Alternative Two Step Purification of a Soluble Receptor Fc Fusion Biologic Using Mixed-Mode Chromatography

Principal Investigator Heidi Jones Bio-Rad 

Principal ScientistScott Conrad McCarthy PI Celkem

This study examines the purification of a biologic fusion protein, EGFR:Fc, employing non-affinity

mixed-mode chromatography methods. The performance of these approaches is compared with

conventional protein A affinity purification, emphasizing practical considerations within biologic

protein purification workflows. An EGFR:Fc fusion protein, expressed in HEK 293 cells, was

captured and polished using Bio-Rad’s mixed-mode resins Nuvia™ wPrime2A and Nuvia™ HPQ.

Initial capture on Nuvia™ wPrime2A at pH 6.8 resulted in approximately 90% purity, although

minor product-related impurities remained detectable. Further purification on Nuvia™ HPQ

enhanced purity to over 98%, achieving levels comparable to those obtained via Protein A affinity

purification. The two-step non-affinity purification process yielded 70–80% of EGFR:Fc, whereas

protein A affinity purification produced yields exceeded 90%. Both purification strategies

delivered biologics with analogous binding characteristics and impurity profiles. This investigation

offers valuable insights into the application of mixed-mode chromatography for biologic protein

purification.