Primary metabolism in the new human cell line AGE1.HN at various substrate levels: increased metabolic efficiency and α1-antitrypsin production at reduced pyruvate load
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Metabolic responses of the new neuronal human cell line AGE1.HN to various substrate levels were analyzed in this study showing that reduced substrate and especially pyruvate load improves metabolic efficiency, leading to improved growth and α1-antitrypsin (A1AT) production. The adaptation of the metabolism to different pyruvate and glutamine concentrations was analyzed in detail using a full factorial design. The most important finding was an increasingly inefficient use of substrates as well as the reduction of cell proliferation with increasing pyruvate concentrations in the medium. Cultivations with different feeding profiles showed that the highest viable cell density and A1AT concentration (167% of batch) was reached in the culture with the lowest glucose level and without pyruvate feeding. Analysis of metabolic fluxes in the differently fed cultures revealed a more efficient metabolic phenotype in the cultures without pyruvate feeding. The measured in vitro enzyme activities of the selected enzymes involved in pyruvate metabolism were lower in AGE1.HN compared with CHO cells, which might explain the higher sensitivity and different adaptation of AGE1.HN to increased pyruvate concentrations. The results indicate on the one hand that increasing the connectivity between glycolysis and the TCA cycle might improve substrate use and, finally, the production of A1AT. On the other hand, a better balanced substrate uptake promises a reduction of energy spilling which is increased with increasing substrate levels in this cell line. Overall, the results of this study provide important insights into the regulation of primary metabolism and into the adaptation of AGE1.HN to different substrate levels, providing guidance for further optimization of production cell lines and applied process conditions.
KeywordsMammalian cell Human cell Metabolic flux Recombinant protein CHO Physiology
This work has been financially supported by the BMBF project SysLogics–Systems biology of cell culture for biologics (FKZ 0315275A-F). We thank Armin Melnyk for performing enzyme assays, Michel Fritz for valuable support for the HPLC analysis, as well as Judith Wahrheit for fruitful discussions.
- A1AT-Group (1998) Survival and FEV1 decline in individuals with severe deficiency of alpha1-antitrypsin. The Alpha-1-Antitrypsin Deficiency Registry Study Group. Am J Respir Crit Care Med 158(1):49–59Google Scholar
- Bonarius HP, Ozemre A, Timmerarends B, Skrabal P, Tramper J, Schmid G, Heinzle E (2001) Metabolic-flux analysis of continuously cultured hybridoma cells using 13CO2 mass spectrometry in combination with 13C-lactate nuclear magnetic resonance spectroscopy and metabolite balancing. Biotechnol Bioeng 74(6):528–538CrossRefGoogle Scholar
- Gambhir A, Korke R, Lee J, Fu PC, Europa A, Hu WS (2003) Analysis of cellular metabolism of hybridoma cells at distinct physiological states. J Biosci Bioeng 95(4):317–327Google Scholar
- Heinzle E, Matsuda F, Miyagawa H, Wakasa K, Nishioka T (2007) Estimation of metabolic fluxes, expression levels and metabolite dynamics of a secondary metabolic pathway in potato using label pulse-feeding experiments combined with kinetic network modelling and simulation. Plant J 50(1):176–187CrossRefGoogle Scholar
- Kumar N, Gammell P, Clynes M (2007) Proliferation control strategies to improve productivity and survival during CHO based production culture: a summary of recent methods employed and the effects of proliferation control in product secreting CHO cell lines. Cytotechnology 53(1–3):33–46CrossRefGoogle Scholar
- Niklas J, Heinzle E (2011) Metabolic flux analysis in systems biology of mammalian cells. Adv Biochem Eng Biotechnol. doi: 10.1007/10_2011_99
- Nivitchanyong T, Martinez A, Ishaque A, Murphy JE, Konstantinov K, Betenbaugh MJ, Thrift J (2007) Anti-apoptotic genes Aven and E1B-19K enhance performance of BHK cells engineered to express recombinant factor VIII in batch and low perfusion cell culture. Biotechnol Bioeng 98(4):825–841CrossRefGoogle Scholar
- Petrache I, Hajjar J, Campos M (2009) Safety and efficacy of alpha-1-antitrypsin augmentation therapy in the treatment of patients with alpha-1-antitrypsin deficiency. Biologics 3:193–204Google Scholar
- Street JC, Delort AM, Braddock PS, Brindle KM (1993) A 1H/15N n.m.r. study of nitrogen metabolism in cultured mammalian cells. Biochem J 291(Pt 2):485–492Google Scholar