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Scale‐Up of a High Cell Density Continuous Culture with Pichia pastoris X‐33 for the Constitutive Expression of rh‐Chitinase
Author(s) -
Schilling Bernhard M.,
Goodrick Jason C.,
Wan Nick C.
Publication year - 2001
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp010041e
Subject(s) - pichia pastoris , dilution , bioreactor , volume (thermodynamics) , chitinase , steady state (chemistry) , recombinant dna , chromatography , chemistry , continuous stirred tank reactor , sparging , biochemistry , enzyme , thermodynamics , organic chemistry , physics , gene
The feasibility of large‐scale production of recombinant human chitinase using a constitutive Pichia pastoris expression system was demonstrated in a 21‐L continuous stirred tank reactor. A steady‐state recombinant protein concentration of 250 mg/L in the supernatant was sustained for 1 month at a dilution rate of 0.042 h − 1 (equivalent to one volume exchange per day), enabling a volumetric productivity of 144 mg/L d (240 U/L d). The steady‐state dry cell weight concentration in this high cell density culture reached 110 g/L. Considering safety and economical aspects, all large‐scale cultivations were conducted without molecular oxygen supplementation. Conventional air sparging was used instead. The oxygen demand of the process was determined by off‐gas analysis (OUR = 4.8 g O 2 L − 1 h − 1 with k L a = 846 h − 1 ) and evaluated with regard to further reactor scale‐up.