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Enabler for process analytical technology implementation in Pichia pastoris fermentation: Fluorescence‐based soft sensors for rapid quantitation of product titer

Rapid quantitation of product titer is a critical input for control of any bioprocess. This measurement, however, is marred by the myriad components that are present in the fermentation broth, often requiring extensive sample pretreatment before analysis. Spectroscopy techniques such as fluorescence spectroscopy are widely recognized as potential monitoring tools. Here, we investigate the possibility of using fluorescence of the culture supernatant as a potential at‐line monitoring tool to measure the concentration of a recombinant therapeutic protein expressed in a Pichia pastoris fed‐batch fermentation. We propose an integrated method wherein both the target protein and total protein concentrations are predicted using intrinsic riboflavin fluorescence and extrinsic fluorescence, respectively. The root mean square error for estimating the concentrations of the target protein (using riboflavin fluorescence) and total protein (using extrinsic fluorescence) have been estimated to be <0.1 and <0.2, respectively. The proposed approach has been validated for two different biotherapeutic products, human serum albumin and granulocyte colony stimulating factor, that were expressed using Mut+ and Muts strains of P. pastoris, respectively. The proposed approach is rapid (1 min analysis time, 10 min total with at line sampling) and thus could be a significant enabler for process analytical technology implementation in Pichia fermentation.