Liu Na, 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.
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.
J Chromatogr A. 2015 Jul 24;1404:44-50. doi: 10.1016/j.chroma.2015.05.046. Epub 2015 May 28.
In the previous studies on protein adsorption to poly(ethylenimine) (PEI)-grafted Sepharose FF resins, a critical ionic capacity (600mmol/L) of PEI-Sepharose resins was found for the adsorption of bovine serum albumin (BSA), above which both protein capacity and uptake rate increased drastically. In this work, the influence of counterions on the PEI-Sepharose resin with an ionic capacity of 683mmol/L (FF-PEI-L680) was investigated with sodium salts of SCN(-), Cl(-), HPO4(2-) and SO4(2-). Linear gradient elution, batch adsorption and breakthrough experiments showed that counterion preference, effective pore diffusion coefficient (De) and dynamic binding capacity (DBC) values increased in the order of SCN(-), Cl(-), HPO4(2-) and SO4(2-), while static adsorption capacity decreased in this order. It is considered that higher counterion preference of the ion exchange groups resulted in lower protein binding strength and adsorption capacity, while the De value increased due to the enhanced "chain delivery" effect (a kind of surface diffusion). Besides, the DBC value was mainly dependent on De value. In particular, SO4(2-) was the most favorable counterion for the PEI-Sepharose resin, which gave rise to the highest De value (De/D0=1.17, D0 is protein diffusivity in free solution) and DBC value (118mg/mL at a residence time of 2min). Moreover, the effects of counterions on BSA adsorption to DEAE Sepharose FF and Q Sepharose FF, which were non-grafted resins, were also studied for comparisons. It was found that the counterion preferences of the two non-grafted resins were different from each other and also different from that of FF-PEI-L680. The different counterion preferences were attributed to the differences in the ion-exchange ligand chemistries. In addition, the De values for DEAE Sepharose FF and Q Sepharose FF kept unchanged. The low counterion sensitivity of De values could be interpreted as the lack of "chain delivery" effect for the non-grafted resins. The results indicate that protein adsorption and chromatographic performance with PEI-Sepharose can be improved by proper counterions. For the four counterions tested, SO4(2-) was the most favorable for providing the best adsorption and elution outcomes with FF-PEI-L680.
在先前关于蛋白质吸附到聚乙烯亚胺（PEI）接枝的琼脂糖FF树脂的研究中，发现PEI-琼脂糖树脂对于牛血清白蛋白（BSA）吸附的临界离子容量为600mmol/L，高于此值，蛋白质容量和吸附速率均急剧增加。在本工作中，使用SCN(-)、Cl(-)、HPO4(2-)和SO4(2-)的钠盐研究了抗衡离子对离子容量为683mmol/L的PEI-琼脂糖树脂（FF-PEI-L680）的影响。线性梯度洗脱、分批吸附和穿透实验表明，抗衡离子偏好、有效孔扩散系数（De）和动态结合容量（DBC）值按SCN(-)、Cl(-)、HPO4(2-)和SO4(2-)的顺序增加，而静态吸附容量按此顺序降低。据认为，离子交换基团较高的抗衡离子偏好导致较低的蛋白质结合强度和吸附容量，而De值由于增强的“链传递”效应（一种表面扩散）而增加。此外，DBC值主要取决于De值。特别地，SO4(2-)是PEI-琼脂糖树脂最有利的抗衡离子，它产生了最高的De值（De/D0 = 1.17，D0是蛋白质在自由溶液中的扩散率）和DBC值（在停留时间为2分钟时为118mg/mL）。此外，还研究了抗衡离子对未接枝树脂DEAE琼脂糖FF和Q琼脂糖FF上BSA吸附的影响以作比较。发现这两种未接枝树脂的抗衡离子偏好彼此不同，也与FF-PEI-L680的不同。不同的抗衡离子偏好归因于离子交换配体化学的差异。此外，DEAE琼脂糖FF和Q琼脂糖FF的De值保持不变。De值的低抗衡离子敏感性可以解释为未接枝树脂缺乏“链传递”效应。结果表明，通过适当的抗衡离子可以改善PEI-琼脂糖的蛋白质吸附和色谱性能。对于所测试的四种抗衡离子，SO4(2-)最有利于FF-PEI-L680提供最佳的吸附和洗脱结果。
