Rotavirus vaccine compositions and process for preparing the same

Abstract

Invention provides novel rotavirus vaccine compositions comprising rotavirus antigens, stabilizers and buffers. The buffers in the invention are pre-mixed in the rotavirus vaccine compositions to neutralize the high acidic pH of the stomach without requiring separate administration of an antacid before vaccine administration. Vaccine compositions with buffers of the invention are stable liquid rotavirus vaccine formulations for oral administration.

Claims

1. A liquid vaccine formulation comprising: a rotavirus antigen of strain 116E; and a combination buffer system, wherein the combination buffer system is a mixed buffer system comprising ammonium acetate, ammonium bicarbonate and di-ammonium orthophosphate buffer.

2. The vaccine formulation of claim 1, wherein the mixed buffer is present at a concentration range of 0.01 M to 2 M.

3. The vaccine formulation of claim 2, wherein the mixed buffer is present at a concentration of 0.5 M.

4. The vaccine formulation of claim 1, wherein the dose volume is 1 ml, which is sufficient to neutralize the acidity of the gastric environment, wherein said vaccine formulation is capable of eliciting protective immune response against infections caused by rotavirus infections.

5. The vaccine formulation of claim 1, wherein the formulation is stable for at least two years at 2-8 C.

6. The vaccine formulation of claim 1, wherein the formulation is stable for at least 6 months at 25 C.

7. The vaccine formulation of claim 1, further comprising a sugar.

8. The vaccine formulation of claim 7, wherein the sugar comprises from 20% to 75% sucrose.

9. The vaccine formulation of claim 7, wherein the sugar comprises up to 10% lactose.

10. The vaccine formulation of claim 7, wherein the sugar comprises up to 1% trehalose.

11. The vaccine formulation of claim 1, further comprising an albumin.

12. The vaccine formulation of claim 7, wherein the albumin comprises human serum albumin.

13. The vaccine formulation of claim 7, wherein the albumin comprises up to 10% lactalbumin hydrolysate.

14. A liquid vaccine formulation comprising: a rotavirus antigen; and a buffer comprising ammonium acetate, ammonium bicarbonate, and di-ammonium orthophosphate buffer.

15. The vaccine formulation of claim 14, wherein the rotavirus antigen comprises an antigen of rotavirus strain 116E.

16. The vaccine formulation of claim 14, wherein the buffer comprises a concentration of from 0.01 M to 2 M.

17. The vaccine formulation of claim 14, wherein the buffer comprises a concentration of about 0.5M.

18. The vaccine formulation of claim 14, which has a pH from 7 to 8.5.

19. The vaccine formulation of claim 14, which is stable for at least two years at 2-8 C.

20. The vaccine formulation of claim 14, which is stable for at least 6 months at 25 C.

21. A liquid vaccine formulation comprising: a rotavirus antigen; and a magnesium hydroxide carbonate buffer system, wherein the vaccine has a pH from 10 to 10.5.

22. The vaccine formulation of claim 21, wherein the rotavirus antigen comprises an antigen of rotavirus strain 116E.

23. The vaccine formulation of claim 21, wherein the buffer system comprises magnesium hydroxide carbonate dihydrate at a concentration of from 0.01 M to 0.5 M.

24. The vaccine formulation of claim 21, wherein the buffer system comprises magnesium hydroxide carbonate dihydrate at a concentration of about 0.1M.

25. The vaccine formulation of claim 21, which is stable for at least two years at 2-8 C.

26. The vaccine formulation of claim 21, which is stable for at least 6 months at 25 C.

27. The vaccine formulation of claim 1, which comprises a dose volume of between 0.5 ml to 2.0 ml.

28. The vaccine formulation of claim 27, wherein the dose volume is 1.0 ml.

29. The vaccine formulation of claim 27, wherein the dose volume is 2.0 ml.

30. The vaccine formulation of claim 1, which comprises a dose volume sufficient to neutralize the acidity of the gastric environment.

31. The vaccine formulation of claim 1, which is capable of eliciting a protective immune response against infections caused by rotavirus.

32. The vaccine formulation of claim 14, which comprises a dose volume of between 0.5 ml to 2.0 ml.

33. The vaccine formulation of claim 32, wherein the dose volume is 1.0 ml.

34. The vaccine formulation of claim 32, wherein the dose volume is 2.0 ml.

35. The vaccine formulation of claim 14, which comprises a dose volume sufficient to neutralize the acidity of the gastric environment.

36. The vaccine formulation of claim 14, which is capable of eliciting a protective immune response against infections caused by rotavirus.

37. The vaccine formulation of claim 21, which comprises a dose volume of between 0.5 ml to 2.0 ml.

38. The vaccine formulation of claim 37, wherein the dose volume is 1.0 ml.

39. The vaccine formulation of claim 37, wherein the dose volume is 2.0 ml.

40. The vaccine formulation of claim 21, which comprises a dose volume sufficient to neutralize the acidity of the gastric environment.

41. The vaccine formulation of claim 21, which is capable of eliciting a protective immune response against infections caused by rotavirus.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Baby Rossete Rice Assay results of various formulations to study the buffering capacity of the formulations.

(2) FIG. 2: Stability of rotavirus vaccine formulations at 2-8 C. with citrate phosphate buffers.

(3) FIG. 3: Stability of rotavirus vaccine formulations at 25 C. with citrate phosphate buffers.

(4) FIG. 4: Stability of rotavirus vaccine formulations at 37 C. with citrate phosphate buffers.

(5) FIG. 5: Stability Studies of rotavirus vaccine formulations using Citrate Bicarbonate Buffers at 2-8 C.

