Liu Na, Wang Ziyuan, Liu Xiaoguang Margaret, Yu Linling, Sun Yan
Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China; Department of Chemical and Biological Engineering, The University of Alabama, Tuscaloosa, AL, USA.
J Chromatogr A. 2014 Dec 12;1372C:157-165. doi: 10.1016/j.chroma.2014.10.108. Epub 2014 Nov 8.
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.
我们之前研究了用于牛血清白蛋白（BSA）离子交换色谱的聚（乙烯亚胺）（PEI）接枝琼脂糖凝胶FF树脂，发现存在一个临界离子容量（cIC，对于BSA为600mmol/L），高于此值时，BSA吸附容量和摄取速率都会急剧增加。为了扩展PEI接枝树脂的应用，我们在此提议通过用疏水性苯甲酰基修饰接枝的PEI链来开发混合模式色谱（MMC）树脂。用苯甲酸对三种IC值为329至701mmol/L的PEI接枝树脂（FF-PEI-L330、FF-PEI-L520和FF-PEI-L700）进行了修饰。发现每种树脂都有一个可以达到的最大苯甲酰密度（BD），此时BD/残余IC的平均值为1.2。首先用FF-PEI-L700衍生的树脂研究了BD（120 - 400mmol/L）对BSA吸附（在pH 8.0）和洗脱（在pH 3.0）的影响。观察到蛋白质结合容量和回收率都随着BD的增加而增加，表明高BD有利于蛋白质吸附。然而，用酸性缓冲液（pH 3.0）洗脱结合的BSA并不完全。据推测，PEI链是一种pH依赖性阳离子聚电解质，在蛋白质结合条件（高离子强度，IS）下形成了一个塌陷层，而在洗脱时（低pH和IS）它们呈现出伸展的空间结构。这些PEI链结构转变使得孔从加载条件下的开放状态变为洗脱条件下的堵塞状态。孔堵塞被认为是一种链引发的空间位阻（CaSH）效应。因此，由于其高链密度，FF-PEI-L700不适合制造MMC树脂。然后，研究了在最大BD值下PEI密度（初始IC值为330 - 700mmol/L）的影响。结果，在初始IC为330mmol/L且BD为160mmol/L的树脂（B160-PEI330）上，在pH 3.0时获得了完全的BSA回收率。结果表明，在低PEI链密度（IC = 329mmol/L）下，CaSH效应可以忽略不计。此外，在B160-PEI330柱上，在pH 4.0时可以实现γ-球蛋白的选择性洗脱，这表明通过控制洗脱条件从含有白蛋白的混合物中纯化抗体的可能性。最后，B160-PEI330上的吸附平衡和摄取动力学表明，在广泛的IS范围内，对不同蛋白质具有良好的结合性能，表明其作为MMC吸附剂的实用性。
