Novel Multivalent Polysaccharide-Protein Conjugate Vaccine Composition and Formulation Thereof

Abstract

Novel multivalent polysaccharide-protein conjugate vaccine formulation. The formulation is liquid or lyophilized or a Liquid-Lyo combination pentavalent formulation of Neisseria meningitidis serogroup A, C, Y, W and X capsular polysaccharides (Men A, C, Y, W, X), each said polysaccharide being conjugated separately to tetanus toxoid (TT) carrier protein to obtain Men A, C, Y, W, X-TT conjugates, with one or more buffer and with or without an adjuvant along with pharmaceutically acceptable components/excipients.

Claims

1. Novel multivalent polysaccharide-protein conjugate vaccine formulation wherein said formulation is a liquid or lyophilized or a Liquid-Lyo combination pentavalent formulation of Neisseria meningitidis serogroup A, C, Y, W and X capsular polysaccharides (Men A, C, Y, W, X), each said polysaccharide being conjugated separately to tetanus toxoid (TT) carrier protein to obtain Men A, C, Y, W, X-TT conjugates, with one or more buffer and with or without an adjuvant along with pharmaceutically acceptable components/excipients wherein said formulation provides desired osmolality, desired pH, high stability and desired immunogenicity.

2. The novel vaccine formulation as claimed in claim 1 wherein said liquid formulation comprises of: TABLE-US-00019 Ingredient Quantity/Concentration Men A, C, Y, W, X -TT 4-20 g polysaccharide/serogroup/ml Buffer 5-30 mM Phosphate buffered saline Excipient 0-150 mM NaCl 5-30 mM Histidine Adjuvant: Aluminum phosphate as 500-2000 g Al.sup.+++/ml Water (MQW) qs

3. The novel vaccine formulation as claimed in claim 1 wherein said liquid formulation comprises of: TABLE-US-00020 Ingredient Quantity/Concentration Men A, C, Y, W, X -TT 10-20 g polysaccharide/serogroup/ml Buffer 10-25 mM Phosphate buffered saline (pH 7.0 0.2) Excipient 0-150 mM NaCl Adjuvant: Aluminum phosphate as 750-1500 gAl.sup.+++/ml Water (MQW) qs

4. The novel vaccine formulation as claimed in claim 1 wherein said liquid formulation comprises of: TABLE-US-00021 Ingredient Quantity/Concentration Men A, C, Y, W, X -TT 10-20 g polysaccharide/serogroup/ml Buffer: 10 mM PBS (pH 7.0 0.2) Excipient: 0-150 mM NaCl Adjuvant Aluminum phosphate as 1000 gAl.sup.+++/ml Water (MQW) qs

5. The novel vaccine formulation as claimed in claim 1 wherein said liquid formulation comprises of: TABLE-US-00022 Ingredient Quantity/Concentration Men A, C, Y, W, X -TT 10 g polysaccharide/serogroup/ml Buffer: 10 mM PBS (pH 7.0 0.2) Excipients: 0-150 mM NaCl Water (MQW) qs

6. The novel vaccine formulation as claimed in claim 1 wherein said lyophilized formulation comprises of: TABLE-US-00023 Ingredient Quantity/Concentration Men A, C, Y, W, X -TT 10-20 g polysaccharide/serogroup/ml Buffer: 5-20 mM PBS (pH 6.5-7.5) Excipients: 2-10% Diluent qs

7. The novel vaccine formulation as claimed in claim 1 wherein said Liquid-Lyo combination formulation comprises of: TABLE-US-00024 Ingredient Quantity/Concentration Men A, C, Y, W, X -TT 10-20 g polysaccharide/serogroup/ml Buffer: 5-20 mM PBS (pH 6.5-7.5) Excipients: 2-10% Diluent qs

8. The novel vaccine formulation as claimed in claim 1 wherein said Men A, C, Y, W, X-TT conjugates are obtained employing optimized combination of conjugation chemistry.

9. The novel vaccine formulation as claimed in claim 8 wherein said conjugation chemistry to obtain said at least one conjugate is carbamate chemistry.

10. The novel vaccine formulation as claimed in claim 9 wherein said at least one conjugate employing said carbamate chemistry is preferably MenX-TT conjugate.

11. The novel vaccine formulation as claimed in claim 8 wherein said conjugation chemistry to obtain said at least one conjugate is cyanylation chemistry.

12. The novel vaccine formulation as claimed in claim 11 wherein said at least one conjugate employing said cyanylation chemistry is preferably said Men A, C, Y, W-TT conjugates.

13. The novel vaccine formulation as claimed in claim 1 wherein each said capsular polysaccharide is degraded in the size range of 0.380.1 Kd when tested for molecular size distribution using HPLC PWXL4000 and 5000 columns in series, preferably in the size range of 0.380.06 Kd to obtain conjugates with high antigenicity, high immunogenicity and high stability.