(6) FIG. 6 Stability Studies of rotavirus vaccine formulations using Citrate Bicarbonate Buffers at 25 C.

(7) FIG. 7: Stability Studies of rotavirus vaccine formulations using Carbonate Bicarbonate Buffers at 2-8 C.

(8) FIG. 8: Stability Studies of rotavirus vaccine formulations using Carbonate Bicarbonate Buffers at 25 C.

(9) FIG. 9: Stability Studies of rotavirus vaccine formulations using Magnesium Hydroxide Carbonate Buffers at 2-8 C. and 25 C.

(10) FIG. 10: Stability Studies of rotavirus vaccine formulations using Mixed Buffers at 2-8 C.

(11) FIG. 11. Stability of rotavirus vaccine formulations at 25 C. with ammonium based Mixed Buffers.

(12) FIG. 12. Stability of rotavirus vaccine formulations at 37 C. with ammonium based mixed buffers.

(13) FIG. 13: Effect of Different Buffers on ORV formulations on log titer vs. time.

(14) FIG. 14: Effect of Different Buffers on ORV formulations on log loss titer vs. time.

DESCRIPTION OF THE INVENTION

(15) Definitions:

(16) FFU/ml: A measurement of the concentration of the live virus in a given amount of fluid/harvest. This is measured by spreading a known amount of the viral fluid over a layer of cultured cells which are infected by the virus, then counting the number of areas in the culture which look infected.

(17) Target titer: Each single human dose of rotavirus vaccine should contain not less than 10.sup.5.5 ffu/dose to raise the vaccine immunogenecity, till end of shelf life of the vaccine which is referred as the Target Titer.

(18) The required virus bulk is added on the basis of bulk titer and the target titer for the formulation on volume basis. Typical rota virus bulk titers are around 10.sup.7.5 per dose while the formulation titers were targeted around 10.sup.6.0 or 10.sup.5.5 per dose, except a few batches were also formulated around 10.sup.4.0 titer to check the effect of vaccine titer on stability for a given composition. All components are finally expressed in weight/volume percentage terms.

(19) Typical target titer was around 10.sup.6.0 at the time of preparation of the vaccine; the objective being to achieve a vaccine formulation with a titer at the minimum of 10.sup.5.0 after giving an allowance of 0.5 log loss in titer (with a margin for titer estimation error around +/0.30), after 2 years of storage at 2-8 C. Any composition which shows a titer loss greater than 0.3 compared to the zero day titer at 2-8 C. is considered as exhibiting signs of instability. Of course, titer losses are quite high at 25 C. and 37 C. as a function of storage time, but these results are used only for screening purposes.

(20) The total dose volume exemplified in the various formulations ranges from 0.5 ml to 2.0 ml has formulations considering the fact that, the vaccine is able to be easily absorbed by the infant. The excipients (sugars) are dissolved in the desired novel buffer solution(s) as is a common practice in biological preparations while the stabilizers [(Human Serum Albumin (HSA)/Lactalbumin Hydrolysate (LAH)] are made as aqueous solutions (in Water For Injection) and all solutions are sterile filtered. Both these solutions are prepared on weight/volume basis. While vaccine+buffer is known with 2.5 ml as the total volume, based on acid neutralization studies, lower buffer volumes have also been tested. This was also corroborated with information available in public domain on commercial rotavirus vaccines.

(21) As required, detailed embodiments of the present invention are disclosed herein with the help of examples; however, it is to be understood that the disclosed embodiments are merely examples of the invention, which can be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed structure. Further, the terms and phrases used herein are not intended to be limiting but rather to provide an understandable description of the invention.

Example 1: Method of Adaptation of Rotavirus in Presence of Recombinant Trypsin

(22) Small scale studies were conducted using T flasks and CF1 (Cell Factory 1), further scaled upto CF40 to study the comparative infectivity and yields of Rotavirus in Vero cells. Post infection, fluid viral harvest (VH) per ml in terms of yield of harvest was observed after every 48 hours. Three sets of experiments were conducted using T Flasks and Cell Stacks and average of the three experiments was considered and the results are shown in the table below.

(23) TABLE-US-00001 TABLE 1 Yield of viral harvest taken after every 48 hours post infection in presence of recombinant trypsin and trypsin (animal origin). VH Container VH I II VH III Type of Trypsin used used (48 hrs) (96 hrs) (144 hrs) Porcine Trypsin T 175 Flask 6.52 6.97 6.75 Recombinant Trypsin T 175 Flask 6.85 7.08 6.92 Porcine Trypsin CF 1 6.88 7.05 7.12 Recombinant Trypsin CF 1 7.03 7.48 7.21 Porcine Trypsin CF 40 6.83 7.23 6.76 Recombinant Trypsin CF 40 6.95 7.38 6.87

(24) Hence, it is evident from the experiments that Recombinant Trypsin has given comparatively better yields than Porcine Trypsin since Porcine Trypsin contains Chymotrypsin as impurity as well as free of any potential hazards associated with PCV 1 and 2, along with PCBs as well.

Example 2: Buffering Capacity of Various Novel Buffers and Vaccine with Buffer Formulations

(25) An ideal buffer can be selected by measuring the buffering capacity of a particular buffer. Buffering capacity is measured as the amount of buffer which would maintain pH to neutral conditions of a given formulation, in highly acidic environments. Usually the closer the buffered pH is to the pKa, the greater is the buffering capacity. The buffering capacity of various buffers was tested using 34 mEq of Hydrochloric acid. The buffering capacity results acted as a base to include such buffers in vaccine formulations. The buffering capacity of buffers and vaccine formulations were conducted using 34 mEq of Hydrochloric Acid and the well established Baby Rosette Rice Assay undertaken in the prior art.

