Thermostable freeze dried rotavirus vaccine formulation and process to prepare thereof

Abstract

The present invention relates to a thermostable freeze dried rotavirus vaccine formulation and the process of preparing the same. More specifically the present invention discloses multivalent thermostable liquid, powder or cake based rotavirus vaccine formulation prepared using the freeze drying process, such that the said vaccine formulation possess improved heat-stability, easy to use and transport and highly affordable thereby meeting the requirements of developing and low income country's vaccination program. The said freeze dried rotavirus vaccine formulation along with reconstitution buffer is so engineered to be suitable for filling in appropriate packaging containers/ closures so designed such that they reduce the logistics requirement for storage.

Claims

1. A thermostable freeze dried rotavirus vaccine formulation wherein said vaccine formulation comprises at least one rotavirus serotype, and at least one excipient comprising a lyoprotectant, a first class of buffering agent, a bulking agent, and an activating agent, and optionally a salt, an ion and/or a dispersant wherein the lyoprotectant is selected from sucrose, trehalose, sorbitol, and lactose, either alone or in combination; the first class of buffering agent is selected from HEPES, Tris, phosphate, citrate, and Histidine, either alone or in combination; if present, the salt is selected from Sodium chloride, Magnesium chloride, and Potassium chloride, either alone or in combination; if present, the ion is selected from Zn(II) or Ca (II) either alone or in combination; the bulking agent is selected from Polyvinylpyrrolidone (PVP), Dextran, and mannitol, either alone or in combination; if present, the dispersant is polysorbate 20 and/or polysorbate 80; and the activating agent is L-Arginine and/or Glycine, wherein said lyoprotectant is in the range of 1.2% w/v to 6.0% w/v said first class of buffering agent is in the range of 10 mM to 50 mM; said salt is in the range of 0 mM-50 mM; said divalent cation is in the range of 0 mM to 2 mM; said bulking agent is in the range of 2% to 6% w/v; said dispersant is in the range of 0 to 0.025% w/v; and said activating agent is in the range of 10 to 100 mM.

2. The thermostable freeze dried rotavirus vaccine formulation as claimed in claim 1 wherein said lyoprotectant is Sucrose, Trehalose or a combination thereof; said first class of buffering agent is HEPES; said salt is Sodium Chloride (NaCl); said divalent cation is Ca.sup.+2; said first class of bulking agent is PVP; said dispersant is polysorbate 20; and said activating agent is L-Arginine and/or Glycine.

3. The thermostable freeze dried rotavirus vaccine formulation as claimed in claim 1 wherein said rotavirus serotype is selected from at least one rotavirus strain selected from bovine, rhesus, human, ovine, rhesus/human reassortants, bovine/human reassortants and other monovalent or multivalent rotavirus.

4. The thermostable freeze dried rotavirus vaccine formulation as claimed in claim 1 wherein said rotavirus serotype is selected from G1, G2, G3, G4 and P1 Human-Bovine reassortants either alone or in combination.

5. The thermostable freeze dried rotavirus vaccine formulation as claimed in claim 4 wherein said rotavirus serotype having Log.sub.10 concentration as G1: 6.81, G2: 6.92, G3: 6.91, G4: 6. 78 and P1: 6.83 per dose.

6. The thermostable freeze dried rotavirus vaccine formulation as claimed in claim 1 wherein said vaccine formulation does not exceed potency losses exceeding Log.sub.10=[0.5] when stored at 5 +/3 C. up to 2 years and when stored up to 45 C. for at least 18 weeks.

7. An improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 1, said process comprising the steps of: (a) selecting at least one rotavirus serotype; (b) selecting said at least one excipient; (c) adding said selected rotavirus serotype of step (a) to said at least one excipient of step (b) to prepare a liquid feed with optimal potency retention; (d) freeze drying said aqueous liquid feed of step (c) to obtain a freeze dried formulation; (e) milling said freeze dried formulation obtained from step (d) by using suitable milling methods at temperatures ranging from 0 C. to 25 C. to obtain a milled dried formulation; and (f) blending of said milled dried formulation obtained from step (e) with a blending agent individually or in combination with a second class of buffering agent having acid neutralizing capacity to obtain said thermostable freeze dried rotavirus vaccine formulation for appropriate packaging of monodose or multidose, wherein the process results in a potency loss not exceeding Log.sub.10=[0.2].

8. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 7 wherein said liquid feed is having solid contents in the range of 10 g/100 ml to 25 g/100 ml.

9. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 7 wherein said freeze drying process is performed either at clinically relevant specification (1) or at higher (5) rotavirus concentrations.

10. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 7 wherein said liquid feed is having titre value in the range of 5.0E+06 IU/mL to 6.0E+07 IU/ mL each serotype, preferably 5.0E+06 IU/mL to 4.0E+07 IU/mL each serotype.

11. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 7 wherein said freeze drying process results in cumulative potency loss not exceeding Log.sub.10=[0.5] at temperature in the range of 37 C. +/2 C. for at least 30, 60, 90, 180 days and Log.sub.10=[0.5] at temperature in the range of 45 C. +/2 C. for at least 30, 60, 90, 120 days.

12. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 7 wherein said milled freeze dried powder is obtained by milling methods including Sieve milling, Hammer milling, Ball milling, Conical sieve milling and Roller milling wherein, Particle size of said milled freeze dried powder have D.sub.90 value, is not less than 250 m; the preferred temperature range is in range of 20 C.-22 C.; and said milling is performed in presence of a dry gas including Nitrogen, Argon, CO.sub.2, air or combinations thereof.

13. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 7 wherein said vaccine formulation is in dry form such as freeze dried cake, freeze dried powder, milled freeze dried powder, milled and blended freeze dried powder.

14. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 12 wherein said milled freeze dried powder shows glass transition temperature (T.sub.g) in the range of 48.0 +/31 1.0 C.

15. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 7 wherein said residual moisture content of said freeze dried rotavirus vaccine formulation is below 3.0%.

16. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 9 wherein said blending agent is selected from PVP K25, PVP K40, sucrose, mannitol, maltodextrin with DE ranging from 4 to 20, fructose, glucose, lactose either alone or in the combinations providing D.sub.90 >250 m to milled blended freeze dried powder.

17. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 7 wherein said second class of buffering agent being selected from HEPES, Trisodium citrate dihydrate, histidine, calcium carbonate, sodium carbonate, potassium carbonate, sodium bicarbonate, calcium bicarbonate, potassium bicarbonate, aluminum hydroxide, sodium dihydrogen phosphate monohydrate or magnesium hydroxide or combinations thereof.

18. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 7 wherein said second class of buffering agent when used in conjunction with blending agent results in said acid neutralization capacity of the vaccine in the range of 0.3 mEq/dose to 1.0 mEq/dose of powder.

19. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 7 wherein said vaccine is administered orally with or without a reconstitution buffer or with or without water for injection.

20. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 19 wherein said reconstitution buffer is selected from calcium carbonate, sodium carbonate, potassium carbonate, sodium bicarbonate, calcium bicarbonate, potassium bicarbonate, aluminum hydroxide or magnesium hydroxide, sodium dihydrogen phosphate monohydrate, trisodium citrate dihydrate, phosphate citrate, or combinations thereof.

21. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 20 wherein said vaccine formulation when reconstituted with reconstitution buffer results in acid neutralizing capacity in the range of 0.3 mEq/dose to 1.0 mEq/dose.

22. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 21 wherein the palatability of said reconstitution buffer is maintained by adding 0-60% of sugars such as lactose, glucose, sucrose, fructose or the combinations thereof.

23. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 7 further comprising the step of: (a) packaging of said thermostable freeze dried rotavirus vaccine formulation in a container along with said reconstitution buffer comprising said buffering agent having said acid neutralizing capacity in a separate container for reconstitution prior to oral administration.

24. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 20 wherein said vaccine when reconstituted with said reconstitution buffer maintains virus potency losses not exceeding Log.sub.10=[0.5] at storage conditions of 2 C.-8 C. for 48 h and 25 C. for 8 h.

25. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 7 wherein said vaccine formulation dissolves within 60 +/5.0 sec in said reconstitution buffer maintained at 23 C. +/1 C.

26. The improved freeze drying process of preparing thermostable freeze dried rotavirus vaccine formulation as claimed in claim 7, wherein step (d) is performed in vials or in trays.

27. The improved freeze drying process of claim 7, wherein the solid content of liquid feed is 13 g/100 ml (13%).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: Stability data of freeze dried formulation 1004c in vials containing five human bovine serotypes (G1, G2, G3, G4 and P1) for N=1 (), N=2 () and N=3 () at 2 C. to 8 C. storage conditions.

(2) FIG. 2: Stability data of freeze dried formulation 1004c in vials containing five human bovine serotypes (G1, G2, G3, G4 and P1) for N=1 (), N=2 () and N=3 () at 37 C. storage conditions.

(3) FIG. 3: Stability data of freeze dried formulation 1004c in vials containing five human bovine serotypes (G1, G2, G3, G4 and P1) for N=1 (), N=2 () and N=3 () at 45 C. storage conditions.

(4) FIG. 4: Stability data of freeze dried, milled and blended formulation 1004c containing five human bovine serotypes (G1, G2, G3, G4 and P1) for N=1 (), N=2 () and N=3 () at 2 C. to 8 C. storage conditions.

(5) FIG. 5: Stability data of freeze dried, milled and blended formulation 1004c containing five human bovine serotypes (G1, G2, G3, G4 and P1) for N=1 (), N=2 () and N=3 () at 37 C. storage conditions.

(6) FIG. 6: Stability data of freeze dried, milled and blended formulation 1004c containing five human bovine serotypes (G1, G2, G3, G4 and P1) for N=1 (), N=2 () and N=3 () at 45 C. storage conditions.

DETAILED DESCRIPTION OF THE INVENTION

(7) In order to obviate the aforementioned drawbacks in the existing prior art, the present invention relates to a thermostable freeze dried rotavirus vaccine formulation and the process of preparing the same. More specifically the present invention discloses an improved thermostable dried cake or dried powder form of rotavirus vaccine formulation prepared by using an improved freeze drying process. The present invention also relates to liquid or solid rotavirus vaccine formulation prepared using reconstitution of dried cake or dried powder form of vaccine formulation, such that said vaccine formulation have improved heat-stability, easy to transport and use and is affordable. Therefore, said vaccine meets the requirements of developing and low income country's vaccination program.

(8) The invention uses the process of freeze drying for removing water moiety, one of the major sources of instability for aqueous rotavirus vaccine. The water moiety is extracted from the liquid rotavirus vaccine formulation either for individual virus or serotype, or in combination of multiple rotavirus serotypes to form dehydrated thermostable vaccine formulation. These methods of freeze drying results in achieving moisture content below 3.0 percent approximately thereby preventing degradation pathways in rotavirus serotypes associated with the presence of excess water.

(9) Before the preferred embodiment of the present invention is disclosed, it is understood that this invention is not limited to the particulars materials described, as these may vary. It is also understood that the terminology used in herein is for the purpose of describing the particular embodiment only, and is not intended to limit the scope of the present invention in any way.

(10) It must be noted that as used herein, the singular forms a, an and the include plural reference unlike the context clearly dictates otherwise, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

(11) Accordingly, in a preferred embodiment, dehydration is accomplished using freeze drying process in presence of excipients including stabilizers, substituting unstable water interactions with stable interactions of stabilizing ingredients.

(12) In one of the embodiment, the methodology of Design-of-Experiment (DoE) is employed for identification of the potential excipients, their concentrations and combinations thereof that are likely to have a significant impact on the potency loss of viruses in the vaccine formulation during freeze-drying process and other manufacturing steps and subsequent storage for determining the stability loss. In some cases, to minimize number of experiments, successive fractional factorial DoE have also been employed.

(13) The suitability of potential excipients, their concentrations and combinations thereof is determined by various biophysical methods, for example, Differential Thermal Analysis (DTA), Differential Scanning calorimetry (DSC), Freeze Drying Microscopy (FDM) and Impendence measurements. Further, direct potency is also measured by using various methods,for example, Multivalent QPCR-Based Potency Assay (M-QPA) that permits the specific quantitation of each individual reassortant virus in the presence of the other four reassortant viruses. The biophysical methods give an indication of process suitability such as e.g. time and energy inputs required and the latter potency measurement methods gives an indication of the effect of excipients including stabilizers on the potency during processing and subsequently during storage. In some cases, potency change after repeated freeze-thawing of the virus in presence of DoE compositions followed by incubation at elevated temperature i.e. at 25 C. for extended period of time for at least 24 hours and a maximum of up to one week have also been used.

(14) The excipients including various stabilizers are selected from, but not limited to (a) lyoprotectants such as Sucrose, Trehalose, Sorbitol and Lactose, (b) divalent cation more particularly divalent ionic structural stabilizers such as Zn (II) and Ca (II); (c) buffering agents such as HEPES, Tris, Phosphate, Citrate or Histidine; and (d) salts such as Sodium Chloride (NaCl), Magnesium chloride, Potassium cholride; (e) bulking agents such as Polyvinylpyrrolidone(PVP), Dextran,Mannitol; (0 activating agents including L-Arginine and/or Glycine and (g) dispersants such as Tween (e.g. Tween 20 and Tween 80).

(15) In a preferred embodiment, the thermostable freeze dried vaccine preparation contain, lyoprotectants in the range of 1.2% w/v to 6.0% w/v, divalent cations like Zn (II) and Ca (II) in the range of 0 mM to 2 mM, salt is in the range of 0 mM-50 mM, buffering agents in the range of 10 mM to 50 mM, bulking agent in the range of 2% to 6% w/v, activating agents in the range of 10 mM to 100 mM, dispersants in the range of 0% w/v to 0.02% w/v,

(16) In yet another preferred embodiment, excipients including stabilizers, especially the buffering system, are so selected to allow maximum solubility to divalent ions, such as Zn (II) and Ca (II), which are principally required for maximum process stability and also play role in storage stability of rotavirus at elevated temperatures for extended period of time.

(17) In yet another embodiment, the rotavirus vaccine formulation described in this invention can include live attenuated rotavirus of human-bovine reassortment i.e. G1, G2, G3, G4 and P1 as well as other live attenuated rotaviruses from bovine, rhesus, human, bovine, rhesus/human reassortants, or bovine/human reassortants. The other examples of viruses used in this invention, without limitation, may also include other live attenuated viruses including measles, noroviruses, polio and other monovalent or multivalent rotaviruses.

