphate adjuvant in the GBS6 vaccine, in the case of the reconstituted freeze-dried preparation and the liquid preparation, was tested by nephelometry. Both lyophilized and liquid preparations containing 20 mM histidine, 0.02% PS80, 7.0% (w/v) sucrose, and 500 μg/ml aluminum as aluminum phosphate were prepared in low (10 μg/v) ml) and high (50 mcg/ml) doses. Different concentrations of sodium chloride (NaCl) were also tested to determine their effect on antigen binding. The results for lyophilized preparations and liquid preparations are shown in Tables 35 and 36, respectively. Both lyophilized and liquid low dose preparations showed comparable results using NaCl concentrations of approximately 150 mM and above.

Table 35. Percent Antigen Binding to Aluminum Phosphate in Reconstituted Lyophilized Preparations with Different Levels of NaCl

NaCl (mM) Ia (%) Ib (%) II (%) III (%) IV (%) V (%) 10 μg/ml of each c/7.0% sucrose conjugate ~23 40 36 31 34 38 39 ~80 46 45 37 40 44 44 ~150 78 69 63 62 82 84 ~300 77 64 63 60 79 86 50 μg/ml of each c/7.0% sucrose conjugate ~23 21 23 24 18 27 24 ~34 24 23 23 21 27 40 ~150 66 58 52 46 65 67 ~300 65 56 50 48 66 64

Table 36. Binding, in percent, of antigen to aluminum phosphate in liquid preparations with different levels of NaCl

NaCl (mM) Ia (%) Ib (%) II (%) III (%) IV (%) V (%) 10 μg/ml of each conjugate 40 78.4 72.4 62.9 73.3 70.9 70.8 100 61.0 63.3 52.3 51.9 49.8 53.9 150 72.7 77.0 68.1 67.1 64.0 71.0 200 67.7 73.0 58.6 52.3 51.9 60.1 300 78.3 79.2 71.1 57.1 58.0 74.1 50 μg/ml of each conjugate 100 50.5 38.3 49.0 52.8 51.7 37.9 150 49.8 37.6 49.0 50.8 50.9 38.7 200 40.1 35.9 45.3 47.4 44.0 34.7 300 40.3 41.9 50.9 42.2 43.5 37.5

Example 17 Features of resuspension of GBS6 preparations

GBS6 preparations were prepared containing 240 μg/ml (high) or 60 μg/ml (low) total dose of polysaccharide-protein conjugates, 20 mM histidine, 150 mM NaCl, 0.02% PS80 and 0.5 mg/ml AlPO ₄ at pH 6.5. Similar monovalent preparations for each serotype were also prepared at doses of 240 μg/ml (high) or 60 μg/ml (low). Samples were placed in either 1) 2 ml glass vials with a fill volume of 0.73 ml per vial, or 2) BD HYPAK SCF™ PRTC (Becton, Dickinson and Company) 1 ml short glass pre-filled syringes (PFS) with a volume fill 0.58 ml, stoppered with 4432/50 stoppers (West Pharmaceutical Services, Inc.), maintaining a headspace of approximately 5±1 mm above the product. The vial preparations were stored in a vertical position, and the PFS preparations were stored in two different positions, namely, tip down and horizontally, at 5°C.

Resuspension was characterized by the number of shaking required to completely resuspend the preparations after 2 weeks and 3 months of storage. It took less than 5 shakes to resuspend all drugs placed in vials after 2 weeks and 3 months of storage. When stored horizontally, low dose GBS6 in PFS required only 3 shakes after 3 months of storage. However, low dose preparations required approximately 15 shakes to be resuspended when stored in PFS in the tip down position at both 2 weeks and 3 months of storage. In comparison, high dose preparations stored in PFS were difficult to resuspend regardless of storage position. The high dose GBS6 preparation required more than 50 shakes even when stored in a horizontal position. In addition, the number of succussions required to resuspend the drug at a high dose increased over time. Approximately 100 shakes were required to completely resuspend high dose GBS6 preparations stored tip down in the PFS for up to 16 hours after filling (T0), and this number increased to over 200 shakes if stored for 3 months. Finally, some monovalent preparations made using polysaccharide conjugates for serotypes Ia, III, IV, and V were difficult to resuspend after storage in PFS in both positions, and more shaking was required to resuspend over time, indicating that resuspension characteristics monovalent drugs depended on the serotype. Comparison results of resuspension of GBS6 and high and low dose monovalent preparations stored tip down in the PFS at time points T0, 2 weeks and 3 months are shown in Figure 26. Comparison results of GBS6 resuspension and high and low dose monovalent preparations stored in PFS in the tip-down and horizontal position are shown in Figure 27.

High and low dose GBS6 preparations were also tested to determine if long term storage in vials, PFS tip down and PFS horizontal would affect resuspension. In addition, a medium dose formulation (total dose of polysaccharide-protein conjugates 120 μg/mL) was also tested for comparison with low and high dose formulations. It was found that the low dose formulation became more difficult to resuspend after 6 months of storage in tip-down PFS, requiring more than 50 shakes. The medium dose preparation was difficult to resuspend after only 1 month of storage in PFS in the tip down position and required 30 shakes to be resuspended after 1 month of storage in PFS in the horizontal position. The results are shown in Table 37.

Table 37 Comparison of resuspension characteristics of GBS6 preparations at three different doses stored in vials and pre-filled syringes

Container Storage time at 2-8°C (months) Number of shakes for resuspension low dose Average dose high dose PFS (tip down) 6 >50 >50* >50 PFS (horizontal) 6 <10 thirty* >50 Bottle eleven <10 n/t <10

* storage time 1 month;

n/t - not tested.

In these studies, it was determined that the difficulties associated with resuspension can be reduced by storing preparations containing 20 mM histidine, 150 mM NaCl, 0.02% PS80 and 0.5 mg/ml AlPO4 at pH 6.5, during vials or PFS in a horizontal position. However, resuspension can occur within acceptable limits (i.e., using less than 10 shakes) only for preparations stored in the PFS in a horizontal position that contain a total dose of polysaccharide-protein conjugates of 60 μg/ml or less.

Additional studies were conducted to test the effect of three physical properties on resuspension: (1) surface charge or zeta potential using Zetasizer Nano ZS (Malvern Instruments Ltd.); (2) particle size using a Mastersizer 3000 laser diffraction particle size analyzer (Malvern Instruments Ltd.); and (3) the rate of sedimentation, or settling, using a dispersive analyzer LUMISIZER ^® (LUM GmbH). The zeta potential values of AlPO ₄ and GBS conjugates of serotypes Ia, Ib, II, III, IV and V depending on pH are presented in Figures 28 and 29, respectively. The zeta potential and particle sizes of various GBS6 preparations and monovalent GBS preparations are shown in Table 38. The zeta potential values of the 15 preparations were approximately -7 to -10 mV. There was no correlation between surface charge and resuspension for these samples. On the other hand, the particle size affected the resuspension. The particle size in low dose formulations was higher than in high dose formulations for GBS6 and monovalent formulations, and in general, the smaller the particle size, the more difficult it was to uniformly resuspend the formulation.

Table 38 Comparison of Zeta Potential and Particle Size of GBS6 and High and Low Dose Monovalents

A drug Conc. (µg/ml) Zeta potential (mV) Particle size Number of shakes in T0 D10 (µm) D50 (µm) D90 (µm) 1 GBS Hexavalent 60 -7.1 6.3 11.6 24.5 17 2 240 -8.5 4.2 7.1 12.6 96 3 Monovalent type Ia 60 -8.8 6.1 11.8 25.1 16 4 240 -10.0 4.3 7.2 14.3 33 5 Univalent type Ib 60 -8.7 6.7 12.4 23.1 21 6 240 -10.2 4.0 7.2 14.1 27 7 Monovalent type II 60 -8.0 6.7 12.9 25.5 16 8 240 -7.1 3.9 7 15 29 9 Monovalent type III 60 -9.6 eleven 20.8 37.3 16 10 240 -9.6 4.8 9.1 32.6 34 eleven Monovalent type IV 60 -9.0 5.4 9.1 16.4 17 12 240 -7.1 3.8 7.7 22.9 95 13 Univalent type V 60 -7.7 5.3 9.1 17.0 15 14 240 -8.3 4.1 7.2 13.0 62 15 AlPO ₄ placebo 500 -9.1 4.4 7.1 11.6 eleven

The rate of sedimentation of aluminum particles in preparations was measured at time points of 0, 2 weeks and 3 months. High dose preparations showed a slower sedimentation rate than low dose preparations. The sedimentation rate did not change after three months of storage at 5°C. A direct correlation was observed between resuspension and sedimentation rate: at a higher sedimentation rate, it was easier to resuspend the drug. In addition, the low dose formulations, which had larger particles than the high dose formulations, exhibited a faster sedimentation rate and easier resuspension. The data is presented in Figure 30.

Example 18 Effect of GBS6 formulation factors on sedimentation rate

The effect of formulation factors such as antigen concentration, pH and salt concentration on resuspension performance was studied. First, preparations of GBS6 with different concentrations of antigen in histidine buffers with pH 6.5 were investigated. The results of this study showed that a higher concentration of antigen leads to a lower sedimentation rate (see Figure 31; doses are shown for each serotype), which would cause difficulties in resuspension. At low concentrations, the particles were free to settle without interfering with each other due to collisions and/or Brownian motion.