Example 2.1: Buffering Capacity of Various Buffers

(26) Following novel buffers have been checked for their buffering capacities which might be included in the rotavirus vaccine formulations:

(27) TABLE-US-00002 TABLE 2 (a) Buffering capacity of other buffer(s) that may be used as potential vaccine + buffer formulations with viral vaccines. Buffer details 34.8 mEq HCl was used to simulate gastric juice. 0.1M Trisodium Citrate Volume of HCl 0 ml 5 ml 10 ml 15 ml 20 ml 25 ml and 0.3M Potassium Phosphate Volume of buffer 1 ml 1 ml 1 ml 1 ml 1 ml Buffer Combination system pH 7.57 6.89 6.21 5.75 5.11 4.68 0.3M Magnesium Hydroxide Volume of HCl 0 ml 5 ml 10 ml 15 ml 20 ml 25 ml Carbonate Buffer System Volume of buffer 1 ml 1 ml 1 ml 1 ml 1 ml pH 10.5 8.02 7.82 7.68 7.48 6.55 0.2M Sodium Lactate + Volume of HCl 0 ml 5 ml 10 ml 15 ml 20 ml 25 ml 0.1M Sodium Bicarbonate Volume of buffer 1 ml 1 ml 1 ml 1 ml 1 ml Buffer Combination System pH 8.02 7.48 6.92 6.22 5.83 5.01 C 0.2M Sodium Carbonate Volume of HCl 0 ml 5 ml 10 ml 15 ml 20 ml 25 ml and 0.2M Sodium Bicarbonate Volume of buffer 1 ml 1 ml 1 ml 1 ml 1 ml Buffer Combination System pH 9.6 9.03 8.6 8.21 7.84 7.22 60% Sodium lactate solution Volume of HCl 0 ml 5 ml 5 ml Volume of buffer 1 ml 2 ml pH 4.08 6.02 C 0.03M Trisodium Citrate Volume of HCl 0 ml 5 ml 10 ml 15 ml 20 ml 25 ml and 0.3M Sodium Bicarbonate Volume of buffer 1 ml 1 ml 1 ml 1 ml 1 ml Buffer Combination system pH 8.53 8.45 7.51 7.03 6.58 6.02

(28) Conclusions:

(29) Magnesium Hydroxide Carbonate, Carbonate-Bicarbonate Buffer and Citrate-Bicarbonate Buffer Combination Systems are strong buffers and even after addition of 20 ml concentrated Hydrochloric acid (HCl) also, pH came down to around 6.5 to 7.0 from the original pH. Lactate Bicarbonate buffer and 0.05M citrate and 0.1M Bicarbonate Buffers are moderate wherein the pH reaches to 5.5 to 6.0 after addition of 20 ml of HCl. All the above said buffer(s) are able to neutralize 5 ml of HCl where the pH is in the range of 9.0 to around 7.0. since the volume of gastric juice is approximately 3.0 ml to 5.0 ml, all these buffers can be used as buffers for retaining the vaccine pH in the stomach. When these buffers are used for formulation, due to the presence of other excipients along with buffers 1 ml of vaccine is able to neutralize 5 ml of acid, but pH is around 6.0 instead of 7.0. pH 6.0 will not destabilize the viral antigen and hence these buffers can be added to oral vaccine formulations.

(30) Apart from the above mentioned novel buffers ammonium acetate based mixed buffers were checked for buffering capacity Ammonium acetate based mixed buffer combination systems have not been cited in any prior art literature relating to rotavirus vaccine formulations. The buffering capacity of such ammonium based mixed buffers have been evaluated. Mixed Buffers with Ammonium Salts (Ammonium acetate+Ammonium bicarbonate+Di ammonium ortho phosphate of 0.5 M) was checked to confirm its buffering capacity. To 0.5 ml of concentrated Hydrochloric Acid, Mixed Buffer was added from 0.5 ml to 2 ml and pH was checked.

(31) TABLE-US-00003 TABLE 2(c) Buffering capacity of mixed buffer with ammonium salts pH Results (0.5 ml Acid + Buffer) Buffer volume added to 0.5 ml 0.5 ml 1.0 ml 1.5 ml 2 ml of concentrated Hydrochloric Acid. pH of Mixed Buffer (0.5M) 7.25 7.74 7.90 7.98

(32) It is evident from the above table that even after addition of Concentrated Hydrochloric Acid to 0.5 ml of buffer, the buffer is able to retain the pH of 7.25 which indicates that these Ammonium buffers are strong buffers which can resist the pH changes upon the addition of Concentrated Acid and also when the buffer is added progressively from 0.5 ml to 2.0 ml pH, increased in the range of 7.7 to 8.0.

Example 2.2: Buffering Capacity of Various Vaccine and Novel Buffer Formulations

(33) In our efforts to develop stable novel vaccine and buffer compositions, a large number of formulations have been prepared with various buffers at a wide range of concentration. A few guiding factors have been framed while developing such compositions: Acid Neutralizing Capacity or Buffering Capacity of the chosen buffer. Volume of buffer required to be less than 2.0 ml for the prescribed acid system. Stability of the buffer+virus vaccine combination.

(34) For such studies, the pure liquid vaccine compositions without buffers which have been found to be stable at 2-8 C. have served as the base compositions. Again to recapitulate, compositions which show a titer loss less than 0.5 log or those whose final titer is greater than 105.50 are considered to be stable compositions.

(35) 2.2.1.

(36) Buffering capacity using 34 mEq HCl of various vaccine formulations with Citrate Bicarbonate, Carbonate Bicarbonate and Citrate Phosphate Buffer was also tested and provided in the table mentioned below.

