Effect of cell retention techniques in Komagataella phaffii lab-scale continuous processes

Perfusion technologies play a growing role in the implementation of continuous processes for biotherapeutics production in mammalian-based manufacturing. However, their application to alternative production hosts is limited. Cell retention systems are of key importance for the efficiency of perfusion bioreactors. In this study, we investigate two cell retention technologies for the development of lab-scale Komagataella phaffii continuous processes. An acoustic-based process (AP) and a membrane-based process (MP) were developed using an acoustic cell separator (ACS) and a vibrating membrane filtration (VMF) device, respectively. Both systems allowed for continuous cell recycle and production of scFv13R4 antibody fragment for 8 days (AP) and 9 days (MP), without loss in productivity, while maintaining high viability (greater than 90%). Higher volumetric and specific productivities were achieved during the AP process, namely 50.63 ± 1.63 mg L^-1 day^-1 and 1.09 ± 0.07 mg g^-1 day^-1, against the 32.29 ± 1.21 mg L^-1 day^-1 and 0.44 ± 0.02 mg g^-1 day^-1 afforded by the MP process. The VMF device provided 100% separation efficiency with biomass accumulating up to concentrations of 74.1 ± 0.1 g L^-1 dry cell weight (DCW), whereas the acoustic device reached 55.1 ± 0.47 g L^-1 DCW at 98% separation efficiency. The acoustic device showed selectivity towards larger and more complex cells in the yeast population, which might be linked to the observed higher productivities for the AP process. This study discusses the advantages and drawbacks of both cell retention technologies and provides an outlook towards their future investigation in K. phaffii perfusion processes.