Fibronectin splice variant ED B (extracellular domain B) is a promising marker for angiogenesis in growing solid
tumors. Currently, recombinant
antibodies against ED B are being investigated concerning their potential use, for either therapeutic or diagnostic purposes. Single-chain
antibody fragments directed against the ED B can be efficiently expressed in Pichia pastoris; thus, a recombinant strain of the methylotropic yeast P. pastoris was used for this work. Three different forms of scFv
antibody fragment are found in the supernatant from this fermentation: covalent homodimer, associative homodimer, and monomer. Both homodimeric forms can be converted to the monomeric form (under reducing conditions) and be efficiently radiolabeled, whereas the monomeric form of scFv already present in the supernatant cannot. It was also found that the fraction of
protein in the monomeric form is highly dependent on the mode of induction rather than scFv concentration. This suggests that the monomeric form of the scFv present in the supernatant might be a result of events occurring at the expression, secretion, or folding level. A high cell density fermentation protocol was developed by optimizing
methanol induction, yielding the highest scFv
antibody fragment production rate and product quality; cell concentration at the induction point and specific
methanol uptake rate were found to be the most important control variables. A decrease in specific
methanol uptake rate led to a higher specific production rate for the scFv
antibody fragment (5.4 microg g(cell) h(-1)). Product quality, i.e., percentage of product in a homodimeric form, also increased with the decrease in
methanol uptake rate. Furthermore, the volumetric productivity depended on cell concentration at the induction point, increasing with the increase of cell concentration up to 320 g L(-1) wet cell weight (WCW). The reduction of the
methanol feeding rate for induction, and consequently of the
oxygen uptake rate, have important consequences for optimizing product titers and quality and thus on the scale-up of this production process; hence one of the major limitations upon high cell density cultivation in
bioreactors is keeping the high
oxygen transfer rate required. From the results obtained, a scale-up strategy was developed based on the available
oxygen transfer rates at larger scales, allowing the definition of the optimum biomass concentration for induction and
methanol feeding strategy for maximization of product titer and quality.