大孔聚合物层析介质孔结构对蛋白载量的影响

doi:10.7503/cjcu20180304

高等学校化学学报 ›› 2018, Vol. 39 ›› Issue (12): 2673.doi: 10.7503/cjcu20180304

大孔聚合物层析介质孔结构对蛋白载量的影响

李恒¹, 王少云², 方嘉璇¹, 赵岚³, 靳海波¹, 何广湘¹, 郭晓燕¹, 谷庆阳¹, 郝思雯¹, 热孜亚¹, 支伟杰¹, 于洪斌¹, 张荣月¹()

1. 北京石油化工学院化学工程学院, 燃料清洁化及高效催化减排技术北京市重点实验室, 北京 102617
2. 中科森辉微球技术(苏州)有限公司, 苏州 215123
3. 中国科学院过程工程研究所, 生化工程国家重点实验室, 北京 100190

收稿日期:2018-04-17 出版日期:2018-11-15 发布日期:2018-11-15
作者简介:
联系人简介: 张荣月, 男, 博士, 副研究员, 主要从事生物分离层析介质制备及应用方面的研究. E-mail: ryzhang@iccas.ac.cn
基金资助:
北京市自然科学基金面上项目(批准号: 2162013, 2172054)、北京市教委科技面上项目(批准号: KM201710017002)、北京市属高校高水平教师队伍建设支持计划高水平创新团队建设计划项目(批准号: IDHT20180508)、校内学科平台建设项目(批准号: 2018XK002)和北京高等学校高水平人才交叉培养“实培计划”项目(批准号: 17032021006)资助.

Effect of Pore Structure on Protein Capacity in Macroporous Polymer Chromatographic Supports

LI Heng¹, WANG Shaoyun², FANG Jiaxuan¹, ZHAO Lan³, JIN Haibo¹, HE Guangxiang¹, GUO Xiaoyan¹, GU Qingyang¹, HAO Siwen¹, RE Ziya¹, ZHI Weijie¹, YU Hongbin¹, ZHANG Rongyue^1,^*()

1. Beijing Key Labaratory of Fuels Cleaning and Advanced Catalytic Emission Reduction Technology,Beijing Institute of Petro-chemical Technology, Beijing 102617, China
2. Senhui Microsphere Tech.(Suzhou) Co., Ltd. Suzhou 215123, China
3.National Key Lab of Biochemical Engineering, Institute of Process Engineering,Chinese Academy of Sciences, Beijing 100190, China

Received:2018-04-17 Online:2018-11-15 Published:2018-11-15
Contact: ZHANG Rongyue E-mail:ryzhang@iccas.ac.cn
Supported by:
† Supported by the Project of Beijing Natural Science Foundation, China(Nos.2162013, 2172054), the Science and Technology Projects of Beijing Education Commission, China(No.KM201710017002), the Project of Support Plan for the Construction of High Level Teachers and High-Level Innovation Team Building of Beijing Municipal University, China(No.IDHT20180508), the Subject Platform Construction Project, China(No.2018XK002) and 2018BIPT-SPBYSJ of China(No.18032021002).

摘要/Abstract

摘要：

分别以甲基丙烯酸缩水甘油酯和乙二醇二甲基丙烯酸酯作为功能单体和交联剂, 采用悬浮聚合方法制备了大孔聚合物微球. 考察了致孔剂组成对微球的孔径、比表面积的影响, 并用聚乙烯亚胺将微球衍生为阴离子交换层析介质, 考察了微球结构与蛋白载量之间的关系. 结果表明, 微球孔径尺寸随着致孔剂中不良溶剂用量[V(良溶剂)/V(不良溶剂)=1:1~1:3.5]的增加而增大, 而比表面积则呈相反趋势. 离子交换容量(0.11~0.27 mmol/mL)与比表面积(4~38 m²/g)呈正相关, 对应的蛋白静态结合载量亦呈正比关系. 在所考察的孔径范围(301~1524 nm)内, 蛋白动态结合载量先减少后保持稳定, 即当孔径超过410 nm后, 蛋白动态载量值保持在13 mg/mL不变, 表明介质孔径超过此数值后蛋白载量不再受介质的比表面积影响. 此外, 以乙肝病毒表面抗原分子(HBsAg, 22 nm)为探针分子, 利用激光共聚焦显微镜观察了该分子在微球内部的分布, 结果表明, 在该孔径考察范围内, HBsAg均能完全扩散至微球内部.

