Abstract:

Agarose gel microspheres were used to prepare a series of metal-chelating affinity chromatography media by chemically cross-linking the microspheres and ligand attachment. Subsequently, their adsorption properties in model proteins were examined. Firstly, the effects of different reaction conditions on microsphere size were explored. The results showed that under the optimal conditions, the particle size of agarose microspheres was concentrated in the range of 45—160μm, with a yield of 95% and an average particle size of 91.2μm. Then, the crosslinked agarose microspheres 4FF(4 Fast Flow) and 6FF(6 Fast Flow) were prepared from agarose microspheres 4B and 6B, and the cross-linking agent dropping method was regulated to explore the effects of different factors on the mechanical strength of the cross-linked agarose microsphere 4FF. The maximum flow rates of 4FF and 6FF could reach 1190cm/h and 2228cm/h, respectively. In addition, the recovery performance and morphological characterization of the prepared 4FF microspheres were tested, and the retention volumes of different standard proteins were determined with the help of size-exclusion chromatography and the effective distribution coefficients were calculated. Finally, dextran-grafted metal chelating affinity chromatography media were prepared by using cross-linked agarose microspheres 6FF as a matrix, and monoclonal antibody Ig G was used as a model protein to explore the effects of different conditions on the dynamic protein loading of the media. The results showed that the highest dynamic protein loading of the medium could reach 44.8mg/mL gel. The present work provides a new idea for the controllable preparation and modification of agarose microspheres, as well as their application in the separation and purification of biological proteins, and lays a foundation for the subsequent scaled-up production.

Key words: agarose microspheres, metal chelation, chromatographic packing, optimization, protein

摘要：

以琼脂糖凝胶微球为研究对象，通过对微球进行化学交联与配体连接，制备系列金属螯合亲和层析介质，并测试其在模型蛋白中的吸附性能。首先，探究不同工艺条件对微球尺寸的影响。结果表明，在最优条件下，琼脂糖微球粒径集中在45~160μm之间，收率为95%，平均粒径为91.2μm。随后，以琼脂糖微球4B和6B为原料制备交联琼脂糖微球4FF（4 Fast Flow）和6FF（6 Fast Flow），调控交联剂滴加方式，探究不同因素对4FF微球机械强度的影响，所得4FF和6FF的最大流速分别达1190cm/h和2228cm/h。此外，本工作对制备的4FF微球进行复原性能测试及形貌表征，借助凝胶色谱法测定不同标准蛋白的保留体积，并计算有效分配系数。最后，以交联琼脂糖微球6FF为基质，制备葡聚糖接枝型金属螯合亲和层析介质，以单克隆抗体Ig G为模型蛋白，探究不同条件对介质动态蛋白载量的影响。结果表明，介质的最高动态蛋白载量可达44.8mg/mL gel。本工作为琼脂糖微球的可控制备、改性，以及在生物蛋白分离纯化中的应用提供了新思路，也为后续扩大化生产奠定了基础。

关键词: 琼脂糖微球, 金属螯合, 层析填料, 优化, 蛋白质