AU2020204645A1 - Multivalent pneumococcal polysaccharide-protein conjugate composition - Google Patents
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Abstract
C:\NRPortb\DCC\AM4014380_I.DOC-24/11/2011
MULTIVALENT PNEUMOCOCCAL
POLYSACCHARIDE-PROTEIN CONJUGATE COMPOSITION
ABSTRACT
An immunogenic composition having 13 distinct polysaccharide-protein conjugates and
optionally, an aluminum-based adjuvant, is described. Each conjugate contains a
capsular polysaccharide prepared from a different serotype of Streptococcus pneumoniae
(1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F) conjugated to a carrier protein.
The immunogenic composition, formulated as a vaccine, increases coverage against
pneumococcal disease in infants and young children globally, and provides coverage for
serotypes 6A and 19A that is not dependen t on the limitations o f s erogroup c ross
protection.
Description
H:\Interwoven\NRPortbl\DCC\AMT\20420460_.doc-13/07/2020
The present invention relates generally to the field of medicine, and specifically to microbiology, immunology, vaccines and the prevention of infection by a bacterial pathogen by immunization.
This is a divisional of Australian patent application No. 2018213968, which in turn is a divisional of 2016222335, which in turn is a divisional of 2013206844, which in turn is a divisional of 2011253684, which in turn is a divisional of 2006235013, the entire contents of which are incorporated herein by reference. Streptococcus pneumoniae Is a leading cause of meningitis, pneumonia, and severe Invasive disease in infants and young chldren throughout the world. The multivalent pneumocccal poysaccharide vaccines have been licensed for many years and have proved valuable in preventing pneumococcal disease in elderly adults and high-risk patients. However, infants and young children respond poorly to most pneumococcal polysaccharides. The 7-valent pneumococcal conjugate vaccine (7vPnC, Prevnar@) was the first of its kind demonstrated to be highly immunogenic and effective against invasive disease and otitis media in infants and young children. This vaccine is now approved in many countries around the world. Prevnar contains the capsular polysaccharides from serotypes 4, 6B, 9V, 14, 18C, 19F and 23F, each conjugated to a carrier protein designated CRM1 7. Prevnar covers approximately 80-90%, 60-80%, and 40-80% of invasive pneumococcal disease (IPD) In the US, Europe, and other regions of the world, respectively [1,2]. Surveillance data gathered in the years following Prevnar's introduction has clearly demonstrated a reduction of invasive pneumococcal disease in US infants as expected (FIG. 1) [3,4.
Surveillance of IPD conducted In US infants prior to the introduction of Prevnar demonstrated that a significant portion of disease due to serogroups 6 and 19 was due to the 6A (approximately one-third) and 19A (approximately one-fourth) serotypes [5,6]. Pneumococcal Invasive disease surveillance conducted in the US after licensure of Prevnar suggests that alarge burden of diseaseis still attributable to serotypes 6A and 19A (FIG. 1) [3. Moreover, these two serotypes account for more cases of invasive disease than serotypes 1, 3, 5, and 7F combined (8.2 vs. 3.3 cases/100,000 children 2 years and under).. In addition, serotypes BA and 19A are associated with high rates of antibiotic resistance (FIG. 2) [7,8,9]. While it is possible that serogroup cross-protection will result in a decline of serotype 6A and 19A disease as more children are Immunized, there is evidence to suggest that there will be a limit to the decline, and a significant burden of disease due to these serotypes will remain (see below).
Given the relative burden and importance of invasive pneumococcal disease due to serotypes 1, 3, 5, 6A, 7F, and 19A, adding these serotypes to the Prevnar formulation would increase coverage for invasive disease to >90% in the US and Europe, and as high as 70%-80% in Asia and Latin America. This vaccine would significantly expand coverage beyond that of Prevnar, and provide coverage for 6A and 19A that is not dependent on the limitations of serogroup cross-protection.
SUMMARY OF THE INVENTION Accordingly, the present Invention provides generally a multivalent immunogenic composition comprising 13 distinct polysaccharide-protein conjugates, wherein each of the conjugates contains a capsular polysaccharide from a different serotype of Streptococcus pneumoniae conjugated to a carrier protein, together with a physiologically acceptable vehicle. Optionally, an adjuvant, such as an aluminum based adjuvant, is included In the formulation. More specifically, the present invention provides a 13-valent pneumococcal conjugate (13vPnC) composition comprising the seven serotypes in the 7vPnC vaccine (4, 61, 9V, 14, 18C, 19F and 23F) plus six additional serotypes (1, 3, 5, 6A, 7F and 19A).
The present invention also provides a multivalent immunogenic composition, wherein the capsular polysaccharldes are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F. of Streptococcus pneumoniae and the carrier protein is CRM 19r.
The present invention further provides a multivalent immunogenic composition, wherein the capsular polysaccharides are from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9v, 14, 18C, 19A, 19F and 23F of Streptococcus pneumoniae, the carrier protein is CRM 197, and the adjuvant is an aluminum-based adjuvant, such as
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aluminum phosphate, aluminum sulfate and aluminum hydroxide. In a particular embodiment of the invention, the adjuvant is aluminum phosphate.
The present invention also provides a multivalent immunogenic composition, comprising polysacchaide-protein conjugates together with a physiologically acceptable vehicle, wherein each of the conjugates comprises a capsular polysaccharide from a different serotype of Streptococcuspneumoniae conjugated to a cancer protein, and the capsular polysaccharides are prepared from serotye 3 and at least one additional serotype.
In one embodiment of this multivalent immunogenic composition, the additional serotype Is selected from the group consisting of serotypes 1, 4, 5, 6A, 68, 7F, 9V, 14, 18C, 19A, 19F, and 23F. In another embodiment, the carrier protein is CRM 19. In yet another embodiment, the composition comprises an adjuvant, such as an aluminum-based adjuvant selected from aluminum phosphate, aluminum sulfate and aluminum hydroxide. In a particular embodiment, the adjuvant is aluminum phosphate.
The present invention also provides a multivalent immunogenic composition, comprising polysaccharlde-protein conjugates together with a physiologically acceptable vehicle, wherein each of the conjugates comprises a capsular polysaccharide from a different serotype of Streptococcus pneumoniaeconjugated to a carrier protein, and the capsular polysaccharides are prepared from serotypes 4, 6B, 9V, 14,18C, 19F, 23F and at least one additional serotype.
In one embodiment of this multivalent immunogenic composition, the additional serotype is selected from the group consisting of serotypes 1, 3, 5, 6A, 7F, and 19A. In another embodiment, the carrier protein is CRM 7 . In yet another embodiment, the composition comprises an adjuvant, such as an aluminum-based adjuvant selected from aluminum phosphate, aluminum sulfate and aluminum hydroxide. In a particular embodiment, the adjuvant is aluminum phosphate.
The present invention also provides a method of inducing an Immune response to a Strepocccus pneumoniae capsular polysacharide conjugate, comprising administering to a human animmunologically effective amount of any of the immunogenic compositions just described.
The present invention further provides that any of the immunogenic compositions administered is a single 0.5 mL dose formulated to contain: 2 pg of 19 carrier protein; each saccharide, except for 6 at 4 pg; approximately 29 pg CRM 0.125 mg of elemental aluminum (0.5 mg aluminum phosphate) adjuvant; and sodium chloride and sodium succinate buffer as excipients.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts the changes in IPD rates by serotype in US children <2 years of age from baseline (1998/1999) to 2001. FIG. 2 depicts the distribution of pneumococcal isolates with resistance to penicillin (PCN) in children <5 years of age (1998). FIG. 3 depicts the reverse cumulative distribution curves (RCDC) of OPA post-third dose results from the D118-P16 Prevnar trial.
Inclusion of Prevnar Serotypes 4, 6B, 9V, 14, 18C,19F, 23F Data from IPD surveillance between 1995-1998 estimated that the seven serotypes In Prevnar were responsible for around 82% of IPD in children <2 years of age [5]. In Northem California, the site of the efficacy trial, the Prevnar serotypes accounted for 90% of all cases ofIPD in infants and young children [10]. Since introductionof the Prevnar vaccine in 2000, there has been a significant decrease In the overall IPD rates due to a decreasein disease due to the vaccine serotypes [3,4]. Therefore, there is no justification at this time to remove any of the Prevnar serotypes from the next generation of pneumococcal corjugate vaccines but rather to add serotypes to obtain wider coverage.
Inclusion ofSerotypes, 3, 5 and 7F Ir the US, the rate ofIPD caused by serotype I in children under the age of 5 years is <2%, about the same as for each of types 3 and 7F [1,6]. Serotypes 1 and 5 account for higher rates of IPD in US populations at high risk for invasive pneumococcal disease. Specifically, serotype I causes 3.5% of IPD in Alaskan native children <2 years of age, and 18% in children 2-4 years of age [11]. Both serotype I and serotype 5 significantly cause disease in other parts of the world and in indigenous populations In developed countries 12,13,14].
Serotype I may also be associated with more severe disease as compared with other pneumococcal serotypes [15]. This observation is based on the difference in rates of case identification between the US and Europe, and the associated difference in medical practice. Overall, the incidence of IPD is lower in Europe than in the US. However, the percent of IPD caused by serotype 1 in Europe Is disproportionately higher than in the US (6-7%, vs. 1-2%, respectively). In Europe, blood cultures are obtained predominantly from hospitalized children. In the US, it is routine medical practice to obtain blood cultures in an outpatient setting from children presenting with fever >39 0C and elevated white blood cell counts. Given the difference in medical practice, it Is postulated that the lower percent of disease caused by serotype I In the US may be diluted by higher rates of other serotypes causing milder disease, while the higher percent in Europe reflects more serious disease. In addition, seroepidemiology studies of children with complicated pneumonia demonstrate that serotype 1 is disproportionately represented [16,17,18]. This suggests that inclusion of serotype I may reduce the amount of severe pneumococcal disease, as well as, contribute to a total reduction in invasive pneumococcal disease.
The addition of serotypes 3 and 7F will increase coverage against IPD in most areas of the world by approximately 3%-7%, and In Asia by around 9%. Thus, an 11-valent vaccine would cover 50% In Asia and around 80% of IPD in all other regions {1,2]. These serotypes are also important with respect to otls media coverage [19]. In a multinational study of pneumococcal serotypes causing otitis media, Hausdorff et al found serotype 3 to be the 8th most common middle ear fluid
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isolate overall [20]. Serotype 3 accounted for up to 8.7% of pneumococcal serotypes associated with otitis media. Thus, the importance of types -3 and 7FInotitis media, as well as in IPD, warrants their inclusion in a pneumococcal conjugate vaccine.
However, attempts to produce a multivalent pneumococcal conjugate vaccine that exhibits significant immunogenicity with respect to serotype 3 polysacchardes have been unsuccessful. For example, in a study of theimmunogenicity and safety of an I1-valent pneumococcal protein D conjugate vaccine (11-Pn-PD), no priming effect was observed for serotype 3 in infants who had received three doses of the 10 vaccine followed by a booster dose of either the same vaccine or a pneumococcal polysaccharide vaccine (Nurkka et a. (2004) Ped. Inf. Dis. J., 23:1008-1014). In another study, opsonophagocytic assay (OPA) results from infants who had received doses of 11-Pn-PD failed to show antibody responses for serotype 3 at levels comparable to other tested serotypes (Gatchallan et al., 17h Annual Meeting of the Eur. Soc. Paed. Inf. Dis. (ESPID), Poster No. 4, PIA Poster Session 1, Istanbul Turkey, Mar. 27, 2001). In yet another study, which assessed the efficacy of an 11 Pn-PD in the prevention of acuteotitle media, the vaccine did not provide protection against episodes caused by seotype 3 (Prymulametal. www.thelancet.com, Vol. 367: 740-748 (March 4, 2006)). Accordingly, a pneumococcal conjugate vaccine comprising capsular polysacchardes from serotype 3 and capable of eliciting an Immunogenic response to serotype 3 polysaccharides provides a significant improvement over the existing state of the art.
