PROTEIN EXPRESSION FROM MULTIPLE NUCLEIC ACIDS

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 add 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, h) 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

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, which is adapted to growth in suspension culture, adapted to growth in serum-free medium, mycoplasma free, b) providing a nucleic acid comprising a prokaryotic origin of replication, a first nucleic acid sequence conferring resistance to a prokaryotic selection agent, 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, c) transfecting said CHO cell, wherein said transfecting comprises the following steps in the following order: (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, d) cultivating said transfected CHO cell in a medium in the presence of said first and said second 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.

2. The method of claim 1, characterized in that said CHO cell is a CHO K1 cell, or a CHO DG44 cell, or a CHO XL99 cell, a CHO DXB11 cell, and ora CHO DP12 cell.

3. The method of claim 1, characterized in that said second and/or third nucleic acid contains hybrid intronic nucleic acid sequence.

4. The method of claim 1, 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.

5. The method of claim 1, characterized in that said method further comprises: after step b) a step b1): b1) providing a nucleic acid comprising a prokaryotic origin of replication, a first nucleic acid sequence conferring resistance to a prokaryotic selection agent, 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, and further comprises after step c) (iv) the following steps (v) and (vi): (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, and further comprises in step d) that said medium for cultivating said transfected CHO cell further comprises a third eukaryotic selection agent.

6. The method of claim 1, characterized in that step c) and step d) are performed in the same medium.

7. The method of claim 6, characterized in that said medium is a serum-free medium, or a serum-free medium supplemented with defined animal-derived components, or a animal-derived component free medium, a protein-free medium, a protein-free medium supplemented with defined animal-derived components or a defined protein-free medium, or a chemically defined medium.

8. The method of claim 1, characterized in that in said step d) said cultivating is in the presence of the eukaryotic selection agents in a volume of less than 500 liter and that said cultivating is in the absence of said eukaryotic selection agents in a volume of 500 liter or more, and that said recovering of the secreted heterologous immunoglobulin is from the cultivation medium without said eukaryotic selection agents.

9. The method of claim 1, 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.

10. The method of claim 1, characterized in that the productivity of said CHO cell is at least 1.5 g/l of said heterologous immunoglobulin within 21 days as fed-batch cultivation.

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

12. The method of claim 1, characterized in that 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).

13. The method of claim 1, characterized in that said heterologous immunoglobulin is an anti-A antibody.

14. The method of claim 1, characterized in that said heterologous immunoglobulin is an anti-P-selection antibody.

15. The method of claim 1, characterized in that said heterologous immunoglobulin is an anti-IL-13R antibody.

16. The method of claim 1, characterized in that said heterologous immunoglobulin is an anti-CD4 antibody conjugate.

17. A CHO cell obtainable with the following method: a) providing a CHO cell, which is adapted to growth in suspension culture, adapted to growth in serum-free medium, mycoplasma free, b) providing a nucleic acid comprising a prokaryotic origin of replication, a first nucleic acid sequence conferring resistance to a prokaryotic selection agent, 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, c) transfecting said CHO cell, wherein said transfecting comprises the following steps in the following order: (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.

18. An anti-A antibody produced with a method of claim 1.

19. An anti-P-Selection antibody produced with a method of claim 1.

20. An anti-IL-13R antibody produced with a method of claim 1.

21. An anti-CD4 antibody conjugate produced with a method of claim 1.

Description

DESCRIPTION OF THE FIGURES

[0143] FIG. 1 Annotated plasmid map of plasmid p5128.

[0144] FIG. 2 Annotated plasmid map of plasmid p5137.

[0145] FIG. 3 Annotated plasmid map of plasmid p5151.

[0146] FIG. 4 Annotated plasmid map of plasmid p5057.

[0147] FIG. 5 Annotated plasmid map of plasmid p5069.

[0148] FIG. 6 (A) Antibody titers of clones obtained after subcloning with limited dilution and of clones obtained with the method according to the invention; X-axis: (1) G24, (2) limited dilution, (3) method according to the invention; Y-axis: immunoglobulin concentration [g/ml]. [0149] (B) Specific production rates of clones obtained after subcloning with limited dilution and of clones obtained with the method according to the invention; X-axis: (1) G24, (2) limited dilution, (3) method according to the invention; Y-axis: specific production rate [pg/d*cell].

[0150] FIG. 7 SDS-Page after protein-A HPLC purification of the antibody. For the four samples 35-45, 37-65, 39-4 and 43-46 two bands are visible, the upper being the heavy chain, the lower being the light chain. Sample 25g7 is a control antibody with antibody-related side products (above the heavy chain and between heavy and light chain). Samples: (1) Molecular weight marker, (2) 35-45, (3) 37-65, (4) 39-4, (5) 43-16), (6) 25g7, (7) Reference antibody, (8) Medium 25x,

[0151] FIG. 8 Annotated plasmid map of plasmid p6311.

[0152] FIG. 9 Annotated plasmid map of plasmid p5321.

EXAMPLES

[0153] Materials & Methods

[0154] General information regarding the nucleotide sequences of human inununoglobulins light and heavy chains is given in: Kahat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed, Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Amino acids of antibody chains are numbered according to EU numbering (Edelman, G. M., et al., Proc. Natl. Acad. Sci. USA 63 (1969) 78-85; Kahat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., (1991)).

[0155] Recombinant DNA Techniques:

[0156] Standard methods were used to manipulate DNA as described in Sambrook, J., et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold. Spring Harbor, N.Y., 1989. The molecular biological reagents were used according to the manufacturer's instructions.

[0157] Gene Synthesis:

[0158] Desired gene segments were prepared from oligonucleotides made by chemical synthesis. The 100-600 bp long gene segments, which are flanked by singular restriction endonuclease cleavage sites, were assembled by annealing and ligation of oligonucleotides including PCR amplification and subsequently cloned into the pCR2.1-TOPO-TA cloning vector (Invitrogen Corp., USA) via A-overhangs or pPCR-Script Amp SK(+) cloning vector (Stratagem Corp., USA). The DNA sequence of the subcloned gene fragments were confirmed by DNA sequencing.

