Recombinant cell clones having increased stability and methods of making and using the same

A cell culture comprising at least 90% of stable recombinant mammalian cells expressing recombinant product, said stable recombinant cells selected in the absence of selective pressure and stable for more than 40 generations under serum- and protein-free conditions.

A method for preparing a recombinant polypeptide in a cell culture, comprising:

• multiplying a stable mammalian recombinant cell clone containing a sequence encoding the recombinant polypeptide in a serum- and protein-free medium comprising a mixture of amino acids containing L-asparagine, L-cysteine, L-cystine, L-proline, L-tryptophan and L-glutamine, wherein the cell clone is cultured in the absence of selective pressure, preparing a cell culture containing the stable cell clone in a bioreactor, harvesting the recombinant polypeptide from the culture supernatant.

A recombinant CHO cell clone selected in the absence of selective pressure, said cell clone stable in serum- and protein-free medium for at least 40 generations and expressing a recombinant product.

A method of producing a recombinant polypeptide in a cell culture comprising the steps of:

• multiplying a stable starting cell clone expressing a recombinant polypeptide in serum- and protein-free medium in the absence of selective pressure so as to obtain a cell culture,
• culturing said cell culture containing stable cells in a bioreactor so as to obtain a cell culture, and
• harvesting said recombinant polypeptide from a supernatant of said cell culture.

A recombinant CHO cell clone which has been cultivated in serum- and protein-free medium in the absence of selective pressure for at least 40 generations and which expresses a recombinant product.