Optimized parvovirus H-1 production

Patent metadata

Field	Value
Publication number	US-12404498-B2
Application number	US-202117542721-A
Country	US
Kind code	B2
Filing date	Dec 6, 2021
Priority date	Jun 17, 2019
Publication date	Sep 2, 2025
Grant date	Sep 2, 2025

Abstract

The present invention provides a robust single clone Master Cell Bank (MCB) for an optimized production of H-1 parvovirus (H-1 PV) which is suitable to increase infectious parvovirus production compared to standard producer NB-324K mixed cells.

First claim

What is claimed is:

1. A MCS NB 324K cell; wherein cells designated MCS NB 324K that have uniform composition and are from a single source have been deposited with the German Collection of Microorganisms and Cell Cultures (DSMZ) under accession number DSM ACC3353.

2. A single clone master cell bank (MCB) for parvovirus H-1 production comprising MCS NB 324K cells of claim 1 .

3. A process for preparing the single clone MCB of claim 2 , comprising:
(a) growing a NB-324K mixed clone cell population in a cell culture medium,
(b) seeding cells from the mixed clone cell population of step (a) in a 96 well plate, 1 cell per well, and growing the cells so seeded for 20-28 days to obtain cell clones,
(c) from the cell clones of step (b), selecting 2 to 5 cell clones that exhibit a single colony per well,
(d) seeding cells from at least two cell clones selected in (c) in a 96 well plate, 1 cell per well, and growing the cells so seeded for 20-23 days to obtain cell clones,
(e) selecting a cell clone from step (d) based on growth and H-1 parvovirus production, and propagating and passaging cells of that clone for between 15 to 41 passages, and
(f) harvesting and storing cells obtained from the 15 to 41 passages of step (e).

4. The process of claim 3 , wherein the cell culture medium of step (a) is a minimum essential medium (MEM) containing fetal bovine serum (FBS), L-Glutamine and Gentamicin.

5. The process of claim 3 , wherein propagating and passaging of step (e) is carried out in T-flask or roller bottles.

6. The process of claim 4 , wherein propagating and passaging of step (e) is carried out in T-flask or roller bottles.

7. A process of preparing a master seed virus (MSV) composition comprising at least two rounds of transfecting the cells of the single clone MCB of claim 2 with sequenced H-1 parvovirus DNA cloned into a plasmid pUC19 in which the Hindlll site is deleted (pUC19ΔHindIII/H1 plasmid DNA), thereby generating the MSV.

8. The process of claim 7 , wherein transfecting comprises calcium phosphate transfection.

9. A method for producing parvovirus H-1 (H-1PV) comprising:
(a) preparing a master seed virus (MSV) composition comprising at least two rounds of transfecting the cells of the single clone MCB of claim 2 with sequenced H-1 parvovirus DNA cloned into a plasmid pUC19 in which the Hindlll site is deleted (pUC19ΔHindIII/H1 plasmid DNA), thereby generating the MSV;
(b) pre-seeding by infecting cells of the single clone MCB of claim 2 with virus of the MSV of step (a) at a cell density from 2.0 to 5.0×10 4 cells/cm 2 and a MOI of 0.5 to 5×10 −2 PFU/cells;
(c) growing the infected cells of step (b) for about 2 to 6 days;
(d) harvesting cells from the step (c) cells, 2 to 6 days post-infection, centrifuging the harvested cells, and obtaining from the centrifuging a cell pellet;
(e) resuspending the cell pellet of step (d) and subjecting the resuspended cell pellet to a mechanical, physical or chemical cell lysis method for obtaining a parvovirus containing cell lysate;
(f) clarifying the parvovirus containing cell lysate of step (e) by filtration;
(g) treating the clarified parvovirus containing cell lysate of step (f) with DNAse to obtain a treated clarified parvovirus containing cell lysate;
(h) performing chromatography preparation comprising buffer exchange on the treated clarified parvovirus containing cell lysate of step (g) to obtain a chromatography-prepared treated clarified parvovirus containing cell lysate;
(i) removing from the chromatography-prepared treated clarified parvovirus containing cell lysate of step (h) empty particles and impurities by a process comprising chromatography, to obtain a purified parvovirus containing lysate;
(j) performing buffer exchange on the purified parvovirus containing lysate of step (i) and concentrating product therefrom through a desalting column or by a tangential flow filtration process; and
(k) admixing the product of step (j) with a carrier.

10. The method of claim 9 wherein the carrier in step (k) comprises Ringer solution or an admixture of iodixanol and Ringer solution.

11. The method of claim 10 wherein the carrier in step (k) comprises an admixture of iodixanol and Ringer solution.

12. The method of claim 9 , wherein the chromatography in step (h) is anion exchange chromatography.

13. The method of claim 11 , wherein the chromatography in step (h) is anion exchange chromatography.

Assignees

• Deutsches Krebsforschungszentrum Stiftung Des Oeffentlichen Rechts

Inventors

• Leuchs Barbara
• Frehtman Veronika
• MüLLER MARCUS
• Rommelaere Jean
• Dahm Michael
• Krebs Ottheinz

Classifications

• C12N2750/14352

  relating to complementing cells and packaging systems for producing virus or viral particles · CPC title

• C12N2750/14351

  Methods of production or purification of viral material · CPC title

• C12N5/0686

  Kidney cells · CPC title

• C12N7/00 Primary

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title