Early Stage Purification, Process Research and Development, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
Biotechnol Prog. 2010 Sep-Oct;26(5):1322-31. doi: 10.1002/btpr.437.
The potential of cationic polyelectrolytes to precipitate host cell and process related impurities was investigated, to replace one or more chromatography steps in monoclonal antibody purification. The impact of antibody isoelectric point, solution properties (pH and ionic strength), and polyelectrolyte properties (structure, molecular weight and pK(a)) on the degree of precipitation was studied. At neutral pH, increasing solution ionic strength impeded the ionic interaction between the polyelectrolyte and impurities, reducing impurity precipitation. Increasing polyelectrolyte molecular weight and pK(a) enabled precipitation of impurities at higher ionic strength. PoIy(arginine) was selected as the preferred polyelectrolyte in unconditioned cell culture fluid. PoIy(arginine) precipitation achieved consistent host cell protein clearance and antibody recovery for multiple antibodies across a wider range of polyelectrolyte concentrations. Poly(arginine) precipitation was evaluated as a flocculant and as a functional replacement for anion exchange chromatography in an antibody purification process. Upstream treatment of cell culture fluid with poly(arginine) resulted in flocculation of solids (cells and cell debris), and antibody recovery and impurity clearance (host cell proteins, DNA and insulin) comparable to the downstream anion exchange chromatography step.
研究了阳离子聚电解质沉淀宿主细胞和工艺相关杂质的潜力，以替代单克隆抗体纯化过程中的一个或多个色谱步骤。考察了抗体等电点、溶液特性（pH 值和离子强度）以及聚电解质特性（结构、分子量和 pK(a)）对沉淀程度的影响。在中性 pH 下，增加溶液离子强度会阻碍聚电解质与杂质之间的离子相互作用，从而减少杂质沉淀。增加聚电解质的分子量和 pK(a) 可以在更高的离子强度下沉淀杂质。在未调节的细胞培养物中，聚（精氨酸）被选为首选的聚电解质。聚（精氨酸）沉淀可实现多种抗体在更宽的聚电解质浓度范围内一致的宿主细胞蛋白清除率和抗体回收率。聚（精氨酸）沉淀被评估为在抗体纯化过程中用作絮凝剂和阴离子交换色谱的功能替代物。用聚（精氨酸）对细胞培养物进行上游处理可导致固体（细胞和细胞碎片）絮凝，并且抗体回收率和杂质清除率（宿主细胞蛋白、DNA 和胰岛素）与下游阴离子交换色谱步骤相当。
