Characterization of novel mixed-mode protein adsorbents fabricated from benzoyl-modified polyethyleneimine-grafted Sepharose.

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, 600mmol/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 701mmol/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 (120-400mmol/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 330-700mmol/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 330mmol/L and a BD of 160mmol/L (B160-PEI330). The result indicates that the CaSH effect could be ignored at the low PEI chain density (IC=329mmol/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.