(18) In yet another embodiment, particularly preferred freeze dried vaccine is prepared using 54 hour freeze drying cycle or 70 hour freeze drying cycle in vials and trays respectively. In some cases, the freeze drying cycle can be appropriately modified using inputs (glass transition temperature T.sub.g and eutectic melting temperature; T.sub.eu) from various biophysical methods mentioned above and potency losses from M-QPA. In all aspects of invention, freeze drying using above optimized parameters and said compositions results in dried cake or dried solid powder with the residual moisture content of less than 3.0%. The said reduction in residual moisture to below 3% content results in preventing thermal degradation pathways associated with presence of water. The said rotavirus vaccine preparations can be obtained using any appropriate freeze drying method.

(19) In a preferred embodiment, freeze drying formulation ingredients in composition and concentration are selected based on Potency and biophysical measurements to arrive at a formulation affording optimal freeze drying parameters and times with no more than Log.sub.10=[0.2] process loss for the said virus serotypes.

(20) In yet another embodiment, said rotavirus vaccine formulation can be freeze dried in vials in a monodose and/or multidose formulations and the said vaccine is reconstituted with the provided reconstitution buffer prior to administration.

(21) In yet another embodiment, the said rotavirus vaccine formulation is freeze dried in bulk using commercially available freeze drying trays like GORE LYOGUARD Freeze-Drying Tray or stainless steel trays.

(22) In yet another preferred embodiment, said rotavirus vaccine formulation is freeze dried in bulk for example rotavirus vaccine with 5-10 fold higher titers than the clinically relevant specifications are freeze dried thus reducing both the size and number of batches and thereby reducing the overall cost of production.

(23) In order to obtain a powder suitable for filling in the container closure for particularly monodose container, the bulk freeze dried material is milled resulting in the milled freeze dried powder with particle size of D.sub.90 value not less than 250 m. The milling unit process can be accomplished using appropriate milling methods including but not limited to Sieve milling, Hammer milling, Conical sieve Milling, Ball milling and Roller milling at temperatures ranging from 0 C. to 25 C.

(24) In yet another embodiment, the milled freeze dried powder can be blended with suitable blending agents selected from but not limited to PVP K25, PVP K40, sucrose, mannitol, maltodextrin with DE generally ranging from 4 to 20, fructose, glucose, lactose or placebo formulation of same composition or the combinations thereof. The said combinations are so prepared to obtain a powdered dosage form having rotavirus titers equal to clinically relevant minimal concentrations or any desired release specification as well as to engineer powder properties acceptable for accurate and precise powder filling in the predetermined packaging containers.

(25) In yet another embodiment, the freeze drying is performed either at clinically relevant specification or at higher titre of rotavirus scales (5 in glass vials or in bulk using stainless steel trays with a process loss of not more than Log.sub.10=[0.2], cumulative potency (process and stability) losses for the freeze dried rotavirus vaccine serotype in any combination do not exceed Log.sub.10=0.5 irrespective of factors such as starting fill, serotype, process scale, and combinations thereof at 37 C.2 C. (RH 75%5.0%) for at least 30, 90 or 180 days and a cumulative potency (process and stability) losses for the freeze dried rotavirus vaccine serotype in any combination do not exceed Log.sub.10=0.5 irrespective of the factors irrespective of factors such as starting fill, serotype, process scale, and combinations thereof at 45 C.2 C. (RH 75%5.0%) for at least 30, 90 or 120 days.

(26) Rotavirus is acid-labile and gets rapidly inactivated with a half-life of less than 12 minutes at pH 2.0. The rotavirus vaccines are intended to be administered orally, it is crucial to protect rotavirus which may get inactivated by gastric acid present in stomach. The effective way for administering such vaccines is to use antacids or neutralizing buffers before or in combination with the oral vaccine.

(27) In yet another embodiment of the present invention, special provisions are employed to protect the vaccine from stomach acid after oral administration, stomach acid neutralization is accomplished by administering the vaccine with buffering agent with better Acid Neutralization Capacity (ANC). The buffering/antacid component are selected from but not limited to HEPES, Trisodium citrate dihydrate, histidine, calcium carbonate, sodium carbonate, potassium carbonate, sodium bicarbonate, calcium bicarbonate, potassium bicarbonate, aluminum hydroxide, sodium dihydrogen phosphate monohydrate or magnesium hydroxide or combinations thereof.

(28) In yet another embodiment of the present invention, said vaccine formulation is milled and and blended with blending agent and/or buffering agent imparting desired ANC which is in the range of 0.3 mEq/ dose to 1.0 mEq/dose of vaccine powder and moisture content is below 3%.

(29) In yet another embodiment of the present invention, said vaccine formulation is reconstituted with a reconstitution buffer just before oral administration that imparts desired ANC which is in the range of 0.3 mEq/dose to 1.0 mEq/ dose of vaccine.

(30) In another embodiment, to provide the palatability to the formulation, sugars are added in the concentration in the range of 0% w/v to 60% w/v to reconstitution buffer. The sugars are selected from but not limited to lactose, glucose, sucrose, fructose or the combinations thereof.

(31) In all aspects of the present invention vaccine formulations are in dry form i.e. Freeze dried cake/powder, milled dried powder or milled and blended freeze dried powder. The said above formulations so obtained do not exceed potency losses exceeding Log.sub.10=[0.5] when stored at 5 C.3 C. for at least 9 months that may be statistically extrapolated to 2 years) and at 37 C. or 45 C. for at least 18 weeks.

(32) In all aspects of the present invention said powdered vaccine formulations are administered orally by reconstituting with either said reconstitution buffer when blended solely with blending agent or with water for Injection when blended with blending agent and with buffering agents having desired ANC.

(33) Yet another aspect of the present invention provides aseptically reconstituted vaccine preparation that does not result in overall (process+stability) virus potency losses exceeding Log.sub.10=[0.5] when re-constituted in appropriate aqueous reconstitution buffer and stored at 2 C.-8 C. for 48 h.

(34) In yet another embodiment of the present invention, the glass transition temperature (Tg) of milled vaccine is more than 47 C. to provide the thermal stability at higher temperature storage conditions.

(35) In yet another embodiment, the powdered vaccine formulation is dispensed in the final packaging container/closures including sachet or vials supplied with separate reconstitution buffer for reconstitution.

(36) Thus, said vaccine formulation have improved heat- stability, easy to transport and use and is affordable, hence said vaccine meets requirements of developing and low income country's vaccination program.

EXAMPLES

(37) The following examples illustrate but do not limit the scope of this invention.

Example 1

Thermostabilization of Rotavirus Serotypes using Freeze Drying Process

(38) Study 1: Selection of Excipients Including Stabilizers:

(39) To identify among the potential ingredients, their concentrations and combinations thereof that are likely to have a significant impact on the stability of rotavirus during freeze-drying process including further processing steps (process loss) and storage (stability loss) as well as to support optimal and economical freeze-drying process, the methodology of Design-of-Experiment (DoE) is applied. The G1 strain of rotavirus is used as representative strain to report on the virus potency changes. To minimize number of experiments, successive fractional factorial designs are used.