(37) TABLE-US-00004 TABLE 2(b) Buffering capacity of various rotavirus formulations with buffers. Buffering Formulation Capacity Example Volume No. Composition Details Added pH BV-1 Rotavirus antigen 116E 0.5 ml 2.97 Sucrose 40% 1.5 ml 5.01 Trehalose 0.5% 1.7 ml 5.73 Lactose 5.0% Lactalbumin Hydrolyzate 0.5% Recombinant Human Serum Albumin 0.35% 0.03M Trisodium Citrate + 0.3M Sodium Bicarbonate Buffer BV-2 Rotavirus antigen 116E 0.5 ml 5.93 Sucrose 40% 0.8 ml 6.25 Trehalose 0.5% Lactose 5.0% Recombinant Human Serum Albumin 0.35% Lactlabumin Hydrolysate 0.5% 0.1M Trisdoium Citrate + 0.3M Potassium phosphate Buffer BV-3 Rotavirus antigen 116E 0.5 ml 2.94 Sucrose 40% 1.0 ml 3.78 Trehalose 0.5% 1.3 ml 5.76 Recombinant Human Serum Albumin 0.35% Lactalbumin Hydrolysate 0.5% 0.1M Sodium Carbonate - Sodium Bicarbonate Buffer BV-4 Rotavirus antigen 116E 0.5 ml 2.72 Sucrose 40.0% 1.0 ml 3.40 Trehalose 0.5% 1.5 ml 5.63 Lactose 0.5% Recombinant Human Serum Albumin 0.35% Lactalbumin Hydrolysate 1.0% ZnCl.sub.2 3 mM 0.1M Sodium Carbonate - Sodium Bicarbonate Buffer BV-5 Rotavirus antigen 116E 0.5 ml 3.49 Recombinant Human Serum Albumin 1.60% 0.8 ml 5.76 Lactalbumin Hydrolysate 1.0% 1.0 ml 6.62 0.1M Sodium Carbonate - Sodium Bicarbonate Buffer BV-6 Rotavirus antigen 116E 0.5 ml 3.71 Sucrose - 40% 1.0 ml 4.65 Trehalose - 0.5% 1.5 ml 6.05 Lactose - 5.0% Recombinant Human Serum Albumin - 0.35% Lactalbumin Hydrolysate - 0.5% ZnCl2 - 3 mM 0.5M Ammonium based mixed Buffer BV-7 Rotavirus antigen 116E 0.5 ml 3.41 Sucrose - 50% 0.8 ml 4.5 Trehalose - 0.5% 1.0 ml 5.94 Recombinant Human Serum Albumin - 0.35% Lactalbumin Hydrolysate - 1.0% 0.1M Magnesium Hydroxide Carbonate Buffer

(38) 2.2.2.

(39) Baby Rossette Rice Assay: Since Rotavirus vaccine is administered orally, it will get exposed to gastric acidity where the pH is around 1.8 to 2.0 due to which rotavirus antigen will get inactivated. To prevent this inactivation of virus potential, buffers used in formulations retains the formulation at neutral pH levels. Rotavirus inactivation takes place at pH 2.0 or 3.0 but at pH 4.0, no or minimal inactivation takes place. The buffering capacity of a given formulation is defined as the time measured to maintain the pH of the formulation above 4.0 and is evaluated by Baby Rossette Rice assay which is a validated method and referred in the prior art.

(40) Procedure for BRR Assay: In a 50 ml beaker Water For Injection was added to the formulation to a final volume of 10 ml. The beaker was placed in water bath and the temperature maintained at 37 C. The initial pH of the solution was measured and recorded. Thereafter, 4.0 ml of 0.1N Hydrochloric Acid was added to the beaker containing the final volume of 10 ml formulation. At the same time the pump for addition of 0.5 ml/minute of 0.1N HCl using peristaltic pump was switched on. The pH values along the time was recorded per minute, until the pH of 4.0 or above is retained. Stop the clock and pump. (Reference: Geigy scientific Tables, Volume 1, 1981 addition, Page 126). Several rotavirus vaccine formulations with novel buffer-combination systems were checked for buffering capacity (the respective components of the formulation details have been mentioned wherever applicable). Buffering capacity results are mentioned in the table below.

(41) TABLE-US-00005 TABLE 2(d) BRR assay results of novel buffer formulations and reference formulations (FIG. 1) Time (minutes) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 pH of CB 8.58 6.52 6.67 6.90 6.86 6.75 6.63 6.38 6.15 5.80 5.11 4.37 3.69 Buffer pH of 7.78 7.88 6.18 6.02 5.79 5.51 5.24 5.01 4.78 4.57 4.33 4.07 3.75 BV-8 pH of 7.93 6.50 6.40 6.31 6.19 6.07 5.93 5.70 5.44 5.20 4.96 4.74 4.55 4.37 4.15 3.91 BV-9 pH of 6.75 5.40 5.30 5.18 5.06 4.95 4.83 4.69 4.58 4.47 4.36 4.23 4.09 3.98 CPB pH of 7.03 5.53 5.32 5.10 4.62 4.76 4.6 4.43 4.29 4.12 3.97 BV-10 pH of 6.64 5.19 5.05 4.90 4.78 4.63 4.46 4.34 4.26 4.06 3.92 BV-11

(42) Citrate Bicarbonate buffer is 0.03 M Trisodium Citrate and 0.3 M Sodium Bicarbonate Buffer.

(43) Formulation Example BV-8 contains sucrose 40.0% w/v, Trehalose 0.5% w/v, Lactose 5.0% w/v, Lactalbumin Hydrolysate 0.5% w/v, rHSA 0.08% w/v, 0.5 M Di Ammonium hydrogen ortho-phosphate, Ammonium acetate and Ammonium bicarbonate mixed buffer along with rotavirus antigen 116E of a dose volume of 1 ml.

