# US 10,370,692 B2 · Recombinant polypeptide production methods

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- Number: US 10,370,692 B2
- Document ID: US10370692B2
- Status: Granted
- Kind: B2
- Application number: 15/106,205
- Filed: 2014-12-10
- Published: 2016-11-10
- Granted: 2019-08-06
- Earliest claimed: 2013-12-20
- Assignees: Hoffmann-La Roche Inc.
- Applicants: Hoffmann-La Roche Inc.
- Inventors: Erhard Kopetzki; Jens Niewoehner; Peter Maier
- Web page: https://patentgrep.com/patent/US10370692B2

## CPC

- C12P 21/005: {Glycopeptides, glycoproteins}
- C07K 5/1019: {with the first amino acid being basic}
- C07K 14/475: Growth factors; Growth regulators
- C07K 14/48: Nerve growth factor [NGF]
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/2881: {against CD71}
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/21: from primates, e.g. man
- C07K 2317/522: CH1 domain
- C07K 2317/524: CH2 domain
- C07K 2317/526: CH3 domain
- C07K 2317/53: Hinge
- C07K 2317/55: Fab or Fab'
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/40: containing a tag for immunodetection, or an epitope for immunisation
- C07K 2319/50: containing protease site
- C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
- 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)}
- 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

Herein is reported a method for producing a fusion-polypeptide comprising the steps of a) cultivating a mammalian cell comprising a nucleic acid encoding a variant fusion-polypeptide wherein the amino acid sequence of the fusion-polypeptide has been modified by replacing in a pro-fusion-polypeptide the endogenous protease cleavage site between the pro-peptide and the fusion-polypeptide with an exogenous (with respect to the origins of the parts of the fusion-polypeptide) or artificial protease cleavage site, and b) recovering the fusion-polypeptide or fusion-pro-polypeptide from the cell or the cultivation medium and thereby producing the (recombinant) fusion-polypeptide.

## Claims

### Claim 1

A method for producing a recombinant polypeptide, comprising the following steps:
cultivating a mammalian cell comprising a nucleic acid encoding the polypeptide as a variant pro-polypeptide which is a fusion polypeptide of a pro-segment and the polypeptide wherein an enzymatic cleavage site between the pro-segment and the polypeptide but not the pro-segment is replaced by an IgA protease cleavage site,
recovering the variant pro-polypeptide from the cell or the cultivation medium,
cleaving the variant pro-polypeptide, and thereby producing the recombinant polypeptide,
wherein the fusion-polypeptide comprises a biologically active entity, a linker peptide, and a monovalent binding entity which binds to a blood-brain-barrier (BBB) receptor.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the cleavage of the variant pro-polypeptide is during purification of the polypeptide.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the polypeptide is a neurotrophic factor.

### Claim 4 (depends on 1)

The method according to claim 1, wherein an exogenous protease is added to the cultivation medium.

### Claim 5 (depends on 1)

The method according to claim 1, wherein an exogenous protease is co-expressed in the cultivation medium.

### Claim 6

A method for producing a recombinant polypeptide, comprising the following steps:
cultivating a mammalian cell comprising a nucleic acid encoding the polypeptide as a variant pro-polypeptide which is a fusion polypeptide of a pro-segment and the polypeptide wherein an enzymatic cleavage site between the pro-segment and the polypeptide but not the pro-segment is replaced by an IgA protease cleavage site, recovering the variant pro-polypeptide from the cell or the cultivation medium, cleaving the variant pro-polypeptide, and thereby producing the recombinant polypeptide,
wherein the fusion-polypeptide comprises a biologically active entity, a linker peptide, and a monovalent binding entity which binds to a blood-brain-barrier (BBB) receptor, and wherein the linker peptide comprises one or more negatively charged amino acid residues.

### Claim 7 (depends on 6)

The method according to claim 6, wherein the cleavage of the variant pro-polypeptide is during purification of the polypeptide.

### Claim 8 (depends on 6)

The method according to claim 6, wherein the polypeptide is a neurotrophic factor.

### Claim 9 (depends on 6)

The method according to claim 6, wherein an exogenous protease is added to the cultivation medium.

### Claim 10 (depends on 6)

The method according to claim 6, wherein an exogenous protease is co-expressed in the cultivation medium.