Recombinant cell clones having increased stability and methods of making and using the same
AssigneeBaxter Aktiengesellschaft
InventorsManfred Reiter, Wolfgang Mundt, Friedrich Dorner
Life of this patent
Estimating the term…
Abstract
Disclosed are stable recombinant cell clones which are stable in serum- and protein-free medium for at least 40 generations, a biomass obtained by multiplying the stable cell clone under serum- and protein-free culturing conditions, and a method of preparing recombinant proteins by means of the biomass. Furthermore, the invention relates to a method of recovering stable recombinant cell clones.
Claims34
A method for preparing a recombinant factor VIII protein or polypeptide having factor VIII activity from a CHO cell culture wherein said CHO cells have been transformed to express recombinant factor VIII, comprising the steps of
- multiplying a CHO recombinant cell clone stable in serum- and protein-free medium, wherein purified, ultrafiltered soybean peptide is added to the medium, 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 Method of production of a recombinant product under serum and protein-free conditions on a large technical scale, comprising the steps of:
- providing an isolated, stable recombinant cell clone obtainable by cultivating a recombinant original cell clone in serum-containing medium, adapting the cells on serum- and protein free medium, assaying said cell clone for stable producers of said recombinant product and cloning of a stable product—producing cell clone under serum- and protein-free conditions,
- multiplying said stable recombinant cell clone in serum- and protein-free medium, so to obtain a cell culture,
- producing the said cell culture containing stable cells in a bioreactor, and
- harvesting said recombinant product from a supernatant of said cell culture,
- wherein a serum- and protein-free medium is used which contains one or more amino acids selected from the group consisting of L-asparagine, L-cysteine, L-cystine, L-proline, L-tryptophan and L-glutamine.
A serum- and protein-free synthetic medium comprised of minimum medium, soybean peptone, glutamine, sodium hydrogencarbonate, ascorbic acid, ethanol amine, and sodium selenite, and wherein said medium additionally contains a mixture of amino acids selected from the group consisting of L-asparagine, L-cysteine, L-cystine, L-proline, L-tryptophan and L-glutamine.
A serum- and protein-free synthetic medium comprised of minimum medium, purified and ultrafiltrated soybean peptone, glutamine, sodium hydrogencarbonate, ascorbic acid, ethanol amine and sodium selenite.
A serum- and protein-free synthetic medium comprised of minimum medium, purified and ultrafiltrated soybean peptone, glutamine, sodium hydrogencarbonate, ascorbic acid, ethanol amine and sodium selenite, wherein said medium additionally contains a mixture of amino acids selected from the group consisting of L-asparagine, L-cysteine, L-cystine, L-proline and L-tryptophan.
A serum- or protein-free medium comprising at least one amino acid selected from the group consisting of L-asparagine, L-cysteine, L-cystine, L-proline, L-tryptophan and L-glutamine and purified, ultrafiltrated soybean peptone.
Dependencies are inferred from the claim text.