EA040838B1 - STABLE POLYVALENT VACCINE COMPOSITION AND METHOD FOR ITS PRODUCTION - Google Patents

Description

Prerequisites for the creation of the invention

Pneumococcus is the main cause of bacterial pneumonia, meningitis and sepsis in children. Recent estimates of childhood deaths due to pneumococcus range from 0.7-1.0 million annually worldwide. In 2000, about 14.5 million episodes of pneumococcal infection were estimated to have occurred (uncertainty range 11.1-18.0 million).

Pneumococcal disease resulted in approximately 0.826 million infant deaths (0.58-0.926 million) aged 1-59 months, of which 0.091 million (0.063-0.1 million) were HIV positive and 0.735 million (0.51-0. 82 million) were HIV negative.

Polyvalent pneumococcal polysaccharide vaccines, which have been approved for many years, have proven their value in preventing pneumococcal infection in adults, in particular the elderly and people at risk. However, infants and young children do not respond well to unconjugated pneumococcal polysaccharides. The pneumococcal conjugate vaccine, Prevnar®, which contains the seven most commonly isolated serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) that cause invasive pneumococcal infection in young children and infants today, was first approved for use in United States in February 2000

In addition, Prevnar™ 13 (Wyeth) is an approved vaccine containing polysaccharide conjugates from serotypes 6A, 6B, 19A, 19F in addition to 1, 3, 4, 5, 7F, 9V, 14, 18C and 23F.

Synflorix™ (GSK) is another approved vaccine that provides protection against 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 20 and 23F, as well as cross-protection against 19A and 6A.

Vaccine preparations should generally be stable and have a uniform consistency to ensure long shelf life and use in multi-dose packaging. Protein-based vaccines, including polysaccharide-protein conjugates, are subject to protein aggregation and precipitation, which may result in a lower effective total vaccine concentration due to the unavailability of the precipitated protein product. Polysaccharide-protein conjugate vaccines, in particular, have a greater tendency to aggregate than carrier protein alone (see Berti et al., 2004, Biophys J86:3-9). The choice of drug for a polysaccharide-protein conjugate vaccine can significantly affect protein aggregation. See Ho et al., 2001, Vaccine 19:716-725.

Despite the existence of several polyvalent pneumococcal polysaccharide protein conjugate vaccine compositions being developed around the world, there is an ongoing need in the art for vaccine formulations that provide high adsorption of individual conjugates without aggregation/precipitation of immunogenic compositions containing polysaccharide-protein conjugates.

Adjuvants commonly used in such polyvalent pneumococcal vaccines have been aluminum salts such as aluminum hydroxide and aluminum phosphate. Many other experimental adjuvants are known, but aluminum salt adsorption remains the most common vaccine adjuvant formulation. Although their use is widespread, aluminum salts are not always comparable to certain antigens, thus leading to significant variations in percentage adsorption of the polysaccharide-protein conjugate to potassium alum or antigenicity.

The immunological properties and stability of a conjugate vaccine candidate adsorbed on an aluminum adjuvant depend on various parameters such as

i) the antigenicity of each antigen, ii) the type of carrier protein used for conjugation, and iii) the type of adjuvant used.

Most importantly, the extent of antigen adsorption to an adjuvant has previously been stated as one of the key parameters for demonstrating lot-to-lot uniformity during formulation development and possibly influencing the efficacy of a vaccine formulation.

In addition, the percentage adsorption of polysaccharide-protein conjugates may further decrease during storage of the composition or under adverse conditions such as temperature fluctuations. See 54th meeting of the WHO Expert Panel on Biological Standardization, Guidelines for the production and control of pneumococcal conjugate vaccines, 17-21 November 2003, Carl E. Frasch, Session IV: Conjugate Vaccines; Vaccine Technologies II; Portugal, 2008. According to the European Regulatory Authority (EMEA) guidelines for pneumococcal polysaccharide-protein conjugates, adsorption completeness (% free conjugate) should be considered as a key quality control parameter in conjunction with potassium alum content, sterility, identity and free polysaccharide content. See evaluation report for Synflorix 2009, procedure no. EMEA/H/C/000973. WHO recommends maximizing adsorption for potassium alum precipitated antigens (eg, diphtheria and tetanus toxoid), where at least 80% of the antigens in these vaccines are adsorbed.

During the production of the polysaccharide-protein conjugate, the composition may contain accumulations

- 1 040838 particles of the type polysaccharide-polysaccharide, protein-protein or polysaccharide-protein. These particulate buildups are also observed in finished products resulting in rejection of 4 to 10% of filled polysaccharide-protein conjugate vaccine vials, affecting the stability and potency of the conjugate vaccine.

Given the above limitations on aggregation and stability of the polysaccharide and its conjugate, there remains a clear need to reduce aggregation and stabilize the polysaccharide during subsequent processing to the stage of the final composition in the production of pneumococcal conjugate vaccine.

The essence of the invention

The present invention provides improvements in the stability of aluminum salt containing vaccines, and in particular, methods for minimizing aggregation and improving the percentage adsorption of individual conjugates in polyvalent pneumococcal polysaccharide-protein conjugate vaccines. The present inventors have observed that co-adsorption of polysaccharide-protein conjugates and the use of a ratio of polysaccharide and protein greater than 1:1 results in

i) a percentage adsorption of less than 55% for pneumococcal conjugates for serotypes 6A, 9V and 23F; and ii) a percentage adsorption of 80 to 90% for the remaining serotype conjugates, which does not allow complete adsorption for a single serotype conjugate for a given polyvalent anti-pneumococcal conjugate composition.

It was also observed that the composition of the vaccine, prepared using a pH of 6.8 to 7.0, resulted in aggregation of approximately 4 to 10% and less adsorption.

The present invention relates to a method for preparing a composition of a stable polyvalent pneumococcal polysaccharide-protein conjugate vaccine showing optimal adsorption from 75 to 99% for each conjugate, where in said method aggregation is prevented by using one of the following:

a) single or separate adsorption for conjugates that otherwise show relatively low adsorption in combined adsorption;

b) a histidine-succinic acid buffer system, together with a pH shift from neutral pH to acidic pH;

c) the ratio of polysaccharide and protein from 0.6 to 1.4; and/or

d) using a six-blade Rushton turbine agitator in the composition vessel.

