Principles and applications of steric exclusion chromatography

Abstract

We introduce a chromatography method for purification of large proteins and viruses that works by capturing them at a non-reactive hydrophilic surface by their mutual steric exclusion of polyethylene glycol (PEG). No direct chemical interaction between the surface and the target species is required. We refer to the technique as steric exclusion chromatography. Hydroxyl-substituted polymethacrylate monoliths provide a hydrophilic surface and support convective mass transport that is unaffected by the viscosity of the PEG. Elution is achieved by reducing PEG concentration. Selectivity correlates with molecular size, with larger species retained more strongly than smaller species. Retention increases with PEG size and concentration. Salts weaken retention in proportion to their concentration and Hofmeister ranking. Retention is enhanced near the isoelectric point of the target species. Virus binding capacity was measured at 9.9×1012 plaque forming units per mL of monolith. 99.8% of host cell proteins and 93% of DNA were eliminated. Mass recovery exceeded 90%. IgM capacity was greater than 60mg/mL. 95% of host cell proteins were eliminated from IgM produced in protein-free media, and mass recovery was up to 90%. Bioactivity was fully conserved by both viruses and antibodies. Process time ranged from less than 30min to 2h depending on the product concentration in the feed stream.