Pichia pastoris has emerged in the past years as a promising host for recombinant protein and biopharmaceutical production. In the establishment of high cell density fed‐batch biomanufacturing, screening phase and early bioprocess development (based on microplates and shake flasks) still represent a bottleneck due to high‐cost and time‐consuming procedures as well as low experiment complexity. In the present work, a screening protocol developed for P. pastoris clone selection is implemented in a multiplexed microfluidic device with 15 μL cultivation chambers able to operate in perfusion mode and monitor dissolved oxygen content in the culture in a non‐invasive way. The setup allowed us to establish carbon‐limited conditions and evaluate strain responses to different input variables. Results from micro‐scale perfusion cultures are then compared with 1L fed‐batch fermentation. The best producer in terms of titer and productivity is rapidly identified after 12 h from inoculation and the results confirmed by lab‐scale fermentation. Moreover, the physiological analyses of the strains under different conditions suggested how more complex experimental conditions are achievable despite the relatively easy, straight‐forward, and cost‐effective experimental setup. Implementation and standardization of these micro‐scale protocols could reduce the demand for lab‐scale bioreactor cultivations thus accelerating the development of protein production processes.

The methylotrophic yeast Pichia pastoris is known as an efficient host for the production of heterologous proteins. While N‐linked protein glycosylation is well characterized in P. pastoris there is less knowledge of the patterns of O‐glycosylation. O‐glycans produced by P. pastoris consist of short linear mannose chains, which in the case of recombinant biopharmaceuticals can trigger an immune response in humans. This study aims to reveal the influence of different cultivation strategies on O‐mannosylation profiles in P. pastoris. Sixteen different model proteins, produced by different P. pastoris strains, are analyzed for their O‐glycosylation profile. Based on the obtained data, human serum albumin (HSA) is chosen to be produced in fast and slow growth fed batch fermentations by using common promoters, PGAP and PAOX1. After purification and protein digestion, glycopeptides are analyzed by LC/ESI‐MS. In the samples expressed with PGAP it is found that the degree of glycosylation is slightly higher when a slow growth rate is used, regardless of the efficiency of the producing strain. The highest glycosylation intensity is observed in HSA produced with PAOX1. The results indicate that the O‐glycosylation level is markedly higher when the protein is produced in a methanol‐based expression system.