METHODS FOR EXPRESSION AND PURIFICATION OF IMMUNOTOXINS

摘要

In one aspect the present invention relates to a method of expressing an immunotoxin in Pichia pastoris strain mutated to toxin resistance comprising a) growing the Pichia pastoris in a growth medium comprising an enzymatic digest of protein and yeast extract and maintaing a dissolved oxygen concentration at 40% and above; and b) performing methanol induction with a limited methanol feed of 0.5-0.75 ml/min/ 10L of initial volume during induction along with a continuous inusion of yeast extract at a temperature below 17.5 ~C., antifoaming agent supplied up to 0.07%, agitation reduced to 400 RPM, and the induction phase extended out to 163 h. In another aspect, t he present invention relates to a method of purifying a non~glycosylated immunotoxin comprising a) loading a solution containing the non~glycosylated immunotoxin onto a hydrophobic interaction column; b) obtaining a first non- glycosylated immunotoxin containing eluant from the hydrophobic interaction column; c) loading the non-glycosylated immunotoxin containing eluant from step (b) onto an anion exchange column; d) obtaining a second non-glycosylat ed immunotoxin containing eluant from the anion exchange column by eluting the non-glycosylated immunotoxin with a sodium borate solution; e) diluting the concentration of sodium borate inthe second non-glycosylated immunotoxin containing eluant from step (d) to about 50 mM or less; f) concentrating the diluted non-glycosylated immunotoxin containing eluant from step (e) over an anion exchange column; and g) obtaining a purified non-glycosylated immunotoxin from the anion exchange column.