We have previously studied poly(ethylenimine) (PEI)-grafted Sepharose FF resins for ion-exchange chromatography of bovine serum albumin (BSA), and found the presence of a critical ionic capacity (cIC, 600 mmol/L for BSA), above which both BSA adsorption capacity and uptake rates increased drastically. To extend the application of PEI-grafted resins, we have herein proposed to develop mixed-mode chromatographic (MMC) resins by modifying the grafted PEI chains with hydrophobic benzoyl groups. Three PEI-grafted resins with IC values from 329 to 701 mmol/L (FF-PEI-L330, FF-PEI-L520 and FF-PEI-L700) were modified with benzoic acid. It was found that there was a maximum benzoyl density (BD) that could be reached for each resin, at which the average value of BD/residual IC was 1.2. The effect of BD (120–400 mmol/L) on BSA adsorption (at pH 8.0) and elution (at pH 3.0) was first explored with FF-PEI-L700-derived resins. It was observed that both protein binding capacity and recovery increased with increasing BD, indicating that high BD was beneficial in protein adsorption. However, the elution of bound BSA with an acidic buffer (pH 3.0) was incomplete. It was hypothesized that the PEI chains, a pH-dependent cationic polyelectrolyte, formed a collapsed layer at the protein binding condition (high ionic strength, IS), while they exhibited extended spatial structures at elution (low pH and IS). These PEI chain structure transitions made the pores change from an opening state at the loading condition to a blocked state at the elution condition. The pore blocking was regarded as a chain-aroused-steric-hindrance (CaSH) effect. Thus, FF-PEI-L700 was not suitable for fabricating MMC resins due to its high chain density. Then, the effect of PEI density (the initial IC values of 330–700 mmol/L) at the maximum BD values was investigated. Consequently, complete BSA recovery at pH 3.0 was obtained on the resin with an initial IC of 330 mmol/L and a BD of 160 mmol/L (B160-PEI330). The result indicates that the CaSH effect could be ignored at the low PEI chain density (IC = 329 mmol/L). Moreover, selective elution of γ-globulin could be achieved at pH 4.0 on the B160-PEI330 column, indicating the possibility of antibody purification from a mixture containing albumin by manipulating elution conditions. Finally, adsorption equilibria and uptake kinetics onto B160-PEI330 showed favorable binding properties for different proteins at a wide range of IS, indicating its usefulness as an MMC adsorbent.

•γ-Globulin adsorption to PEI-Sepharose was studied and compared with BSA.•γ-Globulin capacity increased with ionic capacity (IC) at IC < 560 mmol/L.•The IC where γ-globulin uptake rate started to hop was earlier than that of BSA.•γ-Globulin uptake rate at IC = 680 mmol/L was four times of that at IC < 460 mmol/L.•Both capacity and uptake rate of γ-globulin decreased drastically at IC > 680 mmol/L.Previously, we studied bovine serum albumin (BSA) uptake to poly(ethylenimine) (PEI)-grafted Sepharose resins, and an ionic capacity (IC) range (600–740 mmol/L) for steep increases of both protein capacity (qm) and effective pore diffusion coefficient (De) was found. In this work, seven PEI-grafted Sepharose FF resins at IC range of 270–1030 mmol/L were synthesized to investigate the effect of protein properties on the adsorption and uptake kinetics using BSA and γ-globulin as two model proteins. For BSA, the change trends of qm and De values with IC were well consistent with the previous results. For γ-globulin, the qm values increased slowly till reaching a maximum value at IC = 560 mmol/L and then decreased rapidly at IC > 560 mol/L. The De values nearly kept unchanged at low ICs (IC < 460 mmol/L), and increased steeply at IC > 460 mmol/L till reaching a maximum at 680 mmol/L (De/D0 = 0.48 ± 0.01). After that increase, the De values for γ-globulin dropped quickly at IC > 680 mol/L, which was not observed for BSA. It is interesting to note that in the narrow IC range of 460–680 mmol/L, the De values of γ-globulin increased dramatically for more than four folds. Moreover, it is notable that the IC range where the hopping of De values occurred for γ-globulin was earlier than that for BSA (460 vs. 560 mmol/L). The earlier hopping of γ-globulin uptake rate was attributed to its larger size and less net charge, which facilitated the happenings of the “chain delivery” effect. The quick drops of both qm and De values for γ-globulin at IC > 680 mmol/L were considered due to its large size, which led to the significant decrease of its effective pore volume. The results indicate that both PEI layer and protein size played important roles in protein adsorption to PEI-grafted resins, and further prove the “chain delivery” effect did contributed significantly to the uptake rate hopping in the PEI-grafted resins. This work could also help the design and selection of resins based on protein characteristics and benefit optimization of practical chromatographic processes for therapeutic proteins with PEI-grafted anion exchangers.