[0159] Protein Determination:

[0160] Protein concentration was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence.

[0161] Antibody Titer Determination:

[0162] Antibody titers were determined either by anti-human Fc ELISA or by Protein A chromatography using the autologous purified antibody as a reference.

[0163] SDS-PAGE

[0164] LDS sample buffer, fourfold concentrate (4): 4 g glycerol, 0.682 g TRIS-Base, 0.666 g TRIS-hydrochloride, 0.8 g LDS (lithium dodecyl sulfate), 0.006 g EDTA (ethylene diamin tetra acid), 0.75 ml of a 1% by weight (WIW) solution of Servo Blue G250 in water, 0.75 ml of a 1% by weight (w/w) solution of phenol red, add water to make a total volume of 10 ml.

[0165] The culture broth containing the secreted antibody was centrifuged to remove cells and cell debris. An aliquot of the clarified supernatant was admixed with volumes (v/v) of 4 LDS sample buffer and 1/10 volume (v/v) of 0.5 M 1,4-dithiotreitol (DTT). Then the samples were incubated for 10 min. at 70 C. and protein separated by SOS-PAGE. The NuPAGE Pre-Cast gel system (Invitrogen Corp.) was used according to the manufacturer's instruction. In particular, 10% NuPAGE Novex Bis-TRIS Pre-Cast gels (pH 6.4) and a NuPAGE MOPS running buffer was used.

[0166] Western Blot

[0167] Transfer buffer: 39 mM glycine, 48 mM TRIS-hydrochloride, 0.04% by weight (w/w) SOS, and 20% by volume methanol (v/v)

[0168] After SDS-PAGE the separated antibody chains were transferred electrophoretically to a nitrocellulose filter membrane (pore size: 0.45 m) according to the Semidry-Blotting-Method of Burnette (Burnette, W. N., Anal. Biochem, 112 (1981) 195-203).

Example 1

[0169] Expression Vector for Expressing an Anti-A Antibody

[0170] An example (preferably monoclonal) antibody for which a cell line for expression can he obtained according to the current invention is an antibody against the amyloid -A4 peptide (anti-A antibody). Such an antibody and the corresponding nucleic acid sequences are, for example, reported in WO 2003/070760 or US 2005/0169925 or in SEQ ID NO: 1 to 12.

[0171] The anti-A antibody expressing Chinese hamster ovary (CHO) cell line was generated by three successive complete transfections and selection campaigns.

[0172] A genomic human -light chain constant region gene segment (C-kappa, C.sub.L) was added to the light chain variable region of the anti-A antibody, while a human 1-heavy chain constant region gene segment (C.sub.H1-Hinge-C.sub.H2-C.sub.H3) was added to the heavy chain variable region of the anti-A antibody. The complete -light and 1-heavy chain antibody genes were then joined with a human cytomegalovirus (HCMV) promoter at the 5-end and a human immunoglobulin polyadenylation signal sequence at the 3-end.

[0173] a) Heavy Chain Expression Cassette

[0174] The transcription unit of the anti-AP antibody heavy chain is composed of the following elements: [0175] the immediate early enhancer and promoterfrom the human cytomegalovirus, [0176] a 5-untranslated region derived from a human antibody germline gene, [0177] the anti-A antibody heavy chain variable domain including a signal sequence derived from a human antibody germline gene, [0178] a human/mouse heavy chain hybrid intron 2 including the mouse Ig heavy chain enhancer element (see e.g. (Neuberger, M. S., EMBO J. 2 (1983) 1373-1378), [0179] the genomic human 1-heavy chain gene constant region, [0180] the human immunoglobulin 1-heavy chain polyadenylation (poly A) signal sequence, [0181] the unique restriction sites AscI and SgrAI at the 5- and 3-end, respectively.

[0182] b) Light Chain Expression Cassette

[0183] The transcription unit of the anti-A antibody light chain is composed of the following elements: [0184] the immediate early enhancer and promoter from the human cytomegalovirus (HCMV), [0185] a 5-untranslated region derived from a human antibody germline gene, [0186] the anti-A antibody light chain variable region including a signal sequence derived from a human antibody germline gene, [0187] a human/mouse -light gene hybrid intron 2 including the mouse Ig -light chain enhancer element (Picard and Schafftier, A lymphocyte-specific enhancer in the mouse immunoglobulin kappa gene. Nature 307(1984) 80-82), [0188] the human -light gene constant region (C-kappa), [0189] the human immunoglobulin -polyadenylation (poly A) signal sequence, [0190] the unique restriction sites Sse8387 and FseI at the 5- and 3-end, respectively.

[0191] c) Expression Plasmids 5128, 5137, and 5151

[0192] For expression and production of the anti-A antibody the light and heavy chain expression cassettes were placed on a single expression vector (heavy chain upstream of light chain in clockwise orientation). Three identical expression vectors were generated differing only in the selectable marker gene included, in particular, in the gene conferring resistance to the selection agent neomycin, hygromycin, or puromycin. The vectors also include a mouse DHFR gene which was not used for selection or amplification.

[0193] The expression vectors contain beside the light and heavy chain expression cassette the following dements: [0194] a selectable marker (either a neomycin, hygrornycin or purornycin resistance gene), [0195] an origin of replication allowing for the replication of the plasmid in E. coli, [0196] a beta-lactaxuase gene which confers ampicillin resistance in E. coli, [0197] a mouse derived DHFR gene.

[0198] The plasmid map of the expression vector 5128 containing a hygrornycin selectable marker gene is shown in FIG. 1. The plasmid map of the expression vector 5137 containing a neomycin selectable marker gene is shown in FIG. 2. The plasmid map of the expression vector 5151 containing a puromycin selectable marker gene is shown in FIG. 3.