Inclusion of Serotypes 6A and 19A .5 a. Epidemiology of Serotypes 6A and 19A Surveillance data in the literature suggest that serotypes 6A and 19A account for more invasive pneumococcl disease in US children <2 years of age than serotypes 1, 3, 5, and 7F combined (FIG. 1) [1,5]. In addition, these serotypes are commonly associated with antibiotic resistance (FIG. 2) and play an important role in otiis media [6,19,20]. The ability of the current Prevnar vaccine to protect against disease due to 6A and 19A is not clear. The rationale for inclusion of 6A and 19A components in a 13vPnC vaccine is discussed below.
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b. Responses to 6A and 19A Induced by.6B and 19F Polysaccharides The licensed unconjugated pneumococcal polysaccharide vaccines (for use in persons at least two years of age) have contained 6A or 6B capsular polysaccharide but not both [21]. Immunogenicity data generated at the time-of formulation of the 23-valent pneumococcal polysaccharde vaccine demonstrated that a 6B monovalent vaccine induced antibody to both the 6A and 6B capsules. The data from several trials assessing IgG and opsonophagocytic assay (OPA) responses In a variety of populations with free polysacchadde and with pneumococcal conjugate vaccines suggested that igG responses to 6A are induced by 6B antigens, but the responses are generally lower, and the OPA activity with 6A organisms is different than with 6B organisms [22,23,24,25]. In addition, subjects responding with high 6B antibody may have little or no activity against 6A.
In contrast to the chemical composition of the 6A and 6B capsular polysacchadides where there exists a high degree of similarity, the 19A and 19F capsules are quite different due to the presence of two additional side chains in the 19A polysaccharide. Not surprisingly, immune responses measured in human volunteers immunized with 19F polysaccharide vaccine showed that responses to 19F were induced in 80% of subjects, but only 20% of subjects had a response to 19A [26]. Low levels of cross-reactive IgG and OPA responses to serotype 19A after immunization with 19F polysaccharide have also been documented in trials with conjugate vaccines as well [24,26].
Internal data on cross-reactive OPA responses to 6A and 19A have been generated from the 7vPnCbrdging trial (D118-P16) conducted in US infants (FIG. 3). These studies are consistent with the findings of others, and demonstrate induction of cross-reactive functional antibody to 6A polysacchaide after immunization with 6B polysaccharide, although at a lower level, and very little functional antibody to 19A after immunization with 19F.
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Impact of 6B and 19F immunization on 6A and19A in Animal Models Animal models have been used to evaluate the potential for cross-protection with polysaccharide immunization. In an otitis media model developed by Giebink et al., chinchillas were Immunized with a tetravalent polysaccharide outer membrane protein (OMP) conjugate vaccine (containing 6B, 14, 19F, 23F sacchardes) or placebo [27]. In this trial there appeared to be some cross-protection for 6A; however this did not reach statistical-significance and the level of protection was lower than with 6B against otitis media. In this same model there was 100% protection against 19F otitis media, but only 17% protection against 19A otitis media.
Saeland et al. used sera from infants immunized with an 8-valent pneumococcal tetanus conjugate vaccine (containing 6B and 19F) to passively immunize mice prior to an intranasal challenge with 6A organisms, In alungInfection model [28]. Of the 59 serum samples, 53% protected mice against bacteremiawith 6B and 37% protected against 6A. Mice passivelyImmunized with sera from infants immunized with four doses of an 11-valent pneumococcal conjugate vaccine (containing 19F conjugated to tetanus toxoid) were given an intranasal challenge with 19A organisms in the same model [29]. Of 100 mice passively immunized and then challenged, 60 mice had no 19A organisms detected in lung tissue, whereas organisms were identified in all mice given saline placebo. However, passive immunization did not protect against challenge with 19F organisms in this model; therefore, the relevance of the model for serogroup 19 Is questionable. In general these models provide evidence of some biological impact of 6Bimmunization on 6A organisms although the effect on the heterologous serotype was not as great as that observed with the homologous serotype. The impact of 19F immunization on 19A organisms is not well understood from these models.
Impact of 6B and 19F Polysacchaide Conjugate Immunization on 6A and 19A Disease in Eficacy/Effectiveness Trials The number of cases of disease clue to the 6B, 6A, I9F and I9A serotypes in ?vPnC and 9vPnC (7vPnC plus serotypes I and 5) efficacy trials is noted in Table I
[30,10,31]. The numbers of invasive disease cases are too small to allow any concusions to be drawn for serotypes 6A and 19A. However, the Finnish otitis media trial generated a large number of pneumococcal isolates [32]. In the per protocol analysis 7vPnC was.84%.(95% ,0 62%, 93%) efficacious against oitis media due to serotype 6B and 57% (95% C 24%, 76%) efficacious against otitis media due to serotype 6A (Table 1). In contrast, serotype-specific efficacy with the 7vPnC was not demonstrated for otitis media due to either 19F or19A.
Table 1. Cases of Pneuimcoccal Disease Due to Serotypes 61, 6A,19F, and 19A in Efficacy Trials with the 7vPnC and 9vPnC Vaccines 6B 6A 19F 19A PnC Contr. PnC Contr. PnC Contr. PnC Contr. Kaiser Efficacy Trial-7vPnc 1 7 0 1 2* 13 0 1 (rro Navajo EfficacyTrial -7vPnC 0 5 1 0 1 1 1 0 (0Th SouthAfricanEfficacyTrial- 1 2 1 0 1 3 1 9vPnC HIV (-) (O -1 SouthAfricanEfficacyTrial- 1 7 3 10 2 3 2 3 9vPncNW(+)(ITT) FinnishotifieMediaTrial- 9* 56 19* 45 43 58 17 26 7vPnC (PP) w8tatistically significant efficacy demonstrated From references 30, 10 and 33, and personal communications Contr = control rIT =Intention to treat analysis PP = per protocol analysis
Post-marketing IPD surveillance data is also available from a case-control trial conducted by the Centers for Disease Control to evaluate the effectiveness of Prevnar [33]. Cases of pneumococcal invasive disease occurring in children 3 to 23 months of age were identified in the surveillance laboratories and matchedwith three .O control cases by age and zip code. After obtaining consent, medical and immunization history (subjects were considered immunized if they had received at least one dose of Prevnar) was obtained from parents and medical providers for cases and controls. The preliminary results were presented at the 2003 ICAAC meeting and a summary of the findings for 6B, 19F, 19A and 6A disease is presented in Table 2. These data indicate that Prevnar is able to prevent disease due to 6A, although at a level that may be somewhat lower-than serotype 6B disease. These data also indicate that the cross-protection for invasive disease due to 19A is limited.
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Table 2. Preliminary results of a Case Control Trial Performed by the CDC (presented at ICAAC, 2003) Serotype Informative Sets, n (5C
Vaccine Type, All 115 94 Vaccine Related, All 36 3086
Non-Vaccine Type, All 43 -4 (-1,48) 6B 27B27 (72,99) 19F197 19 (16,92) 6A 15 87 ____ ___(53,97) ____ ____
19A 16 (-87,40 80) *Vaccine effectiveness comparing vaccinated(>1 dose) vs. unvaccinated, and adjusted for underlying conditions Reference 40 and personal/confidential communication
A published analysis [3] of the use of Prevnar also indicated that serotypes 6B and 19F conferred a moderate reduction in IPD caused by serotypes 6A and 19A among children under two years of age (Table I in [3]). Disease rates among unimmunized adults caused by serotypes 6A, 9A, 9L, 9N, 18A, 18B, 18F, 19A, 19B, 19C, 23A and 23B ("all vaccine-related serotypes") were somewhat reduced (Table 2 in [3]). These data establish that herd immunity from the use.of Prevnar in children under two years of age was modest for serotypes 6A and 19A, and provide a basis for the inclusion of serotypes 6A and 19A In the 13vPnC vaccine of this invention.
Conclusion for addition of 6A and 19A The post-marketing surveillance data and the case-control study results noted in FIG. I and Table 2 with the 7vPnC vaccine suggest that, consistent with the other Information on immune responses and performance in the animals models described above, there may be some cross-protection against 6A disease, but to a lesser extent than to 6B disease. Furthermore, it appears the protection against 19A is limited. Therefore, a 13vPnC vaccine containing serotypes 6A and 19A provides coverage that is not dependent on the limitations of serogroup cross-protection by serotypes 6B and 19F.
Accordingly, the present invention provides a multivalent immunogenic composition comprising 13 distinct polysacchalde-protein conjugates, wherein each of the conjugates contains a different capsular polysaccharide conjugated to a carrier protein, and wherein the capsular polysaccharides are prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and,23F of Streptococcus pneumoniae, together with a physiologically acceptable vehicle. One such carrier protein is the diphtheria toxold designated CRM 7 . The immunogenic composition may further comprise an adjuvant, such as an aluminum-based adjuvant, such as aluminum phosphate, aluminum sulfate and aluminum hydroxide.
Capsular polysacharides are prepared by standard techniques known to those skilled In the art. In the present invention, capsular polysaccharides are prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F of Streptococcus pneumoniae. These pneumococcal conjugates are prepared by separate processes and formulated into a single dosage formulation. For example, in one embodiment, each pneumococcal polysaccharide serotype is grown In a soy based medium. The Individual polysaccharides are then purified through centrifugation, precipitation, ultra-filtration, and column chromatography. The purified polysaccharides are chemically activated to make the saccharides capable of reacting with the carrier protein.
Once activated, each capsular polysaccharide is separately conjugated to a carrier protein to form a glycoconjugate. In one embodiment, each capsular polysaccharde is conjugated to the same carrier protein. In this embodiment, the conjugation is effected by reductive amination.
The chemical activation of the polysacchardes and subsequent conjugation to the canier protein are achieved by conventional means. See, for example, U.S. Pat. Nos. 4,673,574 and 4,902,506 [34,35].
Carrier proteins are preferably proteins that are non-toxic and non reactogenic and obtainable in sufficient amount and purity. Carrier proteins should be amenable to standard conjugation procedures. In a particular embodiment of the present invention, CRM 1 97is used as the carrier protein.
CRM 1 r (Wyeth, Sanford, NC) is a non-toxic variant (i.e., toxoid) of diphtheria toxin isolated from cultures of Corynebacterium diphtheriastrain C7 (p197) grown in 1 is'purified through ultra casamino acids and yeast extract-based medium. CRM97 filtration, ammonium sulfate precipitation, and ion-exchange chromatography. Alternatively, CRM 1 97is prepared recombinantly in accordance with U.S. Patent No. 5,614,382, which is hereby incorporated by reference. Other diphtheria toxoids are also suitable for use as carrier proteins.
Other suitable carrier proteins include inactivated bacterial toxins such as tetanus toxoid, pertussis toxoid, cholera toxoid (e.g., as described in International Patent Application W02004/083251 [38]), E. coflLT, E coli ST, and exotoxin A from Pseudomonas aerugnosa, Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), C5a peptidase from Group A or Group B streptococcus, or Haemophilus influenzae protein D, can also be used. Other proteins, such as ovalbumin, keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or purified protein derivative of tuberculin (PPD) can also be used as carrier proteins.
After conjugation of the capsular polysaccharide to the carrier protein, the polysaccharide-proten conjugates are purified (enriched with respect to the amount of polysaccharide-protein conjugate) by a variety of techniques. These techniques include concentration/diafiltration operations, precipitation/elution, column chromatography, and depth filtration. See examples below.
