# US 8,771,988 B2 · Protein expression from multiple nucleic acids

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- Number: US 8,771,988 B2
- Document ID: US8771988B2
- Status: Granted
- Kind: B2
- Application number: 12/681,781
- Filed: 2008-10-09
- Published: 2010-09-30
- Granted: 2014-07-08
- Earliest claimed: 2007-10-12
- Assignees: Hoffmann-La Roche Inc.
- Applicants: Ulrich Goepfert; Hendrik Knoetgen; Erhard Kopetzki; Anne Stern
- Inventors: Ulrich Goepfert; Hendrik Knoetgen; Erhard Kopetzki; Anne Stern
- Web page: https://patentgrep.com/patent/US8771988B2

## CPC

- C12N 15/85: for animal cells
- C07K 16/18: against material from animals or humans
- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- C07K 16/2812: {against CD4}
- C07K 16/2854: {against selectins, e.g. CD62}
- C07K 16/2866: {against receptors for cytokines, lymphokines, interferons}
- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/21: from primates, e.g. man
- C07K 2317/51: Complete heavy chain or Fd fragment, i.e. VH + CH1
- C07K 2317/515: Complete light chain, i.e. VL + CL
- 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)
- 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)}

## Abstract

The current invention reports a method for the recombinant production of a secreted heterologous immunoglobulin in a CHO cell comprising the following steps: i) providing a CHO cell, which is adapted to growth in suspension culture, adapted to growth in serum-free medium, mycoplasma free, and virus free, ii) providing a vector comprising a prokaryotic origin of replication, a first nucleic acid conferring resistance to a prokaryotic selection agent, a second nucleic acid encoding the heavy chain of said heterologous immunoglobulin, a third nucleic acid encoding the light chain of said heterologous immunoglobulin, a fourth nucleic acid conferring resistance to a eukaryotic selection agent, iii) transfecting said CHO cell, wherein said transfecting comprises a) transfecting said CHO cell with said vector comprising a fourth nucleic acid conferring resistance to a first eukaryotic selection agent, b) selecting a CHO cell by growth in cultivation medium containing said first eukaryotic selection agent, c) transfecting said selected CHO cell with said vector comprising a fourth nucleic acid conferring resistance to a second eukaryotic selection agent different to said first eukaryotic selection agent, d) selecting a CHO cell by selected growth in cultivation medium containing said first and said second eukaryotic selection agent, iv) cultivating said transfected CHO cell in a medium in the presence of said first and second eukaryotic selection agent, under conditions suitable for the expression of said second, and third nucleic acid, and v) recovering said secreted heterologous immunoglobulin from the cultivation medium.