Example 2

[0199] Transfection and Selection of a CHO Cell Expressing an Anti-A Antibody

[0200] Parent CHO-KI cells, pre-adapted to growth in serum-free suspension culture in synthetic animal component free ProCHO4 medium (Cambrex Corp.) containing 8 mM glutamine and 1 HT supplement (Gibco/Invitrogen) were used as host cell line. This supplemented ProCHO4 medium is designated in the following as ProCHO4-complete medium. The adherent growing CHO-KI parent cell line was received from ATTC as ATCC CCL-61.

[0201] The preadapted parent host cells were propagated in suspension in synthetic, animal component-free ProCHO4-complete medium under standard humidified conditions (95%, 37 C., and 5% CO.sub.2). On regular intervals depending on the cell density the cells were splitted into fresh medium. The cells were harvested by centrifugation in the exponential growth phase, washed once in sterile Phosphate Buffered Saline (PBS) and resuspended in sterile PBS.

[0202] Prior to transfection the anti-A antibody expressing plasmids were linearized within the -lactamase gene (E. coli ampicillin resistance marker gene) using the restriction endonuclease enzyme PvuI or AviII. The cleaved DNA was precipitated with ethanol, dried under vacuum, and dissolved in sterile PBS.

[0203] In general, for transfection, the (parent or already transfected) CHO cells were electmporated with 20-50 g linearized plasmid DNA per approximately 10.sup.7 cells in PBS at room temperature. The electroporations were performed with a Gene Pulser XCell electroporation device (Bio-Rad Laboratories) in a 2 mm gap cuvette, using a square wave protocol with a single 180 V pulse. After transfection, the cells were plated out in ProCHO4-complete medium in 96-well culture plates. After 24 h of growth a solution containing one or more selection agents were added (ProCHO4-complete selection medium; G418: 400 g/ml; hygrnmycin 600 g/ml; puromycin: 8 g/ml). Once a week the ProCHO4-complete selection medium was replaced. The antibody concentration of the anti-A antibody was analyzed with an ELISA assay specific for human IgG1 in the culture supernatants.

[0204] For selection of high-yield anti-A antibody production cell lines the productivity was tested in ProCHO4-complete selection medium after propagation in 6-well culture plates, T-flasks and/or Erlenmeyer shake flasks using an anti-human. IgG1 ELISA and/or analytic Protein A HPLC.

[0205] Subclones were obtained by two methods, Limiting Dilution (ID) and Fluorescence Activated Cell Sorting (FACS).

[0206] Limiting Dilution:

[0207] For limiting dilution cells were plated out in ProCHO4-conditioned medium (consisting of 50% (v/v) fresh ProCHO4-complete selection medium and 50% (v/v) ProCHO4-complete conditioned selection medium derived from the cells to be propagated) at a cell density of 0.5-2 cells per 0.1 ml medium per well of a 96-well culture plate. Once a week the medium was replaced by FroCHO4-complete selection medium. The antibody concentration of the anti-A antibody was analyzed by an ELISA assay specific for human IgG1 in the culture supernatants.

[0208] Single Cell Deposition by Flow Cytometry Including Identification and Isolation of Clones:

[0209] The identification and isolation of stably transfected clones was performed with the aid of a cell surface labeling technique using fluorescently tagged Protein A that binds to secreted but still membrane-attached antibodies. The fluorescence intensity of the stained cells was used as criterion for cell selection.

[0210] In the case of fluorescence activated cell sorting the electroporated population of cells were directly seeded into T-flasks in ProCHO4-complete medium. The appropriate selection agent or agents (G418, hygromycin, and/or puromycin) was/were added to the culture one day after transfection and the transfectant pool was expanded.

[0211] Cells from the expanded transfectant pool were first treated with Accumax (PAA Laboratories) for 15 minutes at 37 C. and then passed through a 40 M nylon mesh to remove remaining large cell aggregates. The cells were collected by centrifugation, resuspended in PBS containing 5% FCS (Gibco/Invitrogen) at a cell density of 10.sup.6 to 10.sup.7 cells/nil and incubated for 20 minutes on ice. Thereafter, the cells were stained with 10 ng/ml Protein A Alexa Fluor 488 (Molecular Probes Inc.) in a volume of 8 ml FCS-PBS for 30 minutes on ice in the dark. Afterwards, the cells were washed once with 5% FCS-PBS and once with ProCHO4 medium containing 8 mM Ultra. Glutamine (Cambrex Corp.), 1 HT supplement and 5% FCS. Finally the cells were resuspended in the supplemented ProCHO medium used for washing at a cell density of 10.sup.6 to 10.sup.7 cells/ml and transferred to a BD FACSAria cell sorter (BD Biosciences).

[0212] Single cells were sorted by flow cytometry and deposited in wells of 96-well culture plates containing of ProCHO4-conditioned medium. The selected and deposited cells encompassed cells with the top 10%, 7%, or 4% of fluorescence intensity of the gated live cells. After 48 hours ProCHO4 complete selection medium containing the appropriate selection agent in 2-fold concentration was added to each well. Once a week the medium was replaced with ProCHO4-complete selection medium. The antibody concentration of the anti-All antibody was analyzed with an ELISA assay specific for human IgG1 in the culture supernatants.

[0213] Transfection and Selection Steps:

[0214] For the first transfection and selection step the plasmid 5137 has been used. Plasmid 5137 has been transfected with electroporation into parent canine adapted to growth in ProCHO4-complete medium. The transfected cells were cultivated in ProCHO4-complete medium supplemented with up to 700 g/ml G418 in 96 well plates. The antibody concentration in the culture supernatants was evaluated by an anti-human IgG1 ELISA. Approximately 1000 clones have been tested and the selected of them were further cultivated in 24-well plates, 6-well plates and subsequently in shaker flasks. The growth and productivity of approximately 20 clones was assessed in static and suspension cultures by anti-human IgG1 ELISA and/or analytic protein A HPLC. The best clone (best clone does not denote the most productive clone it denotes the clone with the best properties for the further steps) was subcloned by limited dilution in ProCHO4-conditioned medium supplemented with 700 g/ml G418. The selected clone was named 8C8.