(40) The fractional factorial designs are limited to the 8 factors as shown in the Table 1 and Table 2.

(41) TABLE-US-00001 TABLE 1 Various stabilizers and their concentrations used to design DoE formulations Sugar/ Bulking Buffer NaCl Agent Amino-acids Activating agents Dispersant Factor 1 2 3 4 5 6 7 8 Excipient Tris/*Phos/ NaCl **Sugar L-Arg Gly CaCl.sub.22H.sub.2O ZnCl.sub.2 Tween HEPES (mM) (mM) (mM) (mM) (mM) (mM) (%) Low level 10 0 1.2/2 10 0 0 0 0 (1) Mid level 25 25 3/3 50 50 1 1 0.01 (0) High 50 50 4/4.8 100 100 2 2 0.02 level (1) *Phos = phosphate; **mannitol, dextran or PVP

(42) TABLE-US-00002 TABLE 2 Combinations of buffer and bulking agents used to design DoE formulations Combination 1 2 3 4 5 6 7 8 9 Buffer *Phos *Phos *Phos HEPES HEPES HEPES Tris Tris Tris Bulking ***Dex PVP **Man ***Dex PVP **Man ***Dex PVP **Man agent *Phos = Phosphate; **Man = Mannitol; ***Dex = Dextran

(43) To further simplify the screening design, one buffer and one combination of sugar and bulking agent at a time is selected in a step by step approach. This fractional factorial design yielded total 19 experiments per combination wherein the concentration of buffering agent, salt, sugar: bulking agent ratio, amino acids, activating agent and dispersant are varied. To test the ability of the stabilizer combination for optimal freeze-drying process design and for supporting virus stability during freeze-drying, following screening experiments are conducted:

(44) (a) Differential Thermal Analysis, Freeze Drying Microscopy and Impendence measurements are conducted on placebo compositions to determine critical temperatures influencing freeze-drying process. The mannitol containing DoE set is omitted in initial screening as these formulations required longer freeze-drying cycles.

(45) (b) Freeze-thaw (FT) studies are conducted wherein, G1 serotype at a concentration of 2.0E+07 IU/ml, is subjected to 5 cycles of freezing and thawing followed by incubation at 25 C. for one week. From these experiments, it is observed that sucrose: PVP containing combinations retained much better potency as compared to sucrose: dextran containing combinations for G1 strain.

(46) (c) The selected best performing formulations in terms of potency are evaluated further in actual freeze drying experiments with G1 at a concentration of 7.3 log/mL of aqueous feed as shown in Table 3. The 1 ml liquid feed from these selected formulations is dispensed into 5 ml capacity glass vials and freeze dried using 82 hour to 110 hour cycle characterized by; i. Precooling the pre lyophilized liquid to 10 C. to 12 C. and loaded into the lyophilizer. ii. freezing the pre lyophilized liquid at 40 C. for 3 hour-4 hour (with ramp down of 4 C. to 40 C. within 0.5 to 2.0h) followed by annealing at 20 C. for another 2 h (ramping from 40 C. to 20 C. in the range of 0.5 h-1.5 h. Final freezing at 50 C. for 3 h to 4h. iii. Sublimation of ice at 42 C. for a maximum of 15 h followed by a further sublimation at 40 C. for another 15 h to 90 h or at a temperature range of 38 C. to 35 C. for a maximum of 55 h under the vacuum conditions of 50 mTorr. iv. Secondary drying at 20 C. to 25 C. range for 10 h to 15 h at 50 mTorr vacuum.

(47) Resulting freeze drying samples are evaluated for initial potency (t=0) compared to non-freeze dried aqueous liquid feed samples. Some of the freeze dried samples are incubated at 37 C.2 C. with Relative Humidity (RH) of 75%5% storage conditions for different time points and compared with samples stored at 2 C. to 8 C. storage conditions as shown in Table 4. Among all the formulations evaluated, 1000, 1004, 1005, 1011 & 1013 retained G1 potency is measured up to 4 weeks when stored at 37 C.2 C. (RH 75%5%). Among the formulations with Dextran as a bulking agent, 1030 retained G1 potency up to 4 weeks at 37 C.2 C. (RH 75%5%). However this formulation is observed to be unsuitable for optimal freeze drying based on DTA and the impedance analysis data.

(48) (d) All formulations are also evaluated for the moisture content using Karl Fischer method. The formulations 1005, 1011 & 1013 showed very high moisture content in repeated trials as shown in Table 5. The formulations 1000 and 1004 showed elegant cake appearance as well as low moisture content as compared to the other three formulations. These formulations are also observed to be better in terms of retaining the rotavirus potency at 37 C.2 C. (RH 75%5%). The formulation 1002 although showed a lower moisture content but lost potency at 37 C.2 C. (RH 75%5%) during 2.sup.nd week of storage.

(49) The freeze dried formulations 1000 and 1004 that yielded elegant cakes with low moisture content. It is observed that the process and the stability loss for G1 serotype did not exceed log.sub.10=[0.5] during storage at 37 C.2 C. (RH 75%5%) are used as exemplified formulations in subsequent studies.

(50) Additionally, from statistical treatment of potency and DTA data of DoE formulations an optimized combination of stabilizer emerged comprising of 25 mM HEPES, Sucrose:PVP ratio of 1.6:6.4 (g %: g %), 25 mM NaCl, 50 mM L-Arginine, 100 mM Glycine and 2 mM CaCl2.2H2O.

(51) TABLE-US-00003 TABLE 3 Composition of best performing formulations with respect to potency identified during Freeze-Thaw-Stability experiments PVP- L- Formulat.sup.n HEPES NaCl Sucrose K25 Arginine Glycine CaCl.sub.22H.sub.2O ZnCl.sub.2 Tween- Dextran ID (mM) (mM) (%) (%) (mM) (mM) (mM) (mM) 20 (%) (%) 1000 10 50 1.2 4.8 100 0 2 0 0.02 0 1001 25 25 3 3 50 50 1 1 0.01 0 1002 10 0 1.2 4.8 10 0 0 0 0 0 1004 50 0 4 2 100 0 2 0 0 0 1005 50 50 1.2 4.8 10 100 2 0 0 0 1011 50 50 4 2 100 100 2 2 0.02 0 1013 10 50 4 2 100 100 0 0 0 0 1030 50 50 4 2 100 100 2 2 0.02 2

(52) TABLE-US-00004 TABLE 4 Stability log loss of freeze dried monovalent formulations containing G1 serotype when exposed at 37 C. (RH 75% 5%) for 4 weeks Storage condition 1000 1001 1002 1004 1005 1011 1013 1030 37 C. 0 0.12 0.70 0.19 0 0 0 0 2-8 C. 0 0.21 0.30 0 0 0 0.26 0

(53) TABLE-US-00005 TABLE 5 Percent moisture content of selected freeze dried formulations evaluated using Karl Fischer's method Formulation 1000 1001 1002 1004 1005 1011 1013 1030 Moisture 0.75 1.37 0.82 0.84 4.98 4.84 4.03 1.19 content
Study 2: Selection of Optimal Buffer and Bulking Agent Combination.