14. The novel vaccine formulation as claimed in claim 1 wherein said conjugates are produced having linker arm between said polysaccharide and said carrier protein wherein linker is attached to either said polysaccharide or said carrier protein or both the said polysaccharide and carrier protein.

15. The novel vaccine formulation as claimed in claim 14 wherein said linker is selected from adipic acid dihydrazide or hydrazine.

16. The novel vaccine formulation as claimed in claim 1 wherein said MenX-TT conjugate preferably has hydrazine linker and said MenA, C, Y, W-TT conjugates preferably have adipic acid dihydrazide linker.

17. The novel vaccine formulation as claimed in claim 1 wherein each said conjugate has the carrier protein to polysaccharide ratio between 0.3-1.0.

18. The novel vaccine formulation as claimed in claim 1 wherein said pharmaceutically acceptable excipients are selected from adjuvant, buffer, preservative, stabilizer, surfactant, either alone or in combination.

19. The novel vaccine formulation as claimed in claim 6 wherein said excipient is selected from Sucrose, Maltose, Arginine, Lactose, Sorbitol, Histidine, Glycine, either alone or in variable combinations.

20. The novel vaccine formulation as claimed in claim 6 wherein said diluent is selected from water, 5-20 mM phosphate buffered saline, aluminum phosphate as 500-1500 g Al.sup.+++/ml either alone or in variable combinations.

21. The novel vaccine formulation as claimed in claim 7 wherein said lyophilized (lyo) portion contains MenA-TT conjugate, MenC-TT conjugate either alone or in variable combinations.

22. The novel vaccine formulation as claimed in claim 7 wherein said excipient is selected from Sucrose, Maltose, Arginine, Lactose, Sorbitol, Histidine, Glycine, either alone or in variable combinations.

23. The novel vaccine formulation as claimed in claim 7 wherein said diluent is selected from water, 5-20 mM phosphate buffered saline, aluminum phosphate as 500-1500 g Al.sup.+++/ml containing MenC-TT, MenY-TT, MenX-TT, MenW-TT either alone or in variable combinations.

24. The novel vaccine formulation as claimed in claim 1 wherein said formulation is liquid or lyophilized formulation or a combination thereof with mono- or multi-dose regimen with or without a preservative.

25. The novel vaccine formulation as claimed in claim 1 wherein optimum human dose of serogroups A, C, Y, W and X ranges between 2-10 g polysaccharide per serogroup per dose, preferably 5-10 g each of serogroup A and X polysaccharides and 5 g each of serogroup C, Y and W polysaccharides..

26. The novel vaccine formulation as claimed in claim 1 wherein said desired osmolality of formulation is 240-330 mOsmol/Kg.

27. The novel vaccine formulation as claimed in claim 1 wherein said desired pH of formulation is 6.5-7.5.

28. The novel vaccine formulation as claimed in claim 1 wherein said lyophilized portion of the lyophilized or liquid-lyo formulation has a moisture content not more than 3%.

29. The novel vaccine formulation as claimed in claim 1 wherein said vaccine formulation is stable at high temperature of 372 C. for at least 21 days and at 252 C. for at least 3 months showing less than 40% free polysaccharide.

30. The novel vaccine formulation as claimed in claim 1 wherein said vaccine formulation shows high antigenicity and high immunogenicity.

31. The novel vaccine formulation as claimed in claim 1 wherein said vaccine formulation is equally immunogenic even after exposure at 372 C. for 7 days as compared to the formulation stored at real-time storage conditions.

32. The novel vaccine formulation as claimed in claim 1 wherein said formulation is preferably a liquid pentavalent meningococcal conjugate vaccine formulation with mono- or multi-dose regimen with or without a preservative.

33. The novel vaccine formulation as claimed in claim 1 wherein said formulation and said composition is capable of being used in production of liquid or lyophilized or liquid-lyo pentavalent meningococcal ACYWX-TT combination vaccine.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0036] FIG. 1 a-e depicts Real Time Stability Studies on liquid Pentavalent Meningococcal ACYWX-TT Conjugate Vaccine at 53 C.

[0037] FIG. 2 a-e depicts Accelerated Stability Studies on liquid Pentavalent Meningococcal ACYWX-TT Conjugate Vaccine at 252 C.

[0038] FIG. 3 a-e: Stress Stability Studies on liquid Pentavalent Meningococcal Conjugate Vaccine at 372 C.

[0039] FIG. 4 a-e: depicts Post 2-dose anti-meningococcal mouse IgG and SBA titers against adjuvanted and non-adjuvanted liquid pentavalent meningococcal ACYWX-TT conjugate vaccine in comparison to licensed ACYW conjugate vaccine and/or vehicle control.