The present invention also discloses a method for preparing polysaccharide-protein conjugates with improved immunogenicity and lower free polysaccharide content for pneumococcal polysaccharides containing a phosphodiester linkage, in particular 19A, 19F, 6A and 6B. This conjugation process minimizes the disintegration of the sized polysaccharide mediated by the cyanylating agent intermediate and prevents subsequent aggregation of the polysaccharide-polysaccharide particles, thus stabilizing unstable polysaccharides. The main condition for reduced aggregation can be attributed to the use of a sized polysaccharide in the range of 100-200 kDa and a ratio of polysaccharide to CDAP (cyanylation agent) in the range of (1):(0.8-1).

The immunogenic composition prepared in accordance with the present invention offers reduced aggregation between polysaccharide-polysaccharide and polysaccharide-protein conjugate particles along with improved stability and immunogenicity.

List of figures

Fig. 1 is an HPLC-IA profile of size-sorted 19A PnPs (178 kDa) prior to dimethylaminopyridine (DMAP) treatment;

fig. 2 shows HPLC-IU profile of sized 19A PnPs (70 kDa) after 24 hours (A) and degradation profiles upon treatment with 14.5 kDa dimethylaminopyridine after 72 hours (B);

fig. 3 - HPLC-IA profiles of size-sorted 19A PnPs (178 kDa; A) and time-dependent aggregation of CDAP-activated polysaccharides (3B, 3C and 3D), conjugation method without 10 kDA diafiltration step 3E (conjugate without 10 kDa diafiltration) , 3F (10 kDa diafiltration conjugate), 3G sized 19A/3F1 activated 19A (modified 1:1 Ps:CDAP ratio for 19A);

fig. 4 - HPLC-IA profiles of size-sorted 19F PnPs (157 kDa; A) and time-dependent aggregation of CDAP-activated polysaccharides (B), conjugation method without diafiltration step 10 kDa 4C/4D (modified Ps:CDAP ratio 1:1 for 19F);

Fig. 5 - Rushton turbine mixer with six flat blades;

fig. 6 - adsorption protocol.

Disclosure of invention

The purpose of this invention is to provide improvements in the stability of vaccines containing an aluminum salt, and in particular, methods to minimize aggregation and improve the percentage adsorption of individual conjugates in polyvalent pneumococcal polysaccharide-protein conjugate vaccines.

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The present inventors have observed that co-adsorption of polysaccharide-protein conjugates and the use of a ratio of polysaccharide and protein greater than 1:1 results in

i) a percentage adsorption of less than 55% for pneumococcal conjugates for serotypes 6A, 9V and 23F; and ii) a percentage adsorption of 80 to 90% for the remaining serotype conjugates, which does not allow complete adsorption for a single serotype conjugate for a given polyvalent anti-pneumococcal conjugate composition. It was also observed that the composition of the vaccine, prepared using a pH of 6.8 to 7.0, resulted in aggregation of approximately 4 to 10% and less adsorption.

The polysaccharide was cultivated by the method disclosed in WO 2013088448 A1, wherein said method comprises (a) providing an inoculum of a C-polysaccharide-expressing bacterial strain;

(b) cultivating the strain by fermentation at pH 7.2, where the rate of addition of the original medium is equivalent to the rate of addition of an alkaline medium to maintain a predetermined pH;

c) fermentation of the nutrient medium at 35-38°C with stirring at a frequency of 50-150 rpm and an air flow rate of 0.1-0.5 rpm.

The polysaccharide was purified using the process disclosed in WO 2012127485. Pn-Ps prepared using this process shows a recovery of approximately 60 to 70%, where the contamination of the C-polysaccharide is reduced by 1-5 times compared to the content of C-Ps after hydrophobic interaction chromatography or before ion exchange chromatography, protein contamination is less than 1%, and nucleic acid contamination is less than 1%. This process was carried out at the research, semi-industrial and industrial levels.

With this process, polysaccharide purification can be carried out in 80-90% less time and with 90% less cost when compared to CTAB/alcohol based methods.

According to one important embodiment of the present invention, an improved percentage adsorption of 75 to 95% can be obtained for pneumococcal conjugates by

i) using a ratio of polysaccharide and protein from about 0.8 to 1.4;

ii) using single or separate adsorption for poorly adsorbed pneumococcal conjugates;

iii) maintaining a low pH during composition formation.

According to one aspect of the first embodiment of the invention, the preferred ratio of polysaccharide and protein is 1:1.

According to the second aspect of the first embodiment of the invention, said composition contains at least 2 polysaccharide-protein conjugates with a polysaccharide selected from serotypes 1, 2, 3,4, 5, 6A, 6B, 7F, 8, 9V, 9F, 9N, 12F, 14 , 15V, 17F, 18C, 19A,19F, 20, 22F, 23F, 33F and 45.

According to the third aspect of the first embodiment, the individual adsorption regimen can be applied to any pneumococcal serotype selected from 2, 3, 4, 6A, 8, 9V, 9F, 9N, 12F, 15B, 17F, 18C, 20, 22F, 23F, 33F and 45, preferably for pneumococcal serotypes 6A, 9V and 23F.

According to a preferred aspect of the first embodiment of the invention, the polyvalent pneumococcal conjugate vaccine is 10-valent when pneumococcal serotypes 6A, 9V and 23F are individually adsorbed as a separate mixture and then added to another mixture consisting of pneumococcal serotypes 1, 5, 6B, 7F, 14 , 19A, and 19F, which were co-adsorbed.

According to another preferred aspect of the first embodiment of the invention, the polyvalent pneumococcal conjugate vaccine is 11-, 13-, 15-, 16- or more valent, while at least one pneumococcal serotype selected from group 2, 3, 4, 6A, 8, 9V, 9F, 9N, 12F, 15B, 17F, 18C, 20, 22F, 23F, 33F and 45 are individually adsorbed or adsorbed in a smaller group as a separate mixture and then added to another mixture consisting of pneumococcal serotypes 1, 5, 6B, 7F, 14, 19A and 19F which were co-adsorbed.