(44) Formulation Example BV-9 contains sucrose 30.0% w/v, Trehalose 2% w/v, Lactose 5.0% w/v, Lactalbumin Hydrolysate 1% w/v, rHSA 0.08% w/v, 0.5 M Di Ammonium hydrogen ortho-phosphate, Ammonium acetate and Ammonium bicarbonate mixed buffer along with rotavirus antigen 116E of a dose volume of 1.5 ml.

(45) CPB is 0.35 M Trisodium Citrate and 0.05 M Potassium Phosphate Buffer.

(46) Formulation Example BV-10 contains sucrose 40% w/v, trehalose-0.5% w/v, Lactalbumin Hydrolysate-0.5% w/v, rHSA-0.35% w/v, 0.05M Potassium Phosphate and 0.35M Trisodium Citrate buffer along with rotavirus antigen 116E, up to a dose volume of 2.0 ml.

(47) Formulation Example BV-11 contains sucrose-40% w/v, trehalose-0.5% w/v, Lactalbumin Hydrolysate 0.5% w/v, rHSA-0.35% w/v, 1.1M Potassium Phosphate buffer upto a dose volume of 2 ml.

(48) Conclusions:

(49) 2.5 ml of Citrate Bicarbonate has been also measured separately which were administered to infants separately prior to administration of the vaccine according to earlier practices which maintained pH of 4 and above for about 12 minutes. Formulation examples BV-8 and BV-9 of BBIL are the ammonium based mixed buffer batches and are able to neutralize and maintained the pH above 4.0 for about 15 minutes.

(50) Certain formulation examples BV-10 include buffer combination system prepared wherein the citrate concentration is raised to 0.35 M and Phosphate is reduced to 0.05 M and checked for buffering capacity using 2.0 ml as dose volume. Buffering capacity was observed and found to be 12 minutes. This formulation also is able to neutralize enough acid and shows good buffering capacity. Hence in this buffer 0.35M Citrate was considered.

Example 3: Stability of Rotavirus Vaccine Formulations with Novel Buffers

(51) Among the novel buffers, some of the most advantageous buffers showing better stability compared to others include the Carbonate-Bicarbonate buffer system, Magnesium Hydroxide Carbonate buffer, Citrate-Bicarbonate buffer system, and a mixed buffer with ammonium salts. The final stability of vaccines with these novel buffers along with final rotavirus vaccine formulations and processes of manufacturing the same have been performed.

(52) The experiments include incorporation of Zinc ions for further stability of the final rotavirus vaccine formulations Zinc ions have been observed to enhance the stability of vaccine by interacting with the composition in a manner that is known in the art. Inge Erk et. al Journal of Virology, Vol. 77, No. 6 Mar. 2003, p. 3595-3601 demonstrates that, the major capsid proteins of the VP6 trimers of rotavirus play a significant role in achieving viral flexibility in presence of Zinc ions at a given pH and temperature. The novel formulations of rotavirus vaccine therefore, include Zinc ions also to render better stability to the rotavirus antigen in the final formulations.

(53) The human Rotavirus strain 116E is a naturally attenuated (human-bovine reassortant). Stability studies of the final formulations with the rota virus antigen have been performed at various temperature ranges. Various vaccine formulations have been subjected to temperatures at 2 to 8 C., and 25 C. Observations of the vaccine titer were taken in terms of FFU/dose (focus forming units) which were taken at regular time intervals based upon the protocols. A maximum drop within the range of 0.3 log loss of the vaccine titer was considered to be maintained as a stable rotavirus vaccine formulation with the new buffer systems at 2-8 C. As explained earlier, virus titer of the vaccine is measured as a function of storage time to assess the formulation stability. For each cases, as described in the below examples, it was observed that, after 24 months of storage, at 2-8 C. the vaccine formulation was found to be stable (with a minimum or no loss considering the error bar). However, at room temperature a maximum of 1.5 log loss in vaccine titer was observed for a period of six months. Such a loss in vaccine titer concludes vaccine stability at 25 C. for six months.

Example 3.1: Stability of Rotavirus Formulations with Citrate-Phosphate Buffer Combinations

(54) Formulation Example BV-10 contains sucrose 40% w/v, trehalose-0.5% w/v, Lactalbumin Hydrolysate-0.5% w/v, rHSA-0.35% w/v, 0.05M Potassium Phosphate and 0.35M Trisodium Citrate buffer along with rotavirus antigen 116E, upto a dose volume of 2.0 ml. Formulation Example BV-11 contains sucrose-40% w/v, trehalose-0.5% w/v, Lactalbumin Hydrolysate 0.5% w/v, rHSA-0.35% w/v, 1.1M Potassium Phosphate buffer upto a dose volume of 2 ml.

