Method for purifying the rabies virus
The subject of the invention is a method for purifying the rabies virus, comprising a single ion-exchange chromatography step, said step being cation exchange chromatography according to which: a) the supernatant of a culture of cells infected with this virus is brought into contact with a cation exchange chromatography support comprising a polymethacrylate matrix onto which sulfoisobutyl groups have been grafted such that the rabies virus binds to this support, and; b) the virus is eluted from its support.
1. A method of purifying rabies virus, the method comprising a single ion exchange chromatography purification wherein said ion exchange chromatography purification is a cation exchange chromatography purification comprising
a) applying a supernatant of a culture of cells infected with the rabies virus to a cation exchange chromatography support under conditions in which the rabies virus binds to the support, wherein the support comprises a polymethacrylate matrix onto which sulfoisobutyl groups have been grafted by covalent bonding, and
b) eluting the virus from the support.
2. The method as claimed in claim 1 , wherein the culture supernatant of cells infected with the rabies virus is free of animal serum or free of any serum protein.
3. The method as claimed in claim 2 , wherein the culture supernatant of cells infected with the rabies virus is free of any exogenous protein of animal origin.
4. The method as claimed in claim 3 , wherein the culture supernatant of the cells infected with the rabies virus contains exogenous proteins of non-animal origin at a concentration <15 mg/l.
5. The method as claimed in claim 1 , wherein the culture supernatant of cells infected with the rabies virus is free of any exogenous product of animal origin.
6. The method as claimed in claim 1 , wherein the culture supernatant of cells infected with the rabies virus is a clarified supernatant.
7. The method as claimed in claim 1 , wherein the amount of virus measured in the eluate corresponds to at least 70% of the amount of virus measured in the supernatant that was applied to the chromatographic support.
8. The method as claimed in claim 7 , wherein the amount of total proteins measured in the eluate corresponds to less than 40% of the amount of total proteins measured in the supernatant that was applied to the chromatographic support and the amount of DNA measured in the eluate corresponds to less than 5% of the amount of DNA measured in the supernatant that was applied to the chromatographic support.
9. The method as claimed in claim 7 , wherein the amount of total proteins measured in the eluate corresponds to less than 40% of the amount of total proteins measured in the supernatant that was applied to the chromatographic support and the amount of DNA measured in the eluate corresponds to less than 1% of the amount of DNA measured in the supernatant that was applied to the chromatographic support.
10. The method as claimed in claim 1 , wherein, further comprising treating the eluate, optionally concentrated, with a nuclease.
11. The method as claimed in claim 10 , wherein the nuclease is an endonuclease.
12. The method as claimed in claim 10 , wherein the treated eluate is subjected to ultracentrifugation on a sucrose gradient and recovering the fraction(s) of the gradient that contain(s) the purified virus.
13. The method as claimed in claim 12 , further comprising inactivating the purified rabies virus with a viral inactivation agent.
14. The method as claimed in claim 13 , wherein the viral inactivation agent is β-propiolactone.
15. The method as claimed in claim 13 , wherein the purification is conducted with products of non-animal origin only.
16. A method of manufacturing a rabies vaccine, the method comprising:
a) infecting a culture of cells with the rabies virus,
b) purifying the rabies virus from an infected-cell culture supernatant according to the method as claimed in claim 13 ,
c) mixing the suspension obtained in b) with a storage buffer, and
d) dividing the mixture obtained in c) into a single-dose or multidose vaccine(s).
17. A method for the manufacture of a rabies vaccine, the method comprising:
a) infecting a culture of cells with the rabies virus,
b) purifying the rabies virus from an infected-cell culture supernatant according to the method as claimed in claim 13 ,
c) mixing the suspension of purified virus obtained in b) with a lyophilization buffer,
d) dividing the mixture obtained in c) into a single-dose or multidose vaccine(s), and
e) lyophilizing the vaccine dose(s).