Second, the precipitation patterns of GBS6 preparations were studied at total antigen concentrations of 60 μg/mL and 300 μg/mL in histidine buffers at pH 6.0, 6.5, and 7.0. The results of studying the sedimentation features of GBS6 preparations with a concentration of 60 μg/ml showed that pH has a significant effect on the sedimentation rate. However, pH did not significantly affect the sedimentation rate of the 300 µg/mL GBS6 preparation. The data are shown in Figure 32. The surface charge of AlPO ₄ decreased from -10.3 mV to -7.9 mV when the pH decreased from 7.0 to 6.0 (data not shown). Typically, a high zeta potential would indicate that the particles are completely dispersed in the medium due to the dominant repulsive forces that exist between the particles. In this case, the particles are in suspension as separate units, and the sedimentation rate is often low due to the wide distribution of particle sizes, as a result of which the particles settle at different rates.

Finally, the effect of ionic strength on the physicochemical properties of AlPO ₄ and its subsequent effect on the properties of the slurry was also studied. Prepared GBS6 preparations with three different dose levels (total antigen concentration of 60 μg/ml, 150 μg/ml and 20 μg/ml) and two different concentrations of NaCl (22 mm and 150 mm). Zeta potential values become more negative at low salt concentrations. Preparations of GBS6 in all three doses with a minimum salt concentration (22 mm) were negatively charged. Zeta potential values ranged from about -16 to about -17 mV. In comparison, high and low dose GBS6 preparations with 150 mM NaCl had a zeta potential of approximately -8 mV. Higher salt concentration resulted in a decrease in surface charge and an increase in settling rate, which could lead to greater ease of resuspension. Data are presented in Figure 33 and Table 39.

Table 39. Comparison of zeta potential and particle size of GBS6 preparations at different doses and with different concentrations of NaCl

A drug Zeta potential
(mV) Particle size D10 (µm) D50 (µm) D90 (µm) 1 GBS6, low dose and low salt concentration (total dose of antigen 60 µg/mL c/22 mM NaCl) -15.9 5.4 10.0 20.2 2 GBS6, medium dose and low salt concentration (total dose of antigen 150 µg/mL c/22 mM NaCl) -17.0 3.8 7.8 19.7 3 GBS6, high dose and low salt concentration (total dose of antigen 240 µg/mL c/22 mM NaCl) -17.0 3.9 7.0 14.3 4 GBS6, low dose and high salt concentration (total dose of antigen 60 µg/mL c/150 mM NaCl) -7.1 6.3 11.6 24.5 5 GBS6 high dose and high salt concentration (total dose of antigen 240 µg/mL c/150 mM NaCl) -8.5 4.2 7.1 12.6

Example 19: Features of resuspension of various antigens and aluminum salts

A study was conducted to further investigate whether the correlation between antigen concentration and difficulty in drug resuspension is GBS specific or common to other antigens such as proteins, polysaccharides and non-GBS polysaccharide-protein conjugates. Preparations containing CRM ₁₉₇ ; a combination of polysaccharides from GBS serotypes Ia, Ib, II, III, IV and V; BSA or a combination of 20 pneumococcal polysaccharide-protein conjugates (20vPnC) were formulated at the same high dose (240 μg/ml total antigen dose), in the same buffer matrix, and with 0.5 mg/ml AlPO ₄ adjuvant, as the GBS preparations. tested in Examples 17 and 18. GBS6 formulated with ^Alhydrogel® 85 (Brenntag Biosector A/S) was also included as a comparator. Samples were tested for the following characteristics: 1) resuspension time (time required for pellets to be resuspended in 1.5 ml centrifuge tubes in a hand shaker after 30-45 minutes of centrifugation at 200 rpm), 2) settling in cylinders under visual observation (height of the sediment layer at different time intervals), 3) particle size, 4) surface charge, and 5) particle size distribution using a MICRO-FLOW IMAGING ^® (MFI) Flow DPA 4200 microscope (Brightwell Technologies Inc.).

Different antigens showed different patterns of settling and resuspension (data are shown in Figure 34 and Table 40, respectively). As noted earlier, preparations that settled faster were easier to resuspend. There was no correlation between resuspension and mean particle size or particle size distribution. Particle size distribution data are presented in Figures 35A and 35B. However, GBS6 formulations containing ^Alhydrogel® 85 were the easiest to resuspend and had a significantly larger particle size, higher settling rate, and low surface charge, indicating that particle size and surface charge correlate with ease of resuspension. It should be noted that aluminum hydroxide is positively charged at pH 6.5, while the conjugates are negatively charged, resulting in a stronger electrostatic interaction and higher charge neutralization. In contrast, AlPO ₄ is negatively charged at pH 6.5.

Table 40. Comparison of particle size, resuspension time and zeta potential of different antigenic preparations

Antigen Particle size Resuspension time (sec) Zeta potential(mV) D10 D50 D90 GBS6 4.5 7.4 12.6 75 -8.1 CRM ₁₉₇ 5.1 7.4 11.1 20 -6.4 Polysaccharides GBS 4.8 6.9 9.7 10 -7.1 BSA 4.8 6.9 10.1 15 -6.6 20vPnC 7.7 14.8 28.9 25 -6.9 GBS6+alhydrogel ^® 85 15.0 26.8 46.9 15 -5

Example 20: Modifying the device configuration

The results of the studies indicated that the preparations were easily resuspended in the vial, but not in the PFS, both containers were made of type 1 glass and rubber stoppers were used for them. However, the key difference between the two systems is the presence of headspace, which allows free mixing of the drug and results in a more uniform resuspended drug. Therefore, the effect of headspace as a factor influencing resuspension in PFS was investigated to understand whether more headspace in PFS would result in better mixing. The syringes were filled with high dose GBS6 preparations (with AlPO ₄ ), sealed with various headspaces (2 mm, 6 mm and 10 mm; shown in Figure 36) and stored horizontally at 2-8°C. Resuspension features (number of shakes) were tested after 2 weeks of storage. The data indicated that an increase in headspace resulted in a decrease in the number of shaking required for resuspension. The data are shown in Table 41. Ease of resuspension was associated with increased turbulence due to more headroom, which made mixing easier. These features were also evident in the vials, in which case there were no mixing problems (due to the larger headspace above the product).

Table 41 Effect of headspace increase on resuspension performance of GBS6 preparations

Free space above the product (± 1 mm) Number of shakes* 2 >50 6 17 10 10

Storage time: 2 weeks at 2-8°C;

*Average value for 3 samples.

Example 21 Effect of Aluminum Concentration

Conducted a study to determine the effect of aluminum concentration on the possibility of resuspension of the drug. For this, GBS6 preparations were evaluated with decreasing concentrations of AlPO ₄ . High dose preparations of GBS6 (total antigen dose 240 μg/ml) were prepared in the same buffer matrix with aluminum concentration levels in the range of 0.5-0.05 mg/ml. Samples were placed in BD HYPAK SCF ^TM PRTC (Becton, Dickinson and Company) 1 ml short glass PFS with a fill volume of 0.58 ml; stoppered with 4432/50 stoppers (West Pharmaceutical Services, Inc.) leaving a headspace of approximately ~0.4 mm above the product and stored horizontally. Resuspension was tested after 0, 1, 2 and 4 weeks of storage at 5°C. Total and associated antigenicity for each of the serotypes in individual preparations was analyzed by nephelometry with serotype-specific polyclonal antibodies.

Although the reduction of aluminum levels had a positive effect on resuspension, preparations with aluminum concentrations of 0.3 mg/ml still required more than 20 shakes at time T0 and more than 50 shakes after 2 weeks. For preparations containing 0.05 mg/ml aluminum, approximately 20 shakes were required after 2 weeks of storage in a horizontal position. The data is presented in Figure 37.

A decrease in the amount of antigen associated with aluminum was also observed. The data is presented in Figure 38.

Example 22: Screening of different flocculants

A suspension with very fine particles has a low settling rate and will result in a very dense, low volume precipitate that will be difficult or impossible to resuspend. The resuspension characteristics can be controlled by pharmaceutical manipulations such as the addition of flocculants to neutralize the surface charge on the suspended particles, which allows the formation of larger floes, or clusters, of the particles, thereby increasing the rate of sedimentation and facilitating resuspension. Differences in sedimentation characteristics between non-flocculated and flocculated slurries are shown in Table 42.