(55) Stability Data of BV-10 and BV-11 are Provided Below:

(56) TABLE-US-00006 TABLE 3(a) Stability at 2-8 C. of BV-10 and BV-11 (FIG. 2). Formulation Example No. 0 Day 1 M 3 M 6 M 9 M 12 M BV-10 6.00 6.03 5.98 6.01 5.93 5.98 BV-11 6.38 6.21 6.25 6.11 6.31 6.28

(57) TABLE-US-00007 TABLE 3(b) Stablity at 25 C. of BV-10 and BV-11 (FIG. 3). B. No 0 Day 2 wk 4 wk 8 wk 12 wk 16 wk 24 wk BV-10 6.00 5.93 5.78 5.63 5.45 5.31 4.89 BV-11 6.38 6.01 5.95 5.71 5.52 5.25 4.79

(58) TABLE-US-00008 TABLE 3(c) Stability at 37 C. of BV-10 and BV-11 (FIG. 4). B. No 0 Day 1 wk 2 wk 3 wk 4 wk BV-10 6.00 5.51 4.3 3.98 3.05 BV-11 6.38 5.78 4.85 4.11 3.68

Example 3.2: Vaccine Formulations with 0.03M Citrate and 0.3M Bicarbonate Buffer (CB Buffer)

(59) Trisodium citrate, dehydrate of (Molecular Weight 294.1 gm) to an amount of 9.6 gm and Sodium bicarbonate anhydrous (Molecular Weight. 84.01 gm) to an amount of 25.6 gm was sequentially added to 500 ml in a sterile glass bottle so as to achieve 0.032M of Citrate and 0.3M of Bicarbonate. The components were dissolved completely and the volume was made up to 1 liter with WFI (water for injection). pH was checked and maintained to a level between 8.2 and 8.4. The buffer vessel was then transferred into 2 L Stainless Steel (SS) pressure vessel and connected with filter. The solution was filtered through 0.2 filter under Biosafety Cabinet by using compressed air. The buffer was collected into another sterile bottle and the bottle was labeled and stored at 2-8 C. for future use.

(60) Formulations were made using this (0.032M) Citrate (0.3M) Bicarbonate buffer combination system prepared including Rotavirus antigen 116E at 10.sup.6 ffu/dose and other stabilizers and components as mentioned below at dose volumes as mentioned in the following table corresponding to each formulation (identified by unique formulation number as appropriate allotted by Bharat Biotech International Limited in the tables), and stability of the vaccine formulation at 2 to 8 C., and 25 C. was tested at various time intervals till 2 years approximately which is given in the accompanying graphs (FIGS. 5 and 6).

(61) TABLE-US-00009 TABLE 3(d) Rotavirus Vaccine Formulations with CB Buffers. Formulation No. Dose Vol Formulation details Buffer used BV-12 1.5 ml Rota virus antigen 0.03M Citrate + 116E, Sucrose 0.3M Bicarbonate 71.00%, Trehalose Buffer 0.50%, HSA 0.35%, LAH 1.00% BV-13 1 ml Sucrose 30.00%, 0.03M Citrate + 0.3M Lactose - 5.00%, Bicarbonate Buffer HSA 0.35%, LAH 0.50%, 3 mM Zn BV-14 1.5 ml Sucrose 71.0%, 0.03M Citrate + Trehalose - 0.5%, 0.3M Bicarbonate LAH 0.5% Buffer

(62) TABLE-US-00010 TABLE 3(e) Rotavirus Vaccine Stability at 2-8 C. with CB Buffer (FIG. 1) Formulation No. 0 Day 1 M 4 M 6 M 12 M 18 M 24 M BV-12 6.31 6.12 6.08 6.04 6.18 5.98 6.01 BV-13 6.27 6.03 6.14 6.07 5.97 6.03 5.93 BV-14 6.35 6.19 6.21 6.18 6.21 6.12 6.07

(63) TABLE-US-00011 TABLE 3(f) Rotavirus Vaccine Stability at 25 C. with CB Buffer (FIG. 2) Formulation 24 wk No. 0 Day 2 wk 4 wk 6 wk 10 wk 16 wk (6 M) BV-12 6.31 6.24 5.83 5.89 5.76 5.74 4.94 BV-13 6.27 6.11 5.72 5.83 5.61 5.55 4.72 BV-14 6.35 6.20 6.01 5.93 5.52 5.43 4.89

Example 3.3: Vaccine Formulations with Carbonate and Bicarbonate Buffer(s) (0.2M and 0.1M) (CAB Buffer)

(64) One of the other well suited buffer systems to use for vaccine preparations that will be ingested and assimilated by the human body, is the carbonate-bicarbonate buffer system. This is because this buffer system is responsible for about 80% of extracellular buffering and is present in blood plasma in the form of a combination of carbonic acid (H2CO3) and bicarbonate (HCO3-) to maintain a pH between 7.35-7.45. This system is highly conducive to use in an organic environment as it fulfils the requirements of non-inhibition of enzymatic action, useful alkaline pH range, simplicity and reasonable good stability. This combination buffer system, has not been reported in the prior art, especially at bicarbonate molarities less than 0.15M.

Example 5.1: 0.2 M Carbonate Bicarbonate Buffer

(65) Sodium carbonate anhydrous (Molecular Weight 105.99 gm) to an amount of 1.69 gm and Sodium bicarbonate anhydrous (Molecular Weight. 84.01 gm) to an amount of 15.4 gm was sequentially added to 500 ml in a sterile glass bottle so as to achieve 0.2M of Carbonate and 0.2M of Bicarbonate. The components were dissolved completely and the volume was made up to 1 liter with WFI (water for injection). pH was checked and maintained to a level between 9.2 and 9.5. The buffer vessel was then transferred into 2 L Stainless Steel pressure vessel and connected with filter. The solution was filtered through 0.2 filter under Biosafety Cabinet by using compressed air. The buffer was collected into another sterile bottle and the bottle was labeled and stored at 2-8 C. for future use.

Example 5.2: 0.1 M Carbonate Bicarbonate Buffer

(66) Sodium carbonate anhydrous (MW. 105.99) to an amount of 0.53 gm and Sodium bicarbonate anhydrous (MW. 84.01) to an amount of 7.98 gm was sequentially added to 500 ml in a sterile glass bottle so as to achieve 0.1M of Carbonate and 0.1M of Bicarbonate. The components were dissolved completely and the volume was made up to 1 liter with WFI (water for injection). pH was checked and maintained to a level between 9.2 and 9.5. The buffer vessel was then transferred into 2 L Stainless Steel pressure vessel and connected with filter. The solution was filtered through 0.2 l filter under Biosafety Cabinet by using compressed air. The buffer was collected into another sterile bottle and the bottle was labeled and stored at 2-8 C. for future use.