(54) The optimized combination (1057 as shown in Table 6 of stabilizers obtained by statistical treatment of potency and DTA data of DoE formulations in Example 1, Study 1; is further tested for optimizing buffer and bulking agents by evaluating for potency changes. Three bulking agents and buffer combinations are used to identify best performing combination resulting in various formulations as shown in Table 6. The compositions are formulated with G1 and G3 strains. The target concentration is each strain is 7.0 log/mL. The formulation 1065H is same as 1065 with annealing step added in freeze drying cycle. The freeze drying cycle is 50 h duration characterized by: i. Precooling the freeze dryer shelf to 45 C. prior to vial loading. ii. Freezing step for 1.5 h followed by holding at the same temperature for 30 minutes. iii. Primary drying by raising the temperature to 2.0 C. followed by a ramp down to 30 C. within 2 h. Further ramping down the temperature to 36 C. within 3 h and finally to 38 C. in 5 h and holding the product at this temperature for 23 h followed by a further ramping up to 10 C. in 4 h. The vacuum maintained during all the primary drying steps is 0.080 mBar. iv. Secondary drying by increasing the temperature up to 25 C. within 30 min and holding the product at same temperature for 8 h under zero vacuum.

(55) TABLE-US-00006 TABLE 6 Composition of optimized formulations used to determine the effect of buffer and bulking agent combination on process and longitudinal stability of representative rotavirus serotypes Sucrose: Bulking L- Buffer NaCl agent Arginine Glycine CaCl.sub.22H.sub.2O ZnCl.sub.2 Tween Formulation (mM) (mM) (g %:g %) (mM) (mM) (mM) (mM) (%) 1057 25 25 1.6:6.4 50 100 2 0 0 (HEPES) PVP K-40 1060 25 25 1.6:6.4 50 100 2 0 0 (Tris) PVP K-40 1063 25 25 1.6:6.4 50 100 2 0 0 (Phosphate) PVP K-40 1065 25 25 1.6:6.4 50 100 2 0 0 (Phosphate) Mannitol 1063T 25 25 1.6:6.4 50 100 2 0 0.01 (Phosphate) Mannitol 1065H 25 25 1.6:6.4 50 100 2 0 0 (Phosphate) Mannitol

(56) In some compositions; 1065H, an additional annealing step was added resulting in a 58 h cycle characterized by: i. Precooling the freeze dryer shelf to 45 C. prior to vial loading. ii. Freezing step for 1.5 h followed by holding at the same temperature for 30 minutes. iii. Annealing step by increasing the temperature from 45 C. to 15 C. within 5 minutes and then holding at the same temperature for 2 h followed by refreezing the product to 45 C. for 1 h. iv. Primary drying by raising the temperature to 7.0 C. within 15 minutes followed by a further ramp down to 27 C. within 2 h. The temperature is further decreased to 33 C. within 3 h and finally to 35 C. in 5 h. The product is held at this temperature for 30 h. The vacuum maintained during all the primary drying steps is 0.080 mBar. This step was followed by a further ramp up to 10 C. in 4 h under zero vacuum. v. Secondary drying by raising the temperature to 25 C. within 30 min and holding the product at the same temperature for 8 h under zero vacuum.

(57) The freeze dried formulations are stored at 37 C.2 C. (RH 75%5%) and 2 C.-8 C. for four weeks. The log loss values are analyzed by M-QPA as shown in Table 7. Compositions with PVP as a bulking agent in combination with HEPES as buffering agent resulted in best retention of potency for representative rotavirus serotypes G1 and G3.

(58) The best performing formulations in terms of potency retention, 1057 and 1063, are further evaluated for their ability to support the potency of five (G1, G2, G3, G4 and P1) human bovine rotavirus serotypes under the storage conditions of 2 C.-8 C. and 37 C.2 C. (RH 75%5%). It is found that HEPES containing formulation, 1057, best supported potency of all five serotypes at high temperatures as compared to Phosphate containing 1063 as shown in Table 8. Composition involving phosphate buffer also resulted in precipitating Ca.sup.+2 ions in formulation indicating essential role of Ca.sup.+2 in longitudinal stability.

(59) TABLE-US-00007 TABLE 7 Effect of different Buffer and Bulking Agent combination on the stability loss of G1 and G3 strains Storage 1057 1060 1063 1063T 1065 1065H condition G1 G3 G1 G3 G1 G3 G1 G3 G1 G3 G1 G3 37 C. 0.09 0.09 0.34 0.51 0.10 0.16 0.16 0.10 0.25 0.68 0.83 1.33 2-8 C. 0 0 0 0.16 0 0 0 0 0 0 0 0.07

(60) TABLE-US-00008 TABLE 8 Stability loss of pentavalent rotavirus formulations 1057 and 1063 with different buffering components for 4 weeks Temperature Formulation condition G1 G2 G3 G4 P1 1057 37 C. 0.27 0 0.20 0 0.04 2-8 C. 0.17 0 0.01 0 0 1063 37 C. 0.53 0.68 0.14 0.62 0.45 2-8 C. 0.16 0.17 0 0.24 0.03
Study 3: Formulation of Reconstitution Buffer to Provide Acid Neutralization Capacity (ANC) to Vaccine.

(61) The exemplified lead formulations from above studies did not exhibit adequate ANC of by themselves. Therefore a reconstitution buffer is prepared to provide the desired ANC sufficient to protect the live virus from gastric acid. Each 2.0 mL of reconstitution buffer is formulated with 400 mg of sucrose, 29.8 mg of sodium dihydrogen phosphate monohydrate and 127 mg of trisodium citrate dihydrate in water q.s to 2.0 mL.

(62) The ANC value is determined using method described by United States Pharmacopeia 34 NF 29. The experiment is conducted in triplicate. The results are expressed in terms of mEq per dose. All the above lead formulations when dissolved in the reconstitution buffer are observed to yield an ANC value of 0.8 mEq/ dose of 2.0 mL.

(63) Study 4: Longitudinal studies and Reproducibility for Exemplified Lead Formulations 1000 and 1004 in Vials.

(64) The two exemplified formulations from Study 1, 1000 and 1004 are tested for longitudinal stability at 37 C. and 2 C. to 8 C. storage conditions, incorporating all the five (G1, G2, G3, G4 and P1) human-bovine rotavirus strains as shown in Table 9. The formulation's compositions, storage conditions and potency analysis are similar as described in Study 1. The initial potency for each serotype is 7.0 log /mL A 99 h cycle is used for freeze drying these formulations as described below: i. The pre freeze drying liquid is precooled to 10 C. to 12 C. and loaded into the freeze dryer followed by a freeze drying cycle. ii. The temperature is decreased from 4 C. to 40 C. in 60 minutes. The freezing step is carried out at 40 C. for 3 h followed by annealing at 20 C. for another 2 h. The annealing temperature is obtained within 20 minutes. Temperature is further decreased to 50 C. within 60 minutes followed by holding the vials at the same temperature for 4 h. iii. The primary drying is carried at 42 C. for 10 h followed by a further sublimation at 40 C. for 15 h and then at a temperature of 37 C. for 45 h under the vacuum conditions of 50 mTorr. The temperature is ramped up slowly from 42 C. to 40 C. and 40 C. to 37 C. in 15 minutes. iv. Secondary drying was conducted at 20 C. for 15 h at 50 mTorr vacuum.