[0215] For the second transfection and selection step the plasmid 5128 has been used. Plasmid 5128 has been transfected with electroporation into cell line clone 8C8 cultivated in ProCHO4-complete medium supplemented with 700 g/ml G418. The transfected cells were expanded for about two to three weeks in ProCHO4-conditioned medium supplemented with 200 g/ml G418 and 300 g/ml hygromycin (ProCHO4-double selection medium). Single antibody secreting cells were identified and deposited on the basis of their fluorescence intensity after staining with a Protein A Alexa Pluor conjugate by PACS analysis. The deposited cells were cultivated in ProCHO4-double selection medium in 96 well plates. The antibody concentration in the culture supernatants was evaluated by an anti-human IgGI ELISA. Approximately 500 clones have been tested and the selected of them were further cultivated in 24-well plates, 6-well plates and subsequently in shaker flasks. The growth and productivity of approximately 14 clones was assessed in static and suspension cultures by anti-human IgGI ELISA and/or analytic Protein A HPLC. The selected clone was named 4F5.

[0216] For the third transfection and selection step the plasmid 5151 has been used. Plasmid 5151 has been transfected with electroporation into cell line clone 4F5 cultivated in ProCHO4-double selection medium. The transfected cells were expanded for about two to three weeks in ProCHO4-triple selection medium (ProCHO4-conditioned medium supplemented with 200 g/ml G418 and 300 g/ml hygromycin and 4 g/ml puromycin). Single antibody secreting cells were identified and deposited on the basis of their fluorescence intensity after staining with a Protein A Alexa Fluor conjugate by PACS analysis. The deposited cells were cultivated in ProCHO4-triple selection medium in 96 well plates. The antibody concentration in the culture supernatants was evaluated by an anti-human IgGI ELBA. Approximately 500 clones have been tested and the selected of them were further cultivated in 24-well plates, 6-well plates and subsequently in shaker flasks. The growth and productivity of approximately 10 clones was assessed in static and suspension cultures by anti-human IgGI ELISA and/or analytic protein A HPLC. The selected done was named 20F2.

[0217] Clone 20F2 has been selected based on his growth, productivity, and product quality characteristics after growth in fed-batch suspension culture in ProCHO4-triple selection medium, i.e. in the concomitant presence of the three selecting agents G418, hygromycin, and puromycin.

[0218] Clone Characteristics:

[0219] As can be seen from the following table the doubling time and cell density after three days of cultivation were comparable when the basic cell line CHO-K1 (wild-type) and the selected clones are compared.

TABLE-US-00001 TABLE 1 Growth characteristics Doubling Starting Cell density Viability time cell density at day 3 at day 3 Clone [h] [10.sup.6 cells/ml] [10.sup.6 cells/ml] [%] CHO-K1 22-23 3 18-20 97-98 (wild-type) 8C8 26-28 3 12-15 96-98 4F5 22-24 3 24-27 96-97 20F2 24-26 2 23-26 97-98

Example 3

[0220] Stability of Clone 20F2 Expressing an Anti-A Antibody

[0221] Stability of growth and product formation was evaluated in sequential cell subculture over a time period of 60 days (about 60 generations) in the presence and absence of the selection agents (with and without antibiotics). The cultivation was performed as described above.

TABLE-US-00002 TABLE 2 Characteristics of clone 20F2. Clone 20F2 cultivation in the cultivation in presence of three the absence of Parameter selection agents selection agents Mean value viability [%] 97 97 Mean value doubling time [h] 27 26 Mean value SPR [pg/c/d] 11 9

[0222] Following extensive passage (up to generation 60) no evidence was obtained indicating that the anti-A antibody producing done 20F2 was unstable with respect to cell growth and product formation in the presence or absence of the three selection agents, respectively.

Example 4

[0223] Expression Vector for Expressing an Anti-P-Selectin Antibody

[0224] Another example (preferably monoclonal) antibody for which a cell line for expression can be obtained according to the current invention is an antibody against the human P-Selectin glycoprotein (anti-P-Seiectin antibody). Such an antibody and the corresponding nucleic acid sequences are for example described in WO 2005/100402, or US 2005/0226876 or SEQ ID NO: 13 to 18.

[0225] The anti-P-Selectin antibody expressing Chinese hamster ovary cell line was generated by two successive complete transfections and done selection campaigns.

[0226] A genomic human kappa-light chain constant region gene segment (C-kappa) was added to the light chain variable region of the anti-P-Selectin antibody, whereas a human gamma 4-heavy chain constant region gene segment (C.sub.H}Hinge-C.sub.H2-C.sub.H3) was added to the heavy chain variable region of the anti-P-Selectin antibody. The complete kappa-light and gamma 4-heavy chain antibody genes were then joined with a he cytomegalovirus immediate early promoter and enhancer (CMV IE) at the 5-end and the Simian Virus 40 early polyadenylation (SV 40 early poly A) signal sequence at the 3-end.

[0227] a) Heavy Chain Expression Cassette

[0228] The transcription unit of the anti-P-Selectin antibody heavy chain is composed of the following elements: [0229] the immediate early enhancer and promoter from the human cytomegalovirus (CMV 1E), [0230] a 5-untranslated region (5 UTR), [0231] the coding sequence for the anti-P-Selectin antibody gamma 4-heavy chain including a signal peptide in an intron-exon gene structure, [0232] the SV 40 early poly A signal sequence.

[0233] b) Light Chain Expression Cassette

[0234] The transcription unit of the anti-P-Sel ctin antibody light chain is composed of the following elements: [0235] the immediate early enhancer and promoter from the human cytomegalovirus (CMV IE), [0236] a 5-untranslated region (5 UTR), [0237] the coding sequence for the anti-P-Selectin kappa-light chain in an intron-exon gene structure, [0238] the SV 40 early poly A signal sequence.

[0239] c) Expression Plasmids 5057 and 5069

[0240] For the expression and production of the anti-P-Selectin antibody the light and heavy chain expression cassettes were placed on a single expression vector (light chain upstream of heavy chain). Two identical expression vectors were generated differing only in the selectable marker gene included, in particular, the murine dihydrofolate red uctase (DHFR) gene or a neomycin resistance gene.

