# US 11,306,323 B2 · Expression process

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- Number: US 11,306,323 B2
- Document ID: US11306323B2
- Status: Granted
- Kind: B2
- Application number: 15/838,868
- Filed: 2017-12-12
- Published: 2018-05-10
- Granted: 2022-04-19
- Earliest claimed: 2013-05-03
- Assignees: Fujifilm Diosynth Biotechnologies UK Limited
- Applicants: Fujifilm Diosynth Biotechnologies UK Limited
- Inventors: Fay Louise Saunders; Anna Louise Dodds; Adeline Marie Geraldine Bayard; Bhupendra Vallabh Kara
- Web page: https://patentgrep.com/patent/US11306323B2

## CPC

- C12N 15/85: for animal cells
- C07K 16/065: {Purification, fragmentation}
- C07K 16/3092: {against tumour-associated mucins}
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2319/02: containing a signal sequence
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

A process for the production of a target polypeptide is provided. The process comprises expression of an expression vector for expressing a target polypeptide in a host cell, preferably a mammalian cell, the expression vector comprising an expression cassette comprising a polynucleotide encoding a recombinant polypeptide operably linked to a fibronectin secretion leader sequence; and recovering the target polypeptide.

## Claims

### Claim 1

A process of producing a target polypeptide which comprises:
(a) expressing an expression vector for expressing a target polypeptide in a Chinese hamster ovary (CHO) host cell, the expression vector comprising an expression cassette comprising a polynucleotide encoding a recombinant polypeptide operably linked to a fibronectin secretion leader sequence comprising SEQ ID NO: 2 or a sequence having 90% sequence identity to SEQ ID NO: 2 and retaining the ability to secrete the target polypeptide; and
(b) recovering the target polypeptide.

### Claim 2

A process of producing a target polypeptide which comprises:
(a) transfection or transformation of a Chinese hamster ovary (CHO) host cell with an expression vector for expressing a target polypeptide in a host cell, the expression vector comprising an expression cassette comprising a polynucleotide encoding the target polypeptide operably linked to a fibronectin secretion leader sequence comprising SEQ ID NO: 2 or a sequence having 90% sequence identity to SEQ ID NO: 2 and retaining the ability 10 secrete the target polypeptide;
(b) culturing the host cell under conditions which allow proliferation of the host cell and expression and secretion of the target polypeptide from the host cell; and
(c) recovering the target polypeptide.

### Claim 3 (depends on 1)

The process according to claim 1, wherein the fibronectin secretion leader sequence corresponds to the host cell.

### Claim 4 (depends on 1)

The process according to claim 1, wherein the expression cassette comprises a human elongation factor 1a (hEF1a) promoter.

### Claim 5 (depends on 4)

The process according to claim 4, wherein the expression cassette comprises a polyA sequence.

### Claim 6 (depends on 5)

The process according to claim 5, wherein the poly A sequence is selected from human betaglobin polyA, bovine growth hormone polyA, SV40 early and SV40 late sequences.

### Claim 7 (depends on 1)

The process according to claim 1, wherein two expression cassettes are employed, a first expression cassette comprising a polynucleotide encoding a light chain of a monoclonal antibody, and a second expression cassette comprising a polynucleotide encoding a heavy chain of a monoclonal antibody.

### Claim 8 (depends on 7)

The process according to claim 7, wherein the two expression cassettes comprise the same promoter, secretion leader and polyA sequence.

### Claim 9 (depends on 8)

The process according to claim 8, wherein the promoter is a human elongation factor 1a (hEF1a) promoter, the fibronectin secretion leader is Chinese hamster fibronectin secretion leader and the polyA sequence is bovine betaglobin polyA sequence.

### Claim 10

A process for the production of a target polypeptide, the process comprising culturing a Chinese hamster ovary cell (CHO) transfected with an expression vector comprising an expression cassette comprising a polynucleotide encoding the target polypeptide operably linked to a fibronectin secretion leader sequence comprising SEQ ID NO: 2 or a sequence having 90% sequence identity to SEQ ID NO: 2 and retaining the ability to secrete the target polypeptide.