After the individual glycoconjugates are purified, they are compounded to formulate the immunogenic composition of the present invention, which can be used as a vaccine. Formulation of theimmunogenic composition of the present invention can be accomplished using art-recognized methods. For instance, the 13 individual pneumococcal conjugates can be formulated with a physiologically acceptable vehicle to prepare the composition. Examples of such vehicles include, but are not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.
In certain embodiments, the immunogenic composition will comprise one or more adjuvants. As defined herein, an "adjuvant" is a substance that serves to enhance theImmunogenicity of an immunogenic composition of this invention. Thus, adjuvants are often given to boost the immune response and are well known to the skilled artisan. Suitable adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (defined below) or bacterial cell wall components), such as, for example, (a) MF59 (PCT Publ. No. WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below, although not required)) formulated into submicron particles using a microfluidizer such as Model11OY microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% Squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RIbim adjuvant system (RAS), (Corixa, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of 3-0-deaylated monophosphorylipid A (MPLm) described In U.S. Patent No. 4,912,094 (Corixa), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detoxm); (3) saponin adjuvants, such as Quil A or STIMULONTMQS-21 (Anigencs, Framingham, MA) (U.S. Patent No. 5,057,540) may be used or particles generated therefrom such as ISCOMs (immunostimulatingcomplexes); (4) bacterial lipopolysaccharides, synthetic lipid A analogs such as aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or analogs thereof, which are available from Corixa, and which are described in U.S. Patent No. 6,113,918; one such AGP is 2-[()-3-Tetradecanoyloxytetradecanoylamin]ethyl 2-Deoxy-4-0 phosphono-3-O-[(R)-3-tetradecanoylokyttradeanoy]-2-[(R)-3 tetradecanoyloxytetradecanoylamino]-b-D-glucopyranoside, which is also know as 529 (formerly known as RC529), which is formulated as an aqueous form or as a stable emulsion, synthetic polynucleotides such as olgonucleotides containing CpG motif(s) (U.S. Patent No. 6,207,646); (5) cytokines, such asInterleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.), interferons (e.g., gammainterferon), granulocyte macrophage colony stimulating factor (OM-CSF), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), costimulatory molecules B7-1 and 17-2, etc.; (6) detoxified mutants of a bacterial ADP-ribosylating toxin such as a cholera toxin (CT) either in awild-type or mutant form, for example, where the glutamic acid at amino acid position 29 is replaced by another amino acid, preferably a histidine, in accordance with published international patent application number WO 00/18434 (see also WO 021098368 and WO 02/098369), a pertussis toxin (PT), or an E. coli heat-labile toxin (LT), particularly LT-K63, LT-R72, CT-S109, PT-K9/G129 (see, e.g., WO 93113302 and WO 92/19265); and (7) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanine-2-(1'-2' dipalmitoyl-sn glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
The vaccine formulations of the present invention can be used to protect or treat a human susceptible to pneumococcal infection, by means of administering the vaccine via a systemic or mucosal route. These administrations can include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous routes; or via mucosal administration to the oral/alimentary, respiratory or genitourinary tracts. In one embodiment, intranasal administration is used for the treatment of pneumonia or otitis media (as nasopharyngeal carriage of pneumococcal can be more effectively prevented, thus attenuating Infection at its earliest stage).
The amount of conjugate in each vaccine dose is selected as an amount that induces an immunoprotective response without significant, adverse effects. Such amount can vary depending upon the pnumococcal serotype. Generally, each dose will comprise 0.1 to 100 pg of polysacchaide, particularly 0.1 to 10 pg, and more 5 particularly 1 to 5 pg.
Optimal amounts of components for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects. Following an Initial vaccination, subjects can receive one or several booster immunizations adequately spaced.
In a particular embodiment of the present invention, the 13vPnC vaccine is a stedle liquid formulation of pneumococcal capsular polysaccharides of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F individually conjugated to CRM1 97 . Each 0.5 mL dose is formulated to contain: 2 pg of each saccharide, except for 6B at 4 pg; approximately 29 pg CRM 197 carrier protein; 0.125 mg of elemental aluminum (0.5 mg aluminum phosphate) adjuvant; and sodium chloride and sodium succinate buffer as excipents. The liquid is filled into single dose syringes without a preservative. After shaking, the vaccine is a homogeneous, white suspension ready for intramuscular administration.
The choice of dose level for the 13vPnC vaccine is similar to the marketed 7vPnC vaccine (Prevnar). The 2 pg sacchaide dose level was selected for all serotypes, except for 6B, which is at 4 pg per dose. The 7vPnC vaccine has shown desirable safety,.immunogenicity, and efficacy against IPD in the 2 pg sacchadde dose level for serotypes 4, 9V, 14, 18C, 19F and 23F, and at the 4 pg dose for 6B.
The Immunization schedule can follow that designated for the 7vPnC vaccine. For example, the routine schedule for infants and toddlers against invasive disease caused by S. pneumoniae due to the serotypesIncluded in the 13vPnC vaccine is 2, 4, 6 and 12-15 months of age. The compositions of this invention are also suitable for use with older children, adolescents and adults.
The compositions of this -invention may further include one or more additional antigens for use against otitis media caused by infection with other bacteria. Such bacteria include nontypable HaemophilusInfluenza,.Moraxella catarrhalis (formerly known as Branhamella catarrhalis) and Allolococcus oitdis.
Examples of nontypable Haemophilus influenzae antigens suitable for -inclusion include the P4 protein, also known as protein "e" (U.S. Patent No. 6,601,831; Intemational Patent Application W003/078453), the P6 protein, also known as the PAL or the PBOMP-1 protein (U.S. Patent No. 5,110,908;Intemational Patent Application WO100790), the P5 protein (U.S. Reissue Patent No. 37,741), the Haemophiis adhesion and penetration protein (U.S. Patent Nos. 6,245,337 and 6,676,948), the LKP tip adhesin protein (U.S. Patent No. 5,643,725) and the NucA protein (U.S. Patent No. 6,221,365).
Examples of Moraxella catarrhalis antigens suitable for inclusion include the UspA2 protein (U.S. Patent Nos. 5,552,146, 6,310,190), the CD protein (U.S. Patent No. 5,725,862), the E protein (U.S. Patent No. 5,948,412) and the 74 kilodalton outer membrane protein (U.S. Patent No. 6,899,885).
Examples of Allolococcus otitidis antigens suitable for inclusion include those identified in Intemational Patent Application W003/048304.
The compositions of this invention may also include one or more proteins from Streptococcus pneumoniae. Examples of Streptococcus pneumonae proteins suitable for inclusion include those identified inIntemational Patent Application W002/08385, as well as that described inIntemational Patent Application W002/053761.
The compositions of this invention may further include one or more proteins from Neisseria meningitidis type B. Examples of Neisseria meningitidis type B proteins suitable for inclusion include those identified in international Patent Applications W0031063766, W020041094596, WO01/85772, W002/16612 and WO01/87939.
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The above disclosure generally describes the present invention. A more complete understanding. can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scopeof the invention.
EXAMPLES Example I
Preparation of S. Pneumoniae Capsular Polysaccharide Serotype I
Preparation of Master and Working Cell Banks S. pneumoniae serotype I was obtained from the American Type Culture Collection, ATCC, strain 6301. Several generations of seed stocks were created in order to expand the strain and remove components of animal origin (generations F1, F2, and F3). Two additional generations of seed stocks were produced. The first additional generation was made from an F3 vial, and the subsequent generation was made from a vial of the first additional generation. Seed vials were stored frozen (<-70°C) with synthetic glycerol as a cryopreservative. In addition to frozen vials, lyophilized vials were prepared for the F4 generation. For cell bank preparation, all cultures were grown in a soy-based medium. Prior to freezing, cells were concentrated by centrifugation, spent medium was removed, and cell pellets were re suspended In fresh medium containing a cryopeservative, such as synthetic glycerol.
Fermentation and Harvesting Cultures from the working cell bank were used to inoculate seed bottles containing a soy-based medium. The bottles were incubated at 36C± 2° C without agitation until growth requirements were met. A seed bottle was used to inoculate a seed fermentor containing soy-based medium. A pH of about 7.0 was maintained with sterile sodium carbonate solution. After the target optical density was reached, the seed fermentor was used to inoculate the production fermentor containing soy based medium. The pH was maintained with sterile sodium carbonate solution. The fermentation was terminated after cessation of growth or when the working volume of
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the fermentor was reached. An appropriate amount of sterile 12% deoxycholate sodium was added to the culture to lyse the bacterial cells and release cell associated polysaccharde. After lysing, the fermentor contents were cooled. The pH of the lysed culture broth was adjusted to approximately pH 6.6with acetic acid. The lysate was clarified by continuous flow centrifugation followed by depth filtration and 0.45 pm microfiltration.
In an alternate process, the fermentation pH of about 7.0 was maintained with 3N NaOH. After the target optical density was reached, the seed fermentor was used to inoculate the production fermentor containing soy-based medium. The pH was maintained with 3N NaOH. The fermentation was terminated after cessation of growth or when the working volume of the fermentor was reached. An appropriate amount of sterile 12% deoxycholate sodium was added to the culture to obtain a 0.12% concentration in the broth, to lyse the bacterial cells and release cell associated polysaccharide. After lysing, the fermentor contents were held, with agitation, for a time interval between 8 and 24 hours at a temperature between 70C and 130C, to assure that complete cellular lysis and polysaccharide release had occurred. Agitation during this hold period prevented lysate sediment from settling on the fermentor walls and pH probe, thereby allowing the pH probe integrity to be maintained. Next, the pH of the lysed culture broth was adjusted to approximately pH 5.0 with 50% acetic acid. After a hold timewithout agitation, for a time interval between 12 and 24 hours at a temperature between 15°C and 25°C, a significant portion of the previously soluble proteins dropped out of solution as a solid precipitate with little loss or degradation of the polysaccharide, which remained in solution. The -solution with the precipitate was then clarified by continuous flow centrifugation followed by depth filtration and 0.45 pm microfiltration.
Purification The purification of the pneumococcal polysaccharide consisted of several concentration/diafiltraton operations, precipitation/elution, column chromatography, and depth filtration steps. All procedures were performed at room temperature unless otherwise specified.
Clarified broth from the fermentor cultures ofS. pneumoniae serotype Iwere concentrated and diafiltered using a 100 kDa MWCO (kilodalton molecularweight cutoff) filter. Diafiltraton was accomplished using sodium phosphate buffer at neutral pH. Diafiltration removed the low molecular weight medium components from the higher molecular weight biopolymers such as nucleic acid, protein and polysaccharide. The polysaccharide was precipitated from the concentrated and diafiltered solution by adding hexadecyltrimethyl ammonium bromide (HB) from a stock solution to give a final concentration of 1%HB (w/v). The polysaccharideHB precipitate was captured on a depth filter and the filtrate was discarded. The polysaccharide precipitate was resolubilized and eluted by recirculating a sodium chloride solution through the precipitate-containing depth filter. The filters were then rinsed with additional sodium chloride solution. Sodium Iodide (Nal) was added to the polysaccharide solution from a stock Nal solution to achieve a final concentration of 0.5% to precipitate HB. The precipitate was removed by depth filtration. The filtrate contains the target polysaccharide. The precipitation Vessel and the filter were rinsed with a NaC/Nal solution and the rinse Was combined with the partially purified polysaccharide solution. The filter was discarded. The polysaccharide was then filtered through a 0.2 pm filter. The polysaccharide solution was concentrated on a 30 kDa MWCO ultrafilter and diafiltered with a sodium chloride solution. The partially purified polysaccharide solution was further purified by filtration through a depth filter impregnated with activated carbon. After filtration, the carbon filter was rinsed with a sodium chloride solution. The rinse Is combined with the polysaccharde solution, which is then filtered through a0.2 pm filter. The polysaccharide solution was concentrated on a 30 kDa MWCO ultrafilter and adjusted with a 1M sodium phosphate buffer to achieve a final concentration of 0.025 M sodium phosphate. The pH was checked and adjusted to 7.0 ±0.2. The ceramic hydroxyapatite (HA) column was equilibrated with sodium phosphate buffer containing sodium chloride toobtain the appropriate conductivity (<15 pS). The polysaccharide solution was then loaded onto the column. Under these conditions, impurities bound to the resin and thepolysaccharde was recovered in the flow-through from the column. The polysaccharide solution was filtered through .2pm inline filters located beforeand after the column. The polysaccharide solution was concentrated using a 30 kDa MWCO filter. The concentrate was then diafiltered with WaterforInjection (WFI). The diafiltered polysaccharde solution was filtered through a 0.2 pm membrane filter into polypropylene bottles. Samples were removed for release testing and the purified polysaccharide was stored frozen at -25° t 50C.
