ZIKA VIRUS PURIFICATION

• US 20190008945A1
• Filed: 12/23/2016
• Published: 01/10/2019
• 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, SchlegI, Robert, Weber, Michael, Wruss, Jurgen
• Granted Patent: US 11,331,382 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 improved purification methods for Zika virus vaccines and compositions. Also described are Zika vaccines and methods of producing and administering said Zika vaccines to subjects in need thereof.

Claims

1. Use of an optimized sucrose gradient centrifugation for removal of protamine sulphate from purified infectious Zika virus particles.

2. The use according to claim 1, wherein said optimized sucrose gradient centrifugation comprises a virus comprising fraction in a 10% (w/w) sucrose solution and three further layers of sucrose solutions with different densities, i.e. a first sucrose solution with 15% (w/w) sucrose, a second sucrose solution with 35% (w/w) sucrose, and a third sucrose solution with a 50% (w/w) sucrose.

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 the 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 virus preparation (b);

c) further purifying the Zika virus preparation (b) by an optimized sucrose density gradient centrifugation, wherein the optimized sucrose gradient is provided such that the protamine can be completely or almost completely separated from the Zika 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, preferably below 0.5 μ

g/mL, more preferably below 0.1 μ

g/mL, most preferably below 0.05 μ

g/mL.

5. The process of claim 3 or 4, additionally comprising a further purification step of:

(d) 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 Zika virus particles.

6. The process of any of claims 3 to 5, wherein the residual host cell DNA content of the Zika virus preparation (c) is less than 10 ng/mL and the residual host cell protein content of the final Zika virus preparation (c) is less than 100 ng/mL.

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

8. The process of claim 7, 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.

9. The process of any one of claims 3 to 8, 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 1.6 mg/ml or 2 mg/ml.

10. The process of any one of claims 3 to 9, wherein the enrichment of infectious Zika 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%, preferably at least 80%.

11. The process of any one of claims 8 to 10, wherein the one or more pre-purification step(s) prior to step (b) of any of claims 8 to 11 is performed using a filter having a pore size equal to or less than 1 μ

m, preferably 0.2 μ

m.

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

13. The process of any one of claims 3 to 12, 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-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, a MDSK cell line, a chicken embryonic fibroblast cell line, a duck cell line, and a diploid avian cell line.

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

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

16. The process of any one of claims 3 to 15, wherein the Zika virus comprises SEQ ID NO:

72

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

18. Use of the process according to any one of claims 3 to 17 for manufacturing a composition for immunization against a Zika virus infection.

19. A composition comprising the Zika virus particles obtainable or obtained by the process of any one of claims 3 to 17 for treating and/or protecting from a Zika virus infection.

4. The process of claim 4, wherein said optimized sucrose density gradient centrifugation comprises a Zika virus comprising fraction in a 10% (w/w) sucrose solution and three layers of sucrose with different densities, i.e. a first sucrose solution with 15% (w/w) sucrose, a second sucrose solution with 35% (w/w) sucrose, and a third sucrose solution with a 50% (w/w) sucrose.

20. A Zika virus vaccine comprising an inactivated Zika virus particle grown on Vero cells, wherein the Zika virus vaccine is able to confer seroprotection on at least 70% of subjects that are administered the Zika virus vaccine and comprises minor amounts of protamine sulphate, preferably below the detection limit when measured with HPLC (below LOD) but still detectable by mass spectroscopy;

i.e., trace amounts of PS and/or fragments thereof.

21. The Zika virus vaccine of claim 20, wherein the Zika virus vaccine is able to confer seroprotection on at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, or at least 99% of subjects that are administered the Zika virus vaccine, preferably on at least 80% of subjects.

22. The vaccine of claim 20 or 21, wherein said Zika virus particle has a RNA genome corresponding to the DNA sequence provided by any one of the nucleic acid sequences of SEQ ID NOs:

2-13, or a variant nucleic acid sequence that is at least 88% identical to any one of SEQ ID NOs;

2-13 and able to pack a virulent Zika virus.

23. The vaccine of any one of claims 20 to 22, wherein said Zika virus particle has an E protein selected from the amino acid sequences provided by any one of SEQ ID NOs:

14-69, or a variant amino acid sequence that is at least 95% identical to any one of SEQ ID NOs;

14-69 and able to pack a virulent Zika virus.

24. The vaccine of any one of claims 20 to 23, wherein said Zika virus obtained by culturing on Vero cells is purified by protamine sulfate precipitation and sucrose gradient centrifugation.

25. The vaccine of any one of claims 20 to 24, further comprising an adjuvant.

26. The vaccine of claim 25, wherein said adjuvant is an aluminum salt adjuvant.

27. The vaccine of claim 26, wherein said aluminum salt adjuvant is aluminium hydroxide or aluminium phosphate salt.

28. The vaccine of claim 26, wherein said aluminum salt adjuvant is aluminium hydroxide with less than 1.25 ppb Cu based on the final pharmaceutical composition comprising the Zika virus.

29. The vaccine of any one of claims 20 to 28, further comprising one or more pharmaceutically acceptable excipient(s).