Table 42. Comparison of non-flocculated and flocculated suspensions

not flocculated flocculated 1 Solids present as single particles, low aggregation Solids are present in the form of aggregates 2 Particles exhibit repulsive forces Particles exhibit attractive or weak repulsive forces 3 Sedimentation rate is low and particle sizes are small Sedimentation rate is high, particles form loose aggregates 4 Particles settle in the form of a dense precipitate Particles settle in the form of flakes

The inclusion of flocculants in GBS6 preparations was used as an attempt to improve resuspension performance. The following flocculants and aluminum salts were evaluated for their effect on the rate of sedimentation and resuspension of the high dose GBS6 formulation:

Flocculants :

1. GBS6 control (GBS6 containing 20 mM His buffer, 150 mM NaCl, 0.02% PS80 and 0.5 mg/mL AlPO4 at pH 6.5)

2. Ammonium sulfate (0.25 mg/ml)

3. Ammonium sulfate and KCl (0.25 mg/ml and 20 mM)

4. CaCl ₂ (0.25 mg/ml)

5. SDS (1%)

6. Potassium phosphate (50 mM)

7. NaCl (1M)

Aluminum salts :

1. ^Alhydrogel® 85 (Brenntag Biosector A/S) [Al(OH) ₃ ; 0.25 mg/ml]

2. Adyufos ^® (Brenntag Biosector A/S) (0.5 mg/ml)

3. AlPO ₄ and Al(OH) ₃ (1:1 mixture, 0.25 mg/ml each).

High dose (240 μg/mL) GBS6 preparations were prepared containing 20 mM histidine, 150 mM NaCl, 0.02% PS80, and 0.5 mg/mL AlPO ₄ at pH 6.5, with any of the flocculants listed above, or one of the aluminum salts listed above. Samples were tested for reduction in resuspension time (after centrifuging samples for 45 minutes at 200 rpm in 1.5 ml centrifuge tubes), settling rate, particle size, surface charge, and total and bound conjugates.

Among all the flocculants tested, NaCl and potassium phosphate were able to lead to a strong reduction in resuspension time to less than 20 seconds compared to 180 seconds for the GBS6 control preparation. The data are presented in Figure 39 and Table 42. The settling rate of these two samples was also less than that of other calcium or ammonium salt or SDS formulations. The data are shown in Figure 40. The formulation containing 1 M NaCl had a large particle size and a low surface charge, as expected from flocculation theory. However, the same results were not observed for samples containing 50 mM phosphate, indicating that phosphate promoted resuspension by a different mechanism than NaCl. Data are shown in Table 42.

Of the two aluminum salts tested, Alhydrogel ^® 85 showed better resuspension characteristics than Adjuphos ^® . The main problem with the formulation containing Alhydrogel ^® 85 is that it binds strongly to antigens, as evidenced by the higher antigen binding strength compared to AlPO ₄ . The data are shown in Table 43. There are reports that this phenomenon adversely affects the immune response caused by vaccines. Egan, PM, et al., Vaccine, 27:3175-3180 (2009).

Table 43 Resuspension characteristics of GBS6 preparations with flocculant or aluminum salt

Lot 00708311-114 Particle size (µm) Resuspension time (sec) Zeta potential (mOsm) Binding (%) D10 D50 D90 1 GBS6 control 4.46 7.25 12.27 180 -7.9 40.8 2 0.25 mg/ml ammonium sulfate 4.40 7.23 11.90 47 -7.2 36.8 3 sulfate+KCl 4.62 7.81 15.83 33 -7.8 37.1 4 0.25 mg _CaCl2 4.47 7.34 13.27 90 -6.9 40.5 5 Alhydrogel ^® 85 and AlPO ₄ 4.96 6.95 9.76 40 -9.2 68.2 6 1.0% SDS 5.13 6.94 9.52 155 -20.0 17.6 7 50 mM phosphate buffer 4.53 7.27 12.33 20 -12.5 10.8 8 1 M NaCl 7.10 13.10 25.13 17 -1.5 11.2 9 0.25 mg/ml Alhydrogel ^® 85 12.80 23.20 42.10 10 -5.3 93.1 10 0.25 mg/ml adyufos ^® 2.37 5.27 18.87 120 -8.5 16.2

Example 23 Optimizing NaCl and Phosphate Levels

A study was made to optimize the concentrations of NaCl and potassium phosphate to levels that would correspond not only to the desired characteristics of resuspension, but also to other indicators, such as osmolality and antigen binding to aluminum, which are two important aspects associated with the reactogenicity and efficacy of the vaccine.

Preparations of GBS6 were prepared at a high dose (total dose of antigen 240 μg/ml) containing NaCl at different concentrations (150-690 mm), 20 mm histidine, 0.02% PS80 and 0.5 mg/ml AlPO ₄ at pH 6, 5. Also prepared preparations containing potassium phosphate at different concentrations (10-50 mm) and NaCl at two different concentrations (150 mm and 240 mm) to study the effect of combining two salts. The formulated bulk samples were tested for settling rate, resuspension time after centrifugation, particle size and surface charge.

Increasing the NaCl concentration led to a significant decrease in the resuspension time. The data are shown in Figure 41. There was a sharp decrease in resuspension time in the case of 300 mM NaCl, indicating that the drug had gone from a deflocculated to a flocculated state, and the 300 mM NaCl concentration was the critical coagulation concentration (CCC) for this transition. The CCC was reduced to 240 mM in the presence of 10 mM or more phosphate, indicating that there was a synergistic effect of NaCl and phosphate. The data are presented in Figure 42. Rapid settling and flocculation was observed in samples containing 10-30 mm phosphate with 240 mm NaCl, as shown in Figures 43A and 43B. The zeta potential and particle size data showed that an increase in NaCl concentration leads to a decrease in surface charge and an increase in particle size, which was in favor of the flocculation theory. This trend was not evident in the case of phosphate. The data are shown in Table 44. Based on the results of this study, 240 mM NaCl and 20 mM phosphate were found to be optimal target concentrations.

Table 44 Effect of NaCl and Phosphate on Zeta Potential and Particle Size

Zeta potential Particle size DP (mV) SO D10 D50 D90 150 mM NaCl -9.1 0.5 2.6 5.5 16.3 200 mM NaCl -7.6 0.8 3.3 6.3 12.4 250 mM NaCl -8.2 0.9 3.5 6.6 15.4 300 mM NaCl -5.5 0.5 4.3 7.1 11.9 390 mM NaCl -6.8 0.9 4.6 7.2 11.5 540 mM NaCl -5.1 0.5 5.3 9.3 19.9 690 mM NaCl -3.4 0.5 5.9 10.8 22.9 150 mM NaCl + 10 mM phosphate -8.9 0.4 3.4 6.3 11.1 150 mM NaCl + 20 mM phosphate -8.3 0.8 3.2 6.2 12.6 150 mM NaCl + 30 mM phosphate -9.9 0.9 3.2 6.2 12.2 240 mM NaCl + 10 mM phosphate -6.0 0.4 5.3 6.5 7.9 240 mM NaCl + 20 mM phosphate -10.5 0.9 4.3 7.1 11.6 240 mM NaCl + 30 mM phosphate -5.5 2.1 4.5 7.4 13.4

Additionally, three batches of GBS6 preparation were characterized containing polysaccharide at a concentration of 40 μg/ml for each serotype (total dose of antigen 240 μg/ml), 20 mM phosphate, 240 mM NaCl, 0.02% PS80 and 0.5 mg/ml AlPO ₄ at pH 6.5. A GBS6 preparation containing 20 mM His buffer, 150 mM NaCl, 0.02% PS80 and 0.5 mg/ml AlPO ₄ at pH 6.5 was included as a control. All 3 formulations supplemented with NaCl and phosphate at pH 6.5 were easy to resuspend with an average resuspension time of 16 seconds (versus 120 seconds for control) and had osmolality above 500 mOsm. The amount of associated antigenicity was approximately 20% lower than control for each of the serotypes. Data are shown in Table 45.

Table 45 Characteristics of GBS6 formulations with additional NaCl and phosphate content

Resuspension (sec) Osmo (mOsm) Zeta potential (mV) Antigen binding to AlPO4 (%) Particle size (µm) Ia Ib II III IV V D10 D50 D90 GBS6 control 120 316 -9.1 41 43 51 34 41 51 2.6 5.5 16.3 GBS6 with 20 mM phosphate and 240 mM NaCl at pH 6.5* 16 519 -6.0 24 21 23 21 22 29 5.3 6.5 7.9

*Average of 3 batches.

Example 24 Short Term Stability of GBS6 Preparations Containing 240 mM NaCl and 20 mM Phosphate

GBS6 preparations in three doses (total dose of antigen 60 µg/ml, 120 µg/ml and 240 µg/ml) containing 20 mM phosphate, 240 mM NaCl, 0.02% polysorbate 80 and 0.5 mg/ml AlPO ₄ at pH 6.5, loaded into BD HYPAK SCF™ PRTC (Becton, Dickinson and Company) 1 ml short glass PFS with a fill volume of 0.58 ml and stoppered with 4432/50 stoppers (West Pharmaceutical Services, Inc.) leaving space above the product approximately ~0.4 mm. Resuspension after storage both tip-down and horizontal for 1 month at RT and 3 months at 5°C was monitored for stability.

Data after one month showed that the drug after storage in the PFS in a horizontal position could be resuspended using 10 shakes or less at all three dose levels. However, a preparation stored tip down in the PFS still required 40 or more shakes. Data are shown in Table 46. Total antigenicity and binding to AlPO₄ for each serotype were stable for 1 month at RT. Data are shown in Table 47.

