Method of manufacturing a recombinant polypeptide
We have modified a commercially-available adherent cell culture bioreactor in several ways to increase productivity of cultured cells, while decreasing contamination risk. We found that modifying a commercially-available adherent cell culture bioreactor to provide for slower cell culture medium flow unexpectedly and dramatically increases the productivity of the cultured adherent cells. We also developed a new sampling manifold configuration and new way of taking samples, to reduce contamination risk.
1. A method of manufacturing a recombinant polypeptide comprising:
(a) culturing producer cells comprising a nucleic acid sequence encoding the recombinant polypeptide in adherent mode in an adherent-cell culture bioreactor, wherein the adherent-cell culture bioreactor comprises:
(i) a substrate for adherent cell growth; and
(ii) a feed-in pump and a feed-out pump, wherein the feed-in pump and the feed-out pump are configured to provide a constant-rate output of cell culture media of less than about 50 mL of media per 1,800 cells per day; and
(b) providing a constant-rate output of cell culture media at a pump output volume sufficient to maintain a parameter in the cell culture media selected from the group consisting of: concentration of lactate in said medium of not more than about 1.58 grams/liter; and concentration of glucose in said medium of between about 0.5 and 1.0 grams/liter,
thereby manufacturing the recombinant polypeptide.
2. The method of claim 1 , wherein the providing step comprises providing a volume of at least about 20 liters of cell culture media.
3. The method of claim 1 , wherein the substrate for adherent cell growth comprises at least about 60 m 2 of surface area for adherent cell culture.
4. The method of claim 3 , wherein the substrate for adherent cell growth comprises at least about 400 m 2 of surface area for adherent cell culture.
5. The method of claim 1 , wherein the nucleic acid sequence encoding the recombinant polypeptide is selected from the group consisting of interferon, herpes simplex thymidine kinase, and green fluorescent protein.
6. The method of claim 1 , wherein the substrate for adherent cell growth comprises medical grade polyester microfibers.