# US 9,750,800 B2 · Method for purifying the rabies virus

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- Number: US 9,750,800 B2
- Document ID: US9750800B2
- Status: Granted
- Kind: B2
- Application number: 14/593,337
- Filed: 2015-01-09
- Published: 2015-08-06
- Granted: 2017-09-05
- Earliest claimed: 2009-04-08
- Assignees: Sanofi Pasteur Limited
- Applicants: Virginie Fabre; Celine Rocca; Pierre Riffard; Eric Calvosa
- Inventors: Virginie Fabre; Celine Rocca; Pierre Riffard; Eric Calvosa
- Web page: https://patentgrep.com/patent/US9750800B2

## CPC

- A61K 39/205: Rhabdoviridae, e.g. rabies virus
- A61P 31/14: for RNA viruses
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- A61K 39/12: Viral antigens
- C12N 2760/20134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/20151: Methods of production or purification of viral material
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The subject of the invention is a method for purifying the rabies virus, comprising a single ion-exchange chromatography step, said step being cation exchange chromatography according to which: a) the supernatant of a culture of cells infected with this virus is brought into contact with a cation exchange chromatography support comprising a polymethacrylate matrix onto which sulfoisobutyl groups have been grafted such that the rabies virus binds to this support, and; b) the virus is eluted from its support.

## Claims

### Claim 1

A vaccine comprising purified and inactivated rabies virus, wherein the amount of residual DNA measured by quantitative PCR and the amount of total protein present in one effective dose of vaccine or in one dose of vaccine which contains 2.5 IU as measured according to the European Pharmacopeia monograph number 0216, are, respectively, less than 20 pg and less than 40 μg.

### Claim 2 (depends on 1)

The vaccine according to claim 1, wherein at least 70% of the total amount of proteins present in one effective dose of vaccine is rabies virus proteins.

### Claim 3 (depends on 2)

The vaccine according to claim 2, wherein the vaccine is free of any exogenous product of animal origin.

### Claim 4 (depends on 1)

The vaccine as claimed in claim 1, wherein the amount of residual DNA measured by quantitative PCR is less than 10 μg.

### Claim 5 (depends on 4)

The vaccine as claimed in claim 4, wherein at least 70% of the total amount of proteins present in one effective dose of vaccine is rabies virus proteins.

### Claim 6 (depends on 5)

The vaccine as claimed in claim 5, wherein the vaccine is free of any exogenous product of animal origin.

### Claim 7 (depends on 6)

The vaccine as claimed in claim 6, wherein the vaccine is free of any human protein.

### Claim 8 (depends on 1)

The vaccine as claimed in claim 1, wherein the vaccine is obtained by a method of purifying rabies virus comprising a single ion exchange chromatography purification, wherein said ion exchange chromatography purification is a cation exchange chromatography purification comprising,
a) applying a supernatant of a culture of cells infected with the rabies virus to a cation exchange chromatography support under conditions in which the rabies virus binds to the support, wherein the support comprises a polymethacrylate matrix onto which sulfoisobutyl groups have been grafted by covalent bonding, and
b) eluting the virus from the support.

### Claim 9 (depends on 8)

The vaccine as claimed in claim 8, wherein the culture supernatant of cells infected with the rabies virus is free of animal serum or free of any serum protein.

### Claim 10 (depends on 9)

The vaccine as claimed in claim 9, wherein the culture supernatant of cells infected with the rabies virus is free of any exogenous protein of animal origin.

### Claim 11 (depends on 10)

The vaccine as claimed in claim 10, wherein the culture supernatant of the cells infected with the rabies virus contains exogenous proteins of non-animal origin at a concentration ≦15 mg/l.

### Claim 12 (depends on 11)

The vaccine as claimed in claim 11, wherein the culture supernatant of cells infected with the rabies virus is free of any exogenous product of animal origin.

### Claim 13 (depends on 8)

The vaccine as claimed in claim 8, wherein the culture supernatant of cells infected with the rabies virus is a clarified supernatant.

### Claim 14 (depends on 8)

The vaccine as claimed in claim 8, wherein the amount of virus measured in the eluate corresponds to at least 70% and preferably to at least 80% of the amount of virus measured in the supernatant that was applied to the chromatographic support.

### Claim 15 (depends on 14)

The vaccine as claimed in claim 14, wherein the amount of total proteins measured in the eluate corresponds to less than 40% of the amount of total proteins measured in the supernatant that was applied to the chromatographic support and the amount of DNA measured in the eluate corresponds to less than 5% of the amount of DNA measured in the supernatant that was applied to the chromatographic support.

### Claim 16 (depends on 14)

The vaccine as claimed in claim 14, wherein the amount of total proteins measured in the eluate corresponds to less than 40% of the amount of total proteins measured in the supernatant that was applied to the chromatographic support and the amount of DNA measured in the eluate corresponds to less than 1% of the amount of DNA measured in the supernatant that was applied to the chromatographic support.

### Claim 17 (depends on 8)

The vaccine as claimed in claim 8, wherein the method further comprises treating the eluate, optionally concentrated, with a nuclease.

### Claim 18 (depends on 17)

The vaccine as claimed in claim 17, wherein the nuclease is an endonuclease.

### Claim 19 (depends on 17)

The vaccine as claimed in claim 17, wherein the treated eluate is subjected to ultracentrifugation on a sucrose gradient and recovering the fraction(s) of the gradient that contain(s) the purified virus.

### Claim 20 (depends on 19)

The vaccine as claimed in claim 19, wherein the method further comprises inactivating the purified rabies virus with a viral inactivation agent.

### Claim 21 (depends on 20)

The vaccine as claimed in claim 20, wherein the viral inactivation agent is β-propiolactone.

### Claim 22 (depends on 20)

The vaccine as claimed in claim 20, wherein the purification is conducted with products of non-animal origin only.

### Claim 23 (depends on 20)

A vaccine containing purified and inactivated rabies virus, wherein the vaccine is obtained by a method of manufacturing a rabies vaccine comprising:
a) Infecting a culture of cells with the rabies virus;
b) purifying the rabies virus from an infected-cell culture supernatant according to the method as claimed in claim 20; and
c) mixing the suspension obtained in b) with a storage buffer, and dividing the resultant mixture a single-dose or multi-dose vaccine(s).

### Claim 24 (depends on 20)

A vaccine containing purified and inactivated rabies virus, wherein the vaccine is obtained by a method for the manufacture of a rabies vaccine comprising,
(a) infecting a culture of cells with the rabies virus,
(b) purifying the rabies virus from an infected-cell culture supernatant according to the method as claimed in claim 20,
(c) mixing the suspension of purified virus obtained in b) with a lyophilization buffer,
(d) dividing the mixture obtained in c) into a single-dose or multidose vaccine(s), and
(e) lyophilizing the vaccine dose(s).

### Claim 25 (depends on 3)

The vaccine as claimed in claim 3, wherein the vaccine is free of any human protein.