COMMERCIAL SCALE PROCESS FOR PRODUCTION OF PRRSV

• US 20120213741A1
• Filed: 02/14/2012
• Published: 08/23/2012
• Est. Priority Date: 02/17/2011
• Status: Active Grant

• Current Assignee: Boehringer Ingelheim Vetmedica GmbH (C.H. Boehringer Sohn AG & Co. KG)
• Original Assignee: Boehringer Ingelheim Vetmedica GmbH (C.H. Boehringer Sohn AG & Co. KG)
• Inventors: KROLL, Jeremy, BERRY, Elizabeth Jane, HADDADIN, Fuad Tawfiq, KHAZRAEINAZMPOUR, Ali, MALBURG, Sonia Regina Cantisano, SANDOVAL BASURTO, Edgar Arnulfo, SCHEERER, Stephen
• Granted Patent: US 9,944,902 B2
• Time in Patent Office: Days
• US Class Current: 424/93.6
• CPC Class Codes: A61K 2039/552 Veterinary vaccine
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/14 for RNA viruses
• CPC Class Codes: C12N 2770/10021 Viruses as such, e.g. new isolates, mutants or their genomic sequences
• CPC Class Codes: C12N 2770/10034 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2770/10051 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

Abstract

The present invention describes an efficient commercial scale production method for the preparation of PRRS virus.

Claims

1. A method for the commercial scale production of porcine reproductive and respiratory syndrome virus (PRRSV) comprising:

a) concurrently seeding a large scale culture media with a mammalian cell line that is permissive to PRRSV infection into a bioreactor and infecting said mammalian cells with a PRRSV.

b) propagating virus for 5 to 7 days post infection;

c) performing a first harvesting step by removing the media from said bioreactor and isolating propogated virus therefrom;

d) replenishing the media in said bioreactor and propagating virus for 1 to 4 days;

e) performing a second harvesting step by removing the media from said bioreactor and isolating propogated virus therefrom;

f) replenishing the media in said bioreactor and propagating virus for 1 to 4 days and

g) performing a third harvesting step by removing the media from said bioreactor and isolating propogated virus therefrom.

2. The method of claim 1, further comprising at least one re-feeding and harvest steps subsequent to the third harvesting step comprising replenishing the media in said bioreactor and propagating virus for 1 to 4 days and performing a fourth harvesting step by removing the media from said bioreactor and isolating propogated virus therefrom

3. The method of claim 1, wherein the target multiplicity of infection (MOI) is 0.01 to 0.30 and the cell planting density.

4. The method of claim 1, wherein said mammalian cells are planted at a density of about 7×

10⁸ to 1.0×

10⁹ per 300 L bioreactor.

5. The method of claim 4, wherein said cell planting density is about 1.0×

10⁹ per 300 L bioreactor.

6. The method of claim 5, wherein said MOI is about 7×

10⁸ virus particles.

7. The method of claim 1, comprising monitoring the dextrose concentration of said media wherein said first harvest step is performed on the first day when the dextrose concentration of the media decreases to less than 0.1 g/L.

8. The method of claim 1, wherein said second harvest is performed 1 or 2 days post-refeeding with media.

9. The method of claim 1, wherein said third harvest is performed 1 to 4 days post the second refeeding with media.

10. The method of claim 1, wherein the culture media is added to the bioreactor on the day prior to or on the same day prior to addition of said mammalian cell line and said PRRS.

11. The method of claim 1, wherein the culture media is added to the bioreactor one day prior to the addition of said mammalian cell line and said PRRS.

12. The method of claim 1, wherein the temperature of said bioreactor is set at between 34°

C. and 38°

C.

13. The method of claim 1, wherein said media comprises 5% v/v irradiated fetal calf serum.

14. The method of claim 1, wherein said PRRSV is selected from the group consisting of PRRSV strain 94881, VR 2332, Lelystad virus strain (Lelystad Agent (CDI-NL-2.91), or other strains such as those deposited under the Accession Numbers ECACC 04102703, ECACC 04102702, ECACC 04102704, CNCM Accession No. I-1140, CNCM Accession No I-1387, CNCM Accession No I-1388, ATCC VR 2332, VR 2385, VR 2386, VR 2429, VR 2474, and VR 2402;

CNCM I-1102, CNCM I-1140, CNCM I-1387, CNCM I-1388, or ECACC V93070108;

ATCC deposit VR-2332, ATCC deposit VR-2368;

ATCC VR-2495;

ATCC VR 2385, ATCC VR 2386, ATCC VR 2429, ATCC VR 2474, and ATCC VR 2402.

16. A PRRSV MLV comprising a PRRSV produced according to the method of any of claim 1 formulated with an acceptable adjuvant or carrier for delivery to a pig.

15. A commercial-scale production method for the preparation of a PRRSV comprising:

a. a concurrent seeding of both mammalian cells permissive to infection by said PRRSV and said PRRSV into a bioreactor containing a media suitable for the growth of said cells; and

b. performing three consecutive harvesting steps wherein PRRSV is harvested wherein after each of the first and second harvests, the media is replenished, and wherein;

i. the first harvest is performed on the first day that the dextrose concentration of the media decreases to less than 0.1 g/L;

ii. the second harvest is performed 1 or 2 days after addition of the media following the first harvest; and

iii. the third harvest is performed between 1 and 4 days after addition of the media following the second harvest.

18. A PRRSV MLV comprising a PRRSV produced according to the method of any of claim 15 formulated with an acceptable adjuvant or carrier for delivery to a pig.

17. A method for the commercial scale production of porcine reproductive and respiratory syndrome virus (PRRSV) comprising:

a) concurrently seeding a large scale culture media with a mammalian cell line that is permissive to PRRSV infection into a roller bottles and infecting said mammalian cells with a PRRSV.

b) propagating virus for 5 to 7 days post infection;

c) performing a first harvesting step by removing the media from said roller bottle and isolating propogated virus therefrom;

d) replenishing the media in said roller bottle and propagating virus for about 2 days;

e) performing a second harvesting step by removing the media from said roller bottle and isolating propogated virus therefrom;

f) replenishing the media in said roller bottle and propagating virus for about 2 days and

g) performing a third harvesting step by removing the media from said bioreactor and isolating propogated virus therefrom.