# US 10,900,024 B2 · Method for purifying viruses or virus-like particles using a crosslinked cellulose hydrate membrane

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- Number: US 10,900,024 B2
- Document ID: US10900024B2
- Status: Granted
- Kind: B2
- Application number: 16/329,512
- Filed: 2017-08-03
- Published: 2019-06-27
- Granted: 2021-01-26
- Earliest claimed: 2016-09-01
- Assignees: Sartorius Stedim Biotech GmbH
- Applicants: Sartorius Stedim Biotech GmbH
- Inventors: Rebecca Petersen; Kathrin Schröder-Tittmann; Louis Villain
- Web page: https://patentgrep.com/patent/US10900024B2

## 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)
- B01D 67/0093: {Chemical modification}
- B01D 71/10: Cellulose; Modified cellulose
- B01J 20/24: Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
- B01J 20/28033: {Membrane, sheet, cloth, pad, lamellar or mat}
- B01D 2323/30: Cross-linking
- B01J 20/00: Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- C12N 2710/00051: Methods of production or purification of viral material
- C12N 2750/14051: Methods of production or purification of viral material
- C12N 2795/00051: 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)
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS

## Abstract

The present invention relates to a method for purifying viruses or virus-like particles using a crosslinked cellulose hydrate membrane and to a kit for purifying viruses or virus-like particles and the use thereof.

## Claims

### Claim 1

A method for purifying viruses or virus-like particles from a solution, comprising the following steps:
a) providing a mixture M composed of a first solution and a second solution, the first solution containing the viruses or the virus-like particles to be purified and impurities, and the second solution containing a polyalkylene glycol having two or three carbon atoms in the repeating unit,
b) loading a crosslinked cellulose hydrate membrane with the mixture M from step a), with the viruses or the virus-like particles attaching to the outer and inner membrane surfaces and being retained, and
c) eluting the viruses or virus-like particles retained in the crosslinked cellulose hydrate membrane using a solution which contains no polyalkylene glycol having two or three carbon atoms in the repeating unit or which contains at least 50% less polyalkylene glycol having two or three carbon atoms in the repeating unit than the mixture M from step a), with the result that the viruses or the virus-like particles are obtained in purified form in the eluate.

### Claim 2 (depends on 1)

The method as claimed in claim 1, wherein an unpurified, clarified bioprocess solution is used as the first solution.

### Claim 3 (depends on 1)

The method as claimed in claim 1, further comprising a step d) which is carried out between step b) and step c), in which step d) the loaded crosslinked cellulose hydrate membrane is washed with a solution which contains as much polyalkylene glycol having two or three carbon atoms in the repeating unit as the mixture M from step a).

### Claim 4 (depends on 1)

The method as claimed in claim 1, further comprising a step e), in which step e) the crosslinked cellulose hydrate membrane is sanitized before the loading according to step b) by means of alkaline solution.

### Claim 5 (depends on 1)

The method as claimed in claim 1, further comprising a step f), in which step f) the crosslinked cellulose hydrate membrane is equilibrated before the loading according to step b), in which it is flushed with a buffer-containing solution, which contains polyalkylene glycol having two or three carbon atoms in the repeating unit, until a constant conductivity and/or a constant UV signal is measured in the permeate.

### Claim 6 (depends on 1)

The method as claimed in claim 1, wherein the first and second solutions, which contain the viruses or virus-like particles to be purified and impurities as well as polyalkylene glycol having two or three carbon atoms in the repeating unit, are mixed with the aid of a mixer.

### Claim 7 (depends on 1)

The method as claimed in claim 1, wherein the contact time of the viruses or the virus-like particles with the second solution, which contains polyalkylene glycol having two or three carbon atoms in the repeating unit, is less than one minute before the loading step.

### Claim 8 (depends on 1)

The method as claimed in claim 1, wherein the concentration of polyalkylene glycol having two or three carbon atoms in the repeating unit in the mixture M which is contacted with the crosslinked cellulose hydrate membrane is from 1 to 20% by weight, based on the total mass of the mixture M.

### Claim 9 (depends on 1)

The method as claimed in claim 1, wherein the polyalkylene glycol having two or three carbon atoms in the repeating unit is selected from the group consisting of polyethylene glycol and polypropylene glycol or mixtures thereof.

### Claim 10 (depends on 1)

The method as claimed in claim 1, wherein polyethylene glycol (PEG) having an average molar mass of from 600 to 10 000 daltons is selected.

### Claim 11 (depends on 1)

The method as claimed in claim 1, wherein the recovery rate for purified viruses or virus-like particles in the eluate is more than 50%.

### Claim 12 (depends on 1)

The method as claimed in claim 1, wherein the concentration of the viruses or the virus-like particles in the eluate is elevated by a factor of from 5 to 10 in comparison with the starting solution.

### Claim 13 (depends on 4)

The method as claimed in claim 4, further comprising a step f), in which step f) the crosslinked cellulose hydrate membrane is equilibrated after the sanitization according to step e), in which it is flushed with a buffer-containing solution B, which contains polyalkylene glycol having two or three carbon atoms in the repeating unit, until a constant conductivity and/or a constant UV signal is measured in the permeate.