FORM-2
THE PATENTS ACT, 1970
(39 OF 1970)
AND
THE PATENTS RULES, 2003
(As Amended)
COMPLETE SPECIFICATION
(See section 10; rule 13)
"Lyophilized Vaccine"
Serum Institute of India Ltd., a corporation organized and existing under the laws of India, of 212/2, Off Soli Poonawalla Road, Hadapsar, Pune 41] 028 Maharashtra India.
The following specification particularly describes the nature of this invention and the manner in which it is to be performed:

Field of invention:
The invention relates to pharmaceutical compositions , such as vaccines,and methods of making and using such compositions.
Background art
Lyophilization is an approach involved in the processing of some vaccine products, and is essentially a freeze-drying process that, under low pressures, removes water through sublimation, and leaves the product as a dried cake with a small amount of moisture.A critical factor impacting the efficacy of the lyophilization process is the formulation of the vaccine. Typically, a vaccine formulation will contain any or all of the following components: a bulking agent (e.g., a sugar), a stabilizer (e.g., a sugar or a protein), and a buffer. The development of effective and efficient processing methods and formulations is therefore of great importance to the development of clinically useful and commercially successful vaccines, including rabies virus vaccines, as discussed above.
RabAvert, Rabies Vaccine, is a freeze-dried vaccine obtained by growing the fixed-virus strain Flury LEP in primary cultures of chicken fibroblasts. The vaccine is lyophilized after addition of a stabilizer solution which consists of buffered polygeline (processed gelatin) and potassium glutamate.One dose of reconstituted vaccine contains less than 12 mg polygeline (processed bovine gelatin), less than 0.3 mg human serum albumin, 1 mg potassium glutamate and 0.3 mg sodium EDTA.
Lyssavac-HDC, a human diploid cell rabies vaccine, formulation of said vaccine comprises gelatin, L-cysteine and potassium phosphate as stabilizers.

CN 1712068 discloses a lyophilized or liquid rabies vaccine of SNK-CTN virus, wherein formulation comprises human serum albumin and aluminium hydroxide.
US 69884422 provides viral vaccine stabilizer comprising human serum albumin (HSA) lactose (e.g., about 2-10% or 4%) and/or mannitol (e.g., about 2-10% or 5%), and/or histidine (e.g., about 1-20 mM or 10 mM) and/or potassium glutamate (e.g., about 20-80 mM or 50 mM).
Role of Human serum albumin (HSA) for effective inhibition of protein surface adsorption and general stabilization of proteins during lyophilization is known.However previously disclosed USA based lyophilized rabies virus compositions have been ineffective.
The inventors of the instant invention have surprisingly found a lyophilization stabilizer composition for rabies virus,said stabilizer comprises of a mixture of one or more polymers and one or more saccharides.
Summary of the invention
The invention pertains to lyophilized virus formulations obtained by a short lyophilization cycle, in particular rabies vaccine comprising an advantageous and novel stabilizer composition.
The invention provides compositions including rabies virus, one or more stabilizers, one or more crystallizing agent agents, one or more sugars, and one or more buffer components.
DESCRIPTION OF THE DRAWINGS
Fig.l Lyophilization graph

Features of the invention will be better understood through a study of the following detailed description of a specific embodiment of the invention. The scope of the invention is limited only through the claims appended hereto.
Detailed description
The compositions of the instant invention can include rabies virus,Minimum Essential Medium,one or more stabilizers, one or more crystallizing agent agents,one or more sugars, and one or more buffer components.
A preferred embodiment of the instant invention is that the formulation contains Earle's Minimum Essential Medium (MEM),trehalose and HSA . Here Minimum Essential Medium (MEM) can consist of various amino acids, vitamins and salt which can protect antigen during various drastic changes of lyophilization. Here trehalose can act as a lyoprotectant to protect the virus surface antigen during freezing and drying and is can also be a very good bulking agent providing structural integrity to virus surface protein.HSA can be used as a lyoprotector and cryoprotector.
The said stabilizer mixture can be prepared in MEM of concentration ranging from 0.5% to 2%. Preferably the concentration is 0.5%.
In one example, the stabilizer is human serum albumin (HSA) (e.g., non-recombinant human serum albumin (HSA) or recombinant human serum albumin (rHA)) (e.g., about 0.05-2.0% or 1%).
Examples of crystallizing agents that can be included in the compositions of the invention are amino acids (e.g.,

