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

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/m L)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/m L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(HBs Ag,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,HBs Ag均能完全扩散至微球内部.

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 m^2/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.