We claim:
1. A process for purification of influenza virus from allantoic fluid, comprising a step of chromatography using strong anion exchanger quaternary ammonia bound to regenerated cellulose membrane.
2. The process as claimed in claim 1, wherein the process comprises the use of Sartobind Q single step chromatography.
3. The process as claimed in claim 1, wherein the allantoic fluid is subjected to inactivation by an inactivating agent, prior to the step of chromatography.
4. The process as claimed in claim 1, wherein the process comprises inactivation step by an inactivating agent, after the step of chromatography.
5. The process as claimed in claim 3 and 4, wherein the inactivating agent is formalin.
6. The process as claimed in claim 5, wherein the concentration of formalin used is 0.005-0.1%.
7. The process as claimed in claim 6, wherein the concentration of formalin used is 0.01 -0.05%.
8. The process as claimed in claim 7, wherein the concentration of formalin used is 0.02%.
9. The process as claimed in claim 1, wherein the conductivity of the allantoic fluid used for chromatography is not more than 22 mS/ cm.
10. A process according to claim 1, wherein the virus preparation is further treated with a splitting agent by passing through hydrophobic interaction chromatography column.
11. The process as claimed in claim 10, wherein the splitting agent is Triton X- 100.
12. The process as claimed in claim 11, wherein the concentration of Triton X- 100 used is 0.1-1.0%.
13. The process as claimed in claim 12, wherein the concentration of Triton X- 100 used is 0.5%.
14. The process as claimed in claim 10, wherein the virus preparation to be treated with splitting agent has a conductivity not more than about 3 mS/ cm
15. The process as used in claim 10, wherein the hydrophobic interaction chromatography column used is the Toyopearl Phenyl 600 M (HIC) column.
16. A process for purification of influenza virus comprising the steps of
a. propagating candidate influenza virus strains, in embryonated hens' eggs
b. providing a harvested mixture of cultured influenza virus in allantoic fluid
c. optionally adding a salt to the allantoic fluid
d. optionally carrying out one or more clarification steps or filtration steps to
separate whole virus from non-virus material
e. optionally carrying out one or more concentration steps
f. carrying out column chromatography using strong anion exchanger having
quaternary ammonia bound to regenerated cellulose membrane.
g. splitting of the whole virus using a suitable splitting agent
h. carrying out hydrophobic interaction chromatography
i. optionally carrying out one or more further purification and/or concentration steps
17. The process as claimed in claims 1 and 16, for purification of influenza virus, to provide highly pure viral antigen, for use in the preparation of a vaccine formulation for inducing immunological response in a subject, by administering immunologically active amount of the antigen.
18. A process for preparing influenza virus vaccine formulation comprising the steps of
a. propagating candidate influenza virus strains, in embryonated hens' eggs,
b. providing a harvested mixture of cultured influenza virus in allantoic fluid,
c. optionally adding a salt to the allantoic fluid,
d. optionally carrying out one or more clarification steps or filtration steps to
separate whole virus from non-virus material,
e. optionally carrying out one or more concentration steps,
f. carrying out column chromatography using strong anion exchanger having
quaternary ammonia bound to regenerated cellulose membrane,
g. splitting of the whole virus using a suitable splitting agent,
h. carrying out hydrophobic interaction chromatography,
i. optionally carrying out one or more further purification and /or concentration
steps, j. providing a pure fully liquid low dose antigenic component, k. providing a fully liquid oil in water emulsion component, 1. optionally providing other carriers, excipients, adjuvants and diluents
wherein, the components of steps j) to 1) are mixed during manufacture and formulated in a single unit.
19. The process as claimed in claim 18, for purification of influenza virus, to provide
highly pure viral antigen, for use in the preparation of a vaccine formulation for inducing
immunological response in a subject, by administering immunologically active amount of
the antigen.
20. A fully liquid influenza vaccine formulation comprising
a. one or more highly pure, low dose split influenza virus antigen component(s)
b. an emulsion comprising squalene
c. Sodium chloride
d. Potassium chloride
e. Potassium dihydrogen phosphate
f. Disodium hydrogen phosphate
g. Magnesium chloride
h. Thiomersal
wherein, the dose of haemagglutinin (HA) is less than 8 micrograms or less than 4 micrograms and wherein components a) to h) are mixed during manufacture and formulated in a single unit.
21. The vaccine formulation as claimed in claim 20, for inducing immunological response in a subject, by administering immunologically active amount of the antigen.