Characterization The 1H-NMR data was consistent with the chemical structure by the assignment of signals assigned to the protons of the polysaccharide molecule. The IH-NMR spectrum showed a series of well-resolved signals (protors from the methyl group) for the quantitation of the O-acety functional group in the polysaccharide. The identity of the monovalent polysaccharide was confirmed by countercurrent immunoelectrophoresis using specific antisera. High performance gel-filtration chromatography coupled with refractive index and multiangle laser light scattering (MALLS) detectors was used in conjunction with the sample concentration to calculate the molecular weight. Size exclusion chromatography media (CL-4B) was used to profile the relative molecular size distribution of the polysaccharide.
Example 2
Preparation of Serotype I Pneumococcal Saccharide - CRMI 1 Conjugate
Activation and Conjugation
Containers of purified polysaccharide were thawed and combined in a reaction vessel. To the vessel, 0.2 M sodium carbonate, pH 9.0 was added for partial deacetylation (hydrolysis) for 3 hours at 50°C. The reaction was cooled to 20°C and neutralization was performed by 0.2 M acetic add. Oxidation in the presence of sodium perodate was performed by incubation at 2-8C, and the mixture was stirred for 15-21 hours.
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The activation reaction mixture was concentrated and diafiitered 1x with 0.9% NaCl using a 30K MWCO membrane. The retentate was 0.2. pm filtered. The activated saccharide was filled Into 100 mL glasslyophilization bottles and shell frozen at -75°C and lyophilized. "Shell-freezing" Is a method for preparing samples for lyophilization (freeze drying). Flasks are automatically rotated by motor driven rollers in a refrigerated bath containing alcohol or any other appropriate fluid. A thin coating of product Is evenly frozen around the inside "shell" of a.flask, permitting a greater volume of material to .be safely processed during each freeze-drying run. These automatic, refrigerated units provide a simple and efficient means of pre-freezing many flasks at a time, producing the desired coatings Inside, and providing sufficient surface area for efficient freeze-drying. Bottles of lyophilized material were brought to room temperature and resuspended in CRM 7 solution at a saccharide/protein ratio of 2:1. To the saccharideprotein mixture IM sodium phosphate buffer was added to a final 0.2M Ionic strength and a pH of 7.5, then sodium cyanoborohydride was added. The reaction was Incubated at 23°C for 18 hours, followed by a second incubation at 37°C for 72 hours. Following the cyanoborohydride incubations, the reaction mixture was diluted with cold saline followed by the addition of IM sodium carbonate to adjust the reaction mixture to pH 9.0. Unreacted aldehydes were quenched by addition of sodium borohydride by incubation at 230C for 3-6 hours. The reaction mixture was diluted 2-fold with saline and transferred through a 0.45 - 5 pm prefilter into a retentate vessel. The reaction mixture is diafiltered 30x with 0.15 M phosphate buffer,pH 6, and 20x with saline. The retentate was filtered through a 0.2 pm filter. The conjugate solution was diluted to a target of 0.5 mg/mL in 0.9% saline, and then sterile filtered into final bulk concentrate (FBC) containers in a Class 100 hood. The conjugate was stored at 2 - 8°C.
Characterization Size exclusion chromatography media (CL-4B) was used to profile the relative molecular size distribution of the conjugate.
The identity of the conjugate was confirmed by the slot-blot assay using specific antisera. The saccharide and protein concentrations were determined by the uronic acid and Lowry assays, respectively. The ratio of saccharide to protein in the covalently bonded conjugate complex was obtained by the calculation: pg/mL saccharide Ratio pg/mL protein O-acetyl content was measured by the Hestrin method (Hestrin et. al., J. Biol. Chem. 1949, 180, p. 249). The ratio of O-acetyl concentration to total saccharide concentration gave pmoles of 0-acetyl per mg of sacchaide.
Example 3
Preparation of S.Pneumoniae Capsular Polysaccharide Serotype 3
Preparation of Master and Working Cell Banks S. pneumoniae serotype 3 was obtained from Dr. Robert Austrian, University of Pennsylvania, Philadelphia, Pennsylvania. For preparation of the cell bank system, see Example 1. Fermentation and Harvesting Cultures from the working cell bank were used toInoculate seed bottles containing soy-based medium. The bottles were incubated at 360C ± 20 C without agitation until growth requirements were met. A seed bottle was used to inoculate a seed fermentor containing soy-based medium. A pH of about 7.0 was maintained with sterile sodium carbonate solution. After the target optical density was reached, the seed fermentor was used to inoculate an intermediate seed fermentor. Afterthe target optical density was reached, the intermediate seed fermentor was used to inoculate the production fermentor. The pH was maintained with sterile sodium carbonate solution. The fermentation was terminated after the working volume of the fermentor was.reached. An appropriate amount of sterile 12% sodium deoxycholate was added to the culture to lyse the bacterial cells and release cell-associated polysaccharide. After losing, the fermentor contents were cooled. The pH of the lysed culture broth was adjusted to approximately pH 6.6 with acetic acid. The lysate was clarified by continuous flow centrifugation followed by depth filtration and 0.45 Pm microfiltration.
Purification The purification of the pneumococcal polysaccharide consisted of several concentration/diafiltraton operations, precipitation/elution, column chromatography, and depth filtration steps. All procedures were performed at room temperature unless otherwise specified. Clarified broth fron the fermentor cultures of S. pneumoniae serotype 3 were concentrated and diafiltered using a 100 kDa MWCO filter. Diafiltration was accomplished using sodium phosphate buffer at neutral pH. Diafiltration removed the low molecular weight medium components from the higher molecular weight biopolymers such as nucleic acid, protein and polysaccharde. Prior to the addition of hexadecyltrimethyl ammonium bromide (HB), a calculated volume of a NaCl stock solution was added to the concentrated and diaffitered polysaccharde solution to give a final concentration of 0.25 M NaCl. The polysaccharde was then precipitated by adding HB from a stock solution to give a final concentration of 1% HB (w/v). The polysaccharide/HB precipitate was captured on a depth filter and the filtrate was discarded. The polysaccharde precipitate was resolubilized and eluted by recirculating a sodium chloride solution through the precipitate-containing depth filter. The filters were thn rinsed with additional sodium chloride solution. Sodium iodide (Nal) was added to the polysaccharide solution from a stock Nal solution to achieve a final concentration of 0.5% to precipitate HB. The precipitate was removed by depth filtration. The filtrate contained the target polysaccharde. The precipitation vessel and the filter were rinsed with a NaC/Nal solution and the rinse was combined with the partially purified polysaccharide solution. The filter was discarded. The polysaccharide was then filtered through a 0.2 pm filter. The polysaccharide solution was concentrated on a 30 kDa MWCO ultrafilter and diafiltered with a sodium chloride solution. The partially purified polysaccharide solution was further purified by filtration through a depth filter impregnated with activated carbon. After filtration, the carbon
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filter was rinsed with a sodium chloride solution. The rinse was combined with the polysaccharide solution, which was then filtered through a 0.2 pm filter. The polysaccharide solutionwas concentrated on a 30 kDa MWCO ultrafilter and adjusted with a IM sodium phosphate buffer to achieve a final concentration of 0.025M sodium phosphate. The pH was checked and adjusted to 7.0 ±0.2. The ceramic hydroxyapatite (HA) column. was equilibrated with sodium phosphate buffer containing sodium chloride'to obtain the appropriate conductivity (15 pS). The polysaccharide solution was then loaded onto the column. Under these conditions, impurities bound to the resin and the polysacchadde was recovered In the flow-through from the column. The polysaccharide was flushed through the column with buffer and was filtered through a 0.2pm filter, The polysacchadde solution was concentrated using a 30 kDa MWCO filter. The concentrate was then diafiltered with WFI. The diafiltered polysaccharde solution was filtered through a 0.2 prn membrane fiter into stainless steel containers. Samples were removed for release testing and the purified polysaccharide was stored frozen at -25± 5°C.
Characterization The 1H-NMR data was consistent with the chemical structure by the assignment of signals assigned to the protons of the polysaccharide molecule. The identity of the monovalent polysaccharide was confirmed by countercurrent immunoelectrophoresis using specific antisera. High performance gel filtration chromatography, coupled with refractive index and multiangle laser light scattering (MALLS) detectors, was used in conjunctionwith Z5 the sample concentration to calculate the molecular weight. Size exclusion chromatography media'(CL-4B) was used to profile the relative molecular size distribution of the polysaccharde.
Example 4
Preparation of Serotype 3 Pneumococcal Saccharde - CRM19 Conjugate
Activation and Conjugation
Containers of purified serotype 3 saccharide were thawed and combined in a reaction vessel. To the vessel, WF and 2M acetic acid were added to a final concentration of 0.2M and 2mg/mL saccharide. The temperature of the solution was raised to 850C for one hour to hydrolyze the polysaccharide. The reaction was cooled to <25 0C and IM magnesium chloride was added to a final concentration of 0.1M. Oxidation in the presence of sodium periodate was performed by incubation for 16-24 hours at 23°C. The activation reaction mixture was concentrated and diafiltered 1Ox with.WFl using a100K MWCO membrane. The retentate was filtered through a0.2-pmfilter. For compounding, 0.2M sodium phosphate, pH 7.0, was added to the activated saccharide to a final concentration of 10mM and a pH of 6.0-6.5. CRM1er carder protein was mixed with the saccharide solution to a ratio of 2g of saccharde per Ig of CRM1 97. The combined saccharide/protein solution was filled into 100 mL glass lyophilization bottles with a 5OmL target fill, shell-frozen at -75°C, and lyophilized. Bottles of co-lyophilized saccharide/protin material were brought to room temperature and resuspended In 0.1M sodium phosphate buffer, pH 7.0, to a final saccharde concentration of 20 mg/mL. The pH was adjusted to 6.5 and then a 0.5 molar equivalent of sodium cyanoborohydride was added. The reaction was incubated at 370C for 48 hours. Following the cyanoborohydride incubation, the reaction mixture was diluted with cold 5mM succinate/0.9% saline buffer. Unreacted aldehydes were quenched by the addition of sodium borohydride and incubation at 23°C for 3-6 hours. The reaction mixture was transferred through a 0.45-5 pm prefilter into a retentate vessel. The reaction mixture was diailtered 30x with 0.1M phosphate buffer (pH 9), 20x with 0.15M phosphate butter (pH 6), and 20x with 5mM succinate/0.9% saline. The retentate was filtered through a 0.2-pm filter.