[0241] The expression vectors contain beside the light and heavy chain expression cassette the following elements: [0242] a selectable marker, either the murine DHFR gene or a gene conferring resistance to the selection agent neomycin under the control of the SV40 early promoter and origin, [0243] an origin of replication allowing for the replication of the plasmid in E. coli taken from pUC19 (pUC origin), [0244] a beta-lactarriase gene which confers ampicillin resistance in E. coli.

[0245] The plasmid map of the expression vector 5057 containing the murine DHFR marker gene is shown in FIG. 4. The plasmid map of the expression vector 5069 containing a neomycin selectable marker gene is shown in FIG. 5.

Example 5

[0246] Transfection and Selection of a CHO Cell Line Expressing an Anti-P-Selectin antibody

[0247] CHO-K1 cells, pre-adapted to growth in serum-free suspension culture in protein-free HyQ SFM4CHO medium (Hyclone, Cat. No. SH30549) supplemented with defined animal-derived components (cholesterol from ovine wool and cod-liver oil) were used as the host cell line. The cells were propagated in shake flasks in protein-free HyQ SFM4CHO medium under standard humidified conditions (95%, 37 C., and 5% CO.sub.2) and under constant agitation at 150 rpm/min. Depending on the cell density the cells were split into fresh medium.

[0248] The adherent CHO-K1 cell lines had been obtained from the American Type Culture Collection as ATCC CCL-61.

[0249] First Transfection and Selection

[0250] Prior to transfection the expression plasmid 5057 was linearized within the beta-lactamase gene using the restriction enzyme PvuI. The cleaved DNA was purified using QiaQuick spin columns (Qiagen) according to the manufacturer's recommendations.

[0251] Transfection was carried out by electroporation using Gene Pulser XCell (BIO-RAD) and 0.2 cm-cuvettes (BIO-RAD, Cat. No. 165-2086). For transfection 10.sup.6 to 10.sup.7 CHO-K1 cells were harvested by centrifugation, resuspended in PBS, transferred to the cuvette and mixed with 20-50 g linearized plasmid DNA. The cells were exposed to a single square wave pulse (160 V, 15 ms) and subsequently diluted in HyQ SFM4CHO medium to a density of approx. 410.sup.5 cells/ml and seeded in a T75 cell culture flask. After 48 hours of propagation without the supplementation of a selection agent, the cells were diluted in HyQ SEM4CHO medium supplemented with 200 nM MTX to a density of 10.sup.4 to 10.sup.5 cells/ml and seeded in 96-well plates with 3-7000 cells per well. After approx. two weeks, fresh medium was added per well and after additional two weeks the culture medium was completely replaced by fresh medium. Four days later the culture supernatants were tested for antibody production by anti-human Fc ELISA. In total approximately 600 clones were screened.

[0252] 45 clones with antibody titers of more than 10 g/ml were picked and transferred to 48-well plates. The clones were expanded to shaker flasks over additional passages and subsequently transferred to serum free production medium for the final productivity assessment. A 125 ml shaker flask was inoculated with 10.sup.5 to 10.sup.6 cells/ml in medium supplemented with 200 nM MTX. Viable cell density and viability were monitored over one week Antibody titers were measured by Protein A chromatography on the final day. Based on these data, clone G24 was selected for further development. G24 reached a maximal viable cell density of 3.310.sup.6 cells/ml. The antibody titer was 402 g/ml. The average specific production rate (SPR) was 28 pg/(cell*d).

[0253] Second Transfection and Selection:

[0254] Clone G24 was subjected to a second transfection. For the second transfection plasmid 5069 was used. Linearization and purification of the plasmid as well as electroporation of G24 were performed as described for the first transfection. After 48 hours of propagation without selection pressure, the cells were diluted in HyQ SFM4CHO medium supplemented with 200 nM MTX and 400 g/ml G418 to a density of 10.sup.3 to 10.sup.4 cells/ml and seeded in 96-well plates with 500 cells per well. After approx. two weeks, fresh medium was added per well and after an additional week the culture medium was completely replaced by fresh medium. Four days later the culture supernatants were tested for antibody production by anti-human Fc ELISA. In total approximately 220 clones were screened.

[0255] Then 13 clones with antibody titers of more than 150 g/ml were picked and transferred to 24-well plates. The clones were expanded to shaker flasks over additional passages and subsequently transferred to serum free production medium for the final productivity assessment. A shaker flask was inoculated with 10.sup.5 to 10.sup.6 cells/ml in 50 ml medium supplemented with 200 nM MTX and 400 g/ml G418. Viable cell density and viability were monitored over one week. Antibody titers were measured by Protein A chromatography on the final day. Based on these data, clone G24_x6 was considered the best clone. G24_x6 reached a maximal viable cell density of 3.010.sup.6 cells/ml. The antibody titer was 685 g/ml. The average specific production rate (SPR) from was 48 pg/(cell*d.).

[0256] Limiting Dilution:

[0257] To compare the method according to the invention with simple subcloning with respect to their effect on productivity we subjected done G24 to limited dilution or single cell deposition in 96-well plates.

[0258] For limiting dilution the cells were seeded in 96-well plates in HyQ SFM4CHO medium supplemented with 50% (v/v) conditioned medium, 10% PCS and 200 nM MTX at 0.5 cells/well. Alternatively 1 cell/well was deposited in 96-well plates by PACS. After 10 days, fresh HyQ SFM4CHO medium, 200 nM MTX without FCS was added per well and after an additional week the culture medium was completely replaced by HyQ SFM4CHO medium, 200 nM MTX. Four days later the culture supernatants were tested for antibody production by anti-human Fc ELISA. In total approximately 230 clones were screened.

[0259] Eleven subclones with antibody titers of more than 130 g/ml were transferred to 24-well plates. After passages in 6-well plates, the clones were transferred to shaker flasks and subsequently transferred to serum free production medium for the final productivity assessment. A shaker flask was inoculated with 10 to 10.sup.6 cells/ml in medium supplemented with 200 nM MTX. Viable cell density and viability were monitored over one week. Antibody titers were measured by Protein A chromatography on the final day. Based on these data G24_13 was considered the best done. G24_13 reached a maximal viable cell density of 3.610.sup.6 cells/ml. The antibody titer was 472 g/ml. The average the specific production rate (SPR) was 31 pg/(cell*d).