(65) The aqueous liquid feed solid contents are in the range of 10.00 g/100mL to 25.00 g/100 mL for optimal drying, yield and or cake appearance.

(66) Process loss is calculated as a difference of potency between samples obtained before and after freeze drying. Stability log loss per 30 days at 37 C.2 C. with % RH 75%5% is calculated by linear regression of potency data of samples incubated at 37 C.2 C. and % RH 75%5% for at least 30 days with 7 day sampling intervals.

(67) TABLE-US-00009 TABLE 9 Summary of the process loss, stability loss and the cumulative loss of pentavalent composition in 1000 and 1004 formulations. n = 1, 2 and 3 are the three independent manufacturing batches. Stability Stability loss Cumulative loss data (log10) per (process loss + Stability obtained Process loss 30 days at 37 C. loss at 37 C. for 30 days) Formulation G1 G2 G3 G4 P1 G1 G2 G3 G4 P1 G1 G2 G3 G4 P1 1000 (n = 1) 0.18 0.0 0.03 0.0 0.11 0.02 0.10 0.06 0.01 0.28 0.20 0.10 0.09 0.01 0.39 1000 (n = 2) 0.01 0.0 0.01 0.0 0.0 0.02 0.04 0.11 0.0 0.19 0.03 0.04 0.12 0.0 0.19 1000 (n = 3) 0.15 0.07 0.0 0.0 0.09 0.12 0.17 0.06 0.12 0.21 0.26 0.24 0.06 0.12 0.30 1004 (n = 1) 0.01 0.0 0.09 0.01 0.0 0.09 0.0 0.16 0.11 0.12 0.10 0.0 0.25 0.12 0.12 1004 (n = 2) 0.0 0.0 0.0 0.0 0.12 0.0 0.0 0.06 0.0 0.0 0.0 0.0 0.06 0.0 0.12 1004 (n = 3) 0.03 0.0 0.0 0.03 0.14 0.05 0.06 0.07 0.0 0.03 0.08 0.06 0.07 0.03 0.17
Study 5: Longitudinal Thermostabilty and Reproducibility for Formulation 1004 in Vials.

(68) The exemplified formulation 1004 is tested for longitudinal stability and process reproducibility under the storage conditions of 37 C.2 C. (RH 75%5%), 45 C.2 C. (RH 75%5%) and 2 C.-8 C. The formulation is incorporated with all the five strains at the log concentrations per dose/ml as G1: 6.81; G2: 6.92; G3: 6.91; G4: 6.78 and P1: 6.83 (1 concentrations). A 63 h freeze drying cycle is developed and optimized for preparation of this formulation and is found to support freeze drying of full load in the freeze dryer (>680 vials per batch). The details of the freeze drying cycle are mentioned below: a. The pre freeze drying liquid is precooled to 10 C. to 12 C. and loaded into the freeze dryer followed by a freeze drying cycle. b. The temperature is ramped down from 4 C. to 40 C. within 60 minutes and the freezing step is carried out at 40 C. for 2 h followed by annealing for another 2 h at 20 C. The annealing temperature is brought within 60 minutes. A further ramp down is carried out up to 40 C. within 60 minutes. The final freezing is carried out at 40 C. for 1.5 h. c. The temperature is brought up to 33 C. within 4 h and primary drying is carried out at 33 C. for 33 h followed by a temperature ramp to 10 C. within 6 h and then to 0 C. and 25 C. within 3 h each. A 50 mTorr of vacuum is maintained during the primary drying steps. d. The secondary drying is carried out at 25 C. for 5 h at 0 mTorr.

(69) The final residual moisture content of all the three independent manufacturing batches is below 2.0%. The formulation retained its potency at 37 C.2 C. (RH 75%5%), 45 C.2 C. (RH 75%5%) and 2 C.-8 C. for at least 12 weeks. Process loss is calculated as a difference of potency between samples before and after freeze drying.

(70) Stability loss (log loss) is calculated by linear regression of potency data of samples incubated under different storage conditions. The values mentioned in the brackets represent the potency log loss values at 45 C.2 C. (RH 75%5%) for 12 weeks as shown in Table 10.

(71) TABLE-US-00010 TABLE 10 Summary of the log process loss, log stability loss and the log cumulative loss of pentavalent composition in 1004 formulation (37 C./2-8 C./45 C.). N = 1, 2 and 3 are three independent manufacturing batches Rota- virus Sero- N = 1 N = 2 N = 3 type (% MC: 1.22) (% MC: 1.10) (% MC: 0.68) Process G1 0.0 0.0 0.04 Loss G2 0.03 0.0 0.02 G3 0.01 0.0 0.11 G4 0.0 0.0 0.06 P1 0.16 0.11 0.12 Stability G1 0.11/0.11/0.12 0.06/0/0.38 0/0/0.20 Loss G2 0.06/0.05/0.19 0.12/0.03/0.25 0.09/0/0.29 G3 0/0/0.08 0.02/0/0.14 0.08/0.03/0.29 G4 0.04/0.02/0.27 0 0.17/0.04/0.24 P1 0.09/0.08/0.29 0.14/0/0.39 0.33/0.13/0.23 Cumulative G1 0.11/0.11/0.12 0.06/0/0.38 0.04/0.04/0.24 loss G2 0.09/0.08/0.22 0.12/0.03/0.25 0.11/0.02/0.31 G3 0.01/0.01/0.09 0.02/0/0.14 0.19/0.14/0.40 G4 0.04/0.02/0.27 0 0.23/0.10/0.30 P1 0.25/0.24/0.45 0.25/0/0.5 0.45/0.25/0.35

Example 2

Designing Scalable and Reproducible Freeze Drying Process for Thermostabilization of Rotavirus Vaccine

(72) Study 1: Optimization of Formulation

(73) This formulation 1004 exhibited suboptimal cake properties at pilot scales with higher titres (>2000 doses) and is further optimized by varying bulking agent concentrations and the sugar: bulking agent ratios so as to increase the solid contents of the formulation generating three variants of 1004 as shown in Table 11. These modified formulations supported a freeze drying cycle of 54 h duration involving following steps; i. precooling a pre freeze-drying liquid to 10 C. to 12 C. and loading into the freeze dryer ii. Freezing the pre freeze-drying liquid at 35 C. (temperature ramp of 4 C. to 35 C.: 2 h) for 1 h and at 45 C. for 1.5 h (temperature ramp of 35 C. to 45 C.: 0.5 h) followed by annealing at 20 C. for another 2 h (temperature ramp of 45 C. to 20 C.: 1 h). Final freezing at 40 C. for 1.5 h (temperature ramp of 20 C. to 40 C.: 0.5 h) iii. Primary drying at 32 C. for 1 h and at 28 C. for 24 h (temperature ramp from 40 C. to 32 C. and from 32 C. to 28 C.: 1 h each) under 50 mTorr vacuum. Temperature ramp up from 28 C. to 0 C.: 10 h) followed by a secondary drying at 25 C. for 5 h under 0 mTorr vacuum (Temperature ramp of 0 C. to 25 C.: 2 h).