According to another preferred aspect of the first embodiment of the invention, the polyvalent pneumococcal conjugate vaccine is 16-valent when at least one pneumococcal serotype selected from the group 2, 3, 4, 6A, 9V, 12F, 15B, 18C and 23F is individually adsorbed in less group as a separate mixture, and then added to another mixture consisting of pneumococcal serotypes 1, 5, 6B, 7F, 14, 19A and 19F, which were co-adsorbed.

The second embodiment of the present invention is that the aggregation of the composition of the polyvalent pneumococcal polysaccharide-protein conjugate can be completely prevented by

i) shifting the pH from neutral pH to acidic pH; and ii) using a histidine-succinic acid buffer combination.

According to one aspect of the second embodiment, said shift may occur from 6.8 to a pH selected from 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8 and 5, 9, but not limited to them. More preferably from 6.8 to a pH selected from 5.4, 5.5, 5.6, 5.7 and 5.8.

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According to another aspect of the second embodiment, said histidine succinic acid buffer system may have a concentration of 1 mM to 200 M. A preferred concentration is at least 1 mM (e.g., maximum 200 mM, 150 mM, 100 mM, 90 mM, 80 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, etc.). More preferably, the concentration of the buffer system histidine-succinic acid in the composition ranged from 10 to 40 mm.

A third embodiment of the present invention is that flocculation or aggregation in the total volume of pneumococcal conjugates can be prevented by using Rushton flat impellers instead of magnetic, axial and radial type agitators in the composition vessels. The stability of the immunogenic composition of the present invention is easily determined using standard techniques that are well known and are standard for those skilled in the art. For example, the immunogenic composition is analyzed for percent adsorption of conjugates, stability, aggregation, immunogenicity, particulate formation, protein loss (concentrations), and the like. by means of methods including, but not limited to, ELISA, light scattering, optical density, centrifuge speed settling, centrifuge equilibrium settling, CD circular dichroism (CD), Lowry method, bicinchoninic acid BCA (BCA) assay, and the like. In a preferred embodiment, the present invention provides a novel enzyme-linked immunosorbent assay (ELISA) method that directly quantifies the amount of conjugated/bound polysaccharide without affecting the antigenicity of the conjugates in polyvalent pneumococcal conjugate vaccines. The same method can be used to quantify the amount of non-adsorbed conjugate in the composition matrix as an identifying parameter for percentage adsorption where conjugates show an adsorption of more than 70%.

Preferably, this ELISA method can use a preliminary step before analysis, comprising desorption of the conjugate from the potassium alum adjuvant without affecting the antigenicity of the carrier protein, as well as the conjugated polysaccharide. In particular, the dissolution of conjugate samples adsorbed on potassium alum is achieved using hydroxide sodium and citric acid. The carrier protein may be selected from, but is not limited to, CRM197, P4, diphtheria toxoid, tetanus toxoid, tetanus toxoid fragment C, pertussis toxoid, Haemophilus influenzae protein D, heat-labile E. coli enterotoxins, heat-stable E. coli enterotoxins, and exotoxin A. Pseudomonas aeruginosa, outer membrane complex C, porins, transferrin-binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesive A (PsaA), PhtD protein, pneumococcal surface proteins BVH-3 and BVH-11, protecting antigen 3A (PA ) anthrax strain, detoxified edema-inducing factor, anthrax lethal factor, egg white, keyhole limpet hemocyanin GLU (KFH), human serum albumin, bovine serum albumin BSA (BSA) and tuberculin powder purified from medium proteins (PPD), in particular CRM197 or P4.

In a preferred embodiment, said polyvalent composition may contain

i) at least one polysaccharide-protein conjugate having CRM197 as carrier protein, at least one polysaccharide protein with TT as carrier protein; or ii) at least one polysaccharide-protein conjugate having CRM197 as carrier protein, at least one polysaccharide protein with DT as carrier protein; or iii) at least one polysaccharide-protein conjugate having CRM197 as carrier protein, at least one pneumococcal surface protein A (PsaA) polysaccharide protein as carrier protein; or iv) at least one polysaccharide-protein conjugate having CRM197 as carrier protein, at least one polysaccharide protein with TT as carrier protein, at least one polysaccharide protein with DT as carrier protein; or

v) at least one polysaccharide-protein conjugate having CRM197 as carrier protein, at least one polysaccharide protein with TT as carrier protein, at least one polysaccharide protein with pneumococcal surface adhesin A (PsaA) as carrier protein; or vi) at least one polysaccharide-protein conjugate having CRM197 as carrier protein, at least one polysaccharide protein with DT as carrier protein, at least one polysaccharide protein with pneumococcal surface adhesin A (PsaA) as carrier protein.

In another embodiment, the preferred serotype 3 conjugated carrier protein is CRM-197, serotype 4 is TT or DT, and serotype 18C is CRM197.

Another embodiment of the invention contains the use of PsaA as a carrier protein in the final composition. PsaA can also be used as an adjuvant in the final formulation.

In some embodiments, the implementation of the polyvalent composition of the present invention may contain a surfactant, preferably polysorbate 20. In some embodiments

- 4 040838 implementation of the final concentration of polysorbate 20 in the composition is from 0.01 to 10% wt./about. compositions. In yet other embodiments, the implementation of the final concentration of polysorbate 20 in the composition is 0.01% wt./about. compositions. In other embodiments, the implementation of the final concentration of polysorbate 20 in the composition is 0.05% wt./about. compositions. In still other embodiments, the implementation of the final concentration of polysorbate 20 in the composition is 0.1% wt./about. compositions. In another embodiment, the final concentration of Polysorbate 20 in the composition is 1.0% w/v. compositions. In yet another embodiment, the final concentration of Polysorbate 20 in the composition is 10.0% w/v. compositions.

Compositions of the present polyvalent vaccine may contain, but are not limited to, preservatives selected from the group of mercury-containing preservatives (eg, thimerosal), 2-phenoxyethanol, methyl parabens, propyl parabens, and benzyl alcohol (or mixtures thereof).