关键词: 大孔聚合物微球, 层析介质, 蛋白载量, 高通量

Abstract:

Macroporous microspheres were prepared through suspension polymerization, based on a copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, which were used for functional monomer and crosslinking agent, respectively. The effect of porogen on microspheres structure was evaluated in terms of pore size and surface area. The anion exchanged supports were prepared through derivation of microspheres with poly(ethylene imine). The relation of the microspheres structure and the protein capacity was examined on these anion exchanged media. The results indicated that the pore size of microspheres increased with the poor solvent in the porogen(good solvent/poor solvent=1:1—1:3.5), however, the surface area showed a contrary trend. The ion exchanged capacity(0.11—0.27 mmol/mL) increased with the surface area of the microspheres(4—38 m²/g), and the responding static binding capacity of proteins also show a positive correlation with the surface area. The dynamic binding capacity of proteins firstly increased and then retained a changeless value in the pore range of 301—1524 nm. This value was retained at 13 mg/mL when the pore size was more than 410 nm. It indicated that the surface area could not influence the dynamic binding capacity while the pore size of media was beyond some value. Furthermore, the large biomolecular transport in the microspheres was observed through laser scanning confocal microscopy. The results indicated that hepatitis B virus surface antigen(HBsAg) could enter freely the microsphere with all of above pore size. The above results provide a reference for fabrication of the chromatographic supports.

Key words: Macroporous polymer microsphere, Chromatographic media, Protein capactiy, High through-put

中图分类号:

TrendMD:

李恒, 王少云, 方嘉璇, 赵岚, 靳海波, 何广湘, 郭晓燕, 谷庆阳, 郝思雯, 热孜亚, 支伟杰, 于洪斌, 张荣月. 大孔聚合物层析介质孔结构对蛋白载量的影响. 高等学校化学学报, 2018, 39(12): 2673.

LI Heng,WANG Shaoyun,FANG Jiaxuan,ZHAO Lan,JIN Haibo,HE Guangxiang,GUO Xiaoyan,GU Qingyang,HAO Siwen,RE Ziya,ZHI Weijie,YU Hongbin,ZHANG Rongyue. Effect of Pore Structure on Protein Capacity in Macroporous Polymer Chromatographic Supports. Chem. J. Chinese Universities, 2018, 39(12): 2673.

图/表 4

Fig.1 Scheme for the preparation and modification of a copolymer of glycidyl methacrylate(GMA) and ethylene glycol dimethacrylate(EDMA) support(PGMA-EDMA-PEI)

Fig.2 Morphology of microspheres by porogen with different compositionsV(DCM)/V(OA): (A) 1:1; (B) 1:1.5; (C) 1:2; (D) 1:2.5; (E) 1:3; (F) 1:3.5.

Table 1 Proteins binding capacity of microspheres with different structure

V(Dichloromethane)/V(n-octanol)	1:1	1:1.5	1:2	1:2.5	1:3	1:3.5
Pore size/nm	301±0.2	375±0.2	410±0.2	751±0.2	1120±0.2	1524±0.2
Surface area/(m²·g^-1)	38±1	30±1	29±1	25±1	14±1	4±1
Porosity(%)	85±0.1	86±0.1	89±0.1	87±0.1	82±0.1	83±0.1
Ion exchange capacity/(mmol·mL^-1)	0.27±0.01	0.25±0.01	0.25±0.01	0.23±0.01	0.16±0.01	0.11±0.01
Static binding capacity^a/(mg·mL^-1)	62±1	38±1	37±1	31±1	18±1	16±1
Dynamic binding capacity^b/(mg·mL^-1)	21±1	15±1	13±1	13±1	13±1	13±1

Fig.3 Distribution of HBsAg in the supports with different pore sizes(A) HP-DEAE(30—50 nm); (B) 301 nm; (C) 375 nm; (D) 410 nm; (E) 751 nm; (F) 1120 nm; (G) 1524 nm.

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大孔聚合物层析介质孔结构对蛋白载量的影响

Effect of Pore Structure on Protein Capacity in Macroporous Polymer Chromatographic Supports

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