title: Metabolism of PER.C6 cells cultivated under fed-batch conditions at low glucose and glutamine levels.
abstract: This is the first study to examine PER.C6 cell glucose/energy and glutamine metabolism with fed-batch cultures at controlled low glutamine, low glucose, and simultaneous low glucose and low glutamine levels. PER.C6(TM) cell metabolism was investigated in serum-free suspension bioreactors at two-liter scale. Control of glucose and/or glutamine concentrations had a significant effect on cellular metabolism leading to an increased efficiency of nutrient utilization, altered byproduct synthesis, while having no effect on cell growth rate. Cultivating cells at a controlled glutamine concentration of 0.25 mM reduced q(Gln) and q(NH(4)(+)) by approximately 30%, q(Ala) 85%, and q(NEAA) 50%. The fed-batch control of glutamine also reduced the overall accumulation of ammonium ion by approximately 50% by minimizing the spontaneous chemical degradation of glutamine. No major impact upon glucose/energy metabolism was observed. Cultivating cells at a glucose concentration of 0.5 mM reduced q(Glc) about 50% and eliminated lactate accumulation. Cells exhibited a fully oxidative metabolism with Y(O(2)/Glc) of approximately 6 mol/mol. However, despite no increase in q(Gln), an increased ammonium ion accumulation and Y(NH(4)(+)/Gln) were also observed. Effective control of lactate and ammonium ion accumulation by PER.C6 cells was achieved using fed-batch with simultaneously controlled glucose and glutamine. A fully oxidative glucose metabolism and a complete elimination of lactate production were obtained. The q(Gln) value was again reduced and, despite an increased q(NH(4)(+)) compared with batch culture, ammonium ion levels were typically lower than corresponding ones in batch cultures, and the accumulation of non-essential amino acids (NEAA) was reduced about 50%. In conclusion, this study shows that PER.C6 cell metabolism can be confined to a state with improved efficiencies of nutrient utilization by cultivating cells in fed-batch at millimolar controlled levels of glucose and glutamine. In addition, PER.C6 cells fall into a minority category of mammalian cell lines for which glutamine plays a minor role in energy metabolism.
authors.affiliation: Fermentation and Cell Culture, Bioprocess R&D, Merck Research Laboratories, Merck & Co., Inc., 770 Sumneytown Pike, WP17-201 P.O. Box 4, West Point, Pennsylvania 19486, USA. luis_maranga@merck.com
authors.forename: Luis
authors.initials: L
authors.lastname: Maranga
authors.forename: Charles F
authors.initials: CF
authors.lastname: Goochee
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000596:Amino Acids / Q000378:metabolism; D000641:Ammonia / Q000378:metabolism; D019149:Bioreactors; D002452:Cell Count; D018929:Cell Culture Techniques; D002461:Cell Line, Transformed; D002470:Cell Survival; D002472:Cell Transformation, Viral; D003470:Culture Media / Q000737:chemistry; D016895:Culture Media, Serum-Free; D004734:Energy Metabolism; D005947:Glucose / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism*; D006801:Humans; D019344:Lactic Acid / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D012160:Retina / Q000166:cytology / Q000196:embryology / Q000254:growth & development* / Q000378:metabolism* / Q000821:virology; D013997:Time Factors
chemical_list: D000596:Amino Acids; D003470:Culture Media; D016895:Culture Media, Serum-Free; D005973:Glutamine; D019344:Lactic Acid; D000641:Ammonia; D005947:Glucose; D010100:Oxygen