Abstract:

Aiming at the process optimization of chromatographic separation, the high-throughput screening methods of protein adsorption with microtiter filter plate were established for initial resin screening, adsorption properties evaluation, adsorption isotherms and adsorption kinetics measurement, and adsorption and elution conditions optimization. Firstly, the operation parameters of 96-well filter plate were optimized, and with two ion exchangers and two mixed-mode resins as the model, the bovine serum albumin adsorption was investigated with different resins and liquid conditions by means of microtiter filer plate. The binding capacity maps were obtained and the appropriate adsorption-desorption conditions were determined. Further, the microtiter filter plate was applied to measure the adsorption isotherms and adsorption kinetics with four resins at specific adsorption conditions, and the adsorption parameters were obtained. Finally, the elution conditions were optimized using microtiter filter plate. The microscale results were comparable to that with packed-bed separation, indicating that the methods developed in the present work were practicable. The results demonstrated that the high-throughput process development of protein adsorption with microtiter filer plate was feasible and can be used to fast screen the best reins and liquid conditions. New methods showed the advantages of low resource consumption, large experimental fluxes, short developing period, wide application and high stability, which would be one new method for development of the protein separation process.

Key words: high-throughput screening , microtiter filer plate, chromatography, adsorption, protein separation

摘要：

针对色谱分离过程优化，建立了基于微孔过滤板的蛋白吸附高通量筛选方法，用于介质初筛、吸附性能考察、吸附等温线和吸附动力学测定、吸附和洗脱条件优化等。首先优化了96孔过滤板的操作参数，以2种离子交换介质和2种混合模式介质为典型代表，采用微孔过滤板方法考察了不同介质和液相条件下牛血清白蛋白的吸附，得到结合载量分布图，确定了合适的蛋白吸附和解吸条件。进一步测定了4种介质在特定吸附条件下的吸附等温线和吸附动力学曲线，获得吸附相关参数。最后，采用微孔过滤板进行了洗脱条件优化，并与填充柱色谱分离进行比较，验证了方法的可靠性。结果表明，基于微孔过滤板的蛋白吸附高通量筛选是切实可行的，可以快速筛选色谱介质和液相，优化蛋白分离条件，具有资源消耗小、实验通量大、研发周期短、适用性广、稳定性高的特点，是蛋白色谱分离过程优化的一种新方法。

关键词: 高通量筛选, 微孔过滤板, 色谱分离, 吸附, 蛋白分离