In a preferred embodiment of the present invention, said polyvalent pneumococcal polysaccharide-protein conjugate vaccine composition, preferably 10- or 16-valent, may contain aluminum phosphate adsorbed conjugates, histidine, succinic acid, sodium chloride, polysorbate 20 and thiomersal. The vaccine composition of the present invention may contain the step of adding an aluminum salt adjuvant in an amount of 20-375 µg, 20-300 µg, 20-200 µg, 25-150 µg Al ⁺⁺⁺ per 0.5 ml dose.

Usually immunogenic compositions are prepared for injection in the form of liquid solutions or suspensions; may also be prepared in solid forms for dissolution or suspension in liquid binders prior to administration. The drug can also be emulsified or placed in liposomes for enhanced adjuvant effect. Direct administration of the composition is typically parenterally (eg, by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or injected into the interstitial space of a tissue). The compositions can also be applied to the affected area. Other routes of administration include oral and inhalation administration, suppositories, transdermal or transcutaneous injection with a needle, and needleless syringes. Dosage may be on a single dose schedule or a multiple dose schedule (eg, including booster doses). Preferably, the vaccines of the present invention may be stored in solution or lyophilized, where the lyophilized vaccine composition of the present invention may be presented as a 1, 5 or 10 dose preparation with a diluent containing aluminum phosphate gel and NaCl.

Another embodiment of the invention comprises the use of a Rushton turbine mixer with flat blades in the vessel of the composition (see Fig. 5).

Examples

Example 1 Fermentation.

The method contains:

(a) providing an inoculum of a strain of bacteria expressing the C-polysaccharide;

(b) cultivating the strain by fermentation at pH 7.2, where the rate of addition of the original medium is equivalent to the rate of addition of an alkaline medium to maintain a predetermined pH;

c) fermentation of the nutrient medium at 35-38°C with stirring at a frequency of 50-150 rpm and an air flow rate of 0-0.5 rpm.

Example 2 Purification of a capsular polysaccharide.

Purification of pneumococcal capsular polysaccharide serotype 19F (ion exchange chromatography after hydrophobic interaction chromatography).

Purified broth 5L from fermented cultures of pneumococcus serotype 19F was concentrated and diafiltered to 500 ml using a 100 kDa HOMM membrane.

Diafiltration was performed using 25 mM neutral pH sodium phosphate buffer followed by diafiltration with water for injection WFI. Nuclease was added to the polysaccharide solution to achieve a final solution concentration of 8 U/mL. Enzymatic treatment was carried out at 370°C for 10±2 h with stirring.

Ammonium sulfate was added to the polysaccharide solution with nuclease to a saturation of 50% and kept at 2-8°C for 12±2 hours (except for serotypes 5 and 4). The mixture was subjected to centrifugation. The precipitate in the test tube (separated phase) was removed. The solution (~500 ml) was subjected to 100 kDa diafiltration using NaCl followed by chilled WFI. This diafiltrated solution containing the high salt buffered polysaccharide was loaded onto a hydrophobic interaction chromatography column.

The hydrophobic interaction chromatography column (300 ml) was conditioned with 50% saturation ammonium sulphate buffer, and then the polysaccharide solution (500 ml) was placed on the column at a pH of 6 to 8, preferably 6 to 7. The column was then washed buffer containing ammonium sulfate 50% saturation. Under these conditions, the polysaccharide was recovered by flow and conditioning washing from the column.

The polysaccharide solution was then concentrated using a 100 kDa HOMM filter and

- 5 040838 then subjected to diafiltration using NaCl and water for injection (WFI). An ion exchange chromatography column (300 ml) (strong base anion exchange resin) was conditioned with 20 mM sodium phosphate buffer, and then the polysaccharide solution (500 ml) was placed on the column with a pH of 6 to 8, preferably 6.5 to 7.5 . The column was then washed with buffer. The adsorbed polysaccharides were recovered from the adsorbent by stepwise gradient elution using 1.0 M NaCl (different polysaccharides were recovered from the adsorbent at different ionic strengths of NaCl).

The polysaccharide solution was then concentrated using a 100 kDa HOMM filter and then diafiltered using water for injection (WFI). The diafiltered polysaccharide solution was passed through a 0.22 µm membrane filter into polypropylene bottles. The purified polysaccharide was stored frozen at (-20)+50°C.

The process described above was also applied to serotypes 4, 6A, 6B, 7F, 9V, 10A, 14, 18C, 19A, 19F and 23F.

Results.

The evaluation of the results of C-polysaccharide after hydrophobic interaction chromatography and after ion exchange chromatography was carried out by the method of NMR spectrum H1/P31. The result of the process was a reduction in the content of contaminants by 2-3 times.

Example 3 Sized polysaccharides.

A homogenizing (microfluidic) device was used to reduce the molecular weight of the polysaccharide prior to the activation step. For 19A, the size reduction was performed at 24-28 ksi, while the size reduction for 19F was performed at 26-30 ksi, where the number of passes was from 1 to 3. The sized polysaccharide was subjected to diafiltration and concentration and then filtering through a 0.22 µm filter. The sized polysaccharide was then subjected to HPLC-IU to evaluate the average molecular weight.

Example 4 Conventional conjugation process.

Conjugation of the polysaccharide to the carrier protein was performed using the CDAP conjugation method of Lis et al. (Vaccine 26: 190-198, 1996). The mechanically reduced polysaccharides (with the exception of 6A, which was used in native form or size graded 6A) were dissolved in 2M NaCl. CDAP (in acetonitrile) from a 100 mg/ml stock solution was added to the polysaccharide solution according to the polysaccharide:CDAP ratio.

Approximately 1 min later, 2M NaOH was added to obtain a certain activation pH. The activation of the polysaccharide was carried out with this pH for 4-10 min at a temperature of 22°C. CRM-197 (the amount depends on the initial Ps/protein ratio) was added to the activated polysaccharide and the coupling reaction was performed at a certain pH for 3-8 hours depending on the serotype. The reaction was then stopped with glycine for 1 hour at 220°C and overnight at 120°C. The conjugates were then purified by diafiltration from 300 kDa to 500 kDa followed by 100 kDa diafiltration. Then the content of polysaccharide and protein in the purified conjugates, which passed through a filter of 0.22 μm, was determined.