[0260] Table 3 summarizes the productivity data of best performing subclone G24_13 and the best performing done G24_x6 obtained with the method according to the invention in comparison to their parental done G24. With the method according to the invention a clone with volumetric and specific productivity increased by more than 50% can he obtained whereas after subdoning only a minor increase of both parameters was observed.

[0261] FIG. 6 shows an overview of the volumetric (A) and specific (B) productivities of all subclones of G24 that had been investigated in shake flasks. As can be seen, the average volumetric and specific productivity of the clones obtained with the method according to the invention was significantly higher than after subcloning.

TABLE-US-00003 TABLE 3 Productivity of the best producing clones compared to the parental clone G24. G24_x6 (method G24_13 according to G24 (Subclone) the invention) Antibody concentration 402 472 685 in the supernatant [g/ml] SPR pg/(cell*d)] 28 31 48 Max. cell density 33 36 30 [10.sup.5/ml]

[0262] Clone Characteristics:

[0263] As can be seen from the following table the doubling time and the cell density after three days of cultivation were comparable when the one-time transfected cell line G24 and the selected clones are compared.

TABLE-US-00004 TABLE 4 Growth characteristics Doubling Starting Cell density Viability time cell density at day 3 at day 3 Clone [h] [10.sup.6 cells/ml] [10.sup.6 cells/ml] [%] G24 29 0.3 0.7 91 G24_13 27 0.3 2.0 91 G24_x6 24 0.3 2.5 93

Example 6

[0264] Transfection and Selection of a CHO Cell Line Expressing an Anti-P-Selectin Antibody

[0265] CHO-PG44 cells pre-adapted to growth in serum-free suspension culture in protein-free. HyQ SFM4CHO medium (Hyclone, Cat. No. SH30549) were used as the host cell line. The host cell line was cultured in commercial medium HyQ SFM4CHO-utility (Hyclone, Cat. No. SH30516) during transfections, screening and subcloning steps.

[0266] First Transfection and Selection

[0267] Prior to transfection the expression plasmid 5057 (FIG. 4) was linearized within the beta-lactarnase gene using the restriction enzyme PvuI.

[0268] The transfection of the host cell line was performed by nucleotransfection provided by AMAXA (Nucleofector Kit T, Cat. No. VCA-1002. Transfection program U-17). Cells were cultured in medium supplemented with 10% fetal calf serum for 48 h after transfection.

[0269] Transfected cells were plated on 96-well plates with 1000 cells per well in medium supplemented with 10% fetal calf serum in the presence of 40 nM methotrexate (MTX) as selection agent and incubated for approx. three weeks.

[0270] Antibody concentration was determined by ELISA in the supernatant of the 96-well plates. About 400 primary clones were screened. Twenty-four clones with the highest antibody productivity were transferred to 24-well plates and cultivated in the presence of the selection agent without supplementation with fetal calf serum.

[0271] Product quality was analyzed by Western Blotting detecting light and heavy antibody chains. Nine clones which showed the highest productivity and which expressed antibody without detectable antibody derived side products (Western blot) were expanded into shake flasks.

[0272] Productivity was analyzed in batch shake flasks after 7 and 10 days of incubation. Product quality was assessed by SOS-PAGE after Protein-A HPLC purification (FIG. 7). Best product concentration was reached with done 43-16. Best specific productivity per cell was achieved with clone 35-45. Both clones showed no detectable side products in the SOS-PAGE. Both clones were selected for subcloning by limiting dilution.

[0273] Parental clones 35-45 and 43-16 were subcloned by limiting dilution on 96-well plates in commercial HyQ medium supplemented with 5% (v/v) fetal calf serum in the presence of 20 nM MTX. After 20 days of incubation antibody production was screened by ELISA. Best subdones in terms of productivity were expanded to shake flasks and subsequently transferred to serum free production medium for the final productivity assessment. The two best subclones, 35-45-F2 and 43-16-A10, of the parental clones 35-45 and 43-16 were assessed in standard batch shake flask assay. Productivity was 270 g/ml and 185 g/ml after 7 days and 337 g/ml and 343 g/ml after 10 days, respectively.

[0274] Second Transfection and Selection:

[0275] Subclone 43-16-A10 was transfected with the expression vector p5069 (FIG. 5) using the nudeofection method (Amaxa Nudeofector Kit T, VCA-1002, Transfection program U-17). The second transfection was also carried out in Hydone medium: HyQ SFM4CHO-utility (Cat. No. SH30516) supplemented with 10% fetal calf serum and 20 nM MTX. Two days after the second transfection cells were transferred to 96-well plates with 1000 cells per well. As second selection agent 250 g/ml G418 was added.

[0276] After cultivation for two weeks more than 2000 primary wells were screened by antibody titer determination by anti-human Fc ELISA. Fifty dories with highest productivity were transferred into 24-well plates and screened a second time by anti-human Fc ELISA three days later. All clones were transferred to 6-well plates and screened by anti-human Fc ELISA three days later. The six clones with the best productivity were directly subcloned from the 6-well plate stage.

[0277] Limiting Dilution:

[0278] The best parental clones of the second transfection and selection round 43-16A10_S1, 43-16A10_S13, 43-16A10_S14, 43-16A10_S19, 43-16A10_S24, 43-16A10_S43 were subdoned by limiting dilution. The product quality of the twelve best subclones was assessed in SDS-PAGE and Western-Blotting from the 24-well stage. No unwanted antibody related side products were detected.

