Alkaline treatment enhances mass transfer in Protein A affinity chromatography

Cycle stability is important for preparative chromatography resins. Up to 200 cycles have been reported for Protein A affinity resins when used under optimized operating conditions. Through engineered ligands, alkaline resistant Protein A resins are available that can withstand repeated cleaning-in-place cycles with even 1 M NaOH. This enables an increase of purification cycles through the reduction of fouling while maintaining high binding capacities. Previously, non-intuitive changes in dynamic binding capacity after alkaline treatment have been observed for these novel Protein A resins, where sharper breakthrough curves and increased capacities were reported. In this work, we have systematically investigated resins with both low and high alkaline stability and studied the changes in static and dynamic binding capacities and elution behavior. We propose that the observed mass transfer increases of up to 40% are due to a switch in diffusion mechanism, as shown by confocal laser scanning microscopy. Based on our results, only a small window of alkaline treatment conditions exists, where dynamic binding capacity can be increased. Our findings may help to explain previous findings and observations of others.