[0040] FIG. 5 a-e: depicts Post 2-dose anti-meningococcal rabbit IgG and SBA titers against Adjuvanted and non-adjuvanted liquid pentavalent meningococcal ACYWX-TT conjugate vaccine in comparison to licensed ACYW conjugate vaccine or vehicle control.

[0041] FIG. 6 a-e: Post 3-dose anti-meningococcal mouse IgG and SBA titers against adjuvanted liquid pentavalent meningococcal ACYWX-TT conjugate vaccine in comparison to a VVM7 adjuvanted liquid pentavalent vaccine and licensed ACYW conjugate vaccine or vehicle control.

DETAILED DESCRIPTION OF INVENTION WITH NON-LIMITING EXAMPLES AND ILLUSTRATIONS

[0042] Accordingly, the present invention provides a novel polysaccharide-protein conjugate vaccine composition and formulation thereof. More particularly, the present invention relates to a conjugate vaccine composition comprising of polysaccharide-protein conjugates produced using conjugation chemistry. The conjugation chemistry used includes combination of optimized carbamate chemistry and cyanylation chemistry. The composition of present invention is capable of being used in production of a pentavalent combination vaccine.

[0043] The polysaccharides used for production of conjugate of the present invention are obtained through an animal component free fermentation process.

[0044] The novel vaccine formulation of present invention comprises of polysaccharide-protein conjugates along with pharmaceutically acceptable components/excipients. All the conjugates in vaccine composition and formulation have same carrier protein. The present invention provides a pentavalent vaccine formulation.

[0045] The novel polysaccharide-protein conjugate vaccine composition and formulation of present invention comprises of five individual polysaccharide-protein conjugates. The polysaccharide is selected from gram negative bacteria Neisseria meningitidis serogroup A, C, Y, W and X capsular polysaccharides. The carrier protein used for preparing all the conjugates is Tetanus Toxoid (TT).

[0046] The capsular polysaccharide is degraded to smaller sizes suitable for conjugation with carrier protein to obtain conjugates with high antigenicity, high immunogenicity and high stability. The capsular polysaccharide is degraded in the size range of 0.380.1 Kd, preferably 0.380.06 Kd when tested for molecular size distribution using HPLC PWXL4000 and 5000 columns in series. The polysaccharide and carrier protein are activated before conjugation. The conjugates are produced having linker arm between polysaccharide and protein moities. The linker is attached to either polysaccharide or carrier protein or both the polysaccharide and protein.

[0047] The novel multivalent polysaccharide-protein conjugate vaccine formulation of the present invention comprises of meningococcal serogroups A, C, Y, W, X polysaccharides each individually conjugated to tetanus toxoid (Men ACYWX-TT) wherein serogroup X polysaccharide is conjugated to tetanus toxoid using optimized carbamate chemistry and serogroup A, C, Y and W polysaccharides are conjugated using optimized cyanylation chemistry. Each said conjugate has the protein to polysaccharide ratio between 0.3-1.0.

[0048] The novel multivalent polysaccharide-protein conjugate vaccine formulation of the present invention comprises of meningococcal serogroups A, C, Y, W, X polysaccharides each individually conjugated to tetanus toxoid (Men ACYWX-TT) mixed with one or more buffer and one or more pharmaceutically acceptable excipients, with or without adjuvant.

[0049] The pharmaceutically acceptable excipients can be adjuvant, buffer, preservative, stabilizer, surfactant, either alone or in combination. The formulation of present invention is a liquid or lyophilized formulation or a liquid-lyo combination with mono- or multi-dose regimen with or without a preservative. The vaccine formulation of the present invention is stable at high temperatures. Considering 40% free PS as the maximum target, the formulation shows stability at the high temperature of 372 C. for at least 21 days, at 252 C. for at least 3 months and at 53 C. for at least 9 months.

[0050] In one of the best embodiments, the novel pentavalent liquid polysaccharide-protein conjugate vaccine formulation of the present invention comprises of:

TABLE-US-00001 Ingredient Quantity/Concentration Men ACYWX-TT 4-20 g polysaccharide/serogroup/ml Buffer 5-30 mM Phosphate buffered saline (pH 7.0 0.2) Excipient 0-150 mM NaCl sufficient to maintain osmolality between 240-330 mOsmol/Kg 5-30 mM Histidine Adjuvant: Aluminum phosphate as 500-2000 g Al.sup.+++/ml Water (MQW) qs
The ingredients are mixed by stirring at room temperature for 0.5-2 hours and followed by filling in vials and storage at 2-8 C.