Table 46 Comparison of resuspension characteristics of PFS preparations stored horizontally and tip-down

Dose pH Storage time at 2-8°C (months) Horizontal (number of shakes) Tip down (number of shakes) Low 6.5 1 month 3 40 Medium 6.5 8 >45 high 6.5 7 >50 high 6.0 10 50

Table 47 Overall antigenicity of GBS preparations

Dose Time point Total antigenicity (µg/ml) Ia Ib II III IV V high 0 40 41.2 41.1 42.0 40.4 39.1 1m RT 44.8 43.1 44.6 44.3 43.7 44.6 Medium 0 18.3 17.9 19.0 18.2 18.4 17.9 1m RT 19.5 19.3 21.7 20.4 20.0 20.6 Low 0 10.4 11.6 9.5 8.6 9.2 9.9 1m RT 9.0 9.1 9.5 9.2 9.0 9.3 % Binding to AlPO ₄ high 0 30.8 24.6 26.2 18.7 22.3 31.2 1m RT 33.3 29.7 33.3 19.9 25.9 35.0 Medium 0 39.9 40.0 29.1 20.0 25.5 37.9 1m RT 45.0 40.5 39.9 33.6 34.0 40.8 Low 0 49.3 52.5 38.0 25.3 31.2 45.2 1m RT 51.5 42.8 41.4 30.1 37.6 45.3

Example 25: Application of Design of Experiment (DoE) and Planned Quality (QbD) concepts for GBS preparations with flocculants in PFS

The design of experiment (DoE) concept was applied to determine the optimal GBS6 preparation for PFS. Three factors were assessed using response surface methodology (Box-Behnken statistical plan): 1) pH (6.0, 6.5 and 7.0); 2) NaCl concentration (220 mM, 240 mM and 260 mM) and 3) phosphate concentration (15 mM, 20 mM and 25 mM). Otherwise, all preparations contained a dose of 240 mg/ml (60 μg/ml for each serotype), 0.02% PS80, approximately 10 mM histidine transferred from the drug substance, and 0.5 mg/ml AlPO ₄ . A total of 15 formulations were tested for ease of resuspension (see Table 48).

Table 48 Drugs in the DoE Study

A drug NaCl (mM) Na-phosphate (mM) pH 1 240 20 6.5 2 260 15 6.5 3 240 15 7.0 4 220 25 6.5 5 260 25 6.5 6 240 25 6.0 7 240 20 6.5 8 240 15 6.0 9 220 20 7.0 10 240 20 6.5 eleven 260 20 6.0 12 240 25 7.0 13 220 15 6.5 14 220 20 6.0 15 260 20 7.0

To account for any associated variability, studies were performed in triplicate and by two operators. Samples were loaded into BD HYPAK SCF™ PRTC (Becton, Dickinson and Company) 1 ml short glass PFS with a 0.58 ml fill volume and sealed with 4432/50 stoppers (West Pharmaceutical Services, Inc.) leaving a headspace of approximately ~ 0.4 mm. Filled syringes were stored horizontally at 2-8°C and 25°C for 3 months. Resuspension was monitored only in the case of storage at 5°C, and the concentration by nephelometry was controlled in the case of storage at 5°C and 25°C. The resuspension results are shown in Table 49.

Table 49 Number of Shakings Required for Resuspension at 3 Months

A drug Operator 1* Operator 2* 1 eleven 8 2 8 6 3 7 4 4 eleven 8 5 8 8 6 19 12 7 7 7 8 19 14 9 10 6 10 8 6 eleven eleven 13 12 8 4 13 eleven eleven 14 28 17 15 5 5

*Average value for 3 samples.

The statistical program JMP (SAS Institute) was used to analyze the data and predict the optimal and most reliable drug, which would require ≤10 shakes to resuspend. The recommendations of the JMP profiling system based on resuspension data obtained by one of the operators are shown in Figure 45. pH and salt concentration were significant individual factors influencing the number of shakes. In addition, the interaction between phosphate and pH was important. Based on the desirability index, the following conditions allow easier resuspension (number of shakes ≤10): pH 7.0±0.5, 245±20 mM NaCl and 20±5 mM phosphate. Similar results were obtained in the analysis of data obtained by the second operator.

However, when using the recommended drug parameters, the degree of binding was lower. As shown in Figure 46, conjugate binding for each of the GBS serotypes varied between 10-20%. In addition, the recommended preparation had a higher osmolality (approximately 500 mOsm/kg), which, however, was still within the acceptable range for the vaccine.

Example 26 Al(OH) GBS6 Preparations ₃

As described in Example 22, Al(OH) ₃ GBS6 preparations had excellent resuspension characteristics, but there was stronger antigen binding, which could adversely affect the immune response. Accordingly, experiments were performed to characterize the binding of Al(OH) ₃ to GBS conjugates and to modulate the adsorption strength of Al(OH) ₃ .

High dose GBS6 preparations (total dose of conjugates 240 µg/mL) containing 20 mM histidine, 150 mM NaCl and 0.02% PS80 at pH 6.5 were prepared with different concentrations of Al(OH) ₃ (0.7 mg/mL). ml, 0.6 mg/ml, 0.5 mg/ml, 0.4 mg/ml, 0.3 mg/ml, 0.2 mg/ml, 0.15 mg/ml, 0.1 mg/ml and 0.05 mg/ml). Binding was determined by measuring total and supernatant antigenicity by nephelometry. As shown in Figure 47, Al(OH) ₃ binding plateaued at 0.5 mg/ml Al(OH) ₃ for all six serotypes. The adsorption capacity and adsorption coefficient were determined by the Langmuir formula. Data are shown in Table 50, with data for AlPO ₄ as comparator.

Table 50 Binding Characteristics of AlPO GBS Conjugates ₄ and Al(OH) ₃

Serotype GBS adsorption capacity
µg serotype antigen/mg Al Adsorption coefficient
ml/mg protein With AlPO ₄ Ia 39.5 1488.2 Ib 38.6 1786.2 II 41.8 377.6 III 25.6 1649.8 IV 29.8 1407.6 V 41.7 1403.5 With Al(OH) ₃ Ia 109.9 5352.9 Ib 117.6 3400.0 II 161.3 3263.2 III 122.0 2411.8 IV 138.9 3428.6 V 147.1 2956.5

To modulate the strength of Al(OH) ₃ binding, the buffer in the preparation was replaced by sodium phosphate in order to replace surface hydroxyls on aluminum hydroxide with phosphate and reduce ligand exchange. GBS6 preparations containing 150 mM NaCl, 0.02% PS80 and 0.15 mg/mL Al(OH) ₃ at pH 6.5 were prepared with 3 different concentrations of phosphate (10 mM, 20 mM and 30 mM) and in different doses (total dose of conjugates 15 µg/ml, 30 µg/ml, 60 µg/ml, 90 µg/ml, 120 µg/ml, 150 µg/ml and 210 µg/ml). Binding was determined based on total and supernatant antigenicity. The adsorption capacity and adsorption coefficient were determined by the Langmuir formula. Data are shown in Table 51, with data for AlPO ₄ as comparator.

Table 51. Effect of phosphate concentration on the binding of GBS conjugates to Al(OH) ₃

Serotype GBS Conc. Na-phosphate (mM)* Adsorption coefficient (ml/mg) Adsorption capacity (µg serotype antigen/mg Al) Binding to Al (%)** % Extraction*** Ia 0 5353 110 >95 14 10 890 32 66 103 20 665 26 52 101 thirty 429 19 45 109 Preparation with AlPO ₄ 40 1488 46 on Ib 0 3400 118 >95 6 10 1622 56 66 93 20 520 21 63 105 thirty 338 15 44 103 Preparation with AlPO ₄ 39 1786 50 on II 0 3263 161 >95 2 10 1333 86 80 99 20 852 79 75 105 thirty 711 67 66 103 Preparation with AlPO ₄ 42 378 57 on III 0 2412 122 >95 10 10 878 37 66 104 20 478 23 47 107 thirty 207 7 thirty 109 Preparation with AlPO ₄ 26 1650 37 on IV 0 3429 139 >95 4 10 2571 62 73 102 20 1509 39 63 104 thirty 1114 29 47 95 Preparation with AlPO ₄ thirty 1408 44 on V 0 4957 147 >95 3 10 2040 65 77 104 20 1982 45 64 106 thirty 1758 47 58 95 Preparation with AlPO ₄ 42 1404 53 on

*AlPO ₄ formulation contains ~10 mM histidine transferred from the drug substance;

**Formulation binding to AlPO ₄ is the average of 3 batches;

***Extraction conditions: 0.1 N NaOH, RT, rotation for 24 hours.

The binding capacity and binding ratio of GBS6 with Al(OH) ₃ were 3-4 times higher than with AlPO ₄ . However, the aluminum hydroxide adsorption strength for GBS6 was modulated by introducing phosphate into the preparation. Phosphate concentration had a negative effect on the strength and capacity of adsorption. Phosphate also affected the binding, in percent, of the antigen, however, the level was still higher than in the case of AlPO ₄ . In addition, GBS could be fully extracted from aluminum in the presence of phosphate. Based on the results obtained, it was determined that 20 mM phosphate, 150 mM NaCl and 0.2% PS80 at pH 6.5 were optimal values for use with aluminum hydroxide.

Aspects of the invention

The following paragraphs describe additional embodiments of the invention.

