# US 2018/0187166 A1 · Method for large scale production and purification of parvovirus

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- Number: US 2018/0187166 A1
- Document ID: US20180187166A1
- Status: Published application
- Kind: A1
- Application number: 15/739,684
- Filed: 2016-06-22
- Published: 2018-07-05
- Earliest claimed: 2015-06-23
- Assignees: Deutsches Krebsforschungszentrum
- Applicants: Deutsches Krebsforschungszentrum
- Inventors: Barbara Leuchs; Mandy Roscher; Marcus Müller; Jean Rommelaere
- Web page: https://patentgrep.com/patent/US20180187166A1

## CPC

- C12N 7/02: Recovery or purification
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- G01N 33/56983: {Viruses}
- C07K 16/081: DNA viruses
- C12N 2750/14323: Virus like particles [VLP]
- C12N 2750/14351: Methods of production or purification of viral material
- C12N 2750/14361: Methods of inactivation or attenuation
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Described is a reproducible, effective and scalable process for parvovirus production including characterization strategies, preferably production of H-1PV.

## Claims

### Claim 1

A method for producing empty inactive or full active parvovirus particles, said method comprising:
(a) providing the producer cell line NB-324K;
(b) growing the cell line under suitable conditions and infecting the cells at a cell density from 2.0 to 5.0×104 cells/cm2 with the parvovirus at a MOI of 0,5 to 2×10-2 PFU/cells;
(c) harvesting the cells 2 to 6 days post-infection and obtaining a cell pellet by centrifugation;
(d) subjecting the resuspended cell pellet to a mechanical, physical or chemical cell lysis method for obtaining a parvovirus containing cell lysate;
(e) sonicating the cell lysate and subjecting it to DNAse treatment;
(f) clarifying the DNAse-treated parvovirus harvest by filtration; and
(g1) purifying the parvovirus by two successive density gradient ultracentrifugations, wherein the first gradient is a Iodixanol/PBS step gradient and the second gradient is a Iodixanol/Ringer step gradient or a Iodixanol/Ringer continuous gradient for obtaining full active parvovirus particles in one fraction and empty parvovirus particles in another fraction.

### Claim 2 (depends on 1)

The method of claim 1, wherein the cell density of step (b) is from 3.0 to 4.0×104 cells/cm2.

### Claim 3 (depends on 1)

The method of claim 1, wherein for step (f) a 0.2-μm filter with prefilter is used.

### Claim 4 (depends on 1)

The method of claim 1, wherein the producer cell line NB-324K is characterized by
(a) a viability of at least 95%;
(b) a passage number below 20; and/or
(c) lack of mycoplasma contamination.

### Claim 5 (depends on 1)

The method of any one of claim 1 4, wherein virus production is performed in a collection system.

### Claim 6 (depends on 5)

The method of claim 5, wherein the collection system is a 10-layer cell culture chamber.

### Claim 7 (depends on 1)

The method of claim 1, wherein the parvovirus is H1-PV.

### Claim 8 (depends on 1)

The method of claim 1 further comprising determining the ratio of native parvovirus capsids to non-assembled capsid proteins or denatured capsids.

### Claim 9 (depends on 8)

The method of claim 8, wherein the ratio is determined by using a monoclonal antibody.

### Claim 10 (depends on 9)

The method of claim 9, wherein the monoclonal antibody is the antibody BL-H1 (DSM ACC 3030).

### Claim 11

(canceled)

### Claim 12

(canceled)