(74) The final residual moisture content of all the above three formulations is below 1.0% (<1.0%) with elegant cakes however 1004b and 1004c were better in terms of cake appearance throughout the shelf-life. Each formulation is freeze dried in vials with all five (G1, G2, G3, G4 and P1) human-bovine rotavirus serotypes using above cycle and are evaluated for potency changes when stored at 37 C.2 C. (RH 75%5%), and 2 C. to 8 C. The process loss is calculated as a difference of potency between samples before and after freeze drying. The stability loss (log loss) per 30 days at 2 C. to 8 C. and at 37 C. 2 C. (RH 75%5%) is calculated by linear regression of potency data of samples incubated under this storage condition. The initial log potency values per ml of liquid feed are G1: 6.81; G2: 6.92; G3: 6.91; G4: 6.78 and P1: 6.83. All modified formulations showed elegant cakes and show potency exhibited process, stability and cumulative process loss that did not exceed Log.sub.10=0.5 for 30 days as shown in Table 12.

(75) TABLE-US-00011 TABLE 11 Composition of optimized formulations used to determine the effect of bulking agent concentrations and the sugar:bulking agent ratios HEPES Sucrose:PVP K-25 L-Arginine CaCl.sub.22H.sub.2O Formulation (mM) (gm %:gm %) (mM) (mM) 1004a 50 4.0:4.0 100 2 1004b 50 4.0:6.0 100 2 1004c 50 6.0:4.0 100 2

(76) TABLE-US-00012 TABLE 12 Summary of the process loss, stability loss and the cumulative loss of pentavalent composition in 1004a, 1004b and 1004c formulations. Process loss Stability log loss per 30 Cumulative log loss per 30 Formul.sup.n G1 G2 G3 G4 P1 G1 G2 G3 G4 P1 G1 G2 G3 G4 P1 1004a 0.18 0.06 0 0.03 0 37 C. 0.10 0 0 0 0 0.28 0.06 0 0 0 2-8 C. 0.01 0 0 0 0.02 0.19 0.06 0 0 0 1004b 0.01 0.11 0.16 0 0.08 37 C. 0.20 0 0.03 0.14 0.07 0.21 0.11 0.19 0.14 0.15 2-8 C. 0.17 0 0.03 0.05 0.04 0.18 0.11 0.19 0.05 0.12 1004c 0 0.14 0 0.06 0 37 C. 0.23 0.02 0.06 0.06 0.08 0.23 0.16 0.06 0.12 0.08 2-8 C. 0.15 0 0 0.02 0.02 0.15 0.14 0 0.09 0.02
Study 2: Longitudinal Thermostability and Reproducibility for Formulation 1004c in vials.

(77) The exemplified formulation 1004c is tested for longitudinal stability and process reproducibility at clinically relevant concentrations of rotavirus, under the storage conditions of 37 C.2 C. (RH 75%5%) and 2 C. to 8 C. for 8 weeks. The formulation is incorporated with all the five strains at the log concentrations per dose per ml as G1: 6.81; G2: 6.92; G3: 6.91; G4: 6.78 and P1: 6.83 (1 concentrations). A 54 h freeze drying cycle as mentioned in Example 2 of Study 1, is used for preparation of this formulation and is found to support freeze drying of full load in the freeze dryer (>680 vials per batch). The total solid content is 13 g/100 ml in aqueous liquid feed.

(78) Process loss is calculated for three independent manufacturing batches (N=1, 2, 3), as a difference of potency between samples before and after freeze drying.

(79) Stability loss (log loss) at 37 C.2 C. with %RH 75%5% is calculated by linear regression of potency data of samples incubated at 37 C.2 C. and % RH 75%5% as shown in Table 13. The moisture content of all the three manufacturing batches are below 2.0% (<2.0%).

(80) TABLE-US-00013 TABLE 13 Summary of the process loss, stability loss and the cumulative loss of pentavalent composition in 1004c formulations in three independent manufacturing batches Process loss Stability log loss per 8 Cumulative log loss per 8 Formulat.sup.n G1 G2 G3 G4 P1 G1 G2 G3 G4 P1 G1 G2 G3 G4 P1 N = 1 0 0.14 0 0.06 0 37 C. 0.43 0.04 0.11 0.11 0.14 0.43 0.18 0.11 0.17 0.14 2-8 C. 0.29 0 0 0.04 0.04 0.29 0 0 0.10 0.04 N = 2 0.11 0.12 0.12 0.15 0.12 37 C. 0.11 0.21 0.12 0.02 0.16 0.22 0.33 0.24 0.18 0.28 2-8 C. 0.03 0.06 0 0 0 0.14 0.17 0 0 0 N = 3 0.10 0.07 0.07 0.12 0.13 37 C. 0.21 0.18 0.16 0.10 0.13 0.31 0.25 0.23 0.22 0.27 2-8 C. 0.07 0.17 0.04 0.08 0.03 0.17 0.24 0.11 0.21 0.17
Study 3: Titer scale up, Longitudinal studies and Reproducibility for 1004c formulation in vials.

(81) The composition for the formulation is same as that used in Example 2 of Study 1 except that the initial human-bovine rotavirus serotype concentrations is increased 5 times (5) with the log values per ml per 5 doses as G1: 7.51; G2: 7.62; G3: 7.61; G4: 7.47; P1: 7.52 in vials. The total solid content is 13 g/100 ml in aqueous liquid feed for all the three independent manufacturing batches N=1, 2 and 3. The solution pH is adjusted to 6.100.1. The 54 h freeze drying cycle (similar to Study 1 of Example 2) is used for preparation of this formulation in Type-I glass vials with 3 mL capacity.

(82) A negligible process loss is observed when compared to the pre- freeze drying liquid control. The longitudinal studies are conducted under the storage conditions of 2 C.-8 C., 37 C.2 C. (RH 75%5%) and 45 C.2 C. (RH 75%5%). The data is depicted in FIG. 1, FIG. 2 and FIG. 3 respectively. Based on the linear regression of this data, it is observed that the freeze dried rotavirus formulation is stable at all these storage conditions for almost 252 days (9 months). Slope values for the samples stored at 2 C. to 8 C. showed no loss. Arrhenius kinetics simulations of the data indicated that expected time period for stability, as indicated by Log.sub.10=0.5 potency loss for any of the five serotypes within this formulation, at 37 C.2 C. (RH 75%5%) can be 23 months. Similar treatment of the data showed that at 45 C.2 C. (RH 75%5%) storage conditions, a loss of Log.sub.10=0.5 in the potency is observed within 9 months and is confirmed in real time.

(83) Study 4: Bulk freeze drying of exemplified formulation 1004c in trays

(84) The formulation 1004c is freeze dried in Stainless Steel (SS) trays at 5 concentrations of rotavirus. A 380 mL of pre freeze drying liquid is added into SS tray thus giving the liquid height same as that obtained in vials in Study 2 and 3 of Example2. Three independent manufacturing batches N=1, 2 and 3 were prepared.

