Minimization of aggregation of secreted bivalent anti-human T cell immunotoxin in Pichia pastoris bioreactor culture by optimizing culture conditions for protein secretion.
In a bioreactor culture of genetically engineered Pichia pastoris secreting a bivalent immunotoxin, 64% of the secreted immunotoxin was present in aggregate forms and this resulted in a loss of bioactivity. Biochemical analyses of the secreted immunotoxin and an in vitro aggregation study using purified monomeric immunotoxin suggested that aggregation was primarily an extracellular event. By employing limited methanol feeding at 0.75 mlmin(-1) per 10l initial medium, oxygen consumption was reduced, permitting a lowering of the bioreactor agitation speed from 800 to 400 rpm. By increasing the anti-foam reagent to 0.6 mll(-1), the thickness of the air/liquid interfacial foam layer was reduced by 80%. These steps reduced the immunotoxin aggregates from 64% to 5%. Consequently immunotoxin purification yield was increased from 53.0% to 73.8%. Simultaneously this methodology enhanced immunotoxin secretion to 120 mgl(-1) at 163 h of methanol induction in a toxin resistant production strain. We conclude that minimizing shearing force and reducing the air/liquid interfacial foam area are crucial factors in reducing hydrophobic protein aggregation upon secretory expression in yeast bioreactor cultures.
Woo
Jung Hee
JH
Section on Biophysical Chemistry, Laboratory of Molecular Biology, National Institute of Mental Health, Bldg. 10 Rm. 3D46, 10 Center Drive, Bethesda, MD 20892-1216, USA. jwoo@swmail.sw.org
Liu
Yuan Yi
YY
Neville
David M
DM
D019149:Bioreactors; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000254:growth & development*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D013601:T-Lymphocytes
D000922:Immunotoxins; D011994:Recombinant Proteins