Reduced oxygen supply increases process stability and product yield with recombinant Pichia pastoris.
A single-chain antibody fragment directed against fimbriae of enterotoxigenic Escherichia coli was produced by recombinant Pichia pastoris under control of the methanol-inducible AOX1 promoter. In high-cell-density cultivation on defined medium, methanol-limited and methanol-saturated conditions were compared. After batch and fed-batch phase on glycerol, the methanol concentration was controlled to 1% (v/v) or methanol was fed with an exponentially increasing rate. Whereas methanol limitation impaired cell integrity and product quality, finally yielding no active product as a result of degradation, oxygen limitation was acceptable. To postpone the onset of limitation, the inlet air was enriched by pure oxygen. Because of faster methanol consumption, however, the process became sensitive to fluctuations in the feeding rate, and complete arrest of metabolism encountered upon small perturbations shortened the active production period. Without additional oxygen supply, the process was robust. Loss of culture integrity was monitored by flow cytometry and was found to precede changes in metabolic rates; it can thus serve as a sensitive indicator of forthcoming problems. Single-step downstream processing from the culture supernatant by His-affinity chromatography was efficient when antifoam agent that coagulates upon pH titration was omitted and yielded 1 g of purified lyophilized product from 6 L initial culture volume.
Trentmann
Oliver
O
Novoplant GmbH, Am Schwabeplan 1b, D-06466 Gatersleben, Germany.
Khatri
Narendar K
NK
Hoffmann
Frank
F
D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010100:Oxygen / Q000378:metabolism*; D010101:Oxygen Consumption / Q000502:physiology; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*
D007128:Immunoglobulin Fragments; D010100:Oxygen