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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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Abstract

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.

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Acknowledgments

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.

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Authors and Affiliations

  1. Biochemical Engineering Institute, Saarland University, 66123, Saarbrücken, Germany

    Jens Niklas, Christian Priesnitz & Elmar Heinzle

  2. ProBioGen AG, 13086, Berlin, Germany

    Thomas Rose & Volker Sandig

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  1. Jens Niklas
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  2. Christian Priesnitz
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Correspondence to Elmar Heinzle.

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Table S1

Stoichiometric matrix of the metabolic network model applied for metabolic flux analysis (PDF 10 kb)

Table S2

Profile of total growth and total uptake (negative values)/production (positive values) of metabolites during the cultivation of AGE1.HN in media with different glutamine (Gln) and pyruvate (Pyr) concentrations. aQuotient of totally produced lactate and total glucose that was taken up (moles per mole). bC-mol of produced extracellular lactate per C-mol of consumed glucose and pyruvate (C-mol/C-mol). VCD viable cell density (106 cells/ml), TCD total cell density (106 cells/ml); concentration changes for all metabolites are given in millimolar. Glc glucose, Lac lactate, Pyr pyruvate (standard abbreviations for amino acids). NH +4 , ammonia in milligrams per liter (PDF 60 kb)

Table S3

Metabolic fluxes in AGE1.HN cells upon different substrate feeding (feed 1–feed 4). Same experiment as in Figs. 4, 5, and 6 (PDF 11 kb)

Fig. S1

Growth and metabolic profiles of cultivations in which different pyruvate and glutamine concentrations were applied (JPEG 123 kb)

High Resolution (TIFF 1036 kb)

Fig. S2

Amino acid profiles of the feeding experiments (experiment described and analyzed in Figs 4, 5, and 6) (JPEG 64 kb)

High Resolution (TIFF 453 kb)

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Niklas, J., Priesnitz, C., Rose, T. et al. 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. Appl Microbiol Biotechnol 93, 1637–1650 (2012). https://doi.org/10.1007/s00253-011-3526-6

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