The conjugate solution was diluted to a sacchaide target of 0.5 mg/mL, and then sterile filtered IntoFBC containers in a Class 100 hood. The conjugate was stored at 2 - 8°C. Characterization Size exclusion chromatography media (CL-4B) was used to profile the relative molecular size distribution of the conjugate. The identity of the conjugate was confirmed by the slot-blot assay using specific antisera. The saccharide and protein concentrations were determined by the Anthrone and Lowry assays, respectively. The ratio of saccharde to protein in the covalently bonded conjugate complex was obtained by the calculation:
pg/mL saccharide Ratio= pg/mL protein
Example 5 Preparation of S. Pneumoniae Capsular Polysaccharide Serotype 5
S. pneumoniae serotype 5 was obtained from Dr. Gerald Schiffman of the State University of New York, Brooklyn, New York. For preparation of the cell bank system, see Example 1. For fermentation, harvesting, purification and characterization of the polysaccharide, see Example 1.
Alternate Fermentation Process
Cultures from the working cell bank were used to Inoculate seed bottles containing a soy-based medium and a 10mM sterile NaHCO3 solution. The bottles were incubated at 36°C± 20 C without agitation until growth requirements were met. A seed bottle was used to inoculate a seed fermentor containing soy-based medium and a 10mM sterile NaHCO solution. A pH of about 7.0 was maintained with 3N NaOH. After the target optical density was reached, the seed fermentor was used to inoculate the production fermentor containing soy-based medium with a 10mM
NaHCOs concentration. The pH was maintained with 3N NaOH. The fermentation was terminated after cessation of growth or when the working volume of the fermentor was reached. An appropriate amount of sterile 12% sodium deoxycholate was added to the culture to obtain a 0.12% concentration in the broth, to lyse the bacterial cells and release cell-associated polysaccharide. After lysing, the fermentor contents wer held, with agitation, for a time interval between 8 and 24 hours at a temperature between 70C and 130C to assure that complete cellular lysis and polysaccharde release had occurred. Agitation during this hold period prevented lysate sediment from settling on the fermentor walls and pH probe, thereby allowing the pH probe integrity to be maintained. Next, the pH of the lysed culture broth was adjusted to approximately pH 4.5 with 50% acetic acid. After a hold time without agitation, for a time interval between 12 and 24 hours at a temperature between 15C and 25°C, a significant portion of the previously soluble proteins dropped out of solution as a solid precipitate with little loss or degradation of the polysaccharide, 16 which remained in solution. The solution with the precipitate was then clarified by continuous flow centrifugation followed by depth filtration and 0.45 pm microfiltration.
Example 6
Preparation of Serotype 5 Pneumococcal Saccharlde - CRM 1 r Conjugate
Activation and Conjugation .
Containers of serotype 5 saccharlde were thawed and combined in a reaction vessel. To the vessel, O.1M sodium acetate, pH 4.7, was added followed by oxidation in the presence of sodium periodate by incubation for 16-22 hours at 23°C. The activation reaction mixture was concentrated and diafiltered 1Ox with WF using a100K MWCO membrane. The retentate was filtered through a0.2 pm filter. The serotype 5 activated saccharide was combined with CRM 1er at a ratio of 0.8:1. The combined saccharidelprotein solution was filled into 100 mL glass lyophilization bottles (50 mL target fill), shell-frozen at -75°C, and co-lyophilized. Bottles of co-lyophilized material were brought to room temperature and resuspended in 0.1M sodium phosphate, pH 7.5, and sodium cyanoborohydride was added. The reaction was incubated at 30°C for 72 hours, followed by a second addition of cyanoborohydride and incubated at 300C for 20-28 hours.
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Following the cyanoborohydride Incubations, the reaction mixture was diluted 2--fold with saline and transferred through a 0.45-5 pm prefilter into a retentate vessel. The reaction mixture was diafiltered 30x with 0.01M phosphate buffer, pH 8, 20x with 0.15M phosphate buffer, pH 6, and 20x with saline. The retentate was filtered through a0.2 pm filter., The conjugate solution was diluted to a saccharde.target of 0.5 mg/mL, and then sterile filtered into FBC containers in a Class 100 hood. The conjugate was stored at 2- 8°C. For the characterization of the conjugate, see Example 2.
Example 7 Preparation of S. Pneumonlae Capsular Polysaccharide Serotype 6A
S. pneumoniae serotype 6A was obtained from Dr. Gerald Schiffman of the State University of New York, Brooklyn, New York. For preparation of the cell bank system, see Example 1. For fermentation, harvesting and purification of the polysaccharide, see Example 1, except that during purification, the 30 kDa MWCO concentration step, prior to the chromatography step, is omtted.
Example 8 Preparation of Serotype 6A Pneumococcal Saccharide - CRM 19 7Conjugate
Activation and Coniuqation Serotype 6A polysaccharide is a high molecular weight polymer that had to be reduced in size prior to oxidation. Containers of serotype 6A sacchaide were thawed and combined in a reaction vessel. To the vessel, 2 M acetic acid was added to a final concentration of 0.1 M for hydrolysis for 1.5 hours at 60°C. The reaction was cooled to 23°C and neutralization was performed by adjusting the reaction mixture with I M NaOH to pH 6. Oxidation in the presence of sodium perodate was performed by incubation.at 230 C for 14-22 hours. The activation reaction mixture was concentrated and diafiltered 1Ox with WFI using a1OOK MWCO membrane. The retentate was filtered through a 0.2 pm filter. Serotype 6A was compounded with sucrose and filled into 100 mL glass lyophilization bottles (5OmL target fill) and shell-frozen at -750 C andlyophilized.
Bottles of lyophilized material were brought to room temperature and resuspended in dimethylsulfoxide (DMSO) at a saccharidelprotein ratio of 1:1. After addition of sodium cyanoborohydride, the reaction mixture was incubated at 23°C for 18 hours. Following the cyanoborohydride incubation, the reaction mixture was diluted with cold saline. Unreacted aldehydes were quenched by addition of sodium borohydride by Incubation at 230C for 3-20 hours. The diluted reaction mixture was transferred through a 5 pm prefilter Into a retentate vessel. The reaction mixture was diafiltered 1Ox with 0.9% NaCl and 30x with succinate-buffered NaCL The retentate was filtered through a 0.2 pm filter. The conjugate solution was diluted to a saccharide target of 0.5 mg/mL, and then sterile filtered into FBC containers in a Class 100 hood. The conjugate was stored at 2 --8C. For the characterization of the conjugate, see Example 2.
Example 9 Preparation of S. Pneumoniae Capsular Polysaccharide Serotype 7F
S. pneumoniae serotype 7F was obtained from Dr. Gerald Schiffman of the State University of New York, Brooklyn, New York. For preparation of the cell bank system, and for fermentation and harvesting of the polysaccharide, see Example 3. For an alternate fermentation and harvesting process, see the alternate process described in Example 1.
Purification The purification of the pneumococcal polysaccharide consisted of several concentrationldiafiltration operations, precipitation/elution, column chromatography, and depth filtration steps. All procedures were performed at room temperature unless otherwise specified. Clarified broth from fermentor cultures of S. pneumoniae serotype 7F were concentrated and diafiltered using a 100 kDa MWCO filter. Diafiltration was accomplished using sodium phosphate buffer at neutral pH. Diafiltration removed the low molecular weight medium components from the higher molecular weight biopolymers such as nucleic acid, protein and polysaccharide.
Serotype 7F does not form a precipitate with HB. Instead, impurities were precipitated from the concentrated and dafiltered solution by adding the HB from a stock solution to a final concentration of 1% HB. The precipitate was captured on a depth filter and the filter was discarded. The polysacharde was contained in the filtrate. Sodium iodide (Nal) was added to the polysaccharde solution from.a stock Nal solution to achieve a final concentration of 0.5% to precipitate HB. The precipitate was removed by depth filtration. The filtrate contained the target polysaccharde. The precipitation vessel and the filter were rinsed with a NaC/Nal solution and the rinses were combined with the partially purified polysaccharide solution. The filter was discarded. The polysaccharlde was then filtered through a 0.2pm filter. The polysaccharide solution was concentrated on a 30 kDa MWCO ultrafilter and diafiltered with a sodium chloride solution. The partially purified polysaccharide solution was further purified by filtration through a depth filter impregnated with activated carbon. After filtration, the carbon filter was rinsed with a sodium chloride solution. The rinse was combined with the polysaccharide solution, which was then filtered through a 0.2 pm filter. The polysaccharide solution was concentrated on a 30 kDa MWCO ultrafilter and adjusted with a IM sodium phosphate buffer to achieve a final concentration of 0.025M sodium phosphate. The pH was checked and adjusted to 7.0i 0.2. The ceramic hydroxyapatite (HA) column was equilibrated with sodium phosphate buffer containing sodium chloride to obtain the appropriate conductivity (15 pS). The polysaccharide solution was then loaded onto the column. Under these conditions, impurities bound to the resin and the polysaccharide was recovered in the flow-through from the column. The polysaccharide was flushed through the column with buffer and was filtered through a0.2 pm filter. The polysaccharide solution was concentrated using a 30 kDa MWCO filter. The concentrate was then diafiltered with WFI. The diafiltered polysaccharide solution was filtered through a 0.2 pm membrane filter into stainless steel containers. Samples were removed for release testing and the purified polysaccharide was stored at 2° - 80C. For characterization of the polysaccharide, see Example 3.
Example 10
Preparation of Serotype 7F Pneumococcal Saccharide - CRM 1 97Conjugate
Activation and Conjugation Oxidation in the presence of sodium perodate was performed by incubation for 16-24 hrs at 23°C. The activation reaction mixture was concentrated and diafiltered 1x with 10mM NaOAc, pH 4.5, using a OOK MWCO membrane. The retentate was filtered through a 0.2 im filter. Serotype 7F was filled into 100 mL glass yophilization bottles (50 mL target fill) and shell-frozen at -75°C andlyophilized. Bottles of lyophilized serotype 7F and CRM197 were brought to room temperature and resuspended in DMSO at a saccharide/protein ratio of 1.5:1. After the addition of sodium cyanoborohydride, the reaction was incubated at 230C for 8 10 hours. Unreacted aldehydes were quenched by the addition of sodium borohydride by incubation at 230C for 16 hours. The reaction mixture was diluted 10-fold with cold saline and transferred through a 5 pm prefilter into a retentate vessel. The reactionmixture was diafiltered IOx with 0.9% saline and 30x with succinate-buffered saline. The retentate was filtered through a 0.2 pm filter. The conjugate solution was diluted to a saccharde target of 0.5 mg/mL0.9% saline, and then sterile filtered into FBC containers in a Class 100 hood. The conjugate was stored at 2 - 8°C. For characterization of the conjugate, see Example 4.
Example 11 Preparation of S. Pneumoniae Capsular Polysaccharde Serotype 19A
S. pneumoniae serotype 19A was obtained from Dr. Gerald Schiffman of the State University of New York, Brooklyn, New York. For preparation of the cell bank system, see Example 1. For fermentation, harvesting and purification of the polysaccharide, see Example 7. For characterization, see Example 3.
Example 12 Preparation of Serotype 19A Pneumotoccal Saccharde - CRM 1 r Conjugate
Activation and Conjugation Containers of serotype 19A saccharide were thawed and combined in a reaction vessel. Sodium acetate was added to 10 mM (pH 5.0) and oxidation was carried out in the presence of sodium periodate by incubation for 16-24 hrs at 23C. The activation reaction mixture was concentrated and diafiltered lox with 10mM acetate, pH 5.0, using alOOK MWCO membrane. The retentate was filtered through a 0.2 pm filter. The activated saccharide was compounded with sucrose followed by the addition of CRM 197 . The serotype 19A activated saccharide and CRM 19 7 mixture (0.8:1 ratio) was filled into 100 mL glass lyophilization bottles (50 mL target fill) and shell-frozen at -750 C andlyophilized. Bottles of lyophilized material were brought to room temperature and resuspended in DMSO. To the saccharide/protein mixture, sodium cyanoborohydride (100 mgml) was added. The reaction was incubated at 23C for 15 hours. Following the cyahoborohydride incubation, unreacted aldehydes were quenched by the addition of sodium borohydride by Incubation at 230 C for 3-20 hours. The reaction mixture was diluted 10-fold with cold saline and transferred through a 5 pm prefilter into a retentate vessel. The reaction mixture was diafiltered 1Ox with 0.9% NaCl, 0.45-pm filtered, and 30xwith diailtration using 5mM succinate 0.9% NaCl buffer, pH 6. The retentate was filtered through a 0.2 pm filter.
