PURIFICATION OF FLAVIVIRUSES
The present invention provides a method to prepare purified enveloped (e.g., flavivirus) viral particle preparations employing ion exchange chromatography and tangential flow filtration.
1 . A method for the purification of a flavivirus viral particle from a host cell culture comprising the steps of:
a. recovering flavivirus viral particles from a host cell culture;
b. subjecting the flavivirus viral particles obtained from step (a) to tangential flow filtration;
c. applying the retentate from the tangential flow filtration step to an anion exchange chromatography resin;
d. eluting flavivirus viral particles from the anion exchange chromatography resin; and
e. recovering purified flavivirus viral particles.
2 . The method of claim 1 wherein the flavivirus viral particles are recombinant flavivirus viral particles.
3 . The method of claim 2 wherein the flavivirus viral particles comprise replication defective pseudoinfectious virus particles or chimeric flaviviruses comprising capsid and non-structural sequences of a yellow fever virus and pre-membrane and envelope sequences of a second, different flavivirus.
4 . (canceled)
5 . The method of claim 1 wherein the flavivirus viral particles comprise flaviviruses transmitted via an arthropod vector.
6 . The method of claim 5 wherein the flaviviruses are selected from the group consisting of yellow fever viruses, Japanese encephalitis viruses, dengue viruses, West Nile viruses, and tick-borne encephalitis viruses.
7 . (canceled)
8 . The method of claim 6 wherein the yellow fever viruses comprise YF-17D virus.
9 . The method of claim 1 wherein the host cell culture comprises mammalian cell culture.
10 . The method of claim 1 wherein the tangential flow filtration comprises filtration through one or more hollow fiber TFF cassettes or through a flat sheet media.
11 . The method of claim 10 wherein the one or more hollow fiber TFF cassettes comprises a media selected from the following polysulfone, polyethersulfone, modified polyethersulfone or mixed cellulose ester medias.
12 - 13 . (canceled)
14 . The method of claim 1 wherein the anion exchange chromatography resin comprises immobilized positively charged moieties or one or more anionic exchange monolithic columns.
15 . The method of claim 14 wherein the immobilized positively charged moieties comprise quaternary amino groups or diethylaminoethane groups.
16 - 17 . (canceled)
18 . The method of claim 14 wherein the one or more anionic exchange monolithic columns comprises low-shear convective interaction media with a quaternary amine functional group.
19 . The method of claim 1 wherein the recovering of flavivirus viral particles from the host cells comprises decanting medium from the host cell culture.
20 . A pharmaceutically acceptable dosage form of flavivirus viral particles produced in a host cell culture, said flavivirus viral particles isolated by a method comprising the steps of:
a. recovering flavivirus viral particles from a host cell culture;
b. subjecting the flavivirus viral particles solution obtained from step (a) to tangential flow filtration;
c. applying the retentate from the tangential flow filtration step to an anion exchange chromatography resin;
d. eluting flavivirus viral particles from the anion exchange column;
e. recovering purified flavivirus viral particles; and
f. suspending purified flavivirus viral particles in a pharmaceutically acceptable carrier.
21 . The pharmaceutically acceptable dosage form of claim 20 wherein the flavivirus viral particles are recombinant flavivirus viral particles.
22 . The pharmaceutically acceptable dosage form of claim 21 wherein the flavivirus viral particles comprise replication defective pseudoinfectious virus particles or chimeric flaviviruses comprising capsid and non-structural sequences of a yellow fever virus and pre-membrane and envelope sequences of a second, different flavivirus.
23 . (canceled)
24 . The pharmaceutically acceptable dosage form of claim 21 wherein the flavivirus viral particle are flaviviruses transmitted via an arthropod vector.
25 . The pharmaceutically acceptable dosage form of claim 24 wherein the flaviviruses are selected from the group consisting of yellow fever viruses, Japanese encephalitis viruses, dengue viruses, West Nile viruses, and tick-borne encephalitis viruses.
26 . (canceled)
27 . The pharmaceutically acceptable dosage form of claim 25 wherein the yellow fever viruses comprise YF-17D virus.
28 . The pharmaceutically acceptable dosage form of claim 20 wherein the host cell culture comprises mammalian cell culture.
29 . The pharmaceutically acceptable dosage form of claim 20 wherein the tangential flow filtration comprises filtration through one or more hollow fiber TFF cassettes or through a flat sheet media.
30 . The pharmaceutically acceptable dosage form of claim 29 wherein the one or more hollow fiber TFF cassettes comprises a media selected from the following polysulfone, polyethersulfone, modified polyethersulfone or mixed cellulose ester medias.
31 - 32 . (canceled)
33 . The pharmaceutically acceptable dosage form of claim 20 wherein the anion exchange chromatography resin comprises immobilized positively charged moieties or one or more anionic exchange monolithic columns.
34 . The pharmaceutically acceptable dosage form of claim 33 wherein the immobilized positively charged moieties comprise quaternary amino groups or diethylaminoethane groups.
35 - 36 . (canceled)
37 . The pharmaceutically acceptable dosage form of claim 33 wherein the one or more anionic exchange monolithic columns comprises low-shear convective interaction media with a quaternary amine functional group.
38 . The pharmaceutically acceptable dosage form of claim 20 wherein the quantity of host cell DNA in said composition is less than 10 ng host cell DNA per dose, one dose being defined as 500 μL of 2E+7 PFU/mL.