C1. An immunogenic polysaccharide-protein conjugate comprising a Group B Streptococcus (GBS) capsular polysaccharide and a carrier protein, wherein the capsular polysaccharide has a sialic acid level greater than about 60%.

C2. An immunogenic conjugate according to Claim C1 wherein the capsular polysaccharide is selected from the group consisting of polysaccharides of serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII and IX.

C3. The immunogenic conjugate of claim C2, wherein the capsular polysaccharide is serotype Ia.

C4. The immunogenic conjugate of claim C2, wherein the capsular polysaccharide is serotype Ib.

C5. The immunogenic conjugate of claim C2, wherein the capsular polysaccharide is serotype II.

C6. The immunogenic conjugate of claim C2, wherein the capsular polysaccharide is serotype III.

C7. The immunogenic conjugate of claim C2, wherein the capsular polysaccharide is serotype IV.

C8. The immunogenic conjugate of claim C2, wherein the capsular polysaccharide is serotype V.

C9. The immunogenic conjugate of claim C2, wherein the capsular polysaccharide is serotype VI.

C10. The immunogenic conjugate of claim C2, wherein the capsular polysaccharide is serotype VII.

C11. The immunogenic conjugate of claim C2, wherein the capsular polysaccharide is serotype VIII.

C12. The immunogenic conjugate of claim C2, wherein the capsular polysaccharide is serotype IX.

C13. The immunogenic conjugate of any one of C1-C12, wherein the capsular polysaccharide has a sialic acid level greater than about 95%.

C14. An immunogenic conjugate according to any one of C1-C13, wherein the capsular polysaccharide has a sialic acid level of about 100%.

C15. The immunogenic conjugate of any one of C1-C14, wherein the capsular polysaccharides have at least about 0.6 mM sialic acid per mM polysaccharide.

C16. An immunogenic conjugate according to any one of C1-C15, wherein the capsular polysaccharides have at least about 0.65 mM sialic acid per mM polysaccharide.

C17. The immunogenic conjugate of any one of C1-C16, wherein the capsular polysaccharides have at least about 0.7 mM sialic acid per mM polysaccharide.

C18. The immunogenic conjugate of any one of C1-C17, wherein the capsular polysaccharides have at least about 0.75 mM sialic acid per mM polysaccharide.

C19. The immunogenic conjugate of any one of C1-C18, wherein the capsular polysaccharides have at least about 0.8 mM sialic acid per mM polysaccharide.

C20. The immunogenic conjugate of any one of C1-C19, wherein the capsular polysaccharides have at least about 0.85 mM sialic acid per mM polysaccharide.

C21. The immunogenic conjugate of any one of C1-C20, wherein the capsular polysaccharides have at least about 0.9 mM sialic acid per mM polysaccharide.

C22. The immunogenic conjugate of any one of C1-C21, wherein the capsular polysaccharides have at least about 0.95 mM sialic acid per mM polysaccharide.

C23. The immunogenic conjugate of any one of C1-C22, wherein the capsular polysaccharide has a molecular weight of from about 5 kDa to about 1000 kDa.

C24. The immunogenic conjugate of any one of C1-C23, wherein the capsular polysaccharide has a molecular weight of from about 25 kDa to about 750 kDa.

C25. An immunogenic conjugate according to any one of C1-C24, wherein the capsular polysaccharide has a molecular weight of from about 25 kDa to about 400 kDa.

C26. The immunogenic conjugate of any one of C1-C25, wherein the capsular polysaccharide has a molecular weight of from about 25 kD to about 200 kD.

C27. An immunogenic conjugate according to any one of C1-C25, wherein the capsular polysaccharide has a molecular weight of from about 100 kDa to about 400 kDa.

C28. An immunogenic conjugate according to any one of C1-C27, wherein the molecular weight of the conjugate is from about 300 kDa to about 20,000 kDa.

C29. An immunogenic conjugate according to any one of paragraphs C1-C28, wherein the molecular weight of the conjugate is from about 1000 kDa to about 15000 kDa.

C30. An immunogenic conjugate according to any one of C1-C29, wherein the molecular weight of the conjugate is from about 1000 kDa to about 10,000 kDa.

C31. The immunogenic conjugate of any one of C1-C30, wherein the capsular polysaccharide has an O-acetylation level of from about 0% to about 40%.

C32. The immunogenic conjugate of any one of C1-C31, wherein the capsular polysaccharide has an O-acetylation level of less than about 5%.

C33. The immunogenic conjugate of any one of C1-C32, wherein the capsular polysaccharide has an O-acetylation level of less than about 4%.

C34. The immunogenic conjugate of any one of C1-C33, wherein the capsular polysaccharide has an O-acetylation level of less than about 3%.

C35. The immunogenic conjugate of any one of C1-C34, wherein the capsular polysaccharide has an O-acetylation level of less than about 2%.

C36. The immunogenic conjugate of any one of C1-C35, wherein the capsular polysaccharide has an O-acetylation level of less than about 1%.

C37. The immunogenic conjugate of any one of C1-C36, wherein the capsular polysaccharide contains at least about 0.1 mM O-acetate per mM saccharide repeat unit.

C38. The immunogenic conjugate of any one of C1-C37, wherein the capsular polysaccharide contains at least about 0.2 mM O-acetate per mM saccharide repeat unit.

C39. The immunogenic conjugate of any one of C1-C38, wherein the capsular polysaccharide contains at least about 0.3 mM O-acetate per mM saccharide repeat unit.

C40. The immunogenic conjugate of any one of C1-C39, wherein the capsular polysaccharide contains at least about 0.35 mM O-acetate per mM saccharide repeat unit.

C41. An immunogenic conjugate according to any one of C1-C40, wherein the capsular polysaccharide contains approximately 0.4 mM O-acetate per mM saccharide repeating unit.

C42. The immunogenic conjugate of any one of C1-C41, wherein the capsular polysaccharide contains less than about 0.01 mM O-acetate per mM saccharide repeat unit.

C43. The immunogenic conjugate of any one of C1-C42, wherein the capsular polysaccharide contains less than about 0.05 mM O-acetate per mM saccharide repeat unit.

C44. The immunogenic conjugate of any one of C1-C43, wherein the capsular polysaccharide contains less than about 0.04 mM O-acetate per mM saccharide repeat unit.

C45. The immunogenic conjugate of any one of C1-C44, wherein the capsular polysaccharide contains less than about 0.03 mM O-acetate per mM saccharide repeat unit.

C46. The immunogenic conjugate of any one of C1-C45, wherein the capsular polysaccharide contains less than about 0.02 mM O-acetate per mM saccharide repeat unit.

C47. An immunogenic conjugate according to any one of C1-C46, wherein all polysaccharides are separately conjugated to a carrier protein.

C48. The immunogenic conjugate of any one of C1-C47, wherein the carrier protein is CRM ₁₉₇ or tetanus toxoid.

C49. The immunogenic conjugate of any one of C1-C48, wherein the carrier protein is CRM ₁₉₇ .

C50. A method for isolating a capsular polysaccharide, which includes reacting an organic reagent with a cell broth containing a capsular polysaccharide-producing bacterium.

C51. The method of claim C50 wherein the bacterium is not lysed.

C52. The method according to C50 or C51, wherein the bacterium is killed by heating.

C53. The method of any one of C50-C52 further comprising the step of centrifuging to obtain a cell paste.

C54. The method according to any one of paragraphs C50-C53, further comprising the step of filtering.

C55. The method of claim C54 wherein said filtration step is diafiltration.

C56. The method of any one of C50-C56, wherein the capsular polysaccharide-producing bacterium is selected from the group consisting of Streptococcus agalactiae , Streptococcus pneumoniae , Staphylococcus aureus , Neisseria meningitidis, Escherichia coli, Salmonella typhi, Haemophilus influenzae, Klebsiella pneumoniae, Enterococcus faecium, and Enterococcus faecalis .

C57. The method of claim C56, wherein the bacterium is Streptococcus agalactiae.

C58. The method of any one of C50-C57, wherein said organic reactants are derivatized hydroxylamine compounds.

C59. The method of any one of C50-58, wherein the hydroxylamine is any of the hydroxylamines shown in Table 2 of Example 2.

C60. The method of any one of C50-C59, wherein the hydroxylamine is selected from the group consisting of dibenzylhydroxylamine; diethylhydroxylamine; hydroxylamine; ethylenediamine; triethylenetetramine; 1,1,4,7,10,10-hexamethyltriethylenetetramine and 2,6,10-trimethyl-2,6,10-triazoundecane.

C61. The method of any one of C50-C60, wherein the concentration of hydroxylamine is from about 5 mM to about 200 mM.

C62. The method of any one of C50-C61, wherein the pH of the reaction mixture is from about 5.5 to about 9.5.

C63. The method according to any one of items C50-C62, in which the reaction occurs at a temperature from about 20°C to about 85°C.

C64. The method of any one of C50-C63, wherein the reaction time is from about 10 hours to about 90 hours.

C65. A method for producing an immunogenic polysaccharide-protein conjugate according to any one of C1-C49, wherein the capsular polysaccharide is isolated by a method according to any one of C50-C64.

C66. An immunogenic polysaccharide-protein conjugate comprising a capsular polysaccharide obtained by the method of any one of C50-C64.