The conjugate solution was diluted to a target of 0.5 mg/mL using 5mM succinate/0.9% saline, and then sterile filtered into FBC containers In a Class.100 hood. The conjugate was stored at 2 - 8°C. For characterization of the conjugate, see Example 4.
Example 13 Preparation of S. Pneumoniae Capsular Polysaccharide Serotypes 4,61, 9V, 14,18C, 19F and 23F
Preparation of theS. pneumoniae Seed Culture S. pneumoniae serotypes 4, 6B, 9V, 18C, 19F and 23F were obtained from Dr. Gerald Schiffrman, State University of New Yorl, Brooklyn, New York. S. pneumoniae serotype 14 was obtained from the ATCC, strain 6314. Separately, one vial of each of the desired serotypes of Streptococcus pneumonae was used to start a fermentation batch. Two bottles containing a soy based medium and phenol red were adjusted to a pH range of 7.4 ± 0.2 using sodium carbonate, and therequired volume of 50% dextrosel% magnesium sulfate solution was then added to the bottles. The two bottles were inoculated with different amounts of seed. The bottles were incubated at 36± 2°C until the medium turned ) yellow. Following incubation, samples were removed from each bottle and tested for optical density (OD) (0.3 to 0.9) and pH (4.6 to 5.5). One of the two bottles was selected for inoculation of the seed fermentor. Soy-based medium was transferred to the seed fermentor and sterilized. Then a volume of 50% dextrose/1% magnesium sulfate solution was added to the fermentor. The pH and agitation of the seed fermentor were monitored and controlled (pH 6.7 to 7.4). The temperature was maintained at 36°± 20C. The seed inoculum (bottle) was aseptically connected to the seed fermentor and the inoculum was transferred. The fermentor was maintained in pH control and samples were periodically removed and tested for OD and pH. When the desired OD of 0.5 at 600 nm was reached, the intermediate fermentor was inoculated with the fermentation broth from the seed fermentor. Soy-based medium was transferred to the intermediate fermentor and sterilized. Then a volume of 50% dextrose/1% magnesium sulfate solution was added to the fermentor. The pH and agitation of the intermediate fermentor were monitored and controlled (pH 6.7 to 7.4). The temperature was maintained at 36° 2°C. The contents of the seed fermentr were transferred to theintermediate fermentor. The fermentor was maintained in pH control and -samples were periodically removed and tested for OD and pH. When the desired OD of 0.5 at 600 nm was reached, the production fermentor was inoculated with the fermentation broth from the Intermediate fermentor. Soy-based medium was transferred to the production fermentor and sterilized. Then a volume of 50% dextrose/1% magnesium sulfate solution was added to the fermentor. The pH and agitation of the production fermentor were monitored and controlled (pH 6.7 to 7.4). The temperature was maintained at 360 2°C. The fermentor was maintained In pH control and samples were periodically removed and tested for OD and pH, until the fermentation was complete. Deoxycholate sodium was added to the fermentor to a final concentration of approximately 0.12% w/v. The culture was mixed for a minimum of thirty minutes and the temperature set point was reduced to10C. The culture was incubated ovemight and following confirmation of inactivation, the pH of the culture was adjusted to between 6.4 and 6.8, as necessary, with 50% acetic add. The temperature of the fermentor was increased to 200 ± 5°C and the contents were transferred to the clarification hold tank. The contents of the clarification hold tank (including the cellular debris) were processed through a centrifuge at a flow rate between 25 and 600 liters per hour (except Serotype 4, wherein the cell debris was discarded and the flow rate tightened to- between 25 and 250 liters per hour). Samples of the supernatant were removed andtestedforOD. The desired OD during the centrifugation was<0.15. Initially, the supematant was recirculated through a depth filter assembly until an OD of 0.05 ± 0.03 was achieved. Then the supernatant was passed through the depth filter assembly and through a 0.45 pm membrane filter to the filtrate hold tank. Subsequently, the product was transferred through dosed pipes to the purification area for processing. All of the above operations (cntrifugatlon, filtration and transfer) were performed between 10°C to 30°C.
For an alternate fermentation and harvesting process for serotypes 4 and 6B, see the alternate process described in Example 1.
Purification The purification of each pneumococcal polysaccharide consisted'of several concentration/diafiltration operations, precipitation/elutlon, column chromatography, and depth filtration steps. All procedures were performed at room temperature unless otherwise specified. Clarified broth from the fermentor cultures of the desired S. pneumonlae serotype was concentrated and diafiltered using a 100 kDa MWCO fiter. Diafiltration was accomplished using sodium phosphate buffer at pH < 9.0. Diafiltration removed the low molecular weight medium components from the higher molecular weight biopolymers such as nucleic acid, protein and polysaccharide. The polysaccharide was precipitated from the concentrated and diafiltered solution by adding HB from a stock solution to give a final concentration of 1% HB (wv) (except Serotype 23F, which had a final concentration of 2.5%). The polysaccharide/HB precipitate was captured on a depth filter and the filtrated was discarded. (Note: Serotype 14 does not precipitate; therefore the filtrate was retained.) The polysaccharide precipitate was resolubilized and eluted by recirculating a sodium chloride solution through the precipitate-containing depth filter. The filters were then rinsed with additional sodium chloride solution. Sodium iodide (Nal) was added to the polysaccharide solution from a stock Nal solution to-achieve a final concentration of 0.5% to precipitate HB (except for Serotype 68, which had a final concentration of 0.25%). The precipitate was removed by depth filtration. The filtrate contained the target polysaccharide. The filter was discarded. The polysaccharide was then filtered through a 0.2pm filter. The polysaccharide solution was concentrated on a 30 kDa MWCO ultrafiter and diaflftered with a sodium chloride solution. The partially purified polysaccharide solution was further purified by filtration through a depth filter impregnated with activated carbon. After filtration, the carbon filter was rinsed with a sodium chloride solution. The rinse was combined with the polysaccharide solution, which was then filtered through a0.2pm filter.
The polysaccharide solution was concentrated on a 30 kDa MWCO utrfilter and the filter was rinsed with'a sodium chloride solution. The pH was checked and adjusted to 7.0* 0.3. The ceramic hydroxyapatite (HA) column was equilibrated with sodium phosphate buffer containing sodium chloride until the pH is 7.0 h 0.3 and the conductivity was 26 + 4pS. The polysaccharide solution was then loaded onto the column. Under these conditions, impurities bound to the resin and the polysaccharide was recovered in the flow through from the column. The polysaccharide solution was filtered through a 0.2pm filter. The polysaccharide solution was concentrated using a 30 kDa MWCO filter. The concentrate was then diafiltered with WFI until the conductivity was <15pS. The dlafiltered polysaccharde solution was filtered through a 0.2pm membrane filter into bulk containers and stored at 2-80C.
Example 14 Preparation of Pneumococcal Saccharide - CRMI, Conjugates For Serotypes 4, 613, 9V, 14, 18C, 19F and 23F
Activation Process The different serotype saccharides follow different pathways for activation (hydrolysis or no hydrolysis prior to activation) and conjugation (aqueous or DMSO reactions) as described In this example. Polysaccharide was transferred from the bulk containers to the reactor vessel. The polysaccharde was then diluted in WFI and sodium phosphate to a final concentration range of 1.6 - 2.4 mg/mL.
Step 1. For serotypes 6B, 9V, 14, 19F and 23F, pH was adjusted to pH 6.0 0.3. For serotype 4, hydrochloric acid (0.01 M final acid concentration) was added and the solution was incubated for 25 - 35 minutes at 450 ± 20C. Hydrolysis was stopped by cooling to 21 - 250C and adding 1M sodium phosphate to a target of pH 6.7 ± 0.2. An in-process test was done to confirm an appropriate level of depyruvylation.
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For serotype 18C, glacial acetic acid (0.2M final acid concentration) was added and the solution was incubated for 205 - 215 minutes at 940±20C. Temperature was then decreased to 21 - 25°C and 1 - 2 M sodium phosphate was added to a target of pH 6.8 ± 0.2.
Step 2: PeriodateReaction The required sodium periodate molar equivalents for pneumococcal saccharde activation was determined using total saccharide content (except for serotype 4). For serotype 4, a ratio of 0.8-1.2 moles of sodium perodate per mole of saccharde was used. With thorough mixing, the oxidation reaction was allowed to proceed between 16 to 20 hours at 21 - 250C for all serotypes except 19F for which the temperature was s 15°C.
Step 3: Ultrafiltration The oxidized saccharide was concentrated and diafiltered with WFI (0.01 M sodium phosphate buffer pH 6.0 for serotype 19F) on a 100 kDa MWCO ultrafilter (5 kDa ultrafilter for 18C). The permeate was discarded and the retentate was filtered through a 0.22 r filter.
Step 4: Lyophilization For serotypes 4, 9V, and 14 the concentrated saccharde was mixed with CRM 197 carrier protein, filled into glass bottles, shell-frozen and stored at5 -65°C. The frozen concentrated saccharide-CRM 197was lyophilized and then stored at -25° 50C. For serotypes 6B, 19F, and 23F a specified amount of sucrose was added which was calculated to achieve a 5% ± 3% sucrose concentration in the conjugation reaction mixture. Serotype 18C did not require sucrose addition. The concentrated saccharide was then filled into glass bottles, shell-frozen and stored at<s -650C. The frozen concentrated saccharide was lyophilized and then stored at -250±50C.
Conjugation Process Two conjugation processes were used: aqueous conjugation for serotypes 4, 9V, 14 and 18C, and DMSO conjugation for serotypes 6B, 19F and 23F.
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Aqueous Conjugation Step 1: Dissolution For serotypes 4, 9V and 14, the yophilized activated saccharide-CRM 197 mixture was thawed and equilibrated at room temperature. The lyophilized activated saccharide-CRM 1 was then reconstituted in .M sodium phosphate buffer at a typical ratio of: * IL of bufferper 16 -24 g of sacchardde for seroype-4 and 9V * 1L of buffer per 6 - 10 g of saccharlde for serotype 14
The reaction mixture was incubated at 370 ± 2°C until total dissolution for the serotype 9V and at 23°0± 2°C for serotypes 4 and 14. For serotype 18C, the lyophilized saccharde was reconstituted in a solution of CRM 1 97in IM dibasic sodium phosphate at a typical ratio of 0.11 L of sodium phosphate per 1 L of CRM 1er solution. The reaction mixture (8-12 g/L saccharide concentration) was incubated at 23± 2C until total dissolution. The pH was tested as an in-process control at this stage.