(85) However for optimal cake properties and moisture content the bulk freeze drying in trays required the freeze drying cycle of 70 h duration. This freeze drying cycle involve following steps; i. precooling a pre freeze-drying liquid to 10 C. to 12 C. and loading into the freeze dryer ii. freezing pre freeze-drying liquid at 35 C. for 1 h (Temperature ramp of 4 C. to 35 C.: 2 h) and at 45 C. for 1.5 h (Temperature ramp of 35 C. to 45 C.: 0.5 h) followed by annealing at 20 C. for another 2 h (Temperature ramp of 45 C. to 20 C.: 1 h). Final freezing at 40 C. for 1.5 h (Temperature ramp of 20 C. to 40 C.: 0.5 h) iii. Primary drying is carried at 32 C. for 1 h and at 28 C. for 34 h (Temperature ramp of 40 C. to 32 C. and 32 C. to 28 C.: 1 h each) under a vacuum of 50 mTorr. iv. Secondary drying of cake at 25 C. for 5 h under 0 mTorr vacuum conditions. (Temperature ramp of 28 C. to 0 C.: 15.5 h and from 0 C. to 25 C.: 2 h)

(86) The final residual moisture content of all the above mentioned three independent manufacturing batches is below 3% (<3.0%) thereby preventing degradation pathways in rotavirus serotypes associated with the presence of excess water. An elegant cake is observed in trays with no collapse which is confirmed visually.

(87) Study 5: Processing of Bulk Freeze Dried Cake to Obtain Free Flowing Powders.

(88) In order to obtain a powder suitable for filling in the final packaging container/closure the bulk freeze dried pentavalent rotavirus vaccine cake is milled using Ball milling in 250 mL or 2.0 L stainless steel gas tight vessel in a

(89) Turbula Mixer (Willy A. Bachofen AG, Switzerland. Type T2F No. 131266). The vaccine cake and the steel balls are added to obtain a weight ratio of 1:10. The turbula mixer is operated for 105 sec at a speed of 72 rpm. Milled cake is checked visually for completion and further milling of 30 seconds is performed as required. All sample preparation steps prior to enclosing in container is carried out under low RH conditions (<5%) in a nitrogen purged glove box.

(90) Micronized pentavalent rotavirus freeze dried vaccine is blended with Maltodextrin DE 6-8 (Glucidex 6D) as a blending agent using a 250 mL or 2.0 L stainless steel gas tight vessel in Turbula Mixer (Willy A. Bachofen AG, Switzerland. Type T2F No. 131266). The blending is conducted at 23 rpm for 15 minutes followed by a further 2-3 minutes at 32 rpm. The milled and blended formulations are characterized for Particle size distribution, dissolution time, pH after reconstitution, residual moisture content and the glass transition temperature (T.sub.g). The T.sub.g is obtained in a step-scan DSC with a series of micro-steps with temperature increase at a particular ramp rate followed by an isothermal period.

(91) Following observations are made on the blended powder; i. The particle sizes characterized by D.sub.50 and D.sub.90 of the milled powder, reported as mean volume diameter, are observed to be 85 and 291 m respectively whereas the blending agent showed particle sizes of 91 and 270 m respectively. ii. Both the milled freeze dried powder and the blending agent showed irregular shaped particles when observed microscopically. iii. Milled samples exhibited residual moisture in a range of 2.0% to 2.5% whereas the final blended powders showed low moisture content of 1.0% to 1.5%. iv. The pH of pre-freeze drying liquid is 6.100.1. The milled and blended freeze dried pentavalent rotavirus vaccine powder is reconstituted with the reconstitution buffer and the reconstituted vaccine exhibited an Acid Neutralization Capacity (ANC) of 0.80 mEq/dose of 2 ml. The pH of vaccine after reconstitution was observed to be 6.100.1. v. The Modulated Differential Scanning calorimetry (MDSC) showed a T.sub.g as a sharp transition with an onset as a reversing event at 48 C.1 C. for the milled dried powder for three independent tray dried manufacturing batches. vi. The dissolution time for the single dose pentavalent rotavirus vaccine blended powder (130 mg) in 2.0 mL of reconstitution buffer maintained at 23.0 C.1.0 C. is observed to be 605.0 seconds with manual gentle shaking of vial. At buffer temperatures maintained at 2 C. to 8 C., it took almost 2 minutes to dissolve the pentavalent rotavirus vaccine completely with gentle manual shaking.
Study 6: Longitudinal Stability Studies and Reproducibility Studies for Milled and Blended Freeze Dried Lead Formulation.

(92) The milled and blended freeze dried exemplified formulation 1004c was aliquoted into type-I glass vials and stored at 37 C.2 C. (RH 75%5%) and 45 C.2 C. (RH 75% 35 5%). At the time of sampling, the freeze dried vaccine is reconstituted with the reconstitution buffer followed by potency analysis. Process loss is calculated as a difference of potency between samples before and after freeze drying in three independent manufacturing batches. Stability loss is calculated by linear regression of potency data of samples incubated at this storage condition for at least 26 weeks. i. Exemplified formulation 1004c exhibited cumulative potency losses that are reproducibly statistically equal to 0.0 even after 26 weeks at 2 C. to 8 C. storage conditions as shown in FIG. 4. ii. Exemplified formulation 1004c exhibited cumulative potency losses that are significantly and reproducibly lower than 0.5 log even after 26 weeks at 37 C.2 C. (RH 75%5.0%) storage conditions as shown in FIG. 5. iii. In case of 45 C.2 C. (RH 75%5%) storage conditions, the potency values reproducibly reached equal to 0.5 log loss; for three independent manufacturing batches within 18 weeks as shown in FIG. 6.
Study 7: Post Reconstitution Stability Studies for Lead Formulation 1004c

(93) The freeze dried pentavalent rotavirus vaccine is reconstituted with the reconstitution buffer. Aliquots of 200 L each are dispensed into eppendorfs tubes with 1.5 mL capacity and are incubated at 2 C.-8 C., 25 C., 37 C. and 70 C. storage conditions in the incubators. The samples are collected at 0 h, 8 h, 11 h, 35 h and 48 h time intervals and are evaluated for the potency. The potency of rotavirus remaining at above conditions is determined by comparing the IU/dose of the freeze dried vaccine incubated under above mentioned conditions with samples that are incubated at 70 C.

(94) The data obtained from these studies show potency is retained for 8-11 hours 25 C. for all the five strains as shown in Table 14. At 2 C. to 8 C. and 70 C. storage conditions, the reduction in the potencies is negligible.

(95) TABLE-US-00014 TABLE 14 Log.sub.10 potency values of five rotavirus serotypes when reconstituted with reconstitution buffer and incubated under different temperatures Rotavirus strains G1 G2 G3 G4 P1 Time 25 C. 2-8 C. 70 C. 25 C. 2-8 C. 70 C. 25 C. 2-8 C. 70 C. 25 C. 2-8 C. 70 C. 25 C. 2-8 C. 70 C. 0 h 6.85 6.85 6.85 6.95 6.95 6.95 7.02 7.02 7.02 6.73 6.73 6.73 6.76 6.76 6.76 8 h 6.80 6.80 6.90 7.02 7.03 7.06 6.99 7.06 7.03 6.72 6.78 6.77 6.72 6.76 6.70 11 h 6.80 6.81 6.85 6.99 7.00 7.03 6.90 6.95 6.99 6.71 6.81 6.76 6.69 6.72 6.71 35 h 6.74 6.81 6.82 6.94 6.95 7.00 6.73 6.84 6.95 6.67 6.64 6.72 6.66 6.68 6.67 48 h 6.74 6.74 6.82 6.92 6.94 6.98 6.64 6.95 7.01 6.56 6.68 6.73 6.66 6.68 6.69