C67. An immunogenic composition comprising an immunogenic polysaccharide-protein conjugate according to any one of C1-C49 or C66.

C68. Immunogenic composition containing polysaccharide-protein conjugates containing capsular polysaccharides from group B streptococcus (GBS) serotype IV and at least one additional serotype selected from the group consisting of Ia, Ib, II, III, V, VI, VII, VIII and IX.

C69. The immunogenic composition of claim C68, wherein at least one additional serotype is serotype Ia.

C70. The immunogenic composition of claim C69, further comprising a conjugate containing a capsular polysaccharide from GBS serotype Ib.

C71. An immunogenic composition according to C69 or C70, further comprising a conjugate containing a capsular polysaccharide from GBS serotype II.

C72. An immunogenic composition according to any one of C69-C71, further comprising a conjugate containing a capsular polysaccharide from GBS serotype III.

C73. An immunogenic composition according to any one of C69-C72, further comprising a conjugate containing a capsular polysaccharide from GBS serotype V.

C74. An immunogenic composition according to any one of C69-C73, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VI.

C75. An immunogenic composition according to any one of C69-C74, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VII.

C76. An immunogenic composition according to any one of C69-C75, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VIII.

C77. An immunogenic composition according to any one of C69-C76, further comprising a conjugate containing a capsular polysaccharide from GBS serotype IX.

C78. The immunogenic composition of claim C68, wherein at least one additional serotype is serotype Ib.

C79. The immunogenic composition of claim C78, further comprising a conjugate containing a capsular polysaccharide from GBS serotype II.

C80. An immunogenic composition according to C78 or C79, further comprising a conjugate containing a capsular polysaccharide from GBS serotype III.

C81. An immunogenic composition according to any one of C78-C80, further comprising a conjugate containing a capsular polysaccharide from GBS serotype V.

C82. An immunogenic composition according to any one of C78-C81, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VI.

C83. An immunogenic composition according to any one of C78-C82, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VII.

C84. An immunogenic composition according to any one of C78-C83, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VIII.

C85. An immunogenic composition according to any one of C78-C84, further comprising a conjugate containing a capsular polysaccharide from GBS serotype IX.

C86. The immunogenic composition of claim C68, wherein at least one additional serotype is serotype II.

C87. The immunogenic composition of claim C86, further comprising a conjugate containing a capsular polysaccharide from GBS serotype III.

C88. An immunogenic composition according to C86 or C87, further comprising a conjugate containing a capsular polysaccharide from GBS serotype V.

C89. An immunogenic composition according to any one of C86-C88, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VI.

C90. An immunogenic composition according to any one of C86-C89, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VII.

C91. An immunogenic composition according to any one of C86-C90, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VIII.

C92. An immunogenic composition according to any one of C86-C91, further comprising a conjugate containing a capsular polysaccharide from GBS serotype IX.

C93. The immunogenic composition of claim C68, wherein at least one additional serotype is serotype III.

C94. The immunogenic composition of claim C93, further comprising a conjugate containing a capsular polysaccharide from GBS serotype V.

C95. An immunogenic composition according to C93 or C94, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VI.

C96. An immunogenic composition according to any one of C93-C95, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VII.

C97. An immunogenic composition according to any one of C93-C96, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VIII.

C98. An immunogenic composition according to any one of C93-C97, further comprising a conjugate containing a capsular polysaccharide from GBS serotype IX.

C99. An immunogenic composition according to C68, wherein at least one additional serotype is serotype V.

C100. The immunogenic composition of claim C99, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VI.

C101. An immunogenic composition according to any one of C99 or C100, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VII.

C102. An immunogenic composition according to any one of C99-C101, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VIII.

C103. An immunogenic composition according to any one of C99-C102, further comprising a conjugate containing a capsular polysaccharide from GBS serotype IX.

C104. The immunogenic composition of claim C68, wherein at least one additional serotype is serotype VI.

C105. The immunogenic composition of claim C104, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VII.

C106. An immunogenic composition according to any one of C104 or C105, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VIII.

C107. An immunogenic composition according to any one of C104-C106, further comprising a conjugate containing a capsular polysaccharide from GBS serotype IX.

C108. The immunogenic composition of claim C68, wherein at least one additional serotype is serotype VII.

C109. The immunogenic composition of claim C108, further comprising a conjugate containing a capsular polysaccharide from GBS serotype VIII.

C110. An immunogenic composition according to any one of C108 or C109, further comprising a conjugate containing a capsular polysaccharide from GBS serotype IX.

C111. The immunogenic composition of claim C68, wherein at least one additional serotype is serotype VIII.

C112. The immunogenic composition of claim C111 further comprising a conjugate comprising a capsular polysaccharide from GBS serotype IX.

C113. The immunogenic composition of claim C112, wherein at least one additional serotype is serotype IX.

C114. Immunogenic composition containing polysaccharide-protein conjugates containing capsular polysaccharides from GBS serotypes Ia, Ib, II, III and IV.

C115. Immunogenic composition containing polysaccharide-protein conjugates containing capsular polysaccharides from GBS serotypes Ia, Ib, II, III and V.

C116. Immunogenic composition containing polysaccharide-protein conjugates containing capsular polysaccharides from GBS serotypes Ia, Ib, II, III, IV and V.

C117. An immunogenic composition containing polysaccharide-protein conjugates containing at least four GBS capsular polysaccharides of serotypes selected from the group consisting of serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII and IX.

C118. The immunogenic composition of claim C117 comprising GBS capsular polysaccharides of at least five serotypes.

C119. An immunogenic composition according to C117 or C118, comprising GBS capsular polysaccharides of at least six serotypes.

C120. An immunogenic composition according to any one of C117-C119, comprising GBS capsular polysaccharides of at least seven serotypes.

C121. An immunogenic composition according to any one of C117-C120, comprising GBS capsular polysaccharides of at least eight serotypes.

C122. An immunogenic composition according to any one of C117-C121, comprising GBS capsular polysaccharides of at least nine serotypes.

C123. An immunogenic composition according to any one of C117-C122, comprising GBS serotype V capsular polysaccharide.

C124. An immunogenic composition according to any one of C117-C123, characterized by the absence of immune interference.

C125. The immunogenic composition of any one of C67-C124 further comprising a pharmaceutically acceptable excipient, buffer, stabilizer, adjuvant, cryoprotectant, salt, divalent cation, nonionic detergent, free radical induced oxidation inhibitor, carrier, or a mixture thereof.

C126. The immunogenic composition of any one of C67-C125 further comprising a buffer.

C127. The immunogenic composition of claim C126 wherein the buffer is selected from the group consisting of HEPES, PIPES, MES, Tris (trimethamine), phosphate, acetate, borate, citrate, glycine, histidine, and succinate.

C128. The immunogenic composition of claim C127, wherein the buffer is histidine.

C129. The immunogenic composition of any one of C67-C128 further comprising a surfactant.

C130. The immunogenic composition of claim C129, wherein the surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid esters, polysorbate-80, polysorbate-60, polysorbate-40, polysorbate-20, and polyoxyethylene alkyl esters.

C131. The immunogenic composition of claim C130, wherein the surfactant is polysorbate-80.

C132. The immunogenic composition of any one of C67-C131 further comprising an excipient.

C133. The immunogenic composition of claim C132, wherein the excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melisitose, dextran, mannitol, lactitol, palatinite, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerin monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerin, propylene glycol, water, ethanol.

C134. The immunogenic composition of claim C133, wherein the excipient is sodium chloride.

C135. The immunogenic composition of any one of C67-C134 further comprising an adjuvant.

C136. The immunogenic composition of claim C135, wherein the adjuvant is an aluminum-based adjuvant or QS-21.

C137. The immunogenic composition of claim C136 wherein the aluminum adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxyphosphate and aluminum hydroxide.

C138. The immunogenic composition of claim C137, wherein the adjuvant is aluminum phosphate.

C139. The immunogenic composition of claim C138, wherein the adjuvant is aluminum hydroxyphosphate.

C140. An immunogenic composition according to any one of C67-C139, comprising a buffer, a surfactant, an excipient, and optionally an adjuvant, wherein the pH of the composition buffer is from about 6.0 to about 7.0.

C141. An immunogenic composition according to any one of C67-C140, comprising histidine, polysorbate-80, sodium chloride, and optionally aluminum phosphate, wherein the pH of the composition buffer is from about 6.0 to about 7.0.

C142. An immunogenic composition according to any one of C67-C141, comprising from about 10 mM to about 25 mM histidine, from about 0.01% to about 0.03% (v/w) polysorbate-80, about 10 mM to about 250 mM sodium chloride, and optionally about 0.25 mg/ml to about 0.75 mg/ml aluminum as aluminum phosphate.

C143. An immunogenic composition according to any one of C67-C142, containing a dose of from about 5 µg/ml to about 50 µg/ml.

C144. The immunogenic composition of any one of C67-C143, optionally lyophilized in the presence of at least one excipient.

