# US 11,078,294 B2 · Prevention of disulfide bond reduction during recombinant production of polypeptides

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- Number: US 11,078,294 B2
- Document ID: US11078294B2
- Status: Granted
- Kind: B2
- Application number: 17/124,314
- Filed: 2020-12-16
- Published: 2021-04-01
- Granted: 2021-08-03
- Earliest claimed: 2007-07-09
- Assignees: Genentech, Inc.
- Applicants: Genentech, Inc.
- Inventors: Yung-Hsiang Kao; Michael W. Laird; Melody Trexler Schmidt; Rita L. Wong; Daniel P. Hewitt
- Web page: https://patentgrep.com/patent/US11078294B2

## CPC

- C07K 16/2887: {against CD20}
- A61K 39/39591: {Stabilisation, fragmentation}
- A61P 5/00: Drugs for disorders of the endocrine system
- A61P 37/02: Immunomodulators
- C07K 1/14: Extraction; Separation; Purification
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- 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)
- 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

## Abstract

The invention concerns methods for preventing the reduction of disulfide bonds during the recombinant production of disulfide-containing polypeptides. In particular, the invention concerns the prevention of disulfide bond reduction during harvesting of disulfide-containing polypeptides, including antibodies, from recombinant host cell cultures.

## Claims

### Claim 1

A method for producing an antibody, comprising expressing the antibody in a Chinese Hamster Ovary (CHO) recombinant host cell culture, and following a production phase of the cell culture, sparging the pre-harvest cell culture fluid of the recombinant host cell with air to inhibit reduction of a disulfide bond in the antibody during processing,
wherein the antibody is a therapeutic monoclonal antibody, and wherein the air sparging is continued until the amount of dissolved oxygen (dO2) in the pre-harvest cell culture fluid is at least 10%.

### Claim 2 (depends on 1)

The method of claim 1, wherein the antibody expressed in the recombinant host cell is produced at a scale of greater than 5,000 L.

### Claim 3 (depends on 1)

The method of claim 1, wherein the air sparging is continued until the pre-harvest cell culture fluid is at least 30% saturated with air.

### Claim 4 (depends on 3)

The method of claim 3, wherein the antibody expressed in the recombinant host cell is produced at a scale of greater than 5,000 L.

### Claim 5 (depends on 1)

The method of claim 1 wherein the air sparging is continued until the pre-harvest cell culture fluid is between 100% saturated to 30% saturated with air.

### Claim 6 (depends on 2)

The method of claim 2, wherein the amount of dissolved oxygen (dO2) in the pre-harvest cell culture fluid is at least 30%.

### Claim 7 (depends on 1)

The method of claim 1, further comprising preparing the harvested cell culture fluid of the recombinant host cell and recovering the antibody from the harvested cell culture fluid.

### Claim 8 (depends on 1)

The method of claim 1, further comprising preparing the harvested cell culture fluid and purifying the antibody from the harvested cell culture fluid.