Methods for purification of viruses
The present invention provides methods for the purification of cell-associated viruses from adherent cells (e.g., MDCK or Vero cells). In particular, the present invention provides purification methods for the production of immunogenic compositions comprising a live attenuated cell-associated virus (e.g., an attenuated respiratory syncytial virus (RSV) or cold-adapted, and/or temperature sensitive influenza virus) that result in levels of host cell DNA (HCD), host cell protein (HCP) and non-specific endonuclease (e.g., Benzonase), which are below the specifications required by regulatory agencies. The immunogenic compositions can be used to actively immunize subjects or to generate antibodies for a variety of uses, including passive immunization and diagnostic immunoassays.
1. A method of purifying respiratory syncytial virus from virus-infected cells, wherein the method comprises:
a) clarifying a viral harvest to form a clarified viral harvest, wherein clarifying comprises:
i. filtering the viral harvest by direct flow filtration (DFF) through at least one clarification filter having a pore size of between about 3 micrometers to about 10 micrometers;
ii. treating the viral harvest with a non-specific endonuclease; and
iii. filtering the viral harvest by DFF through at least one clarification filter having a pore size of between about 0.45 micrometers to about 3 micrometers;
b) stabilizing the clarified viral harvest with a stabilization buffer comprising sucrose, phosphate or Tris with NaCl salt or KCl salt to form a stabilized viral harvest;
c) concentrating the stabilized viral harvest by tangential flow filtration (TFF) and performing buffer exchange by diafiltration (DF) using a hollow fiber cartridge with a molecular weight cut off (MWCO) of 500 kDa to form a concentrated viral harvest; and
d) after (c), sterilizing the concentrated viral harvest by DFF through at least one clarification filter having a pore size of between about 0.8 micrometers to about 0.45 micrometers to form a sterilized viral harvest; whereby the respiratory syncytial virus is purified from the virus-infected cells and has a final viral infectivity titer of at least 5.7 log 10 FFU/mL.
2. The method according to claim 1 , wherein the method further comprises a step of purification by ion-exchange chromatography.
3. The method according to claim 1 , wherein an exchange buffer of step (c) comprises about 10-25% w/v sucrose, about 150 mM to 10 mM potassium phosphate and about 175 mM to 50 mM KCl pH 7.0-7.4.
4. The method according to claim 1 , wherein the respiratory syncytial virus is is rA2cp248/404/1030ASH.
5. The method according to claim 1 , wherein the viral harvest is stabilized with a buffer comprising about 150 mM to 50 mM potassium phosphate, about 250 mM to 175 mM sucrose, and about 175 mM to 125 mM NaCl or KCl, pH 7.0-7.4.
6. The method according to claim 1 , wherein step (c) results in a 5× concentration and least 5 diavolumes of buffer are exchanged.
7. The method according to claim 1 , wherein the non-specific nuclease is exposed to the viral harvest for between about 120 minutes and 210 minutes.
8. The method according to claim 1 , wherein the respiratory syncytial virus is a live respiratory syncytial virus.
9. The method according to claim 1 , wherein the respiratory syncytial virus is a live attenuated respiratory syncytial virus.