C145. The immunogenic composition of claim C144, wherein at least one excipient is selected from the group consisting of starch, glucose, lactose, sucrose, trehalose, raffinose, stachyose, melisitose, dextran, mannitol, lactitol, palatinite, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerin monostearate, talc, glycine, arginine, lysine, sodium chloride (NaCl), skimmed milk powder, glycerin, propylene glycol, water and ethanol.

C146. The immunogenic composition of claim C145, wherein at least one excipient is sucrose.

C147. An immunogenic composition according to any one of paragraphs C144-C146, containing from about 1% (w/v) to about 10% (w/v) of at least one excipient.

C148. An immunogenic composition according to any one of paragraphs C144-C147, containing an additional excipient.

C149. The immunogenic composition of claim C148, wherein the additional excipient is mannitol or glycine.

C150. An immunogenic composition according to item C148 or C149 containing from about 1% (w/v) to about 10% (w/v) of an additional excipient.

C151. An immunogenic composition according to any one of C143-C150, reconstituted with water, water for injection (WFI), adjuvant suspension or saline.

C152. An immunogenic composition according to any one of C67-C151 for use as a medicine.

C153. An immunogenic composition according to any one of C67-C152 for use in a method for inducing an immune response against GBS in a patient.

C154. The immunogenic composition of claim C153, wherein the patient is a woman planning a pregnancy or a pregnant woman.

C155. The immunogenic composition of claim C154, wherein the woman is in the second half of her pregnancy.

C156. The immunogenic composition of claim C155, wherein the pregnant woman is at least 20 weeks pregnant.

C157. The immunogenic composition of claim C156, wherein the pregnant woman is at least 27 weeks to 36 weeks pregnant.

C158. The immunogenic composition of claim C157, wherein the patient is an adult 50 years of age or older.

C159. The immunogenic composition of claim C158, wherein the patient is an adult 65 years of age or older.

C160. The immunogenic composition of claim C159, wherein the patient is an adult 85 years of age or older.

C161. An immunogenic composition according to any one of C153-C160, wherein the patient is immunocompromised.

C162. The immunogenic composition of claim C161, wherein the patient has a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease, or liver disease.

C163. The immunogenic composition of any one of C153-162, wherein the group B streptococcus is Streptococcus agalactiae .

C164. A method of inducing an immune response against group B streptococcus, comprising administering to a patient an effective amount of an immunogenic composition according to any one of C67-C150.

C165. A method of preventing, or alleviating, a disease or condition associated with group B streptococcus in a patient, comprising administering to the patient an effective amount of the immunogenic composition of any one of C67-C151.

C166. The method according to C164 or C165, wherein the patient is a woman planning a pregnancy or a pregnant woman.

C167. The method of claim C166, wherein the woman is in the second half of her pregnancy.

C168. The method according to C166 or C167, wherein the pregnant woman is at least 20 weeks pregnant.

C169. The method of any one of C166-C168, wherein the pregnant woman is at least 27 weeks to 36 weeks pregnant.

C170. The method of C164 or C165, wherein the patient is an adult 50 years of age or older.

C171. The method of claim C170, wherein the patient is an adult 65 years of age or older.

C172. The method of C170 or C171, wherein the patient is an adult 85 years of age or older.

C173. The method of any one of C164-C172, wherein the patient is immunocompromised.

C174. The method of claim C173, wherein the patient has a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease, or liver disease.

C175. The method of any one of C164-C174, wherein the group B streptococcus is Streptococcus agalactiae .

C176. An antibody that binds the capsular polysaccharide in an immunogenic conjugate of any one of C1-C49 or C66.

C177. A composition containing an antibody according to item C176.

C178. A method for producing antibodies, comprising administering to a patient an immunogenic composition according to any one of C67-C151.

C179. Antibody obtained by the method according to item C178.

C180. A method for creating passive immunity in a patient, comprising the steps of:

(a) obtaining an antibody preparation using an immunogenic composition according to any one of paragraphs C67-C151; And

(b) administering the antibody preparation to the patient to confer passive immunity.

C181. A process for preparing an immunogenic polysaccharide-protein conjugate according to any one of C1-C49 or C66, comprising the steps of:

(a) reacting the GBS capsular polysaccharide with an oxidizing agent to produce an activated polysaccharide; And

(b) reacting the activated polysaccharide with a carrier protein to produce a polysaccharide-protein conjugate.

C182. The method of claim C181, wherein step (b) is performed in a polar aprotic solvent.

C183. The method of claim C182 wherein the solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), sulfolane, dimethylformamide (DMF), and hexamethylphosphoramide (HMPA).

C184. The method of claim C183, wherein the solvent is dimethyl sulfoxide (DMSO).

C185. The method of any one of C181-C184, wherein the polysaccharide is reacted with 0.01-10.0 molar equivalents of an oxidizing agent.

C186. The method of any one of C181-C185, wherein the oxidizing agent is a periodate.

C187. The method of claim C186 wherein the periodate is sodium periodate.

C188. The method according to any one of C181-C187, wherein the duration of the oxidation reaction in step (a) is from 1 hour to 50 hours.

C189. The method of any one of C181-C188, wherein the oxidation reaction temperature is maintained at about 2°C to about 25°C.

C190. The method of any one of C181-C189, wherein the oxidation reaction is carried out in a buffer selected from the group consisting of sodium phosphate, potassium phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), and Bis-Tris.

C191. The method of claim C190, wherein the buffer has a concentration of from about 1 mM to about 500 mM.

C192. The method of any one of C181-C191, wherein the oxidation reaction is carried out at a pH of from about 4.0 to about 8.0.

C193. The method of C181 wherein the oxidant is 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO).

C194. The method according to C193, wherein N-chlorosuccinimide (NCS) is the co-oxidant.

C195. The method of any one of C181-C194, wherein step (a) further comprises quenching the oxidation reaction by adding a quencher.

C196. The method of any one of C182-C195, wherein the polysaccharide concentration is from about 0.1 mg/mL to about 10.0 mg/mL.

C197. The method of any one of C181-C196, wherein the activated polysaccharide has an oxidation state of 5 to 25.

C198. The method of any one of C181-C197, further comprising the step of lyophilizing the activated polysaccharide.

C199. The method of claim C198, wherein the activated polysaccharide is lyophilized in the presence of a saccharide selected from the group consisting of sucrose, trehalose, raffinose, stachyose, melisitose, dextran, mannitol, lactitol, and palatinite.

C200. The method of any one of C181-C199, wherein step (b) comprises:

(1) combining the activated polysaccharide with a carrier protein, and

(2) reacting the combined activated polysaccharide and carrier protein with a reducing agent to obtain a GBS capsular polysaccharide-carrier protein conjugate.

C201. The method of claim C200, wherein the concentration of activated polysaccharide in step (2) is from about 0.1 mg/mL to about 10.0 mg/mL.

C202. The method according to C200 or C201, wherein the starting ratio (by weight) of activated polysaccharide and carrier protein is between 5:1 and 0.1:1.

C203. The method of any one of C200-C202, wherein the reducing agent is selected from the group consisting of sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride, or zinc in the presence of Bronsted or Lewis acids, pyridine-borane, 2-picoline-borane, 2,6-diborane -methanol, dimethylamine-borane, tert-BuMe ⁱ PrN-BH ₃ , benzylamine-BH ₃ or 5-ethyl-2-methylpyridine-borane (PEMB).

C204. The method according to C203, wherein the reducing agent is sodium cyanoborohydride.

C205. The method of any one of C200-C204, wherein the amount of reducing agent is from about 0.1 to about 10.0 molar equivalents.

C206. The method according to any one of C200-C205, wherein the reduction reaction time in step (2) is from 1 hour to 60 hours.

C207. The method of any one of C200-C206, wherein the reduction reaction temperature is maintained at 10°C to 40°C.

C208. The method of any one of C181-C207, further comprising the step (step (c)) of capping unreacted aldehydes by adding a borohydride.

C209. The method of C208, wherein the amount of borohydride is from about 0.1 to about 10.0 molar equivalents.

C210. The method of claim C208, wherein the borohydride is selected from the group consisting of sodium borohydride (NaBH ₄ ), sodium cyanoborohydride, lithium borohydride, potassium borohydride, tetrabutylammonium borohydride, calcium borohydride, and magnesium borohydride.

C211. The method of claim C292, wherein the borohydride is sodium borohydride (NaBH ₄ ).

C212. The method of any one of C207-C211, wherein the duration of the capping step is from 0.1 hour to 10 hours.

C213. The method of any one of C207-C212, wherein the temperature during the capping step is maintained at about 15°C to about 45°C.

C214. The method of any one of C181-C213, further comprising the step of purifying the polysaccharide-protein conjugate.

C215. The method of any one of C181-C214, wherein the polysaccharide-protein conjugate contains less than about 40% free polysaccharide based on total polysaccharide.

C216. The method of any one of C181-C215, wherein the ratio (by weight) of polysaccharide to carrier protein in the conjugate is from about 0.5 to about 3.0.

C217. The method of any one of C181-C216, wherein the degree of conjugation of the conjugate is between 2 and 15.