[0051] In another embodiment, the novel pentavalent liquid polysaccharide-protein conjugate vaccine formulation of the present invention comprises of:

TABLE-US-00002 Ingredient Quantity/Concentration Men ACYWX-TT 10-20 g polysaccharide/serogroup/ml Buffer 10-25 mM Phosphate buffered saline (pH 7.0 0.2) Excipient 0-150 mM NaCl to maintain osmolality between 240-330 mOsmol/Kg Adjuvant: Aluminum phosphate 750-1500 gAl.sup.+++/ml Water (MQW) qs
The ingredients are mixed by stirring at room temperature for 0.5-2 hours and followed by filling in vials and storage at 2-8 C.

[0052] One of the best embodiments of the formulation with adjuvant providing desired osmolality and high stability and desired immunogenicity comprises of:

TABLE-US-00003 Ingredient Quantity/Concentration Men ACYWX-TT 10-20 g polysaccharide/serogroup/ml Buffer: 10 mM PBS (pH 7.0 0.2) Excipient: 0-150 mM NaCl Adjuvant Aluminum phosphate as 1000 g Al.sup.+++/ml Water (MQW) qs

[0053] The ingredients are mixed by stirring at room temperature for 0.5-2 hours and followed by filling in vials and storage at 2-8 C.

[0054] Another embodiment of the formulation without adjuvant providing desired osmolality, high stability and desired immunogenicity comprises of

TABLE-US-00004 Ingredient Quantity/Concentration Men ACYWX-TT 10 g polysaccharide/serogroup/ml Buffer: 10 mM PBS (pH 7.0 0.2) Excipients: 0-150 mM NaCl Water (MQW) qs

[0055] The ingredients are mixed by stirring at room temperature for 0.5-2 hours and followed by filling in vials and storage at 2-8 C.

[0056] The formulations of the present invention with or without adjuvant have been tested for osmolality, stability and immunogenicity. The formulations have been exposed to high temperatures of 372 C. (VVM) for 21 days to check the rise in the free polysaccharide content. The free polysaccharide content in the pentavalent vaccine formulation has been separated by deoxycholate (DOC) precipitation and filtration method and estimated by High performance anion exchange chromatography with pulsed amperometric detector (HPAEC-PAD) method.

[0057] The formulation is stable at high temperatures. Considering 40% free PS as the maximum target, the formulation shows stability at the high temperature of 372 C. for at least 21 days, at 252 C. for at least 3 months and at 53 C. for at least 9 months.

[0058] The formulation of the present invention is in liquid or lyophilized form or a combination thereof. The present invention also provides the optimum dosage of each of the conjugates in the vaccine composition and formulation. The optimum dose is 5-10 g of serogroup A and X polysaccharide and 5 g of serogroup C, Y and W polysaccharide per human dose without adjuvant or with 500 g Al.sup.+++ in form of aluminum phosphate adjuvant per human dose.

Example 1: Preparation of Placebo Liquid Formulations

[0059] NaCl has been mixed with different buffers to achieve the osmolality of the buffer in the range of 30030 mOsmol/Kg. A placebo formulation without any meningococcal serogroup (active antigen) has been prepared by mixing Aluminum phosphate with different Buffers and NaCl to know the osmotic strength of the formulation as per Table 1 below:

TABLE-US-00005 TABLE 1 Matrix for the preparation of placebo liquid formulation Alumimum Formula- Strength Strength phosphate tion No. Buffer type of NaCl of buffer content(as Al.sup.+++) MLF1 Sodium chloride 0.85% 1 mg/ml (NaCl) MLF2 2-(N-morpholino) 100 mM 25 mM 1 mg/ml ethanesulfonic acid (MES) MLF3 Phosphate 100 mM 25 mM 1 mg/ml buffered saline (PBS) MLF4 L-Histidine 100 mM 25 mM 1 mg/ml MLF5 MES & Histidine 100 mM 25 mM 1 mg/ml MLF6 PBS & Histidine 100 mM 25 mM 1 mg/ml
The above formulations (placebo) have been analyzed for Osmolality and pH to determine the suitable concentration of NaCl to achieve the desired osmolality in the final formulations. The osmolality of the formulations is provided in Table 2.

TABLE-US-00006 TABLE 2 Osmolality and pH of the Placebo liquid formulations Osmolality Formulation No. (mOsmol/Kg) pH MLF1 417 6.64 MLF2 357 6.77 MLF3 367 6.97 MLF4 346 6.69 MLF5 352 6.78 MLF6 353 6.85

[0060] Table 2 has suggested that the osmolality of the formulations of Table 1 has been higher than the expected limits of 240-330 mOsmol/Kg, indicating to reduce the strength of NaCl in order to reduce osmolality. The molar strength of NaCl has been reduced to achieve desired osmolality of the formulations.

