# US 2018/0023067 A1 · Method For Producing A Recombinant Protein Of Interest

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- Number: US 2018/0023067 A1
- Document ID: US20180023067A1
- Status: Published application
- Kind: A1
- Application number: 15/549,720
- Filed: 2016-02-09
- Published: 2018-01-25
- Earliest claimed: 2015-02-09
- Assignees: Sandoz Ltd.
- Applicants: Sandoz AG
- Inventors: Sascha Keller; Anna Justyna Jasiak; René Funke
- Web page: https://patentgrep.com/patent/US20180023067A1

## CPC

- C12N 9/506: {derived from RNA viruses}
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12P 21/06: produced by the hydrolysis of a peptide bond, e.g. hydrolysate products (preparing foodstuffs by protein hydrolysis A23J3/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

Disclosed is a method for producing a recombinant protein of interest, characterised in by the following steps: (a) providing a fusion protein comprising an Npro autoprotease moiety and a protein of interest moiety in inclusion bodies, (b) solubilising the fusion protein in the inclusion bodies by subjecting the inclusion bodies to a solubilisation buffer containing a detergent and wherein the solubilisation buffer contains no chaotropes or chaotropes in a concentration of less than 1.5 M urea (c) refolding the solubilised fusion protein and (d) allowing the fusion protein to be cleaved by the Npro autoprotease moiety under kosmotropic conditions, wherein the recombinant protein of interest is cleaved from the fusion protein, and (e) recovering the protein of interest.

## Claims

### Claim 1

Method for producing a recombinant protein of interest, characterised in by the following steps:
(a) providing a fusion protein comprising an Npro autoprotease moiety and a protein of interest moiety in inclusion bodies,
(b) solubilising the fusion protein in the inclusion bodies by subjecting the inclusion bodies to a solubilisation buffer containing a detergent and wherein the solubilisation buffer contains no chaotropes or chaotropes in a concentration of less than 1.5 M urea
(c) refolding the solubilised fusion protein and
(d) allowing the fusion protein to be cleaved by the Npro autoprotease moiety under kosmotropic conditions, wherein the recombinant protein of interest is cleaved from the fusion protein, and
(e) recovering the protein of interest.

### Claim 2 (depends on 1)

Method according to claim 1, characterized in that the inclusion bodies were generated in a recombinant production system.

### Claim 3 (depends on 1)

Method according to claim 1, characterized in that the detergent is contained in the solubilisation buffer in a concentration of 0.2 to 15% (w/v).

### Claim 4 (depends on 1)

Method according to claim 1, characterized in that steps (c) and/or (d) are performed at kosmotropic conditions that correspond to a urea concentration of 0 to 1.5 M.

### Claim 5 (depends on 1)

Method according to claim 1, characterized in that steps (b), (c) and/or (d) are performed in a buffer.

### Claim 6 (depends on 1)

Method according to claim 1, characterized in that steps (b), (c) and/or (d) are performed in a buffer containing a reducing agent.

### Claim 7 (depends on 1)

Method according to claim 1, characterized in that steps (c) and/or (d) are performed in a buffer containing an ion chelating agent.

### Claim 8 (depends on 1)

Method according to claim 1, characterized in that the detergent in the solubilisation buffer is a non-ionic detergent, or an anionic detergent, or mixtures thereof.

### Claim 9 (depends on 1)

Method according to claim 1, characterized in that steps (c) and/or (d) are performed in a buffer containing a detergent; and/or an amino acid; and/or a carbohydrate; or mixtures thereof.

### Claim 10 (depends on 1)

Method according to claim 1, characterized in that steps (c) and/or (d) are performed in a buffer with a pH of 6 to 9.5.

### Claim 11 (depends on 1)

Method according to claim 1, characterized in that the protein of interest is a protein for therapeutic use in humans.

### Claim 12 (depends on 1)

Method according to claim 1, characterized in that steps (c) and/or (d) are performed in the presence of a buffer comprising NaCl.

### Claim 13 (depends on 1)

Method according to claim 1, characterized in that that steps (c) and/or (d) are performed in the presence of a buffer comprising sucrose, DTT, NaCl, EDTA and a detergent.