Achieving Clinically Relevant Yields of High-Potency CAR-NK Cell Expansion in PBS Vertical-Wheel® Bioreactors through Integrated Perfusion Via XCell® ATF

This white paper presents a closed-system workflow for CAR-NK cell expansion and scale-up using PBS Vertical-Wheel bioreactors integrated with alternating tangential flow perfusion technology. Process-development studies were performed first in PBS-Mini systems to evaluate agitation rates, seeding densities, feeding approaches, and shear-protection strategies before translation into PBS-3 bioreactors. Fed-batch and perfusion-based processes were compared using cell growth, metabolite monitoring, viability, apoptosis assessment, CAR expression, and potency measurements. Perfusion operation increased cell yield compared with fed-batch culture while maintaining cell health and functional activity. The document describes development of scalable manufacturing strategies intended to support clinically relevant CAR-NK cell production and outlines how small-scale optimization can be transferred into larger controlled bioreactor platforms for commercial cell therapy manufacturing.