Table 1 Variants of parameters of the conjugation reaction depending on the serotype for 10 serotypes

Conjugation parameters for 10 serotypes Characteristics Process conditions for different serotypes 1 5 6A 6B 7F 9V 14 19A 19F 23F Conc. Ps (mg/ml) 4.5 5 5 eleven 10 8 10 9.5 9.5 9 Dissolution 2M 2MNaCl 2M 2M 2M 2M 2M 2M 2M 2M PS Time NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl NaCl activation 4 4 4 4 4 4 4 4 10 4 (min.) Ps/CDAP ratio 1:1.2 5 1:1 1:1 1:1.15 1:1.2 5 1:1.2 1:1.1 1:1 1:0.8 1:1.1 Conc. CRM 197 20 20 20 20 20 20 20 20 20 20 (mg/ml) Ratio PS/CRM 0.82 0.75 1.05 0.77 0.85 0.87 0.82 0.86 0.96 0.71 pH ^a : pH ^c : pH 9.5:9 9.5:9.5:9 9.5:9.5: 9.5:9 9.5:9 9.5:9 9.5:9.5: 09:0 9.5:9 9.5:9 5:9.5 5 9.5 5:9.5 5:9.5 5:9.5 9.5 9:09 5:9.5 5:9.5 Free < 1 6.3 2.63 1.57 < 1 NOT DETECTED 2.53 1.5 1.07 2.59 PS (%) WOMAN Free „ /О/ \ NOT DETECTED 1.54 2.2 NOT DETECTED NOT DETECTED NOT DETECTED NOT DETECTED NOT DETECTED 2.2 NOT DETECTED protein (%) WOMAN WOMAN WOMAN WOMAN WOMAN WOMAN WOMAN Distributed no molecular size (%) 70.56 74.93 67.67 76.52 75.68 72.75 76.59 78.1 74.35 72.91

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table 2

Comparison of percentage adsorption of individual serotypes of PCV-10 compositions in the approach to the development process of the composition with one and two mixtures

No. Serotypes of PCV-10 compositions Single mix approach (all antigens together) % adsorption Two mixture approach (mixtures A and B) Exp. -1 Exp. - 2 Exp. - 3 Exp. - 4 Exp. - 5 Exp. - 6 1 Serotype 1 90 85 92 >99 >99 97 2 Serotype 5 84 82 85 90 88 97 3 Serotype 6A 67 51 61 77 71 93 4 Serotype 6B 82 72 83 84 80 86 5 Serotype 7F 83 77 81 87 86 84 6 Serotype 9V 67 43 74 89 85 85 7 Serotype 14 86 83 88 92 91 90 8 Serotype 95 92 97 95 93 85 9 Serotype 19F 91 87 93 91 88 75 10 Serotype 23F 56 52 58 84 81 81

In the single mixture approach, serotypes 6A, 9V and 23F adsorbed poorly in the formulation, while in the two mixture approach, percentage adsorption was >70% for all serotypes.

Table 3

Effect of pH shift on the aggregation of PCV-10 compositions

No. Composition Description % vial rejection due to aggregation (white particles/flocculi) Experiment-7 Experiment -8 Experiment 9 1 PKV-10 Dosage forms without "pH shift" 10% 4% 6% 2 PKV-10 Compositions with "pH offset" from pH 6.8 to pH 5.6-5.8 Experiment-10 Experiment-11 Experiment-12 No aggregation (no rejection during validation) No aggregation (no rejection during validation) No aggregation (no rejection during validation)

Aggregation was not detected at pH 5.6-5.8 for the PCV-10 formulation compared to pH 6.8.

It was found that the ratio of polysaccharide and protein (1:1) has a beneficial effect on the duration of adsorption of the pneumococcal conjugate in the composition.

Table 4

The effect of the ratio of Ps and CRM ₁₉₇ on the adsorption of pneumococcal conjugate (serotype 6A) in the composition

No. Pneumococcal conjugate serotype 6A total volume Ps to protein ratio Adsorption (%) 1 ZPN6ACP1201 1.95 34 2 ZPN6ACP1203 1.51 42 3 ZPN6ACP1204-A 1.32 87 4 ZPN6ACP1204-B 1.15 95 5 ZPN6ACP1205 1.09 86

The influence of the ratio of polysaccharide and protein was observed during the adsorption of conjugates. 6A conjugates with a Ps to protein ratio of >1.5 showed poor adsorption. Most of the serotypes used in the PCV-10 formulation had a Ps:Pr ratio in the range of 0.6-1.3, resulting in optimal adsorption of various serotypes, including serotype 6A. Based on the adsorption data obtained for serotype 6A and the experience with PCV-10 formulation in the development of a 16-valent vaccine formulation, it can be extrapolated that the Ps:Pr ratio of the remaining 6 conjugates in the same range can provide adequate adsorption of conjugates of all 16 serotypes.

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Table 5

Specifications for Rushton Turbine Agitators with Flat Blades for Various Vessels

Geometric volume (l) 14 Working volume (l) 10.0 Blade diameter (mm) 74.7 Blade height (mm) 17.3 Number of blades 6 Stir control (rpm) 50-500 Peripheral speed (m/s) 0.19-1.95

Example 6 ELISA protocol.

Antigen content and percentage adsorption were determined using a modified sandwich ELISA.

The conventional sandwich ELISA has been modified with respect to the following test/assay conditions and therefore has the following advantages:

i) determination of the amount of conjugated polysaccharide in the presence of 9 other conjugated antigens in a 10-valent vaccine, ii) extraction of all ten conjugates from aluminum phosphate gel, where adsorption is more than 80%, iii) preparation of the conjugate occurs even at pH 9 without compromising the antigenicity of the conjugate .

Antigen content and percentage adsorption were determined by ELISA according to the protocol below.

Day 1.

1. The plates were coated with immobilized antibody (anti-carrier protein antibody) and then incubated for 0.5-2 hours at 35±5°C.

