Production methods

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).