Production of recombinant human erythropoietin in Bowes melanoma cells in suspension culture.
Recombinant DNA technology was used to insert a fetal liver genomic library ApaI fragment encoding for human erythropoietin (Epo) into Bowes melanoma cells. The cells expressed the erythropoietin gene, and Epo was secreted into the culture medium together with the normally-secreted tissue plasminogen activator. Attempts to grow the cells in glass spinners in Dulbecco's medium supplemented with fetal bovine serum produced cell aggregates growing in suspension. When calcium-free suspension culture media (Joklik, DME-S, McCoy 5A-S) were used, single cell suspension cultures were obtained and high Epo production observed. When attempts were made to scale up the small glass spinners, poor growth or Epo production occurred unless the vessels were aerated. This was shown to be because of the drop in pH, possibly due to CO2 accumulation, rather than due to oxygen depletion. It was shown that a semi-continuous operation could be achieved in aerated 8-1 spinners fitted with either a conventional stirrer or a vibromixer agitator. The system was scaled up to a 100-1 stainless steel vessel fitted with a vibromixer agitator. This system was operated for over 4 months with weekly harvests producing over 100 million units of Epo in about 1000-1 of culture fluid. Interference by the serum proteins with downstream purification of the hormone from the culture fluid made the use of serum-free media highly desirable. Studies showed that the Epo was produced in serum-free systems containing peptones.
Piasa Biotechnology Inc., Washington, DC 20026-4591.
L
L
Keay
J M
JM
Walsh
R M
RM
Buchholz
J M
JM
Terando
D004274:DNA, Recombinant / Q000235:genetics; D004921:Erythropoietin / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008545:Melanoma / Q000473:pathology; D011994:Recombinant Proteins / Q000096:biosynthesis*; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis; D014407:Tumor Cells, Cultured
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D004921:Erythropoietin; D010959:Tissue Plasminogen Activator