2. The plates were washed 3-6 times in a plate washer.

3. Plates were blocked with blocking buffer (3% BSA in 1X PBS and trisbuffer for serotypes 14 and 9V) and then incubated for 1.5-2.5 hours at 30±5°C.

4. The plates were washed 3 times in a plate washer.

5. Samples have been added to the plates.

6. The plates were kept overnight at 5±3°C.

Day 2

1. The plates were placed at room temperature.

2. First, the antibody was added after washing the plates three times, then incubating at room temperature 25±2° C. for 30 minutes, and then washing.

3. Then the antibody was added and kept at a temperature of 25±2°C for 30 minutes.

4. Added TMB substrate and kept at room temperature for 15-20 min in the dark.

5. Stop reagent has been added.

6. Plates were read at 450 nm.

Sample dissolution procedure without compromising the antigenic determinant of the carrier protein.

The proper amount of 0.5-2M NaOH was added to 2 ml of the vaccine sample. This sample was gently vortexed until the solution became clear. The pH of the solution was adjusted from 9-12 until the solution became clear. The pH of the solution was adjusted back to 6-7.4 with 0.5-2M citric acid. This solution was centrifuged (dissolved samples) at 3000-6000g for 5 min and the supernatant was collected for testing.

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Table 6

Serotype Series 1 Series 2 Content in mcg/ml % adsorption Content in mcg/ml % adsorption Serotype 1 3.87 89 4.66 99.4 Serotype 5 3.57 83 4.03 90.8 Serotype 6A 3.39 83 4.6 68.3 Serotype 6B 8.01 80 6.8 85 Serotype 7F 4.72 97 4.18 84.4 Serotype 9V 4.61 80 3.95 70.4 Serotype 14 5D1 94 5.06 92.5 Serotype 19A 4.42 94 3.65 91.8 Serotype 19F 4.35 82 4.14 81.4 Serotype 23F 3.66 72 4.03 75

Example 7. Pneumococcal conjugate vaccine - 10-valent (PCV-10).

Table 7

Composition of PKV-10

No. Ingredient name Quantity/dose (0.5 ml) 1 Polysaccharide serotype 1, 5, 6A, 7F, 9V, 14, 19A, 19F and 23F* 2 mcg each 2 Polysaccharide serotype 6B* 4 mcg 3 Aluminum Phosphate Adju-Phos® 0.125 mg as A1 ⁺⁺⁺ 4 Sodium chloride 4.5 mg 5 succinic acid 1.18 mg 6 Polysorbate-20 50 mcg 7 Thiomersal (for multi-dose presentation only) 25 mcg 8 L-histidine 1.55 mg

The active ingredients are conjugated to the CRM ₁₉₇ carrier protein.

Example 8. Composition 1 (PKV-16).

Pneumococcal conjugate vaccine - 16-valent (PCV-16).

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Table 8

Composition I PKV-16

No. Compositions Quantity/dose Active ingredients* 1 Serotype 1 2.0 µg 2 Serotype 2 2.0 µg 3 Serotype 3 2.0 µg 4 Serotype 4 2.0 µg 5 Serotype 5 2.0 µg 6 Serotype 6A 2.0 µg 7 Serotype 6B 4.0 µg 8 Serotype 7F 2.0 µg 9 Serotype 9V 2.0 µg 10 Serotype 12F 2.0 µg AND Serotype 14 2.0 µg 12 Serotype 15B 2.0 µg 13 Serotype 18C 2.0 µg 14 Serotype 19A 2.0 µg 15 Serotype 19F 2.0 µg 16 Serotype 23F 2.0 µg Excipients* 17 aluminum phosphate not more than 1.25 mg A1 ³⁺ 18 G istidine 1.55 mg 19 Carrier proteins 11.3-113.3 mcg 20 succinic acid 1.18 mg 21 Sodium chloride 4.5 mg 22 Polysorbate 20 50 mcg 23 Thiomersal** 25 mcg 24 in DI of necessity

^The active ingredients of the vaccine are conjugated to at least one carrier protein selected from CRM ₁₉₇ , TT and DT.

** Added only for multi-dose presentation.

Example 9. Composition 1 (PKV-16).

Table 9

Composition II PKV-16

No. Compositions Quantity/dose Active ingredients* 1 Serotype 1 2.0 µg 2 Serotype 2 2.0 µg 3 Serotype 3 2.0 µg 4 Serotype 4 2.0 µg 5 Serotype 5 2.0 µg 6 Serotype 6A 2.0 µg 7 Serotype 6B 4.0 µg 8 Serotype 7F 2.0 µg 9 Serotype 9V ___________2.0 mcg_________

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10 Serotype 12F 2.0 µg And Serotype 14 2.0 µg 12 Serotype 15B 2.0 µg 13 Serotype 18C 2.0 µg 14 Serotype 19A 2.0 µg 15 Serotype 19F 2.0 µg 16 Serotype 23F 2.0 µg Excipients* 17 aluminum phosphate not more than 1.25 mg A1 ³⁺ 18 G istidine 1.55 mg 19 Carrier proteins 11.3-113.3 mcg 20 succinic acid 1.18 mg 21 Sodium chloride 4.5 mg 22 Polysorbate 20 50 mcg 23 2-phenoxyethanol* * 10 mg 24 in DI

^The active ingredients of the vaccine are conjugated to at least one carrier protein selected from CRM ₁₉₇ , TT and DT.

^^Added only with multi-dose presentation.

Example 10

I) Decay of sized PnPs (19A, 19F, 6A and 6B) in the presence of dimethylaminopyridine.

The sized PnPs in the reaction solution was treated with dimethylaminopyridine at a ratio of 1:1.5 and its degradation profile was checked by HPLC-IU.

Results.

It was observed that only 19A PnPs decomposed in the presence of dimethylaminopyridine, while other PnPs containing phosphodiester (19F, 6A and 6B) remained unchanged. See fig. 1 (without dimethylaminopyridine) and 2 showing PnPs degradation mediated by dimethylaminopyridine.

To minimize this degradation, 19A activated PnPs was diafiltered at 10 kDa using 2M NaCl to remove dimethylaminopyridine formed from the reaction solution prior to conjugation with CRM ₁₉₇ .