[0279] Three suhclones, 43-16-A10-S1-16, 43-16-A10-S24-11, and 43-16-A10-S43-14, were selected according to their productivity in 6-well plates for the expansion in shake flasks. They were transferred to serum free production medium for the final productivity assessment. Their productivity was compared to the subclone after the first transfection, clone 43-16-A10. The productivity was increased twofold for two of the clones after the second transfection and selection, 43-16-A10-S1-16 and 43-16-A10-S24-11, from 221 g/ml after 7 days in the batch shake flask to 436 g/ml and 407 g/ml, respectively. After 10 days incubation in the batch shake flask the productivity increased from 306 g/ml to 683 g/ml and 446 g/ml, respectively.

[0280] The specific productivity per cell increased as well from 17 pg/cell/day for the done 43-16-A10 after the first transfection to 40 pg/cell/day for the first transfected clone 43-16-A10-S1-16 and to 33 pg/cell/day for the second transfected done 43-16-A10-S24-11. The doubling time was not affected by the second transfection. The doubling time for the clone 43-16-A10 after the first transfection was 33 h and it was 32 h for both clones 43-16-A10-S1-16 and 43-16-A10-S24-11.

Examle 7

[0281] Expression Vetor for Expressing an Anti-IL-13R Antibody

[0282] Another example (preferably monoclonal) antibody for which a cell line for expression can be obtained according to the current invention is an antibody binding to the IL-13 Receptor alpha 1 (anti-IL-13R1 anti-IL-13R antibody). Such an antibody and the corresponding nucleic acid sequences are for example described in WO 2006/072564 or SEQ ID NO: 19 to 28.

[0283] A genomic human kappa-light chain constant region gene segment (C-kappa) was added to the light chain variable region of the anti-IL-13R antibody whereas a human gamma 1-heavy chain constant region gene segment (C.sub.H1-Hinge-C.sub.H2-C.sub.H3) was added to the heavy chain variable region of the anti-IL-13R antibody. The expression plasmid 5321 comprises an expression cassette for the anti-IL-13R antibody 1-heavy chain, and the anti-IL-13R antibody -light chain, and a nucleic add encoding the murine DHFR gene. An annotated plasmid map is shown in FIG. 9.

[0284] a) Heavy Chain Expression Cassette

[0285] The transcription unit of the anti-IL-13R antibody conjugate heavy chain is composed of the following dements: [0286] the immediate early enhancer and promoter from the human cytomegalovirus (CMV IE), [0287] a 5-untranslated region (5 UTR), [0288] the coding sequence for the anti-IL-13R antibody gamma 1-heavy chain conjugate including a signal peptide in an intron-exon gene structure, [0289] the human gamma 1-immunoglobulin polyadenylation signal sequence.

[0290] b) Light Chain Expression Cassette

[0291] The transcription unit of the anti-IL-13R antibody light chain is composed of the following elements: [0292] the immediate early enhancer and promoter from the human cytomegalovirus (CMV IE), [0293] a 5-untranslated region (5 UTR), [0294] the coding sequence for the anti-IL-13R kappa-light chain in an intron-exon gene structure, [0295] the human immunoglobulin kappa-polyadenylation signal sequence.

[0296] c) Expression Plasmids

[0297] For the expression and production of the anti-IL-13R antibody conjugate the light and heavy chain expression cassettes were placed on a single expression vector (light chain upstream of heavy chain). Two identical expression vectors were generated differing only in the selectable marker gene included, in particular, the murine DHFR gene and both the murine DHFR gene and a hygromycin resistance gene.

[0298] The expression vectors contain beside the light and heavy chain expression cassette the following elements: [0299] an origin of replication allowing for the replication of the plasmid in E. coli (pUC origin), [0300] a beta-lactamase gene which confers ampicillin resistance in E. coli.

Example 8

[0301] Transfection and Selection of a CHO Cell Line Expressing an Anti-IL-13R Antibody

[0302] For the first transfection and selection step the plasmid 5321 has been used. Plasmid 5321 has been transfected with electroporation into parent cell line adapted to growth in ProCHO4-complete medium. The transfected cells were cultivated in HyQSFMCHO-medium (HyClone) supplemented with up to 200 nM methotrexate in plates. The antibody concentration in the culture supernatants was evaluated by an anti-human IgGI ELISA. The clones have been tested and the selected of them were further cultivated in 24-well plates, 6-well plates and subsequently in shaker flasks. The growth and productivity was assessed in static and suspension cultures by anti-human IgG1 ELISA and/or analytic Protein A HPLC. The best done (best done does not denote the most productive clone it demotes the done with the best properties for the further steps) was selected. The selected done was named 200_019. Productivity was 90 g/ml with an average specific production rate of 7 pg/cell*d after 7 days of cultivation.

[0303] For the second transfection and selection step a plasmid with a DHFR and hygromycin resistance gene has been used. The plasmid has been transfected with electroporation into the selected cell line cultivated in HyQSFMCHO-medium (HyClone) supplemented with up to 200 nM methotrexate. The double selection medium contained in addition 300 g/ml hygromycine B. Single antibody secreting cells were identified and deposited on the basis of their fluorescence intensity after staining with a Protein A Alexa Fluor conjugate by FACS analysis. The selected done was named 5_17_35. Productivity was 150 g/ml with an average specific production rate of 10 pg/cell*d after 7 days of cultivation.

Example 9

[0304] Expression Vector for Expressing an Anti-CD4 Antibody Conjugate

[0305] Another example (monoclonal) antibody for which a cell line for expression can be obtained according to the current invention is an antibody against the human CD4 surface receptor (anti-CD4 antibody) which is conjugated to two to eight antifusogenic peptides. Such an antibody and the corresponding nucleic acid sequences are for example reported in PCT/EP2008/005894 or SEQ ID NO: 29 to 40.

[0306] A genomic human kappa-light chain constant region gene segment (C-kappa) was added to the light chain variable region of the anti-CD4 antibody of SEQ ID NO: 39, whereas a human gamma 1-heavy chain constant region gene segment (C.sub.H1-Hinge-C.sub.H2-C.sub.H3) was added to the heavy chain variable region of the anti-CD4 antibody of SEQ ID NO: 36. The expression piasinid 6311 comprises an anti-CD4 antibody 1-heavy chain, which is joint at the last but one C-terminal amino acid, i.e. the C-terminal lysine residue of the heavy chain is removed, with a nucleic acid encoding an antifusogenic peptide of SEQ ID NO: 41 via the peptidic glycine-serine linker of SEQ ID NO: 42, and a anti-CD4 antibody -light chain, and a nucleic acid conferring resistance to the selectable marker neomycin. An annotated plasmid map is shown in FIG. 8.

