ZIKA VIRUS PURIFICATION

• US 20180362936A1
• Filed: 12/23/2016
• Published: 12/20/2018
• Est. Priority Date: 12/23/2015
• Status: Active Grant

• Current Assignee: Valneva Austria Gmbh (Valneva SE)
• Original Assignee: Valneva Austria Gmbh (Valneva SE)
• Inventors: Barbero Calzado, Jana, Nebenfuhr, Mario, Schlegl, Robert, Weber, Michael, Wruss, Jurgen
• Granted Patent: US 10,744,194 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2039/5252 inactivated (killed)
• CPC Class Codes: A61K 2039/5254 avirulent or attenuated
• CPC Class Codes: A61K 2039/5258 Virus-like particles
• CPC Class Codes: A61K 2039/55505 Inorganic adjuvants
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: A61K 39/39 characterised by the immunostimulating additives, e.g. chemical adjuvants
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: C07K 14/18 Togaviridae; Flaviviridae
• CPC Class Codes: C07K 14/1825 Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
• CPC Class Codes: C12N 2770/24134 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2770/24151 Methods of production or purification of viral material
• CPC Class Codes: C12N 2770/24163 by chemical treatment
• CPC Class Codes: C12N 2770/36151 Methods of production or purification of viral material
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12N 7/02 Recovery or purification
• CPC Class Codes: C12N 7/06 Inactivation or attenuation by chemical treatment
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Described herein are processes for purifying infectious Zika virus particles and uses of protamine in such processes.

Claims

1. Use of protamine, preferably a protamine salt, to separate infectious Zika virus particles from non-infectious Zika virus particles.

2. The use according to claim 1, wherein the protamine salt also facilitates the separation of infectious Zika virus particles from host cell proteins and/or low molecular weight materials.

3. A process of purification of infectious Zika virus particles, comprising the steps of:

a) providing a crude harvest (a) comprising Zika virus particles and impurities, wherein the impurities are generated from growing said Zika virus particles on a cell substrate;

b) reducing impurities from said crude harvest (a) by precipitation with an agent comprising protamine, preferably a protamine salt, more preferably a protamine sulphate, even more preferably a recombinant protamine sulphate, to obtain a Zika virus preparation (b), wherein the enrichment of infectious Zika virus particles in the Zika virus preparation (b) relative to total Zika virus products in the crude harvest (a) is in the range of at least 50% to 95%, preferably at least 80%.

6. The process of any of claims 3 to 5, wherein the crude harvest (a) comprising Zika virus particles and impurities is subjected to one or more pre-purification step(s) prior to step (b).

7. The process of claim 6, wherein the one or more pre-purification step(s) comprises

a) filtration using a filter having a pore size equal to or less than 0.2 μ

m; 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 300 kDa, preferably equal to or greater than 100 kDa.

8. The process of any one of claims 3 to 7, wherein the concentration of protamine sulphate is 0.5 to 3 mg/ml, more preferably 1 to 2 mg/ml, more preferably 1.2 to 1.8 mg/ml, more preferably 1.4 to 1.6 mg/ml, most preferably 2.0 mg/ml.

9. The process of any one of claims 3 to 8, wherein the enrichment of infectious Zika virus particles in the Zika virus preparation (c) or any final Zika virus preparation relative to total Zika virus products in the crude harvest (a) is in the range from at least 50% to 95%, preferably at least 80%.

10. The process of any one of claims 6 to 9, wherein the one or more pre-purification step(s) prior to step (b) of any of claims 6 to 9 is performed using a filter having a pore size equal to or less than 1 nm, preferably 0.2 μ

m.

11. The process of any one of claims 3 to 10, wherein the residual impurity of the Zika virus preparation (c) is less than 10%.

12. The process of any one of claims 3 to 11, wherein the Zika virus is propagated in a cell line selected from the group consisting of an EB66 cell line, a Vero cell line, a Vero-α

His 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, a MDSK cell line, a chicken embryonic fibroblast cell line, a duck cell line, and a diploid avian cell line.

13. The process of claim 12, wherein said cell line is a Vero cell line.

14. The process of any one of claims 3 to 13, wherein said Zika virus is a live virus, an attenuated live virus, a chimeric virus, a modified live virus, or a recombinant live virus.

15. The process of any one of claims 3 to 14, wherein said Zika virus is preferably a Zika virus strain of the Asian lineage or an immunogenic variant thereof.

16. The process of any one of claims 3 to 15, wherein said process resulting in final Zika virus preparation (c) is followed by an inactivation step, wherein the Zika virus is inactivated preferably by formaldehyde.

17. A composition comprising the Zika virus particles obtainable or obtained by the process of any one of claims 3 to 16, wherein the composition contains protamine at levels below detection in size exclusion chromatography.

18. The composition according to claim 17, wherein the composition contains trace amounts of protamine or fragments thereof detectable by mass spectroscopy or other sensitive methods.

19. The composition according to claim 17 or 18 for treating and/or protecting from a Zika virus infection.

20. Use of the process according to any one of claims 3 to 16 for manufacturing a composition for immunization against a Zika virus infection.

4. A process of purification of infectious Zika virus particles, comprising the steps of:

a) providing a crude harvest (a) comprising Zika virus particles and impurities, wherein the impurities are generated from growing said Zika virus particles on a cell substrate;

b) reducing impurities from said crude harvest (a) by precipitation with an agent comprising protamine, preferably a protamine salt, more preferably a protamine sulphate, even more preferably a recombinant protamine sulphate, to obtain a Zika virus preparation (b);

c) further purifying said Zika virus preparation (b) by one or more size exclusion methods such as (i) a sucrose density gradient centrifugation, (ii) a solid-phase matrix packed in a column comprising a ligand-activated core and an inactive shell comprising pores, wherein the molecular weight cut off of the pores excludes the Zika virus particles from entering the ligand-activated core, and wherein a molecule smaller than the molecular weight cutoff of the pores can enter the ligand-activated core and collecting the virus particles, and/or (iii) size exclusion chromatography to obtain a virus preparation (c) comprising the infectious Zika virus particles, wherein the residual host cell DNA of the virus preparation (c) is less than 100 ng/mL and the residual host cell protein and the residual aggregates of infectious virus particles of the final virus preparation (c) is less than 1 μ

g/mL.

5. The process of claim 4, wherein the residual host cell DNA of the Zika virus preparation (c) is less than 10 ng/mL and the residual host cell protein of the final virus preparation (c) is less than 100 ng/mL.