II) Decay and aggregation of sized PnPs (19A, 19F, 6A and 6B) in the presence of CDAP.

The sized PnPs in the reaction solution were treated with CDAP at a ratio of 1:1.5 during activation. It was observed that 50% dimethylaminopyridine was created as an intermediate (measured by RP-HPLC) leading to degradation of PnPs as well as aggregation between activated PnPs after a certain activation time (see Table 2). IU. See fig. 3A (no CDAP), 3B, 3C and 3D (for 19A) and 4A (no CDAP) 4B (for 19F) showing aggregation by CDAP and degradation of PnPs.

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Table 10

Factors responsible for cross-linking and aggregation of polysaccharide-polysaccharide particles. The influence of the duration of CDAP activation on the formation of aggregations

Serotypes Molecular weight of sized/modified PnPs (kDa) [HPLC] Activated Modified PnPs (kDa) [HEPHIU] effects 6A 432 435 No aggregation between activated polysaccharides 6B 131 131 No aggregation between activated polysaccharides 19A 178 254 (after 20 min) Aggregation between activated polysaccharides 487 (after 60 min) Aggregation between activated polysaccharides 1173 (after 120 min) Aggregation between activated polysaccharides 19F 157 205 (after 30 min) Aggregation between activated polysaccharides

III) Prevention of degradation and aggregation of sized PnPs (19A and 19F) with a PnPs:CDAP ratio of 1:1.5 by applying a diafiltration step.

To minimize this degradation and aggregation, 19A activated PnPs was diafiltered at 10 kDa using 2M NaCl to remove dimethylaminopyridine formed from the reaction solution prior to conjugation with CRM ₁₉₇ . See fig. 3E (non-diafiltrated 10 kDa conjugate) and 3F (10 kDa diafiltered conjugate) showing the HPLC-IR profile of the conjugate using 10 kDa diafiltered and activated PnPs and CRM ₁₉₇ . However, the diafiltration step negatively affected the yield of the conjugate, which led to a decrease in the overall yield by 30-40%.

IV) Prevention of degradation and aggregation of sized PnPs (19A and 19F) by reducing the PnPs:CDAP ratio to 1:1 (19A) and 1:0.8 (19F) without the use of a diafiltration step.

The sized PnPs in the reaction solution was treated with CDAP at a ratio of 1:1 and 1:0.8 for 19A and 19A, respectively, and its degradation and aggregation profile was checked by HPLC-IR.

Results.

Altered Ps:CDAP ratios (1:1 for 19A and 1:0.8 for 19F) were observed to prevent degradation and aggregation for both PnPs 19A and PnPs 19F. See fig. 3 (G and H for 19A) and 4 (C and D for 19F). An additional benefit of using the modified ratios was that the 10 kDa diafiltration step was eliminated, resulting in minimal overall yield loss.

It was observed that in the case of serotype 19A, when the duration of CDAP activation was more than 10 minutes, this led to the cross-linking of the activated polysaccharide with the activated polysaccharide, inevitably leading to the formation of polysaccharide-polysaccharide aggregations.

In addition, in the case of serotype 19F, when the duration of CDAP activation was more than 20 minutes, this led to the cross-linking of the activated polysaccharide with the activated polysaccharide, inevitably leading to the formation of polysaccharide-polysaccharide aggregations. In addition, it was found that the duration of the conjugation reaction is longer than the duration required for the cross-linking of the activated polysaccharide, thus leading to the formation of aggregations. See fig. 3 and 4.

However, for other serotypes such as 6APnPs and 6BPnPs, no such cross-linking has been observed.

Example 11 Preparation of PnPs19A and PnPs19F conjugates.

Conjugation of the polysaccharide to the carrier protein was performed using the CDAP conjugation method of Lis et al. (Vaccine 26: 190-198, 1996) with the following changes:

i) to prepare conjugate 19A using a ratio of polysaccharide and CDAP equal to 1:1 at 22°C with an activation period of 4 min and using a ratio of polysaccharide to protein equal to 1:1, ii) to prepare conjugate 19F using a ratio of polysaccharide and CDAP equal to 1:0.8 at a temperature of 22°C with an activation period of 9-10 min and using a ratio of polysaccharide and protein equal to 1:1, for 19F.

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Table 10

Comparison of conjugate results for 19A and 19F with conventional CDAP conjugation method _____________ (lot 1) improved conjugation method (lot 2) _____________

I) Conjugation Reaction Parameters Conjugates Conjugate 19A Conjugate 19F Batch Number Lot 1 Pot 2 Lot 1 Lot 2 Conc. PnPs (mg/ml) 9.5 9.5 9.5 9.5 Dissolution of PnPs 2MNaCl 2MNaCl 2MNaCl 2MNaCl Activation time 4 4 4 9 to 10 (min) Ratio 1.0:1.5 1.0:1, 0 1.0:1.5 1.0:0.8 PnPs/CDAP Conc. CRM (mg/ml) ^v ' 20 20 20 20 Initial ratio 1.0:1.5 1.0:1.0 1.0:1.5 1.0:1.0 PS/CRM197 pH^pH^pffi 9.0:9.0:9.0 9.0:9.0:9.0 9.5:9.5:9.5 9.5:9.5:9.5 II) Final characteristics of the conjugate Conjugates Conjugate 19A Conjugate 19F Series number Lot 1 Pot 2 Lot 1 Pot 2 Final PnPs/CRMi97 Ratio 0.53 0.86 0.52 0.96 CRMi97/PnPs 1.88 1.16 1.92 1.04 Free PnPs (%) 1.9 1.5 1.7 1.07 FREE CRM197 (%) NOT DISCOVERED NOT DISCOVERED NOT DISCOVERED 2.2 Wed molecular size HPLC-1)(kDa) 1081 853 994 847 Wed molecular size (UV/IR/MALS) (kDa) 8212 6012 6393 4773

Results.

