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 / 10 L 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, the present invention relates to a method of purifying a nonglycosylated
immunotoxin comprising a) loading a solution containing the nonglycosylated
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-glycosylated 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 in the 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.