METHOD OF PURIFYING A COMPOSITION COMPRISING A GROUP B ADENOVIRUS
A method of purifying a composition comprising a group B adenovirus, for example comprising a purification step of: subjecting a composition comprising said group B adenovirus to diafiltration employing a diafiltration-buffer with a conductivity of at least 180 mScm −1 , for example a conductivity of 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, or 290 mScm −1 . Also provided is a composition obtained using the purification method disclosed herein.
1 .- 23 . (canceled)
24 . A method for purifying a replication competent group B adenovirus from host cell proteins, comprising a purification step of:
subjecting a composition comprising said group B adenovirus to diafiltration employing a diafiltration-buffer with a high salt concentration, wherein said salt concentration is at least 2 M.
25 . The method according to claim 24 , wherein the diafiltration buffer has a conductivity of at least 180 mScm −1 .
26 . The method according to claim 24 , wherein the buffer comprises a salt selected from a chloride salt, a sulfate salt, and any salt fully soluble and dissociated in water combinations thereof.
27 . The method according to claim 24 , wherein the salt in the diafiltration-buffer comprises one or more of the following: an alkaline earth metal salt, sodium acetate, Tris, Bis-Tris, NaH 2 PO 4 , NaCl or KCl.
28 . The method according to claim 24 , wherein the diafiltration-buffer is selected from: meglumine buffer, Gly-NaCl buffer, TRIS buffer.
29 . The method according to claim 28 , wherein the diafiltration-buffer comprises HEPES.
30 . The method according to claim 24 , wherein the diafiltration-filtration buffer is at a pH in the range 7 to 9.8.
31 . The method according to claim 24 , wherein the diafiltration employs an ultrafiltration membrane at least 300 KDa or greater.
32 . The method according to claim 24 , wherein the diafiltration has a flow rate of 1 to 3 m 2 /s.
33 . The method according to claim 24 , wherein the diafiltration is carried out employing a hollow fibre cartridge or flat membrane cassette filter.
34 . The method according to claim 33 , wherein the TFF is performed using a consistent volume method.
35 . The method according to claim 24 , wherein the diafiltration is performed using at least 8 diavolumes of high salt diafiltration-buffer.
36 . The method according to claim 24 , wherein the diafiltration process comprises two steps.
37 . The method of claim 36 , wherein a first step of the process is diafiltration with the high conductivity diafiltration-buffer.
38 . The method according to claim 36 , wherein a second step of the process is diafiltration with the final formulation buffer.
39 . The method according to claim 24 , wherein only one diafiltration buffer is employed.
40 . The method according to claim 24 , comprising a further purification step comprising subjecting the composition of adenovirus to a chromatography purification.
41 . The method according to claim 40 , wherein the chromatography step employs ion-exchange chromatography.
42 . The method according to claim 24 , wherein the adenovirus purification steps do not employ chromatography.
43 . The method according to claim 24 , wherein the crude cell lysate after addition of an endonuclease is filtered to clarify the adenovirus composition.
44 . The method according to claim 43 wherein a second filter is employed in the clarification.
45 . The method according to claim 24 , which comprises a filtration step comprising passing the adenovirus composition through a 0.2 μm filter.