Step 2: Conjugation Reaction For serotypes 4 and 9V, the conjugation reaction was initiated by adding the sodium cyanoborohydrde solution (100 mg/mL) to achieve 1.0 - 1.4 moles sodium cyanoborohydride per mote of saccharide. The reaction mixture was incubated for 44 - 52 hours at 370 * 2°C. The temperature was then reduced to 230 ± 2C and sodium chloride 0.9% was added to the reactor. Sodium borohydride solution (100 mg/mL) was added to achieve 1.8 - 2.2 molar equivalentsof sodium borohydride per mole saccharde. The mixture was incubated for 3 - 6 hours at 230± 20 C. The mixture was diluted with sodium chloride 0.9% and the reactor was rinsed. The diluted conjugation mixture was filtered using a1.2 pm pre-filter into a holding vessel. For serotypes 14 and 18C, the conjugation reaction was initiated by adding the cyanoborohydride solution (100 mg/mL) to achieve 1.0 - 1.4 moles of sodium cyanoborohydride per mole of saccharde. The reaction mixture was incubated for 12 - 24 hours at 230 ± 2C. The temperature was increased to 370 20C and the reaction was incubated for 72 - 96 hours. The temperature was then reduced to 230
2°C and 0.9% sodium chloride was added to the reactor. Sodium borohydride solution (100mg/mL) was added to achieve 1.8 - 2.2 molar equivalents of sodium borohydride per mole of saccharde. The mixture was incubated for 3 - 6 hours at 23°0 2°C. The mixture was diluted with 0.9% sodium chloride and the reactor was rinsed. The diluted conjugation mixture was then filtered using a 1.2 pm pr-filterinto a holding vessel.
Step 3: Ultrafiltration 100 kDa The diluted conjugation mixture was concentrated and diafiltrated on a 100 kDa MWCO ultrafilter with either a minimum of 15 volumes (serotype 4) or 40 volumes (serotypes 9V, 14, and 18C) of 0.9% sodium chloride.. The permeate was discarded. For serotype 4, the retentate was filtered through a 0.45pm filter. An In-process control (saccharide content) was performed at this step.
Step 4: HA Column Purfication This step was only performed for the serotype 4 conjugate. The HA column was first neutralized using 0.5M sodium phosphate buffer (pH 7.0 ± 0.3) and then equilibrated with 0.9% sodium chloride. The fitered retentate (serotype 4) was loaded onto the column at a flow rate of 1.0 Umin. The column was washed with 0.9% sodium chloride at a flow rate of 2.0 Umin. The product was then eluted with 0.5M sodium phosphate buffer at a flow rate of 2.0 Umin. The HA fraction was then concentrated and diafiltered on a 100 kDa MWCO membrane with a minimum of 20 volumes of 0.9% sodium chloride. The permeate was discarded.
Step 5: Sterile Filtration The retentate after the 100 kDa MWCO diafiltration was filtered through a 0.22pm filter. In-process controls (saccharide content, free protein, free saccharde and cyanide) were performed on the filtered product. In-process controls on filtered retentate were performed to determine whether additional concentration, diafiltration, and/or dilution were needed to meet FBC targets. These and additional tests were repeated in FBC samples.
As necessary, the filtered conjugate was diluted with 0.9% sodium chloride in order to achieve a final concentration of less than 0.55 g/L. Release tests for saccharide content, protein content and saccharide:protein ratio were performed at this stage. Finally, the conjugate was filtered (0.22 pm) and filled into 10 L stainless steel canisters at a typical quantity of 2.64 g/canister. At this stage, yield, saccharde content, protein content, pH, saccharide:protein ratio and lysine content were performed as in-pocess controls. Release testing (appearance, free protein, free saccharide, endotoxin, molecular size determination, residual cyanide, saccharide Identity, CRM197identity)was perfdrmed at this stage.
DMSO Conjugation Step I: Dissolution The lyophilized activated saccharide serotypes 6, 19F, 23F and the yophilized CRM197 carrier protein were equilibrated at room temperature and reconstituted in DMSO. The dissolution concentration typically ranged from 2-3 grams of saccharde (2-2.5 g protein) per liter of DMSO.
Step II: Conjugation Reaction The activated saccharide and CRM 197 carrier protein were mixed for 60 - 75 minutes at 23±2°C at a ratio range of 0.6 g - 1.0 g saccharlde/g CRM191 for serotypes 6B and 19F or 1.2 to 1.8 g accharide/g CRM 191 for serotype 23F. The conjugation reaction was initiated by adding the sodium cyanobohydride solution (100mg/mL) at a. ratio of 0.8 - 1.2 molar equivalents of sodium cyanoborohydride to one mole activated saccharide. WFI was added to thereaction mixture to a target of 1% (v/v) and the mixture was incubated for over 40 hours at 230 20C. Sodium borohydride solution, 100 mg/mL (typical 1.8 - 2.2 molar equivalents sodium borohydride per mole activated saccharide) and WFI (target 5% v/v) were added to the reaction and the mixture was incubated for 3 - 6 hours at 2302°C. This procedure reduced any unreacted aldehydes present on the saccharides. Then the reaction mixture was transferred to a dilution tank containing 0.9% sodium chloride at <150 C.
Step I: 100 kDa Ultrafiltration The diluted conjugate mixture was filtered through a 1.2 pm filter and concentrated and diafiltered on a 100 kDa MWCO membrane with a minimum of 15 volumes of 0.9%'sodium chloride (0.01M sodium phosphate/0.05M NaCl buffer was used for serotype 23F). The permeate was discarded. The retentate was filtered through a 0.45 pm filter. An in-process saccharide content sample was taken at this stage.
Step IV: DEAE Column Purification This step was only performed for serotype 23F. The DEAE column was equilibrated with 0.01M sodium phosphate/0.05M sodium chloride buffer. The filtered retentate (serotype 23F) was loaded onto the column and washed with 0.01M sodium phosphatO.05M sodium chloride buffer. The column was then washedwith 0.01M sodium phosphatel0.9% NaCl buffer. The product was then eluted with 0.01M sodium phosphate/0.5M sodium chloride buffer. StepV: 100kDaUlfrafiltration The retentate from 6B and 19F was concentrated and diafiltered with at least 30 volumes of 0.9% sodium chloride. The permeate was discarded. The eluate from serotype 23Fwas concentrated and diafiltered with a minimum of 20 volumes of 0.9% sodium chloride. The permeate was discarded.
Step W: Sterile Filtration The retentate after the 100 kDa MWCO dialfiltration was filtered through 0.22 pm filter. In-process controls (saccharide content, free protein, free saccharide, residual DMSO and residual cyanide) were performed on the filtered product. In process controls on filtered retentate were performed to determine whether additional concentration, diafiltration, and/or dilution were needed to meet FBC targets. These and additional tests were repeated in FBC samples. As necessary, the filtered conjugate was diluted with 0.9% sodium chloride to achieve a final concentration of less than 0.55 g/L. Release tests for saccharide content, protein content and saccharlde:protein ratio were performed at this stage.
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Finally, the conjugate was filtered (0.22 pm) and filled into 10 L stainless steel canisters at a quantity of 2.64 g/canister. At this stage, yield, saccharide content, protein content, pH, saccharide:protein ratio and lysine content were performed as in process controls. Release testing (appearance, free protein, free saccharide, 5 endotoxin, molecular size determination, residual cyanide, residual DMSO, saccharide identity and CRM197 identity) was performed at this stage.
Example 15 Formulation of a Multivalent Pneumococcal Conjugate Vaccine The final bulk concentrates of the 13 conjugates contain 0.85% sodium chloride. Type 3, 6A, 7F and 19A bulk concentrates also contain -5 mM sodium succinate buffer at pH 5.8. The required volumes of bulk concentrates were calculated based on the batch volume and the bulk sacharide concentrations. After 80% of the 0.85% sodium chloride (physiological saline) and the required amount of succinate buffer were added to the pre-labeled formulation vessel, bulk concentrates were added. The preparation was then sterile filtered through a 0.22 pm membrane into a second container by using a Millipore Durapore membrane filter unit. The first container was washed with the remaining 20% of 0.85% sodium chloride and the solution was passed through the same filter and collected into the second container. The formulated bulk was mixed gently during and following the addition of bulk aluminum phosphate. The pH was checked and adjusted if necessary. The formulated bulk product was stored at 2--8C. The formulated bulk product was filled into Type 1 borosilicate glass syringes obtained from Becton Dickinson. The vaccine was monitored at regular intervals for turbidity to ensure the uniformity of the filling operation. The filled vaccine (Final Product) was stored at 2-80 C.
Example 16 Immunogenicity of the 13-Valent Conjugate Vaccine To date, the preclinical studies performed on the 13vPnC vaccine have been in rabbits. Studies #HT01-0021 and #HTOI-0036 were designed to independently examine the effect of chemical conjugation of capsular polysaccharides (PSs) from S pneumonlae to CRM197 and the effect of aluminum phosphate (AIPO 4) adjuvant on the immune response to the 13vPnC vaccine In rabbits. These effects were characterized by antigen-speciic ELISA for serum IgG concentrations and for antibody function by opsonophagocyticassay (OPA).
Study#HT1-0021
Study #HT01-0021 examined the ability of the 13vPnC vaccine with AIPO 4 adjuvant to elicit vaccine serotype-specific Immune responses. The pneumococcal serotypes represented In the 13vPnC vaccine include types 1, 3, 4, 5, 6A, 6B, 7F, 9V,14,18C,19A,19Fand23F Secondary objectives included an evaluation of the kinetics and duration of the antibody response. New Zealand White rabbits were immunized intramuscularly at week 0 and week 2 with the planned human clinical dose of each polysaccharide (2 pg of each PS, except 4pg of 6B) formulated with or without AIPO4 (100 pg /dose). Sera were collected at various time points. Serotype specific IgG was measured by ELISA and functional activity was assessed by OPA.
Table 3 shows the geometric mean titer (GMT) achieved in pooled serum samples, following two doses of the 13vPnC vaccine. A ratio of the IgG GMTs was used to compare responses from week 4 to week 0. These data demonstrate that the inclusion of AIPO 4 in the 13vPnC formulation elicited higher levels of IgG antibody in comparison to the same vaccine without adjuvant. Although the antibody responses were greater when APO 4 was included in the formulation, these increases were not statistically significant.
Functional antibody responses were also assessed in rabbits following immunization with the two 13vPnC formulations (Table 4). When comparing vaccine formulations with or without adjuvant, higher OPA GMTs were observed in the 13vPnC + APO 4 vaccine treatment group. OPA titers were detected in week 4 serum pools to all vaccine serotypes in both groups. For the majority of the serotypes, OPA titers measured at week 4 were at least 4-fold higher than those at week 0 (baseline).
The kinetic responses to each of the 13vPnC vaccine serotypes were evaluated from serum-pools of both treatment groups. IgG titers to each serotype were measured from blood draws at week 0 and weeks 1, 2, 3, 4, 8, 12, 26, and 39 and then compared. With the exception of serotype 1, antibody responses in animals receiving adjuvanted vaccine were superior to those that received non adjuvanted vaccine and peaked at week.2 of the immunization schedule (data not shown).
Overall, the data indicate that the 13vPnC vaccine formulated with aluminum phosphate is immunogenic in rabbits, eliciting substantial antibody responses to the pneumococcal capsular polysaccharides contained in the vaccine and these responses are associated with functional activity. The responses observed to the seven core serotypes following immunization with 13vPnC + APO 4 are consistent with historical responses of rabbits to the heptavaent formulation.
