亲和层析介质非特异性吸附的定量表征研究

doi:10.11949/0438-1157.20210311

摘要/Abstract

摘要：

针对亲和层析介质的非特异性吸附，基于脉冲响应法，建立了定量表征方法。以牛血清白蛋白、酵母发酵液和CHO细胞培养液作为典型杂质，考察了四种蛋白A亲和层析介质和两种基质微球，在不同pH和盐浓度条件下非特异性吸附。发现介质和基质对杂质均有不同程度的吸附，在酸性条件下非特异性吸附相对较强。对于不同料液，存在不同的杂质吸附机制，可通过静电、疏水作用或两者共同作用结合。两种介质和基质的比较结果表明，琼脂糖基介质的非特异性吸附主要依赖其基质微球与杂质间相互作用，而聚甲基丙烯酸酯基介质对杂质的吸附作用强于其基质微球。结果表明，本文建立的非特异性吸附定量表征方法切实可行，可用于量化表征介质非特异性吸附，探究相关作用机制，为介质研发提供新的分析方法和依据。

关键词: 吸附, 蛋白, 生物分离, 亲和层析介质, 非特异性吸附

Abstract:

For the non-specific adsorption of affinity chromatography resin, a quantitative characterization method was established based on the pulse response method. With bovine serum albumin, yeast broth and CHO cell culture solution as the typical impurities, four Protein A affinity resins and two matrix microspheres were used to investigate the non-specific adsorption behaviors under different pH and salt concentration. It was found that all four resins and two matrix microspheres showed some amount of impurity adsorption. The non-specific adsorption was relatively strong under acidic conditions. Different mechanisms of non-specific adsorption could be identified, i.e. electrostatic interaction, hydrophobic interaction and both. According to the comparison of resins and matrices, the non-specific adsorption of agarose-based resin mainly relied on the interactions between matrix microsphere and the impurities, while the polymer-based reins tested showed stronger adsorption of impurities than its matrix. The results demonstrated that the method developed in the present work is feasible and could be used to quantify non-specific adsorption, explore the mechanisms and provide new approach for resin development.

Key words: adsorption, protein, bioseparation, affinity chromatography resin, non-specific adsorption

中图分类号:

Q 819

范春晖,高宗晔,姚善泾,林东强. 亲和层析介质非特异性吸附的定量表征研究[J]. 化工学报, 2021, 72(10): 5218-5225.

Chunhui FAN,Zongye GAO,Shanjing YAO,Dongqiang LIN. Quantitative characterization of non-specific adsorption on affinity chromatography resins[J]. CIESC Journal, 2021, 72(10): 5218-5225.

图/表 6

表 1 本文表征的Protein A亲和层析介质与基质

Table 1 Protein A affinity chromatography resins and matrices

名称	基质	配基	公司
rProtein A Sepharose 4 Fast Flow (rProA)	交联琼脂糖微球	重组Protein A	GE Healthcare
MabSelect PrismA (PrismA)	高度交联的琼脂糖微球	耐碱性重组Protein A	GE Healthcare
Eshmuno A (EA)	聚乙烯醚微球	重组Protein A	Merck
Toyopearl AF-rProteinA HC-650F (HC-650F)	聚甲基丙烯酸酯微球	重组Protein A	Tosoh
Sepharose 4 Fast Flow (4FF)	交联琼脂糖微球	—	GE Healthcare
Toyopearl HW-65F (HW-65F)	聚甲基丙烯酸酯微球	—	Tosoh

图1 介质非特异性吸附定量表征方法示意图

Fig.1 Scheme of quantitative characterization method for non-specific adsorption of resin

图2 不同上样蛋白浓度（a）和上样蛋白液量（b）对A值测定的影响

Fig.2 Effect of loading protein concentration (a) and protein solution volume (b) on the measurement of A

图3 不同介质和基质微球对BSA吸附A值比较

Fig.3 Comparison of the A value of BSA adsorption with different resins and matrix microspheres under different conditions

图4 不同介质和基质微球对酵母细胞培养液吸附A值比较

Fig.4 Comparison of the A value of yeast broth adsorption with different resins and matrix microspheres under different conditions

图5 不同介质和基质微球对CHO细胞培养液吸附A值比较

Fig.5 Comparison of the A value of CHO broth adsorption with different resins and matrix microspheres under different conditions

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