# US 8,785,173 B2 · Methods for purification of viruses

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- Number: US 8,785,173 B2
- Document ID: US8785173B2
- Status: Granted
- Kind: B2
- Application number: 13/120,638
- Filed: 2009-09-24
- Published: 2012-08-30
- Granted: 2014-07-22
- Earliest claimed: 2008-09-24
- Assignees: MedImmune, LLC
- Applicants: Mark Thompson; Janice Wee; Akanksha Nagpal
- Inventors: Mark Thompson; Janice Wee; Akanksha Nagpal
- Web page: https://patentgrep.com/patent/US8785173B2

## CPC

- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- A61P 31/12: Antivirals
- A61P 31/14: for RNA viruses
- A61P 37/04: Immunostimulants
- A61K 2039/5254: {avirulent or attenuated}
- C12N 2760/16051: Methods of production or purification of viral material
- C12N 2760/18551: Methods of production or purification of viral material
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present invention provides methods for the purification of cell-associated viruses from adherent cells (e.g., MDCK or Vero cells). In particular, the present invention provides purification methods for the production of immunogenic compositions comprising a live attenuated cell-associated virus (e.g., an attenuated respiratory syncytial virus (RSV) or cold-adapted, and/or temperature sensitive influenza virus) that result in levels of host cell DNA (HCD), host cell protein (HCP) and non-specific endonuclease (e.g., Benzonase), which are below the specifications required by regulatory agencies. The immunogenic compositions can be used to actively immunize subjects or to generate antibodies for a variety of uses, including passive immunization and diagnostic immunoassays.

## Claims

### Claim 1

A method of purifying respiratory syncytial virus from virus-infected cells, wherein the method comprises:
a) clarifying a viral harvest to form a clarified viral harvest, wherein clarifying comprises:
i. filtering the viral harvest by direct flow filtration (DFF) through at least one clarification filter having a pore size of between about 3 micrometers to about 10 micrometers;
ii. treating the viral harvest with a non-specific endonuclease; and
iii. filtering the viral harvest by DFF through at least one clarification filter having a pore size of between about 0.45 micrometers to about 3 micrometers;
b) stabilizing the clarified viral harvest with a stabilization buffer comprising sucrose, phosphate or Tris with NaCl salt or KCl salt to form a stabilized viral harvest;
c) concentrating the stabilized viral harvest by tangential flow filtration (TFF) and performing buffer exchange by diafiltration (DF) using a hollow fiber cartridge with a molecular weight cut off (MWCO) of 500 kDa to form a concentrated viral harvest; and
d) after (c), sterilizing the concentrated viral harvest by DFF through at least one clarification filter having a pore size of between about 0.8 micrometers to about 0.45 micrometers to form a sterilized viral harvest; whereby the respiratory syncytial virus is purified from the virus-infected cells and has a final viral infectivity titer of at least 5.7 log10 FFU/mL.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the method further comprises a step of purification by ion-exchange chromatography.

### Claim 3 (depends on 1)

The method according to claim 1, wherein an exchange buffer of step (c) comprises about 10-25% w/v sucrose, about 150 mM to 10 mM potassium phosphate and about 175 mM to 50 mM KCl pH 7.0-7.4.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the respiratory syncytial virus is is rA2cp248/404/1030ASH.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the viral harvest is stabilized with a buffer comprising about 150 mM to 50 mM potassium phosphate, about 250 mM to 175 mM sucrose, and about 175 mM to 125 mM NaCl or KCl, pH 7.0-7.4.

### Claim 6 (depends on 1)

The method according to claim 1, wherein step (c) results in a 5× concentration and least 5 diavolumes of buffer are exchanged.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the non-specific nuclease is exposed to the viral harvest for between about 120 minutes and 210 minutes.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the respiratory syncytial virus is a live respiratory syncytial virus.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the respiratory syncytial virus is a live attenuated respiratory syncytial virus.