Extracellular purine and pyrimidine catabolism in cell culture

Authors

  • Ana V. Carvalhal,

    Corresponding author
    1. Animal Cell Technology Unit, Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica IBET/ITQB, Apartado 12, P-2781-901 Oeiras, Portugal
    • Genentech, Inc, 1 DNA Way, South San Francisco, CA 94080-4990
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  • Sónia Sá Santos,

    1. Animal Cell Technology Unit, Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica IBET/ITQB, Apartado 12, P-2781-901 Oeiras, Portugal
    Current affiliation:
    1. Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa P-1649-028 Lisboa, Portugal
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  • Manuel J. T. Carrondo

    1. Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica IBET/ITQB, Apartado 12, P-2781-901 Oeiras, Portugal
    2. Laboratório de Engenharia Bioquímica, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, P- 2825-114 Monte da Caparica, Portugal
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Abstract

The presence of purines and pyrimidines bases, nucleosides, and nucleotides in the culture medium has shown to differently affect the growth of a Chinese hamster ovary (CHO) cell line producing the secreted form of the human placental alkaline phosphatase enzyme (SEAP; Carvalhal et al., Biotech Prog. 2003;19:69-83). CHO, BHK, as well as Sf9 cell growth was clearly reduced in the presence of purines but was not affected by pyrimidines at the concentrations tested. The knowledge about the mechanisms by which nucleotides exert their effect when present outside the cells remains very incomplete. The catabolism of both extracellular purines and pyrimidines was followed during the culture of CHO cells. Purines/pyrimidines nucleotides added at a concentration of 1 mM to the culture medium decreased to negligible concentrations in the first 2 days. Purine and pyrimidine catabolism originated only purinic and pyrimidic end-products, respectively. The comparison between AMP catabolism in serum-free cultures (CHO cells expressing Factor VII and Sf9 cells) and in cultures containing serum (CHO cells expressing SEAP and BHK cells expressing Factor VII) showed that AMP extracellular catabolism is mediated by both cells and enzymes present in the serum. This work shows that the quantification of purines and pyrimidines in the culture medium is essential in animal cell culture optimization. When using AMP addition as a chemical cell growth strategy for recombinant protein production improvement, AMP extracellular concentration monitoring allows the optimization of the multiple AMP addition strategy for a prolonged cell culture duration with high specific productivity. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011

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