Virus purification

• US 10,537,630 B2
• Filed: 12/23/2016
• Issued: 01/21/2020
• Est. Priority Date: 12/23/2015
• Status: Active Grant

• Current Assignee: Valneva SE
• Original Assignee: Valneva SE
• Inventors: Barbero Calzado, Jana, Nebenfuhr, Mario, Schlegl, Robert, Weber, Michael, Heindl-Wruss, Jurgen
• Primary Examiner(s): Li, Bao Q
• Application Number:
  US16/062,245
• Publication Number:
  US 20180369359A1
• Time in Patent Office: 1,124 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 improved purification methods for virus vaccines and compositions. Also described are Zika, Chikungunya, dengue and yellow fever vaccines and methods of producing and administering said vaccines to subjects in need thereof.

Claims

1. A method for removal of protamine from purified infectious virus particles, comprising a step of sucrose gradient centrifugation, wherein the sucrose gradient comprises a virus comprising fraction in a 10%+/−

1% (w/w) sucrose solution and three further layers of sucrose solutions with different densities, wherein the three further layers of sucrose solutions comprise a first sucrose solution with 15%+/−

1% (w/w) sucrose, a second sucrose solution with 35%+/−

1% (w/w) sucrose, and a third sucrose solution with 50%+/−

1% (w/w) sucrose.

2. A method for purification of infectious virus particles, comprising the steps of

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

b) reducing impurities from said crude harvest (a) by precipitation with an agent comprising protamine to obtain a virus preparation (b);

c) further purifying said virus preparation (b) by sucrose density gradient centrifugation to obtain a virus preparation (c), wherein the sucrose gradient is provided such that the protamine can be completely or almost completely separated from the virus fraction; and

wherein the protamine concentration is reduced by this step to the extent that the protamine concentration in the final drug substance is below 1 μ

g/ml, and wherein said sucrose density gradient comprises a virus comprising fraction in a 10%+/−

1% (w/w) sucrose solution and three layers of sucrose with different densities, wherein the three further layers of sucrose solutions comprise a first sucrose solution with 15%+/−

1% (w/w) sucrose, a second sucrose solution with 35%+/−

1% (w/w) sucrose, and a third sucrose solution with 50%+/−

1% (w/w) sucrose.

3. The method according to claim 2, wherein said virus particles are selected from the group consisting of flaviviruses.

4. The method according to claim 2, additionally comprising a further purification step of (d) further purifying said virus preparation (c) on 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 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, to obtain virus preparation (d).

5. The method according to claim 2, wherein said crude harvest (a) comprising virus particles and impurities is subjected to one or more pre-purification step(s) prior to step (b),

wherein said one or more pre-purification step(s) comprise

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 100 kDa.

6. The method according to claim 2, wherein the concentration of protamine is from 0.5 mg/ml to 3 mg/ml.

7. The method according to claim 2, wherein the enrichment of infectious virus particles in the virus preparation (c) or any final virus preparation relative to total virus products in the crude harvest (a) is in the range from at least 50% to 95%.

8. The method according to claim 2, 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-aHis 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.

9. The method according to claim 2, wherein said infectious virus particles are selected from the group consisting of a live virus, a live attenuated virus, a chimeric virus, a modified live virus or a recombinant live virus.

10. The method according to claim 2, wherein said step resulting in final virus preparation (c) or (d) is followed by an inactivation step.

11. The method according to claim 2, wherein said protamine is selected from the group consisting of a protamine salt, a protamine sulphate and a recombinant protamine sulphate.

12. The method according to claim 10, wherein said inactivation step is a formaldehyde inactivation step.