(67) Formulations were made using this 0.2M Carbonate-Bicarbonate buffer combination system and 0.1M Carbonate-Bicarbonate buffer combination system prepared including Rotavirus antigen 116E at 10.sup.6 ffu/dose and other stabilizers and components as mentioned below at dose volumes as mentioned in the following table corresponding to each formulation (identified by unique formulation numbers as mentioned in Table 3(g)), and stability of the vaccine formulation at 2 to 8 C., and 25 C. was tested at various time intervals till 2 years approximately which is given in the accompanying graph (FIGS. 7 and 8).

(68) TABLE-US-00012 TABLE 3(g) Rotavirus Vaccine Formulations with CAB Buffers. Formulation Dose No. Vol. Formulation Details Buffer used. BV-15 1 ml Sucrose 40%, Trehalose 0.5%, 0.2M Carbonate HSA 0.35%, LAH 1% Bicarbonate Buffer BV-16 1 ml Sucrose 50%, Trehalose 0.5%, HSA 0.2M Carbonate Bicarbonate 0.35%, LAH 1%, 3 mM Zn Buffer BV-17 1 ml Sucrose 50%, Trehalose 0.5%, HSA 0.2M Carbonate Bicarbonate 0.35%, LAH 1% Buffer BV-18 1.5 ml Sucrose 71%, Trehalose 0.5%, HSA 0.1M Carbonate Bicarbonate 0.35%, LAH 1%, Buffer

(69) TABLE-US-00013 TABLE 3(h) Rotavirus Vaccine Formulation Stability at 2-8 C. with CAB Buffers (FIG. 3). B. No. 0 Day 1 M 2 M 3 M 4 M 6 M 9 M 12 M 15 M 18 M 24 M BV-15 6.21 6.21 6.21 6.19 6.11 6.13 6.09 6.09 6.03 5.81 5.80 BV-16 6.36 6.24 6.12 6.18 6.20 6.28 6.25 6.25 5.92 5.93 5.95 BV-17 6.31 6.26 6.12 6.27 6.23 6.24 6.13 6.13 6.07 6.05 5.98 BV-18 6.25 6.21 6.07 6.11 6.11 6.22 6.19 6.21 6.23 6.25 6.13

(70) TABLE-US-00014 TABLE 3(i) Rotavirus Vaccine Formulation Stability at 25 C. with CAB Buffers (FIG. 4). Stability at 25 C. B. No 0 Day 2 wk 4 wk 6 wk 8 wk 10 wk 12 wk 16 wk 24 wk BV-15 6.30 6.21 5.86 5.91 5.87 5.81 5.73 5.68 5.06 BV-16 6.36 6.26 6.09 6.07 6.01 6.04 5.97 5.83 5.22 BV-17 6.31 6.24 6.13 6.05 6.05 6.03 5.96 5.96 5.41 BV-18 6.25 6.24 6.14 6.11 6.01 6.01 5.98 5.98 5.75

Example 3.4: Vaccine Formulations with 0.1M Magnesium Hydroxide Carbonate Buffer (MHC Buffer)

(71) Magnesium Hydorxide Carbonate Dihydrate (Molecular Weight of 314.98 gm) was sequentially added to 500 ml water for injection in a sterile glass bottle so as to achieve 0.1M of Magnesium Hydroxide Carbonate. The components were shaked well for homogeneity, since this salt is water insoluble. The suspension was made to a volume of 1 liter with Water for Injection. dissolved completely and the volume was made up to 1 liter with WFI (water for injection). pH was checked and maintained to a level between 10 to 10.5. The solution was sterilized by autoclaving at 121 C. for 30 minutes. The buffer was collected into another sterile bottle and the bottle was labeled and stored at 2-8 C. for future use.

(72) Formulations using this 0.1M Magnesium Hydroxide Carbonate buffer combination system were prepared including Rotavirus antigen 116E at 10.sup.6 ffu/dose and other stabilizers and components as mentioned below at dose volumes as mentioned in the following table corresponding to each formulation (identified by unique formulation numbers as mentioned in Table 3(j)), and stability of the vaccine formulation at 2 to 8 C., and 25 C. was tested at various time intervals till 2 years approximately which is given in the accompanying graph (FIG. 9).

(73) TABLE-US-00015 TABLE 3(j) Rotavirus Vaccine Formulations with MHC Buffers. Formulation Dose No. volume Formulation details Buffer used BV-19 1 ml Sucrose 40%, Trehalose 0.1M Magnesium 0.5%, HSA 0.35%, LAH Hydroxide 1% Carbonate Buffer

(74) TABLE-US-00016 TABLE 3(k) Rotavirus Vaccine Formulation Stability at 2-8 C. and 25 C. with MHC Buffers (FIG. 5). Stability at 2-8 C. and 25 C. Temperature 0 Day 1 M 4 M 6 M 12 M 18 M 24 M 25 Deg C. 6.75 6.03 5.31 4.24 2-8 Deg C. 6.75 6.61 6.45 6.47 6.39 6.34 6.28

Example 3.5: Vaccine Formulations with 0.5M Mixed Buffer with Ammonium Salts (Mixed Buffer)

(75) Di Ammonium hydrogen ortho phosphate (Mol Wt. 132.06 gm) to an amount of 66.03 gm, Ammonium acetate (Mol Wt. 77.08 gm) to an amount of 38.54 gm and Ammonium bicarbonate (Mol Wt. 79.02 gm) to an amount of 39.51 was added to 500 ml water for injection in a sterile glass bottle so as to achieve 0.5 M of the mixed buffer with the ammonium salts. The components were dissolved completely and the volume was made up to 1 liter with WFI (water for injection). pH was checked and maintained to a level between 7.6 and 7.8. The buffer vessel was then transferred into 2 L Stainless Steel pressure vessel and connected with filter. The solution was filtered through 0.2 filter under Biosafety Cabinet by using compressed air. The buffer was collected into another sterile bottle and the bottle was labeled and stored at 2-8 C. for future use.

