Stable, dried rotavirus vaccine, compositions and process for preparation thereof
The present invention provides novel lyophilized rotavirus vaccine formulations and methods of their preparation. The formulations include vaccine stabilizers, resulting in a vaccine formulation with enhanced stability and minimal loss of potency. The rotavirus vaccine formulations comprise an advantageous ratio of a disaccharide (such as sucrose) to an amino acid (such as glycine). The lyophilization results in a virus formulation with 100% virus preservation and residual moisture from about 0.8% to 1.4%.
1. A lyophilized rotavirus vaccine formulation comprising prior to lyophilization:
a) at least one strain of rotavirus at a concentration from 1×10 5 pfu/mL to 1000×10 5 pfu/mL;
b) sucrose at a concentration of 5% (w/v); and
c) glycine at a concentration of 5% (w/v).
2. The composition of claim 1 , further comprising a biologically effective concentration of a cell culture medium.
3. The composition of claim 2 , wherein said cell culture medium is Minimum Essential Medium (MEM).
4. A method of preparing a stabilized rotavirus vaccine formulation according to claim 1 , comprising:
a) Cultivating at least one strain of rotavirus in MEM additionally containing Glutamine and Sodium Bicarbonate;
b) Preparing a stabilizer comprising, sucrose and glycine;
c) Combining said at least one strain of rotavirus at a concentration from 1×10 5 pfu/mL to 1000×10 5 pfu/mL prior to lyophilization and said stabilizer so as to obtain a mixture of said rotavirus and stabilizer wherein the mixture has sucrose at a concentration of 5% (w/v) and glycine at a concentration of 5% (w/v); and
d) Lyophilizing the mixture of stabilizer and rotavirus for 30 hrs to 60 hrs, thereby producing a lyophilized form of vaccine with a residual moisture content from 0.8% to 1.4%.
5. The method of claim 4 , wherein the lyophilization is performed by:
a) Loading a lyophilizer with the mixture of said rotavirus and stabilizer at temperatures between 25 to −50° C.;
b) Stepwise freezing said mixture of rotavirus and stabilizer with a hold at each temperature, wherein freezing is done below −32° C. at a freezing rate ranging from 0.5 to 5° C./min.;
c) Annealing said mixture of rotavirus and stabilizer at a temperature ranging from −15° C. to −25° C. for 5 hrs followed by freezing at −45° C. for 1 hr;
d) Stepwise raising of the temperature during primary drying for 10 to 30 hrs under a pressure of 55 μbar so as to achieve a sublimation at −32° C.; and
e) providing a secondary drying, wherein said mixture of rotavirus and stabilizer is heated at the rate of 0.5 to 1° C./min, at 25° C. to 30° C. for 6-10 hrs under a pressure of 55 μbar.
6. The method of claim 5 , wherein the lyophilization is performed by:
a) Loading a lyophilizer with the mixture of said rotavirus and stabilizer at temperatures between 20 to 5° C.;
b) Stepwise freezing said mixture of rotavirus and stabilizer with a hold at each temperature, wherein freezing is done below −32° C. at a freezing rate ranging from 0.5 to 1° C./min.;
c) Annealing said mixture of rotavirus and stabilizer at a temperature of −20° C. for 5 hrs followed by freezing at −45° C. for 1 hr;
d) Stepwise raising of the temperature during primary drying for at least 20 hours under a pressure of 55 μbar so as to achieve a sublimation at −32° C.; and
e) providing a secondary drying, wherein said mixture of rotavirus and stabilizer is heated at the rate of 0.5 to 1° C./min, at 25° C. for 8 hrs under a pressure of 55 μbar.
7. The method of claim 5 , wherein the lyophilization cycle duration ranges from 30 to 60 hrs.