Example 2: Preparation of Placebo Liquid Formulation by Reducing the NaCl Strength

[0061] The NaCl strength has been reduced to half as compared to strength of NaCl in Table 1, i.e., form 100 mM to 50 mM to get the osmolality index within the desired range as per Table 3 below:

TABLE-US-00007 TABLE 3 Osmolality and pH of Placebo formulations Alumimum phosphate Results Formulation Buffer Strength Strength content Osmolality No. type of NaCl of buffer (as Al.sup.+++) (mOsmol/Kg) pH MLF1 NaCl 0.42% 1 mg/ml 282 6.90 MLF2 MES 50 Mm 25 mM 1 mg/ml 275 6.62 NaCl 0.9% 289 6.90 Aluminum 2 mg/ml 276 phosphate

[0062] With NaCl close to 50 mM, the osmolality of the formulations achieved in the range of 275 mOsmol/Kg to 282 mOsmol/Kg which is within the range of desired osmolality for the formulations.

Example 3: Preparation of Different Pentavalent Meningococcal Liquid Formulations to Establish the Product Stability in the Presence of Different Excipients and Buffers

[0063] Various pentavalent Meningococcal ACYWX-TT formulations have been prepared by mixing the antigen with the buffers like MES buffer, PBS buffer, Tris Buffer, HEPES buffer and Histidine. A combination of different excipients such as Glycine, Dextrose, Histidine, Mannitol and Polysorbate 80 have been tried to evaluate the stability of the drug product when exposed to a higher temperature of 372 C. The formulations have been prepared as per the Table 4 below:

TABLE-US-00008 TABLE 4 Matrix for the formulation of liquid pentavalent Meningococcal ACYWX-TT conjugate vaccine. Antigen NaCl Aluminum 10 g/ml Formulation Total Total Histidine Dextrose Glycine Tween 80 phosphate of each Code Buffer 60 mM 75 mM 10 mM 1% 10 mM 0.05% 1 mg/ml serogroup MLF-7 Normal saline 0.45% (pH 7.0) MLF-13 PBS MLF-14 (pH 7.0) MLF-21 MES MLF-22 (pH 6.5) MLF-29 Histidine (pH 6.8) MLF-30 TRIS (pH 7.0) MLF-31 HEPES (pH 7.0) MLF-32 PBS/ Hsitidine/ Dextrose/ Glycine/ Tween 80 (pH 7.0)

[0064] The above formulations have been exposed to a temperature of 372 C. for 14 days to evaluate the effect of buffers and excipients in the stability profile of the product. The stability indicating parameter for Meningococcal conjugate formulations is the generation of free PS with time. The samples have been withdrawn on day 7 and day 14 and analyzed by a HPAEC-PAD (Dionex) method for total and free PS %. Results have been compiled for free Polysaccharide generated over time as per Table 5 and 6 below:

TABLE-US-00009 TABLE 5 Free Polysaccharide content in liquid pentavalent Meningococcal ACYWX-TT liquid formulations for Men A, C & Y when exposed to 37 2 C. for up to 14 days. Formu- Men A Free PS % Men C Free PS % Men Y Free PS % lation 7 14 7 14 7 14 Code Initial days days Initial days days Initial days days MLF-7 <1 23 37 20 34 41 14 11 14 MLF-13 2 6 8 22 29 31 11 13 14 MLF-14 <1 4 14 21 23 24 11 12 10 MLF-21 3 12 24 20 32 38 10 10 13 MLF-22 3 14 31 20 30 32 9 12 15 MLF-29 2 7 15 Int. Int. Int. 14 11 12 MLF-30 3 16 30 28 37 32 Int. Int. Int. MLF-31 2 20 35 19 25 42 9 17 12 MLF-32 Int. Int. Int. 12 28 39 Int. Int. Int. Int.: Assay interference

TABLE-US-00010 TABLE 6 Free Polysaccharide content in liquid pentavalent Meningococcal ACYWX-TT conjugate vaccine formulations for Men W & X when exposed to 37 2 C. up to 14 days Formu- Men W Free PS % Men X Free PS % lation 7 14 7 14 Code Initial days days Initial days days MLF-7 18 18 20 <1 <1 <1 MLF-13 18 19 20 <1 <1 <1 MLF-14 18 19 21 <1 <1 <1 MLF-21 20 21 22 <1 <1 <1 MLF-22 19 21 22 <1 3 <1 MLF-29 19 21 23 <1 1 <1 MLF-30 18 18 18 <1 2 1 MLF-31 19 19 21 <1 <1 1 MLF-32 17 18 21 <1 1 3

Example 4: Preparation of Different Pentavalent Meningococcal ACYWX-TT Liquid Formulations Without Aluminum Phosphate

[0065] The pentavalent Meningococcal formulations have been prepared without the addition of Aluminum phosphate. All the formulations have been prepared in PBS. The following matrix has been used to prepare the different formulations:

TABLE-US-00011 TABLE 7 Matrix for the preparation of liquid pentavalent meningococcal ACYWX-TT conjugate vaccine formulations without adjuvant Formu- A, C, Y, W, lation Man- Histi- X-TT (PS per Code PBS NaCl nitol dine MQW conjugate) MLF-36 10 mM 100 mM qs 10 g/ml MLF-38 10 mM 100 mM 10 mM qs 10 g/ml MLF-39 10 mM 100 mM 0.1% qs 10 g/ml

[0066] The above formulations have been analyzed for osmolality and pH to confirm the basic characteristics of the formulation.