about 2-10% or 4%) and/or mannitol (e.g., about 2-10% or 5%), while examples of buffer components that can be included in the compositions are histidine (e.g., about 1-20 mM or 10 mM) and/or potassium glutamate (e.g., about 20-80 mM or 50 mM) . Further, the pH of the compositions can be, e.g., 6-10, 7-9, 7,5-8.5, or 7.9-8.1.
One aspect of the instant invention is that the amino acid is selected from glycine, alanine, glutamine, arginine, lysine, proline, serine and histidine and pharmaceutically acceptable salts thereof. Preferably the amino acid is glycine.
Another aspect of the instant invention is that the saccharide is selected from dextrose, sucrose, lactose, trehalose, cellobiose, raffinose and isomaltose.Preferably, the saccharide is selected from the group consisting of sucrose, lactose ,maltose and trehalose.
When the formulation comprises maltose and HSA the weight ratio of maltose to HSA is suitably from 1:4 to 4:1, or from 1:2 to 2:1, e.g.1:4, 3:2,2:3,4:1, 2:1, 1:2 or 1:1, and preferably the ratio is 1:1.
When the formulation comprises trehalose and HSA, the weight ratio of trehalose to HSA is suitably from 1:4 to 4:1, or from 1:2 to 2:1, e.g.1:4, 3:2, 2:3, 4:1, 2:1, 1:2 or 1:1, and preferably the weight ratio is 1:5.
When the formulation comprises sucrose and HSA, the weight ratio of sucrose to HSA is suitably from 1:4 to 4:1, or from 1:2 to 2:1, e.g.1:4, 3:2, 2:3, 4:1, 2:1, 1:2 or 1:1, and preferably the weight ratio is 3:1 .
Yet another embodiment of the instant invention is that the
said formulation is prepared by mixing virus harvest and
stabilizer in a ratio ranging from 1:1 to 3:1 and
preferably the ratio is 3:1.

Another embodiment of the invention is a lyophilization cycle for formulation without amino acid, that can comprise of following steps a) product loading at precooled shelf temperature of 8°C b) freezing at -50°C for a duration of 5 to 8 hrs , c) optionally annealing at -20 °C for about 5 to 8 hrs d) a primary drying at a temperature ranging from -39 to -25°C, for about 15 to 20 hrs and c) secondary drying at a temperature ranging from about -15 to 37°C for a duration ranging from 4 to 10 hrs.
A preferable embodiment of the invention is a lyophilization cycle for formulation without amino acid, that can comprise of following steps a) product loading at precooled shelf temperature of 8 °C b) freezing at -50°C for a duration of 7 hrs , c) optionally annealing at -20 °C for about 5 to 8 hrs d) a primary drying at -37°C, for about 20 hrs and c) secondary drying at a temperature of
25°C for a duration of 7 hrs.
Yet another embodiment of the invention is a lyophilization cycle for formulation containing a crystallizing agent , the above cycle comprises an optional annealing step at -20°C for about 5 to 8 hrs
According to the instant invention lyophilization cycle is of short duration resulting in an intact homogenous pellet type cake with less than 1% residual moisture and virus loss(post lyophilization) of less than 0.3 logs.Also reconstitution of the said lyophilized product results in rapid and complete dissolution of a lyophilized mass in less than 90 seconds.
Additionally the said lyophilized virus formulation shows more than 3 months stability at 37°C,more than 6 months stability at 25°C and stability atleast for 2 years at 2-8°C

Yet another embodiment of the present invention is that the duration of said short lyophilization cycle can be less than 35 hrs.
Among the strains of virus which can be lyophilized by the process of the present invention there may be mentioned, in particular, rabies virus and especially the rabies strains of the fixed type such as Pasteur CVS or Pitman Moore or modified type such as Flury HEP, Flury LEP, SAD.
Examples:
Example 1
Preparation of stabilizer stock solution
Stabilizer stock solution (4X) in 2X MEM solution was
prepared by dissolving a custom make MEM powder pack in 5
lit of WFI and filtered with 0.2 µ filter. 50 ml of this MEM solution was taken,ingredient was weighed and dissolved in conical flask. Final volume was made to 100 ml again with
MEM solution. This stock solution was filtered using 0. lµ filter.
Example 2
Formulation preparation
150 ml of concentrated harvest was taken in pre sterilized container and 50 ml of filtered stabilizer stock solution was added. Then this active formulation was terminally filtered using 0.45µ filter.

