Gu Feng, Chodavarapu Kiran, McCreary Dennis, Plitt Thomas A, Tamoria Edward, Ni Michelle, Burnham Jennifer J, Peters Michael, Lenhoff Abraham M
W. R. Grace, 7500 Grace Drive, Columbia, MD 21044, United States.
W. R. Grace, 7500 Grace Drive, Columbia, MD 21044, United States.
J Chromatogr A. 2015 Jan 9;1376:53-63. doi: 10.1016/j.chroma.2014.11.082. Epub 2014 Dec 8.
The main objective of our research was to develop silica-based, polymer-functionalized ion exchange materials for single-use bioprocess applications, with the ultimate goal of achieving maximal binding capacity for target proteins. Herein we report the utilization of Grace(®) wide pore silica gel and bonding the silica with cationic polymers. The strong anion exchange materials have been prepared by a two-step process involving initial bonding with two trimethoxysilanes and subsequent aqueous solution radical polymerization with quaternary ammonium ion containing monomers and an azo initiator. Using the binding capacities for bovine serum albumin (BSA), a model protein for the evaluation of the new materials, we optimized the processes with regard to the median pore size of the silica gel, as well as polymer composition and ratios, which were determined by reagent ratios and reaction conditions. The products were also characterized by both chemical and physical methods. It has been found that higher binding capacities are associated with lower ligand density and higher molecular weight for the attached polymers, with over 20% higher in both static and dynamic binding capacity values with the same amount of attached polymers. The advantages of a large pore size distribution and optimal median pore size for the base silica are discussed. Optimal pore size range of 500-1500Å and distribution of Span 90 for over 1.0 give the highest BSA binding capacities. Silica-based strong anion exchange materials showed excellent flow characteristics when packed into a column and were superior to commercial agarose-based strong anion exchange material with respect to dynamic binding capacity, elution of proteins, and baseline separation of a mixture of three model proteins.
我们研究的主要目标是开发用于一次性生物工艺应用的二氧化硅基聚合物功能化离子交换材料，最终目标是实现对目标蛋白质的最大结合能力。在此，我们报告了格雷斯（®）大孔硅胶的利用以及硅胶与阳离子聚合物的键合。通过两步法制备了强阴离子交换材料，第一步是与两种三甲氧基硅烷键合，第二步是与含季铵离子的单体和偶氮引发剂进行水溶液自由基聚合。使用牛血清白蛋白（BSA）的结合能力作为评估新材料的模型蛋白，我们针对硅胶的中值孔径以及聚合物组成和比例优化了工艺，这些由试剂比例和反应条件决定。还通过化学和物理方法对产品进行了表征。已发现较高的结合能力与连接聚合物的较低配体密度和较高分子量相关，在连接聚合物量相同的情况下，静态和动态结合能力值均高出20%以上。讨论了基础硅胶大孔径分布和最佳中值孔径的优势。500 - 1500Å的最佳孔径范围和大于1.0的斯潘90分布给出了最高的BSA结合能力。二氧化硅基强阴离子交换材料填充到柱中时显示出优异的流动特性，在动态结合能力、蛋白质洗脱以及三种模型蛋白混合物的基线分离方面优于市售琼脂糖基强阴离子交换材料。
