Exploiting the metabolism of PYC expressing HEK293 cells in fed-batch cultures.
The expression of recombinant yeast pyruvate carboxylase (PYC) in animal cell lines was shown in previous studies to reduce significantly the formation of waste metabolites, although it has translated into mixed results in terms of improved cellular growth and productivity. In this work, we demonstrate that the unique phenotype of PYC expressing cells can be exploited through the application of a dynamic fed-batch strategy and lead to significant process enhancements. Metabolically engineered HEK293 cells stably producing human recombinant IFNα2b and expressing the PYC enzyme were cultured in batch and fed-batch modes. Compared to parental cells, the maximum cell density in batch was increased 1.5-fold and the culture duration was extended by 2.5 days, but the product yield was only marginally increased. Further improvements were achieved by developing and implementing a dynamic fed-batch strategy using a concentrated feed solution. The feeding was based on an automatic control-loop to maintain a constant glucose concentration. This strategy led to a further 2-fold increase in maximum cell density (up to 10.7×10(6)cells/ml) and a final product titer of 160mg/l, representing nearly a 3-fold yield increase compared to the batch process with the parental cell clone.
Département de Génie Chimique, École Polytechnique de Montréal, C.P. 6079, Succ. Centre-ville, Montréal, Québec, Canada H3C 3A7.
Cédric
C
Vallée
Human Health Therapeutics Portfolio, National Research Council, 6100 Royalmount Avenue, Montréal (Québec), H4P 2R2, Canada.
Yves
Y
Durocher
Département de Génie Chimique, École Polytechnique de Montréal, C.P. 6079, Succ. Centre-ville, Montréal, Québec, Canada H3C 3A7. Electronic address: olivier.henry@polymtl.ca.
Olivier
O
Henry
D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D057809:HEK293 Cells; D006801:Humans; D011766:Pyruvate Carboxylase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D011766:Pyruvate Carboxylase
Fed-batch; HEK293 cells; Pyruvate carboxylase; Recombinant protein