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Pichia pastoris is becoming a desirable host in the biopharmaceutical industry for therapeutics production. It grows on methanol to high cell densities ≥100 g DCW/L and secretes foreign proteins at high titers. However, the culture conditions to reach high cell densities pose a challenge to the processability by primary recovery operations, in particular centrifugation, used for cell removal. This work aims to assess the impact of recombinant P. pastoris strain selection on centrifugal dewatering. Normally, the choice of P. pastoris recombinant strain is based on best target protein expression levels; however, it is unknown whether the choice of strain will have an impact on performance of centrifugation operation. To achieve this aim, a previously developed laboratory ultra-scale down (USD) methodology that successfully predicted centrifugal dewatering of pilot-scale disk-type machines, was used in this work. Two recombinant P. pastoris strains, namely a X-33 and a glycoengineered Pichia strain, were used to perform fermentations secreting different products. The resulting harvested fermentation culture properties were analyzed and the dewatering performances of a pilot- and a large-scale disk-type centrifuge were evaluated using the USD methodology. The choice of P. pastoris strain was found to have a considerable impact on dewatering performance, with P. pastoris X-33 strain reaching better dewatering levels than the glycoengineered strain. The USD method proved to be a useful tool to determine optimal conditions under which the large scale centrifuge needed to be operated, reducing the need for repeated pilot-scale runs during early stages of process development for therapeutic products.

During centrifugation operation, the major challenge in the recovery of extracellular proteins is the removal of the maximum liquid entrapped within the spaces between the settled solids-dewatering level. The ability of the scroll decanter centrifuge (SDC) to process continuously large amounts of feed material with high concentration of solids without the need for resuspension of feeds, and also to achieve relatively high dewatering, could be of great benefit for future use in the biopharmaceutical industry. However, for reliable prediction of dewatering in such a centrifuge, tests using the same kind of equipment at pilot-scale are required, which are time consuming and costly. To alleviate the need of pilot-scale trials, a novel USD device, with reduced amounts of feed (2 mL) and to be used in the laboratory, was developed to predict the dewatering levels of a SDC. To verify USD device, dewatering levels achieved were plotted against equivalent compression (Gtcomp ) and decanting (Gtdec ) times, obtained from scroll rates and feed flow rates operated at pilot-scale, respectively. The USD device was able to successfully match dewatering trends of the pilot-scale as a function of both Gtcomp and Gtdec , particularly for high cell density feeds, hence accounting for all key variables that influenced dewatering in a SDC. In addition, it accurately mimicked the maximum dewatering performance of the pilot-scale equipment. Therefore the USD device has the potential to be a useful tool at early stages of process development to gather performance data in the laboratory thus minimizing lengthy and costly runs with pilot-scale SDC.