C218. A method for producing a polysaccharide-protein conjugate, comprising the steps:

(a) reacting the isolated GBS capsular polysaccharide with an oxidizing agent;

(b) quenching the oxidation reaction of step (a) by adding an quencher to obtain an activated GBS capsular polysaccharide;

(c) combining the activated GBS capsular polysaccharide with a carrier protein,

(d) reacting the combined activated GBS capsular polysaccharide and carrier protein with a reducing agent to produce a GBS capsular polysaccharide and carrier protein conjugate, and

(e) capping unreacted aldehyde by adding sodium borohydride (NaBH ₄ ),

while steps (c) and (d) are performed in DMSO.

C219. An immunogenic composition containing a capsular polysaccharide from group B streptococcus (GBS) conjugated to a carrier protein and an aluminum-based adjuvant.

C220. The immunogenic composition of claim C219, wherein the aluminum adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxyphosphate, and aluminum hydroxide.

C221. An immunogenic composition according to C219 or C220, wherein the adjuvant is aluminum phosphate.

C222. An immunogenic composition according to C219 or C220, wherein the adjuvant is aluminum hydroxide.

C223. An immunogenic composition according to any one of C219-C222, wherein the aluminum-based adjuvant is at a concentration of from about 0.25 mg/mL to about 0.75 mg/mL.

C224. The immunogenic composition of claim C223, wherein the aluminum-based adjuvant is at a concentration of approximately 0.5 mg/mL.

C225. The immunogenic composition of any one of C219-C224, wherein the capsular polysaccharide is selected from the group consisting of polysaccharides of serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII and IX.

C226. The immunogenic composition of any one of C219-C225, which is a hexavalent GBS conjugate composition.

C227. The immunogenic composition of claim C225 comprising capsular polysaccharides from GBS serotypes Ia, Ib, II, III, IV and V.

C228. An immunogenic composition according to any one of C219-C227 placed in a syringe.

C229. The immunogenic composition of claim C228, wherein the syringe is glass.

C230. An immunogenic composition according to any one of C219-C229, placed in a syringe with a headspace of about 1 mm to about 15 mm above the product.

C231. The immunogenic composition of claim C230 placed in a syringe with a headspace of about 2 mm to about 6 mm above the product.

C232. An immunogenic composition according to any one of C219-C231, resuspendable with about 50 shakes or less.

C233. An immunogenic composition according to C232, resuspendable with about 10 shakes or less.

C234. An immunogenic composition according to any one of C219-C233, further comprising a phosphate.

C235. The immunogenic composition of claim C234 wherein the phosphate is at a concentration of from about 15 mM to about 25 mM.

C236. The immunogenic composition of claim C235 wherein the phosphate is at a concentration of approximately 20 mM.

C237. The immunogenic composition of any one of C219-C236 further comprising sodium chloride.

C238. An immunogenic composition according to C219-C237, wherein sodium chloride is present at a concentration of from about 10 mM to about 350 mM.

C239. The immunogenic composition of claim C238, wherein sodium chloride is present at a concentration of about 225 mM to about 265 mM.

C240. The immunogenic composition of claim C239, wherein sodium chloride is present at a concentration of approximately 240 mM.

C241. The immunogenic composition according to paragraph C219-C238, in which sodium chloride is at a concentration of approximately

C242. The immunogenic composition of claim C241 wherein sodium chloride is present at a concentration of approximately 150 mM.

C243. An immunogenic composition according to any one of C219-C242, containing a total dose of conjugates from about 60 µg/ml to about 360 µg/ml.

C244. The immunogenic composition of claim C243, wherein the total dose of the conjugates is from about 120 mg/mL to about 240 μg/mL.

C245. An immunogenic composition according to any one of C219-C244, having a pH value of from about 6.5 to about 7.5.

C246. The immunogenic composition of claim C245 having a pH of about 7.0.

C247. An immunogenic composition comprising about 0.25 mg/ml to about 0.75 mg/ml aluminum phosphate, about 15 mM to about 25 mM phosphate, about 225 mM to about 265 mM sodium chloride, and a total dose of conjugates from about 60 µg/ml to about 360 µg/ml, and having a pH value of from about 6.5 to about 7.5.

C248. An immunogenic composition comprising about 0.25 mg/ml to about 0.75 mg/ml aluminum hydroxide, about 15 mM to about 25 mM phosphate, about 120 mM to about 170 mM sodium chloride, and a total dose of conjugates from about 60 µg/ml to about 360 µg/ml, and having a pH value of from about 6.5 to about 7.5.

C249. An immunogenic composition according to any one of C219-248 for use as a medicine.

C250. An immunogenic composition according to any one of C219-249 for use in a method of inducing an immune response against GBS in a patient.

C251. The immunogenic composition of claim C250, wherein the patient is a woman planning a pregnancy or a pregnant woman.

C252. The immunogenic composition of claim C251, wherein the woman is in the second half of her pregnancy.

C253. The immunogenic composition of claim C252, wherein the pregnant woman is at least 20 weeks pregnant.

C254. The immunogenic composition of claim C253, wherein the pregnant woman is at least 27 weeks to 36 weeks pregnant.

C255. The immunogenic composition of claim C250, wherein the patient is an adult 50 years of age or older.

C256. The immunogenic composition of claim C255, wherein the patient is an adult 65 years of age or older.

C257. The immunogenic composition of claim C256, wherein the patient is an adult 85 years of age or older.

C258. An immunogenic composition according to any one of C250-C257, wherein the patient is immunocompromised.

C259. The immunogenic composition of claim C258, wherein the patient has a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease, or liver disease.

C260. The immunogenic composition of any one of C250-259, wherein the group B streptococcus is Streptococcus agalactiae .

C261. A method of inducing an immune response against group B streptococcus, comprising administering to a patient an effective amount of an immunogenic composition according to any one of C219-C248.

C262. A method of preventing, or alleviating, a disease or condition associated with group B streptococcus in a patient, comprising administering to the patient an effective amount of the immunogenic composition of any one of C219-C248.

C263. The method according to C261 or C262, wherein the patient is a woman planning a pregnancy or a pregnant woman.

C264. The method of claim C263, wherein the woman is in the second half of her pregnancy.

C265. The method according to C263 or C264, wherein the pregnant woman is at least 20 weeks pregnant.

C266. The method of any one of C263-265, wherein the pregnant woman is at least 27 weeks to 36 weeks pregnant.

C267. The method of C261 or C262, wherein the patient is an adult 50 years of age or older.

C268. The method of claim C267, wherein the patient is an adult 65 years of age or older.

C269. The method of C267 or C268, wherein the patient is an adult 85 years of age or older.

C270. The method of any one of C261-C269, wherein the patient is immunocompromised.

C271. The method of claim C270, wherein the patient has a medical condition selected from the group consisting of obesity, diabetes, HIV infection, cancer, cardiovascular disease, or liver disease.

C272. The method of any one of C261-C271, wherein the group B streptococcus is Streptococcus agalactiae .

C273. A method for producing antibodies, comprising administering to a patient an immunogenic composition according to any one of C219-C248.

C274. An antibody produced by the method of claim C273.

C275. A method for creating passive immunity in a patient, comprising the steps of:

(a) obtaining an antibody preparation using an immunogenic composition according to any one of paragraphs C219-C248; And

(b) administering the antibody preparation to the patient to confer passive immunity.

Claims (16)

1. An immunogenic composition containing polysaccharide-carrier protein conjugates, wherein: (i) each of the conjugates comprises a capsular polysaccharide from a different Group B Streptococcus (GBS) serotype conjugated to a carrier protein, the serotypes being serotypes Ia, Ib, II, III, IV, and V, the capsular polysaccharide in each conjugate having a level sialic acid more than 60%, and moreover, the carrier protein contains CRM ₁₉₇ ; And (ii) the immunogenic composition further comprises sodium chloride at a concentration of 150 mM or higher, polysorbate-80, histidine, and optionally an aluminum phosphate adjuvant, wherein the pH of the immunogenic composition is from 6.5 to 7.5; And moreover, the immunogenic composition does not cause a reduced immune response to serotype III. 2. The immunogenic composition according to claim 1, which is a hexavalent GBS conjugate composition. 3. An immunogenic composition according to claim 1, wherein the aluminum phosphate adjuvant is present at a concentration of 0.25 mg/ml to 0.75 mg/ml. 4. An immunogenic composition according to claim 1, wherein the aluminum phosphate adjuvant is present at a concentration of 0.5 mg/ml. 5. Immunogenic composition according to claim 1, placed in a syringe. 6. An immunogenic composition according to claim 1, wherein sodium chloride is present at a concentration of 225 mM to 265 mM. 7. An immunogenic composition according to claim 1, wherein sodium chloride is present at a concentration of 240 mM. 8. An immunogenic composition according to claim 1, wherein sodium chloride is present at a concentration of 200 mM. 9. An immunogenic composition according to claim 1, wherein sodium chloride is present at a concentration of 150 mM. 10. An immunogenic composition according to claim 1, wherein the immunogenic composition contains a total dose of conjugate from 60 µg/ml to 360 µg/ml. 11. An immunogenic composition according to claim 10, wherein the total dose of the conjugate is between 120 µg/ml and 240 µg/ml. 12. An immunogenic composition according to claim 1, wherein the immunogenic composition has a pH of 7.0. 13. The immunogenic composition of claim 1, wherein the group B streptococcus is Streptococcus agalactiae .

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