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Evaluation of Mut+ and MutS Pichia pastoris Phenotypes for High Level Extracellular scFv Expression under Feedback Control of the Methanol Concentration

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Extracellular secretion of over 4 g·L−1 of the A33 scFv antibody fragment was achieved in Pichia pastoris at the 10 L bioreactor scale using minimal medium and feedback control of the methanol concentration. Since methanol acts as both inducer and carbon source, its close regulation is a crucial factor in achieving optimal fermentation conditions. The antibody fragment production levels of both Mut + and Mut S phenotypes were compared in a bioreactor under closed‐loop PID control of the methanol level. As expected, the Mut S phenotype has a growth rate lower than that of the Mut + (0.37 vs 1.05 d−1) when growing under methanol. However, protein productivity and cell yield on substrate are almost double that of the Mut + (18.2 vs 9.3 mg A33 sc per gram of methanol). Induction at wet cell weight of 350 g·L−1 for the Mut S also has a positive effect on the final product concentration. Both Mut + and Mut S phenotypes reach a maximum biomass density around 450 g·L−1 wet cell weight, independent of methanol concentration, reactor scale, or induction density. This reactor configuration allows for reproducible fermentation schemes with different Pichia pastoris phenotypes with AOXpromoters, without prior knowledge of the culture growth parameters.
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Document Type: Research Article

Affiliations: 1: 120 Olin Hall, School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853 2: 321 Stocking Hall, Cornell University-Ludwig Institute for Cancer Research Partnership, Ithaca, New York 14853 3: Ludwig Institute for Cancer Research, New York Branch, New York, New York 10158

Publication date: January 1, 2006

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