Table 3. Rabbit IgG Immune Responses (GMTs) Following Immunization with Two Doses of 13-valent Pneumococcal Glycoconjugate Olluentwith ALPO,? 13vPnC" 13vPnC+ ALPOe Ratio Week 4 Ratio Week4 Ratio. Serotype Week 0 Week 4 Wk4:WkD Week 0 (95%CI) Wk4:WkO Week 0 (95%CI) Wk4:WkO 1 10 <0 . o5,926 11 o11,09122 1 <100 <100 1.0 50 (2,758-12,733) 119 50 (5,327-23,093) 3 1 0 10 Id o6,647 13 816,44328 3 <100 <100 1.0 50 (2,773-15,932) 133 58 (7,09-38106) 284 413,554 27 029,18358 4 <100 <100 1.0 50 (8,031-22,875) 271 50 (15,342-55,508) 584 5- 134 <100 0.4 50 5,859 117 50 (6,9 140) 334 (2,450-14,009) (,5-010 G 14 10 O 7422,415 30 363,73476 6A 141 <100 0.4 74 ($11,987-41,914) 303 83 (21,141-192,146) 6 <100 <100 1.0 57 (3,5184 142 54 (11,2868955) 435 7F 3,859 579 0.2 171 (26931-70,557) 444 143 (46,44 55151) 496 9V 289 995 3.4 205 (7,19334,616) 125 208 (23,256-71,510) 217 14 437 177 0.4 61 416-13,962) 113 70 (9696785) 322 18C <10 < I8 0 10 50 21,283 46 s-35,04070 <100 <100 1.0 50 (15,770-28,725) 426 50 (24,70849,692) 701 19A <100 <100 1.0 121 113,599 (54,518-236,707) 939 99 144 14 280,976 (119,587-660,167) 1 1951
19F 14,365 24,912 <100 <100 1.0 50 (7,346-28,090) 287 50 (9,243-67,141) 498 23 105,323 15,041 23F <100 <100 1.0 50 (1,894-14,962) 106 50 (4,711-48,018) 301 a: GMTs of pooled sera consisted of equal volumes of serumfrom each Indvidual rabbtwithin a group b: Stallstcally different (p=0.022)from treatment groupwIthout ALPO 4
Table 4. . pneumonae OPA GMTs forNZW Rabbit SerumPools Following Immunization'with Two Dosesiof13-valent Pneumococcal Glycoconjugate 13VIRnC* 13vnC+ALPo1 Week0 Week4 Wk :Wko Week 0 Week 4 Wk4ik
1 <8 64 16 <8 64 16 3 <8 8 2 <8 16 4 4 <8 16 4 <8 32 8 5 <8 128 32 <8 512 128 6A 8 128 16 8 512 64 61 <8 256 64 8 1,024 128 7F 8 64 8 8 128 16 9V 8 64 8 8 128 16 14 16 32 2 16 32 2 18C 8 256 32 <8 256 64 19A <8 256 64 <8 1,024 256 19F <8 128 32 <8 512 128 23F 8 64 8 <8 256 64 A: Pools consisted of equal volumes of serum from-individual rabbits within a treatment group (n=12)
Study#HTOI-0036
Study #HT0I-0036 compared rabbit immune responses to the polysaccharides (PSs) contained in the vaccine, after immunization with the 13vPnC vaccine with or without conjugation to the CRM197 protein. New Zealand White rabbits were immunized intramuscularly at week 0 and week 2 with a dose of 2.2 pg of each PS (except 4.4 pg of 6B). Animals received one of three vaccine preparations: (a) 13vPnC (PS directly conjugated to CRM 197), (b) 13vPnPS, (free PS) or (c) 13vPnPS + CRM 197 (free PS mixed with CRM197 ). All vaccine preparations contained AIPO4 as the adjuvant at 125 pg/dose.
Serotype specific- immune responses for all vaccine preparations were evaluated in an IgG ELISA and complement-mediated OPA measuring functional antibody. The immune responses were compared between the treatment groups.
Table 5 presents GMT data obtained from week 4 bleeds analyzed in antigen specific igG ELISAs. Additional analyses show the ratio of GMT values at week 4 to week 0. The data indicate that the conjugate vaccine preparation elicited greater serum IgG titers than free PS or free PS + CRM97 1 vaccine. With the exception of S.
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pneumonae type 14,the 13vPnC vaccne was able to nducefunctional antibodies to the representative strains- of . pneumonae in an OPA (Table 6). After two immunizations with either the 13vPnPS or 13vPnPS + CRM,9 7 vaccine, neither could induce OPA titers - 8-fold at week 4 relative to week 0 for 10 out of the 13 serotypes measured (Table 6).
In conclusion, these results Indicate that conjugation of the 13-valent pneumococcal vaccine polysaccharddes produces higher serum IgG titers and overall greater functional antibody activity than seen with free polysaccharide alone or mixed with unconjugated CRM 197 .
Table 5. Rabbit IgG Responses (GMTs) to PnPS by ELISA Following Immunization with Two Doses of 13-valent Pneumococcal Glycoconjugate 13vPnPS = CRMiis 13vPnPS (free PS) (PS mixedwithCR 1 r) 13vPnC Week 4 Ratio Week4 Ratio Week4 Ratio Serotype Week 0 (95% C) Wk4:WkO Week 0 (95% CO Wk4:WkO Week 0 (95%C) Wk4:Wk0 1 378 2,290 58 395 1, 5.0 472 35,970 762 (843-5,790) (809-4,739) (29,130-44,417) 3 5240 4.2 89 163 (74-358) 1.8 50 10,414 (10,414-16,676) 208.3 (64-908) 4 o379 7. 0607 1. so12,890258 4 50 (150-959) 7.6 50 (313-1,178) 12.1 50 (9A117-18,224) 257.8 226 321 35 264 705.3 5 343 264.5 so02(147-701) 6.4 503626 (24,467-50;824) (113-450) 6A 154 (316688) 3.0 98 (9524 2.1 163 (167,1 328;283) 1,437.1
63 63 727 11.6 74512.0 (384-1,440) 131 599.5 (22,934-49,222) (384-1,375) 7F 7 50061 (39-95) 1. 1.2 50o72 (47-111) 1. 1.4 50o35,702710 (24,350-52,347) 714.0 104 169 50,033 9V 50 (48-195) 2.1 55 (74-390) 3.0 50 (34,765-72,007) 1,000.7 298 195 20,121 (117-757) 4.5 50 (71-535) 3.9 50 (12,087-32,138) 402.4
18C 89 1,555 17.5 66 761 (300-1,935) 11.5 101 71,451 ,641) (32,74-124 707.4 (655-3,688) 19A 19 50o89 (44-179) 1, 1.8 50o80 (39-163) 1. 1.6 50023,48546. (12,857-42,723) 469.7 19F 61 1,362 (559-3,317) 22.3 61 991 223(370-2,654) 16.3 67 19,358 (25&313 288.9
(487.2,420) 2(311,311) (25,134-84,089). 676.1 233731,0511.312163 5.3 68 45,972 67.
C:\NRonbi\DcCAM4014380-I.DOC-24/11/2011
Table 6. 8. pneumoniae OPA Titers for Rabbit Serum Pools FollowingImmunization with Two Doses of 13-valent Pneumococcal Vaccines OPA Titers
No 13vPnPS (free PS) (freePnPSmixdith 13vPnC Treatment CRMiwi)
Serotype WeekO0 Week 4 Wktio Week 4 Ratio Week 4 Ratio Wk4:.WkO Wk4:WkO Wk4:WkO 1 4 16 4 16 4 8 32 3 4 4 1 4 1 4 8 4 4 4 1 4 1 4 64 5 4 32 8 16 4 16 64 GA 8 64 8 32 4 32 664 6B 8 64 8 32 4 32 32 7F 16 32 2 16 1 16 16 9V 16 16 1 32 2 32 8 14 16 16 1 16 1 16 2 18C 4 16 4 16 4 8 64 19A 8 8 1 8 1 16 64 19F 4 4 1 4 1 8 64 23F 16 32 2 16 1 32 32 a: Used as week 0 values for all groups
It should be understood that the foregoing discussion and examples merely present a detailed description of certain embodiments. It therefore should be apparent to those of ordinary scope in the art that various modifications and equivalents can be made without departing from the spirit and scope of the invention.
All journal articles, other references, patents and patent applications that are identified in this patent application are incorporated by reference in their entirety.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
1. Hausdorff WP, Bryant J, Paradiso PR, Siber GR. Which pneumococcal serogroups cause the most invasive disease: implications for conjugate vaccine formulation and use, part I. ClinInfect DIs 2000; 30:100-21.
2. Hausdorff WP, Bryant J, Kloek C, Paradiso PR, Siber GR. The contribution of specific pneumococcal serogroups to different disease manifestations: implications for conjugate vaccine formulation and use, part 1. ClinInfect Dis 2000; 30:122-40.
3. Whitney CG, Farley MM, Hadler J, et al. Decline in invasive pneumococcal disease after the introduction of protein-polysaccharide conjugate vaccine. New Engl JMed 2003; 348(18):1737-46.
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Claims (23)
1. A process for the preparation of a pneumococcal conjugate comprising the steps of: chemically activating a purified capsular pneumococcal polysaccharide by oxidation in the presence of sodium periodate to make said polysaccharide capable of reacting with a carrier protein; and conjugating said activated polysaccharide to said carrier protein by addition of sodium cyanoborohydride to form a glycoconjugate wherein said conjugation is performed in dimethylsulfoxide (DMSO).
2. The process of claim 1, wherein the capsular polysaccharide is from serotype 19A of Streptococcus pneumoniae.
3. The process of claim 1, wherein the capsular polysaccharide is from serotype 7F of Streptococcus pneumoniae.
4. The process of claim 1, wherein the capsular polysaccharide is from serotype 6A of Streptococcus pneumoniae.
5. The process of claim 4, wherein the capsular polysaccharide from serotype 6A is hydrolysed prior to the chemical activation.
6. The process of claim 1, wherein the capsular polysaccharide is from serotype 6B of Streptococcus pneumoniae.
7. The process of claim 1, wherein the capsular polysaccharide is from serotype 23F of Streptococcus pneumoniae.
8. The process of claim 1, wherein the capsular polysaccharide is from serotype 19F of Streptococcus pneumoniae.
9. The process of claim 8, wherein the capsular polysaccharide from serotype 19F is activated at a temperature of about 15°C.
10. The process of any one of claims 1 to 7, wherein the purified capsular pneumococcal polysaccharide is activated at a temperature of about 23°C.
11. The process of any one of claims 1 to 10, wherein the activation takes place over a period of about 16 to about 24 hours.
12. The process of any one of claims 4 to 9, further comprising compounding the activated polysaccharide with sucrose before the conjugation.
:\Interwoven\NRPortbl\DCC\AMT\20420460_I.DOC-9/07/2020
13. The process of any one of claims 1 to 12, further comprising quenching the conjugation by addition of sodium borohydride.
14. The process of any one of claims 1 to 13, wherein the carrier protein is CRM 1e7
. 15. The process of any one of claims 1 to 14, wherein after conjugation, the polysaccharide-protein conjugates are purified.
16. A process for preparing a multivalent immunogenic composition comprising 13 distinct polysaccharide-protein conjugates together with a physiologically acceptable vehicle, wherein each of the conjugates comprises a capsular polysaccharide from a different serotype of Streptococcus pneumoniae conjugated to a carrier protein, wherein the capsular polysaccharides are prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F, wherein the carrier protein is CRM e , wherein after the individual 17
glycoconjugates are purified they are compounded to formulate the immunogenic composition and, wherein said serotype(s) 6A, 6B, 7F, 19A, 19F and/or 23F polysaccharide-protein conjugate(s) is/are produced according to the process of any one of claims 2 to 15.
17. The process of claim 16, wherein said serotypes 6A, 6B, 7F, 19A, 19F and 23F polysaccharide-protein conjugates are produced according to the process of any one of claims 2 to 15.
18. A pneumococcal conjugate prepared by the process of any one of claims 1 to 15.
19. A multivalent immunogenic composition prepared by the process of claim 16 or claim 17.
20. Use of a pneumococcal conjugate prepared by the process of any one of claims 1 to 17, in the manufacture of a vaccine for preventing pneumococcal disease.
21. Use of a multivalent immunogenic composition prepared by the process of claim 16 or claim 17, in the manufacture of a vaccine for preventing pneumococcal disease
22. Use of a pneumococcal conjugate prepared by the process of any one of claims 1 to 18, in the manufacture of a multivalent vaccine for preventing pneumococcal disease.
23. The use of any one of claims 20 to 22, wherein the pneumococcal disease is invasive pneumococcal disease.
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