Virus purification
Described herein are processes for purifying infectious virus particles and uses of protamine in such processes.
1. A method for purifying infectious virus particles, comprising the steps of
(A) providing a crude harvest (a) comprising infectious virus particles, non-infectious virus particles, and impurities, wherein the impurities are generated from growing said virus particles on a cell substrate;
(B) contacting said crude harvest (a) with an agent comprising protamine to obtain a virus preparation (b) comprising infectious virus particles; and
(C) further purifying said virus preparation (b) by one or more size exclusion methods wherein said one or more size exclusion methods comprise
(i) sucrose density gradient centrifugation, and/or
(ii) a solid-phase matrix packed in a column comprising a ligand-activated core and an inactive shell comprising pores, wherein the pores comprise a molecular weight cut-off that excludes the virus particles from entering the ligand-activated core, and wherein a molecule smaller than the molecular weight cut-off of the pores can enter the ligand-activated core, and/or
(iii) size exclusion chromatography;
to obtain a final virus preparation (c) comprising the infectious virus particles, less than 100 ng/mL residual host cell DNA, less than 1 μg/mL residual host cell protein, and less than 1 μg/mL residual aggregates of virus particles are selected from the group consisting of filovirus particles of a flavivirus or alphavirus particles of a alphavirus but;
wherein the virus particles are not of a Japanese encephalitis virus.
2. The method according to claim 1 , wherein the final virus preparation (c) comprises less than 10 ng/mL residual host cell DNA and less than 100 ng/mL residual host cell protein.
3. The method according to claim 1 , wherein said crude harvest (a) is subjected to one or more pre-purification step(s) prior to step (B), wherein the one or more pre-purification step(s) comprise
a) filtration using a filter having a pore size equal to or less than 0.2 μm; and/or
b) digestion of host cell genomic DNA by enzymatic treatment; and/or
c) ultra/diafiltration using a hollow fiber membrane having a pore size equal to or greater than 100 kDa.
4. The method according to claim 1 , wherein the concentration of protamine is between 0.5 mg/ml and 3 mg/ml.
5. The method according to claim 1 , wherein the infectious virus particles in said final virus preparation (c) are enriched by at least 50% to 95% relative to total virus particles in said crude harvest (a).
6. The method according to claim 1 , wherein said virus preparation (c) comprises less than 10% impurities.
7. The method according to claim 1 , wherein said infectious virus particles are propagated in a cell line selected from the group consisting of an EB66 cell line, a Vero cell line, a Vero-αHis cell line, a HeLa cell line, a HeLa-S3 cell line, a 293 cell line, a PC12 cell line, a CHO cell line, a 3T3 cell line, a PerC6 cell line, an MDSK cell line, a chicken embryonic fibroblast cell line, a duck cell line, and a diploid avian cell line.
8. The method according to claim 1 , wherein said infectious virus particles are selected from the group consisting of a live virus, an attenuated live virus, a chimeric virus, a modified live virus and a recombinant live virus.
9. The method according to claim 8 , wherein said infectious virus particles are selected from the group of viruses consisting of Zika virus, Chikungunya virus, yellow fever virus, and Dengue virus.
10. The method according to claim 1 , further comprising a step (D) comprising inactivating the final virus preparation (c).
11. The method according to claim 9 , wherein said Chikungunya virus is an attenuated Chikungunya virus comprising a deletion mutation in the non-structural protein 3 provided by SEQ ID NO: 77 or an immunogenic variant thereof, wherein said immunogenic variant is defined as having at least 80% sequence identity to SEQ ID NO: 77.
12. The method according to claim 1 , wherein said protamine is selected from the group comprising a protamine salt, a protamine sulphate and a recombinant protamine sulphate.
13. The method according to claim 10 , wherein said inactivating step (D) is performed using formaldehyde.
14. The method according to claim 1 , wherein said infectious virus particles are selected from the group consisting of flaviviruses and flaviviruses but not Japanese encephalitis virus.