## Claims

### Claim 1

A method for the recombinant production of a heterologous immunoglobulin in a CHO cell which is secreted to the cultivation medium comprising:
a) providing a CHO cell, wherein said CHO cell is
adapted to growth in suspension culture,
adapted to growth in serum-free medium, and
mycoplasma free,
b) providing a nucleic acid comprising
i) a prokaryotic origin of replication,
ii) a first nucleic acid sequence conferring resistance to a prokaryotic selection agent,
iii) a second nucleic acid sequence encoding the heavy chain of said heterologous immunoglobulin, and a third nucleic acid sequence encoding the light chain of said heterologous immunoglobulin,
whereby a first transfection vector is provided which comprises said provided nucleic acid and an additional fourth nucleic acid sequence conferring resistance to a first eukaryotic selection agent,
whereby a second transfection vector is provided which comprises said provided nucleic acid and an additional fourth nucleic acid sequence conferring resistance to a second eukaryotic selection agent, whereby said second eukaryotic selection agent is different to said first eukaryotic selection agent,
b1) providing a nucleic acid comprising
i) a prokaryotic origin of replication,
ii) a first nucleic acid sequence conferring resistance to a prokaryotic selection agent,
iii) a second nucleic acid sequence encoding the heavy chain of said heterologous immunoglobulin, and/or a third nucleic acid sequence encoding the light chain of said heterologous immunoglobulin,
whereby a third transfection vector is provided which comprises said provided nucleic acid and an additional fourth nucleic acid sequence conferring resistance to a third eukaryotic selection agent, whereby said third eukaryotic selection agent is different to said first eukaryotic selection agent and is also different to said second eukaryotic selection agent,
c) transfecting said CHO cell, wherein said transfecting comprises the following:
(i) transfecting said CHO cell with said first transfection vector,
(ii) selecting a CHO cell transfected in (i) by selected growth in cultivation medium containing a first eukaryotic selection agent to which the first transfection vector confers resistance,
(iii) transfecting said selected CHO cell in (ii) with said second transfection vector,
(iv) selecting a CHO cell transfected in (iii) by selected growth in cultivation medium containing said first eukaryotic selection agent to which the first transfection vector confers resistance and said second eukaryotic selection agent to which the second transfection vector confers resistance,
(v) transfecting said CHO cell selected in (iv) with said third transfection vector,
(vi) selecting a CHO cell transfected in (v) by selected growth in a cultivation medium containing said first eukaryotic selection agent to which the first transfection vector confers resistance and said second eukaryotic selection agent to which the second transfection vector confers resistance and said third eukaryotic selection agent to which the third transfection vector confers resistance,
d) cultivating said transfected CHO cell in a medium in the presence of said first and said second and third eukaryotic selection agent, under conditions suitable for the expression of said second, and/or third nucleic acid, and
e) recovering said secreted heterologous immunoglobulin from the cultivation medium and thereby producing a heterologous immunoglobulin in a CHO cell which is secreted to the cultivation medium;
wherein said resultant CHO cell is stable in the absence of any or all selection agents, as used in the previous steps, for up to generation 60.

### Claim 2 (depends on 1)

The method of claim 1, wherein said CHO cell is selected from the group consisting of a CHO K1 cell, a CHO DG44 cell, a CHO XL99 cell, a CHO DXB11 cell, and a CHO DP12 cell; and wherein further the heterologous immunoglobulin is selected from the group consisting of an anti-AB antibody, an anti P-selection antibody, an anti-IL-13R or antibody and an anti-CD4 antibody conjugate.

### Claim 3 (depends on 2)

The method of claim 2, wherein said second and/or third nucleic acid contains hybrid intronic nucleic acid sequence.

### Claim 4 (depends on 2)

The method of claim 2, characterized in that said first transfection vector and said second transfection vector differ only in the nucleic acid conferring resistance to said eukaryotic selection agent.

### Claim 5 (depends on 2)

The method of claim 2, wherein step c) and step d) are performed in the same medium.

### Claim 6 (depends on 5)

The method of claim 5, wherein said medium is selected form the group consisting of a serum-free medium, a serum-free medium supplemented with defined animal-derived components, an animal-derived component free medium, a protein-free medium, a protein-free medium supplemented with defined animal-derived components, a defined protein-free medium, and a chemically defined medium.

### Claim 7 (depends on 2)

The method of, claim 2, wherein the cultivating of step d) is either in the presence of the eukaryotic selection agents in a volume of less than 500 liter or said cultivating is in the absence of said eukaryotic selection agents in a volume of 500 liter or more, and that the recovering of the secreted heterologous immunoglobulin is from the cultivation medium without said eukaryotic selection agents.

### Claim 8 (depends on 2)

The method of claim 2, characterized in that the productivity of said CHO cells is over 40 generations not less than 70% and not more than 130% of the productivity after 10 generations of cultivation as split-batch cultivation.

### Claim 9 (depends on 8)

The method of claim 8, characterized in that the productivity of said CHO cell is at least 1.5 g/I of said heterologous immunoglobulin within 21 days as fed-batch cultivation.

### Claim 10 (depends on 1)

The method of claim 1, characterized in that said method further comprises:
f) purifying said heterologous immunoglobulin with one or more chromatographic steps.

### Claim 11 (depends on 10)

The method of claim 10, characterized in that said transfected CHO cell of step c) has
a doubling time of 150% or less of the doubling time of the CHO cell selected in substep (ii),
a volumetric yield of at least 125% compared to the volumetric yield of the CHO cell selected in (ii).