Formulation ratio of virus: Stabilizer was the potency of harvest was near about 6-8 IU/ml potency was set in final vial. Example 3 Table 1:Lyophilization cycle kept 75: 25. ? IU/ml, so 4- 5
Precooling
Shelf Precooling Yes 8°C

Freezing
Shelf Temp Control Final T °C Ramp Duration (Min) Soak Duration (Min)
Ramp 1 & Soak 1 -50 30 420
Ramp 2 & Soak 2 - - -
Ramp 3 & Soak 3 - - -

Sublimation
Shelf Temp Control Final T °C Ramp Duration Soak Duration (Min)
Ramp 1 & Soak 1 -39 60 720
Ramp 2 & Soak 2 -30 60 300
Ramp 3 & Soak 3 -25 60 180
Ramp 4 & Soak 4 25 240 -
Ramp 5 & Soak 5
Chamber pressure control 50 ubar for 1620 min.

Secondary Drying
Shelf Temp Control Final T °C Ramp
Duration
(Min) Soak Durat (Min) ion
Ramp 1 & Soak 1 25 - 420
Ramp 2 & Soak 2
Chamber pressure control 25 ubar for 420 min.
Example 4
Table 2: Stabilizer study results
Stabilizer Name Stabilizer details In MEM/WFI Moisture Viral Titer (PRL) Viral Titer(POL) Cake
Characteristi
cs
4 % Sucrose Good cake structure
Stab. "A" +
4 %
Mannitol WFI 4.68 % 2.8 2.22 /4.2 Uniform, Intact
4 % Sucrose + 2.76 % Collapsed cake
Stab. XXB" Glycine + 1% HSA MEM 7.25 % 3.24 3.1 Bubbling,
Irregular
Non-uniform
1 % Good cake
Stab. "C" Trehalose + 5 % HSA MEM 1.05 % 3.32 3.04 Uniform,
Intact
Regular


4 % Sucrose + 2.76 % Cake OK
Stab. "D" Glycine +
1% HSA + 1%
Mannitol WFI 5.72 % 1.68 1.47 Non-uniform
Irregular,
Pores, Dried
4 % Sucrose + 2.76 % Collapsed cake
Stab. "E" Glycine + 1% HSA MEM 4.34 % 3.2 3.06 Non-uniform
cake
Bubbling
4 % Lactose + 2.76 % Collapsed cake
Stab. "F" Glycine + 1% HSA MEM 3.76 % Non-uniform Pours &
powdery
3 % Sucrose + 2.76 % Good cake Uniform,
Stab. "G" Glycine + 1% HSA MEM 3.06 % Minute pores observed
3 % Sucrose + 2.76 % Collapse cake Non-uniform
Stab. "H" Glycine + 1% HSA + MEM 4.7 % 1.27 1.10 Pores & Powdery

1 % Mannitol
3 % Sucrose + 1% HSA + Collapse cake
Stab. "I" 2 % Mannitol MEM 5.7 % Shrinked Highly porous
3.5 % Sucrose + Collapse cake
Stab. "J" 1% HSA +
2.5 % Mannitol MEM 5.1 % Non-uniform
Pores & very
dried
4 % Sucrose Collapse cake
Stab. "K" + 1% HSA +
3 %
Mannitol MEM _ - _ Non-uniform Pores & powdery
Very good cake
Stab. "L" 1 % Maltose + 1% HSA MEM 0.8 % 2.3 2.3 Intact, a
dried ring at
bottom is
observed.
2 % Maltose Completely
Stab. "M" + 1% HSA MEM Collapse cake very dried
3 % Maltose
Stab. "N" + 1% HSA MEM Completely Collapse cake

very dried
Stab. "0" 4 % Maltose + 1% HSA MEM - - - Completely
Collapse cake
very dried,
Pores
Stab. "P" 5 % Maltose + 1% HSA MEM - 2.1 1.2 Cake OK
Non-uniform
Shrinked at
the bottom
1% Collapse cake
Stab. "Q" Trehalose + 1% HSA MEM 3.28 % 3.1 2.7 Non-uniform
Pores & very
dried
0.5 % Very good cake
Stab. "R" Trehalose + 1% HSA MEM 1.20 % 6.5 6.25 structure Uniform,
Intact, Pores
All the Stabilizers are prepared in 4X concentration. Moisture was determined by coulorimetric method
'-'cake is completely collapsed and moisture level was not
detectable.
We i found that t :he formi ulation c omprisi ng 0.5 % Tren alose,

1% Human Serum Albumin (HSA) ,prepared in 0.5% 2X MEM solution was optimal for efficient lyophilization.Here titer loss (post lyophilization) was found to be less than 0.3 logs by ELISA and moisture content less than 1.3%.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative examples and that the present invention may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are/therefore intended to be embraced therein.

