Fed-batch operational strategies for recombinant Fab production with Pichia pastoris using the constitutive GAP promoter

• Operational improvements in Pichia pastoris fed-batch cultures under GAP promoter.
• Systematic study and selection of the best carbon source for PGAP fed-batch processes.
• Glycerol and glucose were selected for batch and fed-batch phases, respectively.
• Evaluation of the effect of specific growth rate on the production parameters.
• Higher process performance was obtained from medium to high μ set-point.

Carbon source and growth rate are two major parameters affecting recombinant protein production in Pichia pastoris. The effect of the most commonly used carbon sources (glycerol or glucose) and the specific growth rate (μ) has been studied on the production of a human antigen-binding fragment (Fab) in this cell factory under the constitutive GAP promoter in fed-batch cultures.Glycerol for batch phase and glucose for fed-batch phase was the most successful carbon source combination. During batch phase, by-products were detected when glucose was used, despite maintaining DO at values higher than 35%. Also, the presence of cell aggregates was detected affecting the reproducibility and operability of the bioprocess.Conversely, glucose was the best substrate for fed-batch phase. When working at C-limiting conditions, neither by-products nor aggregates were detected and Fab production levels were comparable to those obtained with glycerol. In addition, the lower heat yield (YQ/X) and oxygen to biomass yield (YO2/X) for glucose-supported cultures made this substrate the best alternative from an industrial operational point of view.In addition, the effect of specific growth rate on fed-batch Fab production was studied. Medium and high μ (0.10 and 0.15 h−1) set-points showed similar Fab production yield. However, in terms of total and volumetric productivity, higher μ was the best process condition.