# US 8,771,987 B2 · Mammalian cell lines for increasing longevity and protein yield from a cell culture

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- Number: US 8,771,987 B2
- Document ID: US8771987B2
- Status: Granted
- Kind: B2
- Application number: 14/030,526
- Filed: 2013-09-18
- Published: 2014-02-06
- Granted: 2014-07-08
- Earliest claimed: 2004-07-23
- Assignees: Immunomedics, Inc.
- Applicants: Immunomedics, Inc.
- Inventors: David M. Goldenberg; Zhengxing Qu; Chien-Hsing Chang; Edmund A. Rossi; Jeng-Dar Yang; Diane Rossi
- Web page: https://patentgrep.com/patent/US8771987B2

## CPC

- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- C07K 14/005: from viruses
- C07K 14/4747: {Apoptosis related proteins}
- 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 15/85: for animal cells
- C12N 15/86: Viral vectors
- 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 2710/20022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2740/10043: 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)
- 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 transformation under serum-free conditions. The method preferably involves eukaryotic cells, more preferably mammalian cells.

## Claims

### Claim 1

A frozen cell culture comprising:
a) a Bcl-EEE mammalian cell line comprising a first nucleic acid that encodes a mutant Bcl-2 protein, wherein the mutant Bcl-2 protein comprises T69E, S70E and S87E amino acid substitutions, wherein the Bcl-EEE cell line is pre-adapted to grow in serum-free medium; and
b) serum-free cell culture medium;
wherein the cell culture medium containing the cell line is stored frozen.

### Claim 2 (depends on 1)

The frozen cell culture of claim 1, wherein the cell line can be thawed and transfected under serum-free conditions with a second nucleic acid encoding a recombinant protein.

### Claim 3 (depends on 2)

The frozen cell culture of claim 2, wherein the cell line transfected with the second nucleic acid can be cultured under serum-free conditions to produce the recombinant protein.

### Claim 4 (depends on 2)

The frozen cell culture of claim 2, wherein the recombinant protein is an antibody fragment, a growth factor, a protein or peptide hormone, an interleukin, an interferon or an enzyme.

### Claim 5 (depends on 2)

The frozen cell culture of claim 2, wherein the protein is selected from the group consisting of EPO, G-CSF, GM-CSF, EGF, VEGF, thrombopoietin, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, interferon-α, interferon-β, interferon-γ and interferon-λ.

### Claim 6 (depends on 1)

The frozen cell culture of claim 1, wherein the cell line is a myeloma cell line selected from the group consisting of Sp2/0, an Sp2/0 derivative, NSO or YB2/0.

### Claim 7 (depends on 1)

The frozen cell culture of claim 1, wherein the cell line is wherein the cell line is selected from the group consisting of CHO, HEK 293, COS-1, COS-7, HepG2, BHK21, P3X3Ag8.653 and BSC-1.

### Claim 8 (depends on 1)

The frozen cell culture of claim 1, wherein the cell line comprises a second nucleic acid encoding a recombinant protein.

### Claim 9 (depends on 8)

The frozen cell culture of claim 8, wherein the cell line can be thawed and cultured under serum-free conditions to produce the recombinant protein.

### Claim 10 (depends on 8)

The frozen cell culture of claim 8, wherein the recombinant protein is an antibody fragment, a growth factor, a protein or peptide hormone, an interleukin, an interferon or an enzyme.

### Claim 11 (depends on 10)

The frozen cell culture of claim 10, wherein the protein is selected from the group consisting of EPO, G-CSF, GM-CSF, EGF, VEGF, thrombopoietin, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, interferon-α, interferon-β, interferon-γ and interferon-λ.

### Claim 12 (depends on 8)

The frozen cell culture of claim 8, wherein the cell line is a myeloma cell line selected from the group consisting of Sp2/0, an Sp2/0 derivative, NSO or YB2/0.

### Claim 13 (depends on 8)

The frozen cell culture of claim 8, wherein the cell line is wherein the cell line is selected from the group consisting of CHO, HEK 293, COS-1, COS-7, HepG2, BHK21, P3X3Ag8.653 and BSC-1.

### Claim 14 (depends on 1)

The frozen cell culture of claim 1, wherein the medium further comprises at least one caspase inhibitor.

### Claim 15 (depends on 14)

The frozen cell culture of claim 14, wherein said caspase inhibitor is selected from the group consisting of caspase-1, caspase-3, caspase-9, caspase-12 and pan-caspase inhibitors.

### Claim 16 (depends on 14)

The frozen cell culture of claim 14, wherein said caspase inhibitor is selected from the group consisting of Z-VAD-fmk, Ac-DEVD-cho (SEQ ID NO: 7), Aven and XIAP.

### Claim 17 (depends on 1)

The frozen cell culture of claim 1, wherein said medium further comprises at least one growth factor, cytokine or hormone.

### Claim 18 (depends on 17)

The frozen cell culture of claim 17, 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.