TABLE-US-00012 TABLE 8 Results of the liquid pentavalent meningococcal conjugate vaccine formulations without adjuvant Osmolality Formulation Code (mOsmol/Kg) pH MLF-36 289 7.14 MLF-38 315 7.32 MLF-39 295 7.11

[0067] The results obtained meet the desired values of Osmolality and pH.

Example 5: Preparation of Pentavalent Meningococcal ACYWX-TT Liquid Formulations with Higher Dose of Men A & Men X

[0068] The formulation has been prepared with higher dose of 10 g/0.5 ml for serogroups MenA and MenX, while keeping dose of MenC, MenY, MenW at the dose of 5 g/0.5 ml.

TABLE-US-00013 TABLE 9 Matrix for the preparation of formulations with higher MenA and MenX dose Formula- Aluminum A, C, Y, tion phosphate W, X-TT Code PBS NaCl (as Al.sup.+++) MQW conjugates MLF-35 10 mM 100 mM 500 g/ml qs 20 g/ml A &X and 10 g/ml of C, Y &W

[0069] The above formulation has been analyzed for polysaccharide content, Free polysaccharide %, Osmolality and pH to confirm the basic characteristics of the formulation.

TABLE-US-00014 TABLE 10 Results of the liquid pentavalent meningococcal ACYWX-TT formulation having higher MenA & MenX dose Osmolality Formulation Code mOsmol/Kg pH MLF-35 321 7.0

TABLE-US-00015 TABLE 11 Results of the Liquid Pentavalent Meningococcal ACYWX-TT Formulations having all serogroups at 10 g polysaccharide per ml Formulation Alum Osmolality Code pH mg/ml mOsmol/Kg MLF-13 7.06 0.84 320 MLF-15 7.21 0.91 330 MLF-33 7.02 1.04 326 MLF-36 7.14 289 MLF-38 7.32 315 MLF-39 7.11 295

Example 6: Preparation of Different Lyophilized Pentavalent Meningococcal Formulations to Establish the Product Stability in the Presence of Different Excipients and Buffers

[0070] Various pentavalent Meningococcal ACYWX-TT formulations have been prepared by mixing the antigen with the buffers like MES buffer and PBS buffer. A combination of different excipients such as Sucrose, Maltose, Arginine, Lactose, Sorbitol, Histidine, Glycine have been tried to evaluate the pH, osmolality and moisture content at room temperature and to achieve the desired moisture content after lyophilization.

[0071] The desired osmolality and pH has been achieved with 10 mM Sodium Phosphate buffer with different combinations of excipients (alone or in combination) wherein the moisture content after lyophilization is not more than 3% and the cake quality is satisfactory (Table 12).

TABLE-US-00016 TABLE 12 Analysis of various combinations for lyophilized formulation (non-limiting examples): Moisture Formulation 10 mM Phosphate buffer plus Osmolality content code Other Excipients pH (mOsmol/Kg) (%) MF59 Sucrose Lactose Glycine 6.5-7.5 240-330 3 (1%) (4%) (20 mM) MF60 Sucrose Maltose Glycine 6.5-7.5 240-330 3 (1%) (4%) (20 mM) MF61 Sucrose Mannitol Glycine 6.5-7.5 240-330 3 (1%) (4%) (20 mM) MF62 Sucrose Lactose Glycine 6.5-7.5 240-330 3 (2%) (3%) (20 mM) MF63 Sucrose Maltose Glycine 6.5-7.5 240-330 3 (2%) (3%) (20 mM) MF64 Sucrose Mannitol Glycine 6.5-7.5 240-330 3 (2%) (3%) (20 mM) MF65 Sucrose Lactose Glycine 6.5-7.5 240-330 3 (3%) (2%) (20 mM) MF66 Sucrose Maltose Glycine 6.5-7.5 240-330 3 (3%) (2%) (20 mM) MF67 Sucrose Mannitol Glycine 6.5-7.5 240-330 3 (3%) (2%) (20 mM) MF71 Maltose Lactose Glycine 6.5-7.5 240-330 3 (1%) (4%) (20 mM) MF72 Maltose Mannitol Glycine 6.5-7.5 240-330 3 (1%) (4%) (20 mM) MF74 Maltose Mannitol Glycine 6.5-7.5 240-330 3 2%) (3%) (20 mM) MF75 Maltose Lactose Glycine 6.5-7.5 240-330 3 (3%) (2%) (20 mM) MF76 Maltose Mannitol Glycine 6.5-7.5 240-330 3 (3%) (2%) (20 mM) MF77 Maltose Lactose Glycine 6.5-7.5 240-330 3 (4%) (1%) (20 mM) MF82 Lactose Mannitol Glycine 6.5-7.5 240-330 3 (4%) (1%) (20 mM)