We claim,
1. A lyophilized vaccine composition comprising rabies
virus,Earle's minimum essential medium, human serum albumin
and one or more saccharides.
2. The composition of claim 1, wherein the concentration of
Earle's minimum essential medium is between 0.5% to 2%.
3. The composition of claim 1, wherein the human serum albumin is non-recombinant human serum albumin(HSA)or recombinant human serum albumin (rHSA)
4. The composition of claim 1, wherein saccharide is selected from a group consisting of maltose, sucrose, trehalose, dextrose, lactose,raffinose, mannose and xylose
5. The composition of claim 1, wherein said composition
can further comprise of bulking agent ,preferably mannitol.
6. A composition according to claim 1, wherein the said composition can further comprise of amino acids selected from a group of glycine, arginine,lysine,aspartic acid and glutamic acid .
7. The composition according to claim 1, wherein said

composition is selected from a group consisting of :
i)rabies virus, HSA and maltose
ii)rabies virus, HSA and trehalose
iii)rabies virus ,HSA ,sucrose and glycine
8. The composition according to claim 7,wherein the formulation comprises maltose and HSA in the weight ratio between 1:4 to 4:1,preferably 1:1.
9. The composition according to claim 7,wherein the formulation comprises trehalose and HSA in the weight ratio between 1:4 to 4:1,preferably 1:5.
10. The composition according to claim 7,wherein the
formulation comprises sucrose and HSA, the weight ratio of
sucrose to HSA is suitably from 1:4 to 4:1,preferably 3:1.
11. A method of preparing stabilized lyophilized rabies
vaccine formulation according to claim 1, comprising: (a)
providing a virus harvest;(b)preparing a stabilizer stock
solution in Earle's minimum essential medium;(c)add
stabilizer stock solution to concentrated rabies virus
harvest at a ratio ranging from 1:1 to 1:3;and (d)
lyophilizing said vaccine formulation obtained from the
step (c).

12. The method according to claim 11 , wherein said
lyophilization process comprises of a) product loading at
precooled shelf temperature of 8°C b) freezing at -50°C for
a duration of 5 to 8 hrs , c) optionally annealing at -20 °C
for about 5 to 8 hrs d) a primary drying at a temperature
ranging from -3 9 to -25°C, for about 15 to 20 hrs and c) secondary drying at a temperature ranging from about -15 to 37°C for a duration ranging from 4 to 10 hrs.
13. The composition according to claim 12, wherein
lyophilization process comprises of a) product loading at
precooled shelf temperature of 8 °C b) freezing at -50°C
for a duration of 7 hrs , c) optionally annealing at -20 °C
for about 5 to 8 hrs d) a primary drying at -37°C, for
about 20 hrs and c) secondary drying at a temperature of
25°C for a duration of 7 hrs.
14. A lyophilization process of claim 12, wherein the lyophilization cycle duration is in the .range of 15 hrs to about less than 40 hrs.
15. A lyophilization process of claim 14, wherein, the
lyophilization cycle duration is of 35 hrs.
16. A lyophilized product obtained by lyophilization
process of claim 12 or 13.

17. A lyophilized product according to claim 16 , wherein
post lyophilization related virus titer loss is less than
0.3 logs.
18. A lyophilized product according to claim 16 , wherein said moisture content is about 0 to 2%.
19. A lyophilized product according to claim 12 or 13, wherein said product is stable for atleast 2 months of storage at 37°C with a molecular size distribution.
20. A lyophilized product according to claim 12 or 13, wherein said product is stable for atleast 24 months of storage at 2-8°C with a molecular size distribution.
21. A lyophilized product according to claim 16, wherein said product is stable for atleast 6 months of storage at 25°C
22. A lyophilized product obtained by lyophilization
process of claim 12 or 13,wherein reconstitution results in
rapid and complete dissolution of a lyophilized mass in
less than 90 seconds.
23. The composition of claim 1, wherein the said rabies

virus strain is selected from a group consisting of the fixed type such as Pasteur CVS or Pitman Moore or modified type such as Flury HEP, Flury LEP, SAD.