[0307] a) Heavy Chain Expression Cassette

[0308] The transcription unit of the anti-CD4 antibody conjugate hmvy chain is composed of the following elements: [0309] the immediate early enhancer and promoter from the human cytomegalovirus (CMV IE), [0310] a 5-untranslated region (5 UTR), [0311] the coding sequence for the anti-CD4 antibody gamma 1-heavy chain conjugate including a signal peptide in an intron-exon gene structure, [0312] the SAT 40 early poly A signal sequence,

[0313] b) Light Chain Expression Cassette

[0314] The transcription unit of the anti-CD4 antibody conjugate light chain is composed of the following elements: [0315] the immediate early enhancer and promoter from the human cytomegalovirus (CMV IE), [0316] a 5-untranslated region (5 UTR), [0317] the coding sequence for the anti-CD4 kappa-light chain in an intron-exon gene structure, [0318] the SV 40 early poly A signal sequence.

[0319] c) Expression Plasmids

[0320] For the expression and production of the anti-CD4 antibody conjugate the light and heavy chain expression cassettes were placed on a single expression actor (light chain upstream of heavy chain). Three identical expression vectors were generated differing only in the selectable marker gene included, in particular, a neomycin resistance gene, a puromycin resistance gene, and a hygromycin resistance gene.

[0321] The expression vectors contain beside the light and heavy chain expression cassette the following elements: [0322] an origin of replication allowing for the replication of the plasmid in E. coli taken from pUC18 (pUC origin), [0323] a beta-lactamase gene which confers ampicillin resistance in E. coli.

Example 10

[0324] Transfection and Selection of a CHO Cell Line Expressing an Anti-CD4 Antibody Conjugate

[0325] Transfection and Selection Steps:

[0326] For the first transfection and selection step the plasmid 6311 has been used. Plasmid 6311 has been transfected with electroporation into parent cell line adapted to growth in ProCHO4-complete medium. The transfected cells were cultivated in ProCHO4-complete medium supplemented with up to 700 g/ml G418 in 96 well plates. The antibody concentration in the culture supernatants was evaluated by an anti-human IgG1 ELISA. Approximately 5000 clones have been tested and the selected of them were further cultivated in 24-well plates, 6-well plates and subsequently in shaker flasks. The growth and productivity of approximately 15 clones was assessed in static and suspension cultures by anti-human IgG1 ELISA anchor analytic Protein A HPLC. The best done (best done does not denote the most productive clone it denotes the clone with the best properties for the further steps) was subcloned by limited dilution in ProCHO4-conditioned medium supplemented with 700 g/ml G418.

[0327] Subdones were obtained by two methods, Limiting Dilution (LD) and Fluorescence Activated Cell Sorting (PACS).

[0328] Limiting Dilution:

[0329] For limiting dilution cells were plated out in ProCHO4-selection medium at a cell density of 0.5-2 cells per 0.1 ml medium per well of a 96-well culture plate.

[0330] Single Cell Deposition by Flow Cytometry Including Identification and Isolation of Clones:

[0331] In the case of fluorescence activated cell sorting the electroporated population of cells were directly seeded into T-flasks in ProCHO4-complete medium. The appropriate selection agent or agents (G418, hygromycin, and/or puromycin) was/were added to the culture one day after transfection and the transfectant pool was expanded. The growth and productivity of approximately 112 clones was assessed in static and suspension cultures by anti-human IgG1 ELISA and/or analytic Protein A HPLC. The selected clone was named I-17.

[0332] For the second transfection and selection step a plasmid with a hygromycin resistance gene has been used. The plasmid has been transfected with electroporation into cell line clone I-17 cultivated in ProCHO4-complete medium supplemented with 700 g/ml G418. The transfected cells were expanded for about two to three weeks in ProCHO4-conditioned medium supplemented with 200 g/ml G418 and 300 g/ml hygromycin (ProCHO4-double selection medium). Single antibody secreting cells were identified and deposited on the basis of their fluorescence intensity after staining with a Protein A Alexa Fluor conjugate by PACS analysis. The deposited cells were cultivated in ProCHO4-double selection medium in 96 well plates. The antibody concentration in the culture supernatants was evaluated by an anti-human IgG1 ELISA. The selected done was named 24_16.

[0333] For the third transfection and selection step a plasmid with a puromycin resistance gene has been used. The plasmid has been transfected with electroporation into cell line clone 24_16 cultivated in ProCHO4-double selection medium. The transfected cells were expanded for about two to three weeks in ProCHO4-triple selection medium (ProCHO4-conditioned medium supplemented with 200 g/ml G418 and 300 g/ml hygroinycin and 4 g/ml puromycin). Single antibody secreting cells were identified and deposited on the basis of their fluorescence intensity after staining with a Protein A Alexa Fluor conjugate by PACS analysis, The deposited cells were cultivated in ProCHO4-ttiple selection medium in 96 well plates, The antibody concentration in the culture supernatants was evaluated, by an anti-human IgG1 ELISA. The selected clone was named 1_24.

[0334] Clone Characteristics:

[0335] As can he seen from the following table the doubling time and the cell density after three days of cultivation were comparable when the basic cell line CHO-K1 (wild-type) and the selected clones are compared.

TABLE-US-00005 TABLE 5 Growth characteristics Doubling Starting Cell density Viability time cell density at day 3 at day 3 Clone [h] [10.sup.6 cells/ml] [10.sup.6 cells/ml] [%] CHO-K1 22-25 3 18-22 96-98 (pre adapted) I-17 25-30 3 13-15 95-97 24_16 25-30 3 15-16 95-96 1_24 30-32 3 12-14 95-97