Example 7: The Liquid-Lyo Combination Formulation

[0072]

TABLE-US-00017 Ingredient Quantity/Concentration Men A, C, Y, W, X -TT 10-20 g polysaccharide/serogroup/ml Buffer: 5-20 mM PBS (pH 6.5-7.5) Excipients: 2-10% Diluent qs

[0073] In liquid-lyo formulation, the lyophilized (1yo) portion contains MenA-TT conjugate, MenC-TT conjugate either alone or in variable combinations, with excipient being selected from Sucrose, Maltose, Arginine, Lactose, Sorbitol, Histidine, Glycine, either alone or in variable combinations. The diluent is selected from water, 5-20 mM phosphate buffered saline, aluminum phosphate as 500-1500 containing MenC-TT, MenY-TT, MenX-TT, MenW-TT either alone or in variable combinations.

Example 8: Lyophilization Cycles: (Non-Limiting Examples)

[0074] The pentavalent formulation has been lyophilized through freezing, primary drying and secondary drying. The lyophilization cycles are shown in Table 13. Lyophilization cycle-3 is the best mode yielding stable formulation with less than 3% moisture content and a good quality cake.

TABLE-US-00018 TABLE 13 Lyophilization cycles LYOPHILIZATION CYCLE-1 LYOPHILIZATION CYCLE-2 LYOPHILIZATION CYCLE-3 Freezing Freezing Freezing Step Temp Time R/H Step Temp Time R/H Step Temp Time ( C.) (min) ( C.) (min) ( C.) (min) R/H 1 4 60 H 1 4 60 H 1 4 60 H 2 48 250 R 2 48 250 R 2 48 250 R 3 48 5 H 3 48 120 H 3 48 360 H Primary drying Primary drying Primary drying Step Temp Time Vacuum R/H Step Temp Time Vacuum R/H Step Temp Time Vacuum ( C.) (min) (mTor) ( C.) (min) (mTor) ( C.) (min) (mTor) R/H 1 20 90 50 R 1 20 90 50 R 1 28 240 50 R 2 20 15 50 H 2 20 180 50 H 2 20 180 50 R 3 10 90 50 R 3 10 90 50 R 3 20 600 50 H 4 10 30 50 H 4 10 60 50 H 4 10 60 50 R 5 20 300 50 R 5 20 300 50 R 5 10 120 50 R 6 20 10 50 H 6 20 60 50 H 6 25 60 50 R 7 30 30 50 R 7 30 30 50 R 7 30 30 50 R Secondary drying Secondary drying Secondary drying Step Temp Time Vacuum R/H Step Temp Time Vacuum R/H Step Temp Time Vacuum ( C.) (min) (mTor) ( C.) (min) (mTor) ( C.) (min) (mTor) R/H 1 30 810 50 H 1 30 810 50 H 1 30 500 50 H R: Ramp; H: Hold; min: Minutes

Example 9: Stability of Pentavalent Meningococcal ACYWX-TT Liquid Formulations at Stress, Accelerated and Real-Time Conditions

[0075] The stability of pentavalent meningococcal ACYWX-TT conjugate vaccine is tested when exposed at various temperature conditions over the period. The test has been conducted to get the effect of temperatures over certain periods of time. The stability tests have been conducted using three temperature parameters, viz. real-time storage conditions at 53 C., higher temperatures at 252 C. and stress conditions at 372 C. the results are mentioned in FIG. 1-3 (FIG. 1a-1e, 2a-2e, 3a-3e).

[0076] a. real-time storage conditions at 53 C.recommended storage temperature (Real Time Stability Studies) (FIG. 1 a-e).

[0077] b. higher temperatures at 252 C.higher than those recommended for storage (Accelerated Stability Studies) (FIG. 2 a-e).

[0078] c. stress conditions at 372 C.extreme conditions (Stress Stability Studies) (FIG. 3 a-e).

[0079] Samples have been stored for a period of 21 days under stress conditions, for 6 months under accelerated conditions and for 3 years at real time storage conditions (Study ongoing, data available till 9 months). Samples have been withdrawn as per plan and analyzed for the best stability indicating parameter of free PS % content over time.

