Zika virus purification

Patent metadata

Field	Value
Publication number	US-11524064-B2
Application number	US-202016927086-A
Country	US
Kind code	B2
Filing date	Jul 13, 2020
Priority date	Dec 23, 2015
Publication date	Dec 13, 2022
Grant date	Dec 13, 2022

Abstract

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

First claim

What is claimed is:

1. A process for purifying infectious Zika virus particles, wherein said process comprises the steps of:
(i) providing a crude harvest (a) comprising infectious Zika virus particles, non-infectious Zika virus particles, and impurities, wherein the impurities are generated from growing Zika virus particles on a cell substrate;
(ii) contacting said crude harvest (a) with an agent comprising protamine to obtain a Zika virus preparation (b) comprising infectious Zika virus particles, wherein said agent facilitates separation of infectious Zika virus particles from non-infectious Zika virus particles; and
(iii) further purifying said Zika virus preparation (b) by sucrose density gradient centrifugation;
to obtain a final Zika virus preparation (c) comprising infectious Zika 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 Zika virus particles.

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

3. The process according to claim 1 , wherein the final Zika virus preparation (c) comprises less than 10 ng/mL residual host cell DNA, less than 100 ng/mL residual host cell protein, and less than 100 ng/mL residual aggregates of Zika virus particles.

4. The process according to claim 1 , wherein said crude harvest (a) is subjected to a concentration step prior to step (ii), wherein said concentration step comprises ultra/diafiltration using a hollow-fiber membrane having a pore size equal to or greater than 100 kDa.

5. The process according to claim 1 , wherein said agent comprising protamine comprises a protamine salt.

6. The process according to claim 5 , wherein said protamine salt is protamine sulphate.

7. The process according to claim 6 , wherein the concentration of protamine sulphate is between 0.5 and 3 mg/mL.

8. The process according to claim 7 , wherein the concentration of protamine sulphate is 2 mg/mL.

9. The process according to claim 1 , wherein the infectious Zika virus particles in said final Zika virus preparation (c) are enriched by at least 50% to 95% relative to total Zika virus particles in said crude harvest (a).

10. The process according to claim 1 , wherein said final Zika virus preparation (c) comprises less than 10% residual impurities relative to impurities in said crude harvest (a).

11. The process according to claim 1 , wherein said cell line is a Vero cell line.

12. The process according to claim 1 , wherein said Zika virus is a Zika virus strain of the Asian lineage or an immunogenic variant thereof.

13. The process according to claim 12 , wherein said Zika virus strain of the Asian lineage is H/PF/2013 comprising an RNA genome corresponding to the DNA sequence provided by SEQ ID NOs: 13 or 72.

14. The process according to claim 1 , wherein said process resulting in final Zika virus preparation (c) is followed by an inactivation step, wherein the Zika virus is inactivated with formaldehyde.

15. The process according to claim 14 , wherein said formaldehyde inactivation comprises contacting final Zika virus preparation (c) with 0.02% (w/v) formaldehyde at 22° C. for 10 days, followed by neutralization with sodium-metabisulphite.

16. The process according to claim 1 , for manufacturing a composition for immunization against a Zika virus infection.

17. The process according to claim 1 , wherein said sucrose gradient centrifugation is an optimized sucrose gradient centrifugation, comprising a Zika virus fraction provided in a 10%±1% (w/w) sucrose solution and three further layers of sucrose solutions with different densities.

18. The process according to claim 17 , 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.

Assignees

• Valneva Austria Gmbh

Inventors

• Barbero Calzado Jana
• Nebenführ Mario
• Schiegl Robert
• Weber Michael
• Heindl-Wruss Jürgen

Classifications

• A61K2039/5252

  inactivated (killed) · CPC title

• C12N2770/24151

  Methods of production or purification of viral material · CPC title

• A61K2039/55505

  Inorganic adjuvants · CPC title

• C07K14/1825

  Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue · CPC title

• A61K39/12 Primary

  Viral antigens · CPC title