P. pastoris (K. phaffi) perfusion processes: evaluation of cell retention techniques and integrated purification strategies

Pichia pastoris (K. phaffi) emerges as a promising alternative host to overcome limitations in current mammalian-based manufacturing processes. Owing to its intrinsic properties, the yeast host offers many advantages across both upstream and downstream bioprocessing. Extending the benefits associated with process intensification and continuous approaches to alternative hosts presents an opportunity to diversify the production of therapeutic proteins and streamline the cost of current manufacturing processes. Despite extensive research on perfusion processes as a continuous operational strategy for mammalian cell cultures, their application to microbial cultures, particularly P. pastoris, has been limited.

We investigate two distinct cell retention technologies - membrane- and acoustic-based (membrane-free) methods. A comparative study of these methods in high cell density P. pastoris cultivations highlights their impact on cell viability, heterogeneity, and productivity. Understanding perfusion-specific parameters is critical for establishing robust P. pastoris perfusion processes, a knowledge gap not readily transferable from existing fed-batch processes. Establishing P.pastoris perfusion processes as a continuous mode of operation also prompts the re-evaluation of current purification strategies with an integrated approach in mind. We address this topic by presenting case studies on the removal of host cell proteins (HCPs) from the perfusate using a peptide-based adsorbent (PichiaGuard) in a flow-through mode. Additionally, we explore the application of multi-modal cation-exchange membrane chromatography for the direct capture of the product from the perfusate.