Example 10: Immunization of Mice and Rabbits with the Liquid Pentavalent Meningococcal ACYWX-TT Conjugate Vaccine Formulation

[0080] Groups of 8 female mice and 4 female rabbits (6-9 weeks old) have been immunized at 2 week intervals with either novel liquid adjuvanted or non-adjuvanted pentavalent meningococcal ACYWX-TT conjugate vaccine, a vehicle control without bulk conjugates or a licensed ACYW conjugate vaccine. The novel liquid pentavalent meningococcal ACYWX-TT conjugate vaccine has also been used in mouse model after storage at 37 C. for 7 days (VVM7) for evaluation of formulation stability. All immunizations have been performed by administering of vaccine via subcutaneous route in mice and intramuscular route in rabbits. Each mouse has been immunized with formulation equivalent to 1 g polysaccharide per serogroup, whereas each rabbit has been immunized with full intended human dose. Serogroup specific anti-meningococcal IgG antibody titers have been estimated by indirect ELISA and functional antibody titers by serum bactericidal assay in sera collected post 2 and 3 dose. The post 2 and 3 dose results for novel liquid pentavalent meningococcal ACYWX-TT conjugate vaccine indicate significantly high immunogenicity titers as compared to vehicle control in both mouse and rabbit model and non-inferior titers to the licensed vaccine IgG and SBA titers in both animal models (FIG. 4a-4e, 5a-5e, 6a-6e). Further, the novel liquid pentavalent Meningococcal ACYWX-TT conjugate vaccine showed no decrease in immunogenicity titers when exposed to 37 C. for 7 days (FIG. 6a-6e).

Example 11: Determination of Anti-Meningococcal Polysaccharide Serogroup Specific IgG Titers by Indirect ELISA

[0081] Ninety six-well plates (Nunc Maxisorp) have been coated with serogroup specific standard Meningococcal PS by adding 100 l per well mixture of a 5 g/ml PS and m-HSA in PBS buffer, pH 7.30.1. Plates have been incubated overnight at 4 C., and then washed three times with PBS buffer (0.1% Brij 35 in PBS, pH 7.30.1) and blocked with 200 l per well of 5% FBS solution in PBS buffer (0.1% Brij 35 in PBS, pH 7.30.1) for 1 hour at 37 C. Each incubation step has been followed by three PBS buffer wash. Reference and test sera samples have been diluted in PBS buffer (0.1% Brij 35, 5% FBS in PBS, pH 7.30.1), transferred into coated-blocked plates (200 l), and serially twofold diluted followed by overnight incubation at 4 C. Then 100 l per well of optimally diluted peroxidase conjugated anti-mouse/rabbit IgG have been added and left for 1 hour at 25 C. 100 .sub.IA per well of substrate, 3, 3, 5, 5tetramethylbenzidine-H.sub.2O.sub.2 has been added for color development. After 10 minutes of development at 25 C., reaction has been stopped by adding 50 l of 2 M H.sub.2SO.sub.4, and OD has been measured at 450 nm on Micro plate reader. Anti-MenC polysaccharide IgG concentrations (in terms of ELISA Units/ml) for each formulation have been evaluated using Combistat software and the geometric mean concentrations (IgG GMC) have been shown for representative studies and formulation comparisons in FIG. 4-6 (FIG. 4a-e 5a-e, 6a-e).

Example 12: Serum Bactericidal Assay (SBA) for the Serogroup Specific Functional Antibody Titration

[0082] N. meningitidis serogroup specific bacterial stock has been grown overnight on sheep blood agar plate at 37 C. with 5% CO.sub.2. Isolated colonies have been picked and incubated on the surface of another sheep blood agar plate at 37 C. with 5% CO.sub.2. The bacterial growth from second plate have been suspended in optimized SBA buffer for respective serogroup. The optical density (OD.sub.650) of the suspension has been adjusted in working bacterial stock to achieve a colony count of 60-250 per spot in the end of the assay. Quality control (QC) sera and test sera samples have been heat inactivated for 30 min at 56 C. In micro well plate, 20 l of serial two-fold dilutions of test serum has been mixed with 10 l of bacteria at the working dilution and 10 l of baby rabbit complement (Pel-Freez). For negative controls bacteria have been incubated, in a separate well, with active baby rabbit complement without the test serum and with test serum and heat-inactivated baby rabbit complement. The well contents have been mixed by gently tapping the assay plate and incubated the plates for 1 hour at 37 C. with 5% CO.sub.2. Ten L sample from each well plated on blood agar plate by streak plate method. The blood agar plates have been incubated overnight at 37 C. with 5% CO.sub.2 and colonies have been counted. The highest serum dilution showing 50% decrease in colony-forming units after incubation of bacteria with reaction mixture, as compared to respective active complement control has been considered as the SBA titer. The results for representative studies and formulation comparisons are presented in FIG. 4-6 (FIG. 4a-e 5a-e, 6a-e).