# US 7,608,425 B2 · Methods for protein expression in mammalian cells in serum-free medium

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- Number: US 7,608,425 B2
- Document ID: US7608425B2
- Status: Granted
- Kind: B2
- Application number: 11/877,728
- Filed: 2007-10-24
- Published: 2009-04-30
- Granted: 2009-10-27
- Earliest claimed: 2004-07-23
- Assignees: Immunomedics, Inc.
- Inventors: David M. Goldenberg; Chien-Hsing Chang; Edward A. Rossi; Diane Nordstrom
- Web page: https://patentgrep.com/patent/US7608425B2

## CPC

- C07K 14/4747: {Apoptosis related proteins}
- A61P 37/04: Immunostimulants
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/18: against material from animals or humans
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/3007: {Carcino-embryonic Antigens}
- C12N 5/0694: {Cells of blood, e.g. leukemia cells, myeloma cells}
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- A61K 2039/505: {comprising antibodies}
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/31: multispecific
- C12N 2500/90: Serum-free medium, which may still contain naturally-sourced components
- C12N 2501/48: Regulators of apoptosis
- C12N 2510/02: Cells for production
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- 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)
- 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)
- 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)}
- 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)

## Abstract

Disclosed are compositions and methods for increasing the longevity of a cell culture and permitting the increased production of proteins, preferably recombinant proteins, such as antibodies, peptides, enzymes, growth factors, interleukins, interferons, hormones, and vaccines. Cells transfected with an apoptosis-inhibiting gene or vector, such as a triple mutant Bcl-2 gene, can survive longer in culture, resulting in extension of the state and yield of protein biosynthesis. Such transfected cells exhibit maximal cell densities that equal or exceed the maximal density achieved by the parent cell lines. Transfected cells can also be pre-adapted for growth in serum-free medium, greatly decreasing the time required to obtain protein production in serum-free medium. In certain methods, the pre-adapted cells can be used for protein production following transfection under serum-free conditions. In preferred embodiments, the cells of use are SpESF or SpESF-X cells.

## Claims

### Claim 1

A method of protein production comprising:
a) obtaining a cell line comprising a gene encoding a mutant Bcl-2 protein, said protein comprising T69E, S70E and S87E amino acid substitutions;
b) adapting said cell line to grow in serum-free medium to produce a pre-adapted cell line; and
c) transfecting said pre-adapted cell line under serum-free conditions with one or more expression vectors expressing a protein of interest.

### Claim 2 (depends on 1)

The method of claim 1, said transfected cell line is capable of producing the protein of interest without further adaptation to serum free conditions.

### Claim 3 (depends on 1)

The method of claim 1, wherein the expression vector encodes an antibody, humanized antibody, chimeric antibody, human antibody, bispecific antibody, multispecific antibody, multivalent antibody or fragment thereof.

### Claim 4 (depends on 1)

The method of claim 1, wherein the cell line is not a CHO cell line.

### Claim 5 (depends on 1)

The method of claim 1, further comprising freezing the pre-adapted cell line for storage prior to transfection with one or more expression vectors expressing a protein of interest.

### Claim 6 (depends on 1)

The method of claim 1, wherein the expression vector encodes a growth factor, hormone, cytokine, chemokine, interleukin, interferon, an enzyme, a peptide, a vaccine, EPO, G-CSF, GM-CSF, EGF, VEGF, thrombopoietin, IL-1 through IL-31, interferon-alpha, interferon-beta or interferon-gamma.

### Claim 7 (depends on 1)

The method of claim 1, wherein the transfected cell line produces the protein of interest at a yield of at least 150 mg protein/mL of growth medium.

### Claim 8 (depends on 1)

The method of claim 1, further comprising adding one or more growth factors, cytokines or hormones to the medium.

### Claim 9 (depends on 8)

The method of claim 8, wherein the growth factor, cytokine or hormone is selected from the group consisting of erythropoietin, thrombopoietin, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-11, IL-12, IL-13, IL-15, prolactin, growth hormone, G-CSF and GM-CSF.

### Claim 10 (depends on 1)

The method of claim 1, further comprising exposing the cell line to methotrexate to amplify one or more nucleic acid sequences in the cell line.

### Claim 11 (depends on 10)

The method of claim 10, further comprising growing the cell line under conditions that comprise overgrowth of the cells to a viability of 50 to 75%.

### Claim 12 (depends on 11)

The method of claim 11, further comprising allowing the cells to recover in fresh medium.

### Claim 13 (depends on 12)

The method of claim 12, further comprising repeating the overgrowth and recovery of the cell line.