Protein adsorption and chromatography on novel mixed-mode resins fabricated from butyl-modified poly(ethylenimine)-grafted Sepharose

• Novel mixed-mode resins were fabricated by butyl-modification of PEI Sepharose.
• Complete elution was achieved at pH 3.0 for a resin of low butyl density.
• Complete elution was achieved with CHAPS at pH 3.0 for a resin of high butyl density.
• Selective protein elution could be achieved by manipulating CHAPS concentration.
• The high butyl density resin was useful at NaCl concentrations of 0–1.5 mol/L.

We have previously studied benzoyl-modified poly(ethylenimine) (PEI)-grafted Sepharose FF resins for mixed-mode chromatography (MMC), and found PEI-grafted resin with an ionic capacity (IC) of about 300 mmol/L was suitable for fabricating MMC resins to achieve favorable MMC performance. To extend the application of PEI-grafted resins in MMC, we have herein modified a PEI-grafted resin with an initial IC of 315 mmol/L (FF-PEI-L315) with hydrophobic butyl groups, and two MMC resins with residual ICs of 280 and 252 mmol/L were obtained (denoted as Bu30-PEI315 and Bu60-PEI315). Bovine serum albumin (BSA) and γ-globulin adsorption at pH 8.0 and elution at pH 3.0 were first explored on the two MMC resins. It was found that with increasing butyl density, protein binding capacity increased but the elution percentage decreased, indicating that high butyl density was beneficial in protein adsorption but not favorable in elution. It was considered that the butyl density of Bu60-PEI315 was so high that the electrostatic repulsion at pH 3.0 was insufficient to make the protein dissociate from the resin surface. Then, the eluent was switched to 3-[(3-cholamido-propyl)-dimethylammonio]-1-propanesulfonate (CHAPS) solution, and complete BSA recovery was obtained on Bu60-PEI315. Complete γ-globulin recovery was obtained on Bu60-PEI315 at a lower CHAPS concentration than that for BSA. This suggests the possibility of selective elution of the two proteins by manipulating eluent concentration. The results indicate that CHAPS was a suitable eluent for protein recovery and separation with the MMC resin, particularly for the purification of antibody from feedstock containing serum albumin. Finally, adsorption equilibria and uptake kinetics onto Bu30-PEI315 and Bu60-PEI315 were investigated. At 0 mol/L NaCl, BSA binding on the MMC resins was only by electrostatic interaction while γ-globulin showed a bi-modal binding behavior, including electrostatic and hydrophobic interactions. At high NaCl concentrations, both BSA and γ-globulin were bound by hydrophobic interaction. Favorable binding properties for different proteins in a wide range of NaCl concentrations were observed on Bu60-PEI315, indicating its usefulness as an MMC adsorbent.