Production methods
The present invention provides methods of reducing the levels of a titratable selectable pressure required, the number of amplification cycles, and the time taken to generate protein expressing cell lines by altering the codons of the desired open-reading-frames. Through the use of codon adaptation for this purpose the methods of the invention consistently provide sufficient yields in faster time frames saving many weeks in cell line development activities. Furthermore the methods of the invention also generate cell lines with lower concentrations of selection and amplification agent than previously achievable. Accordingly lower levels of selection and amplification marker in the final cells lines are observed.
1. A Chinese Hamster Ovarian (CHO) cell line producing at least one therapeutic protein produced by a method comprising the steps of:
(a) transforming a CHO cell line with a polynucleotide sequence comprising (i) a sequence that encodes the therapeutic protein and has a codon adaptation index (CAI) that is equal to or greater than 0.9 as calculated using EMBOSS codon usage table Ehum.cut, and (ii) a sequence that encodes an amplifiable selection marker which is capable of providing amplification of the polynucleotide sequence;
(b) providing at least one round of amplification in the presence of a selection agent at a concentration of 50% or less than is required to achieve a cell production yield of 0.3-1.5 g/L of the heterologous therapeutic protein in extended unfed batch culture and/or a cell productivity of 10-100 pg/cell/day of the heterologous therapeutic protein in unfed batch culture, compared with a CHO cell line with a sequence that encodes the therapeutic protein and has a codon adaptation index that is less than 0.9 as calculated using EMBOSS codon usage table Ehum.cut; and
(c) selecting the cell line once the cell productivity and/or cell production yield of the therapeutic protein is reached; wherein the CHO cell line has lower levels of the amplifiable selection and amplification marker and copy number of transformed DNA compared to said cell line with a sequence that encodes the therapeutic protein and has a codon adaptation index that is less than 0.9 and is not provided with at least one round of amplification in the presence of a selection agent in accordance with step (b).
2. The CHO cell line of claim 1 comprising a vector comprising at least one expression cassette containing an open reading frame containing the polynucleotide sequence that encodes a therapeutic protein with a CAI score above 0.9, and a second expression cassette containing an amplifiable selection marker.
3. The CHO cell line of claim 1 comprising an amplified expression cassette containing an open reading frame containing the polynucleotide sequence that encodes a therapeutic protein with a CAI score above 0.9 contained within said cell line where it is operatively linked to a second expression cassette containing an amplifiable selection marker.
4. The CHO cell line of claim 1 wherein the cell productivity of the therapeutic protein is in the range of 10-100 pg/cell/day in an unfed batch culture.
5. The CHO cell line of claim 1 , wherein the therapeutic protein is an antibody with a heavy chain, and the cell line produces an antibody composition comprising less than or equal to 5% of Non-glycosylated heavy chain.
6. The CHO cell line of claim 1 , wherein the therapeutic protein is an antibody with a heavy chain, and the cell line produces an antibody composition comprising at least 95% glycosylated heavy chain.
7. The CHO cell line of claim 1 , wherein the therapeutic protein is an antibody with a heavy chain, and the cell line produces an antibody composition comprising at least 96% glycosylated heavy chain.
8. The CHO cell line of claim 1 , wherein the therapeutic protein is an antibody with a heavy chain, and the cell line produces an antibody composition comprising at least 97% glycosylated heavy chain.
9. The CHO cell line of claim 1 , wherein the therapeutic protein is an antibody with a heavy chain, and the cell line produces an antibody composition comprising at least 98% glycosylated heavy chain.
10. The CHO cell line of claim 1 , wherein the therapeutic protein is an antibody and the antibody is monoclonal.