Modified polysaccharide:CDAP ratio, CDAP activation time, and initial polysaccharide:protein ratio have been observed to minimize 4-dimethylaminopyridine-mediated degradation of the sized polysaccharide during activation, and also prevent subsequent polysaccharide-polysaccharide aggregation, thus improving the final performance of the conjugate in in relation to the free polysaccharide content.

The improved conjugation method used to prepare conjugates 19A and 19F resulted in conjugates that did not show a phosphomonoester signal in their respective conjugate profiles ( ^31P proton NMR), indicating that the modified conjugation method was effective in preventing polysaccharide hydrolysis in conjugation reactions. .

In view of the many possible embodiments of the invention to which the principles of the disclosed invention may be applied, it should be recognized that the embodiments shown are only preferred examples of the invention and are not intended to be limiting to the invention. Rather, the scope of the invention is defined by the following claims. Thus, we claim as our invention everything that falls within the scope and spirit of this claim.

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Claims (9)

CLAIM 1. A method for producing a stable polyvalent vaccine composition with a Streptococcus pneumoniae polysaccharide-protein conjugate, which includes at least 10 different Streptococcus pneumoniae polysaccharide-protein conjugates, including the following steps: i) conjugating an anti-pneumococcal polysaccharide derived from Streptococcus pneumoniae with a carrier protein in a ratio of 0.5 to 1.4 and at a pH of 5.2 to 5.9 to obtain a cyanate-activated polysaccharide; ii) individual adsorption of polysaccharide-protein conjugates obtained in step (i), including serotypes 6A, 9V and 23F on the first adjuvant - aluminum salts in histidine-succinic acid buffer with a concentration of 1 to 200 mM and at a pH of 5.6 to 5.8, wherein the amount of said aluminum salt adjuvant is in the range of 20-375 μg Al+++ per 0.5 ml dose of the final vaccine composition; iii) mixing and adsorbing the polysaccharide-protein conjugates obtained in step (i), including serotypes selected from 1, 2, 3, 4, 5, 6B, 7F, 12F, 14, 15B, 18C, 19A, 19F and 22F on the second adjuvant - aluminum salts in a histidine-succinic acid buffer with a concentration of 1 to 200 mm and at a pH of 5.6 to 5.8, in which the amount of the specified aluminum salt adjuvant is in the range of 20-375 μg Al +++ per dose 0, 5 ml of the final composition of the vaccine; and iv) mixing said first adsorbed kit obtained in step (ii) with said second adsorbed kit, in the presence of a surfactant, using a Rushton flat-blade turbine agitator to produce a stable polyvalent Streptococcus pneumoniae polysaccharide-protein conjugate vaccine composition, moreover, the carrier protein is selected from the group including CRM ₁₉₇ , tetanus toxoid (TT) and diphtheria toxoid (DT), Streptococcus pneumoniae polysaccharide-protein conjugates are obtained using cyanylation conjugation chemistry, while the adsorption range is 75-99%, which is measured by the method enzyme-linked immunosorbent assay (ELISA), and the composition shows optimal adsorption for each conjugate and minimal aggregation of the conjugates. 2. The method of claim 1, wherein step (i) includes the following sub-steps: i) interaction of a pneumococcal polysaccharide containing a phosphodiester bond between repeating units having an average size of 130 to 190 kDa, covalently linked to a carrier protein, with a cyanylating agent in a ratio of 1:0.8 to 1:1 at a temperature of 22 to 25°C within 4 to 10 minutes to obtain a cyanate-activated polysaccharide; ii) reacting the cyanate-activated polysaccharide with protein in a 1:1 ratio at pH 9 to 9.5 for 3 to 5 hours, followed by stopping the reaction with glycine. 3. The method of claim 2, wherein the polysaccharide containing the phosphodiester bond between the repeat units is obtained from one of Streptococcus pneumoniae serotype 19A, 19F, 6A, or 6B. 4. The method according to claim 3, in which the polysaccharide is Streptococcus pneumoniae serotype 19A polysaccharide, and the cyanylating agent is 1-cyano-4-dimethylaminopyridine-tetrafluoroborate, mixed in a ratio of 1:1 at a temperature of 22°C for 4 minutes, obtaining cyanate-activated polysaccharide. 5. The method according to claim 3, in which the polysaccharide is the polysaccharide of Streptococcus pneumoniae serotype 19F, and the cyanylating agent is 1-cyano-4-dimethylaminopyridine-tetrafluoroborate, mixed in a ratio of 1:0.8 at a temperature of 22°C for 8-10 min, to obtain cyanate-activated polysaccharide. 6. The method of claim 1, wherein said histidine-succinic acid buffer has a concentration of 10 to 40 mM. 7. A stable polyvalent vaccine composition with a Streptococcus pneumoniae polysaccharide-protein conjugate obtained by the method according to claims 1-6, comprising at least 10 different Streptococcus pneumoniae polysaccharide-protein conjugates having a polysaccharide selected from serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F, 9V, 12F, 14, 15B, 18C, 19A, 19F, 22F, and 23F, wherein at least one polysaccharide-protein conjugate has CRM ₁₉₇ as a carrier protein, at least one polysaccharide-protein conjugate has tetanus toxoid (TT) as carrier protein, and at least one polysaccharide-protein conjugate has diphtheria toxoid (DT) as carrier protein, wherein serotypes 6A, 9V and 23F are individually adsorbed on an aluminum adjuvant, serotypes selected from 1, 2, 3, 4, 5, 6B, 7F, 12F, 14, 15B, 18C, 19A, 19F and 22F are mixed and adsorbed on an aluminum adjuvant, and the adsorption is in the range of 75-99%, while the composition has a buffer histidine succinic acid in a concentration of 1 0 to 40 mM. 8. The composition of claim 7, wherein serotype 3 is conjugated to CRM ₁₉₇ , serotype 18C is conjugated to CRM ₁₉₇ , serotype 4 is conjugated to DT, serotype 15B is conjugated to TT, and serotype 22F is conjugated to TT. 9. Composition according to claim 7, containing 10 different polysaccharide-protein conjugates of Streptococcus pneumoniae, having a polysaccharide from serotypes 1, 5, 6A, 6B, 7F, 9V, 14, 19A, 19F and 23F, which conjugate -