Hydrophilic modification gigaporous resins with poly(ethylenimine) for high-throughput proteins ion-exchange chromatography

• Gigaporous microspheres were hydrophilic modified by poly(ethylenimine) (PEI).
• PEI in the microspheres was in situ branched to resist non-specific adsorption for proteins.
• The modified microspheres gave more than 90% proteins recovery.
• The branched PEI in the microspheres could change its construction in different environment of solutions.
• Dynamic binding capacity of proteins on the modified media didn’t show obviously decrease at the high flow rates.

High hydrophilicity of gigaporous microspheres based on a copolymer of poly(glycidyl methacrylate)-co-divinyl benzene (PGMA-DVB) was successfully realized through coating the branched polyethyleneimine (PEI) in PGMA-DVB microspheres. PEI with various molecules weights and different branching agents were identified in terms of protein recovery as evaluation approach. For this evaluation, PEI600 (Mw = 600) and poly (ethylene glycol) diglycidyl ether (PEGDE, Mw = 400) were used as modification agent and branching agent, respectively. The modified microspheres showed good permeability and revealed a certain mechanical strength. After modification, the protein recovery increased from 40% to >90%. The protein recovery increased with the branched generations and the first and second generations could give the protein recovery of 93% and 96%, respectively. Meanwhile, the ionic capacity also showed a rising trend in the range of 0.11–0.32 mmol/mL with the branched generations. But the dynamic binding capacity of protein (bovine serum albumin, BSA as the model protein) increased at first and then decreased. Analysis of the dry microspheres structure by mercury intrusion method as well as observation of the branched PEI on PGMA-DVB membrane in aqueous solution indicated that excess PEI chains with the extended state in the second generation would block the small pores and decrease the accessible surface area. Therefore, the protein capacity on the second generation, on the contrary, was lower than that on the first generation. Meanwhile, it was found that the PEI chains in the modified microspheres changed their construction from the extended to the collapsed state with increase of NaCl concentration. And the corresponding pore size of the modified microspheres increased with salt concentration through low-field nuclear magnetic resonance. Dynamic binding capacity of proteins on the modified supports did not significantly change with increase of the flow rate. The media showed good performance for separation three model proteins at high flow rate of 1084 cm/h. This modified gigaporous microspheres had a large potential in application for rapid separation of biomolecules.