(76) Formulations using this 0.5 M of Mixed buffer combination system were prepared including Rotavirus antigen 116E at 10.sup.6 ffu/dose and other stabilizers and components as mentioned below at dose volumes as mentioned in the following table corresponding to each formulation (identified by unique formulation numbers as mentioned in Table 3(l)), and stability of the vaccine formulation at 2 to 8 C., was tested at various time for a period of 1 year which is given in the accompanying graphs (FIGS. 10 to 13).

(77) TABLE-US-00017 TABLE 3(l) Rotavirus Vaccine Formulations with Mixed Buffers (Ammonium acetate + ammonium bicarbonate + Di ammonium ortho phosphate) 0.5M Formulation Dose Example No. Vol. Formulation Details Buffer used. BV-20 1 ml 10{circumflex over ()}6.0 ml/1.0 ml (Double 0.5M Mixed buffer concentration of sugars) Sucrose 40.0% Trehalose 0.5% Lactose 5.0% LAH 0.5% rHSA 0.08% BV-21 1.5 ml 10{circumflex over ()}6.0/1.5 ml (Components 0.5M Mixed buffer have been calculated for 1.5 ml) Sucrose 30.0% Trehalose 2.0% Lactose 5.0% rHSA 0.08% LAH 1.0%

(78) TABLE-US-00018 TABLE 3(m) Rotavirus Vaccine Formulation Stability at 2-8 C. with Mixed Buffers (FIG. 6). Stability at 2-8 C. Stability of BV 9 and BV 10 at 2-8 Deg C. B. No 0 Day 1 M 3 M 6 M 9 M 12 M BV-20 6.12 6.17 6.07 6.03 6.08 6.11 BV-21 6.23 6.25 6.25 6.18 6.21 6.23

Example 4: pH Values of Buffers and Vaccine Formulations Before and after Formulations after Stability Period

(79) It is also important to check the pH of the buffers used in different formulations and the corresponding pH values while the buffers are present in the final vaccine formulations. This is to check the given pH ranges of the final vaccine formulation during and after storage period of the vaccine. Such data is presented below. The bicarbonate based systems are above pH 8, which is above the physiological buffer pH. However, the vaccine is stable even over 2 years at 2-8 Deg C. after stability period.

(80) TABLE-US-00019 TABLE 3(n) Buffer pH and vaccine pH before and after formulations at 0 day and 24 months. Formulation Vaccine Vaccine pH Example pH at after Number Buffer used Buffer pH 0 day 24 months BV-12 0.03M Trisodium 8.21 7.61 7.82 Citrate and 0.3M Sodium Bicarbonate Buffer Combination system BV-15 0.2M Sodium 9.51 8.71 8.98 Carbonate and 0.2M Sodium Bicarbonate Buffer Combination System BV-19 0.3M Magnesium 10.1 7.98 8.01 Hydroxide Carbonate Buffer System BV-21 0.5M (Ammonium 7.78 7.62 7.81 acetate + Ammonium bicarbonate + Di ammonium ortho phosphate) mixed buffer

(81) Further, experiments were conducted with fixed formulation components while changing the various buffers, and the stability of the vaccine formulations were studied over prolonged periods of time. Corresponding FIGS. 13 and 14 shows comparative vaccine stability with various novel buffers and fixed formulation components which include sucrose 40% w/v, trehalose 0.5% w/v, LAH 1% w/v, rHSA 0.35% w/v and rotavirus antigen 116E.

REFERENCES

(82) 1. Wainwright, W H. The Development of Live, Attenuated Rotavirus Vaccines: A Manufacturer's Resource Guide. Seattle:PATH; 2006. 2. Bharat Biotech's International PCT publication WO2007/132480 (granted Indian Patent 242868 and granted in UK 0821386.0) and WO2011/07363. 3. U.S. Pat. No. 6,403,098 (Merck) and U.S. Pat. No. 6,616,931 (Merck). 4. Inge Erk et. al Journal of Virology, Vol. 77, No. 6 Mar. 2003, p. 3595-3601. 5. www.ou.edu/research/electron/bmz5364/buffers.html visited Feb. 10, 2012. 6. www.nature.berkeley.edu/soilmicro/methods/phosphate %20buffer.pdf visited Feb. 10, 2012. 7. Guidelines to assure the quality, safety and efficacy of live attenuated rotavirus vaccines (oral) WHO Technical Report Series No 941, 2007. 8. Harry B. Greenberg, Mary K. Estes, Rotaviruses: From Pathogenesis to Vaccination, doi:10.1053/j.gastro.2009.02.076. (Gastroenterology 2009). 8. Penelope H. Dennehy, Rotavirus Vaccines: an Overview, Clinical Microbiology Reviews, January 2008, Vol. 21, No. 1, p. 198-208. 9. Bresee J S, Glass R I, Ivanoff B, Gentsch J. Current status and future priorities for rotavirus vaccine development, evaluation and implementation in developing countries. Vaccine 1999; 17:2207-22. 10. (Ref:Geigy scientific Tables, Volume 1, 1981 addition, Page 126).