Oceanside Process Research & Development, Genentech, Inc., One Antibody Way, Oceanside, CA 92056, United States.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Mar 15;878(9-10):798-806. doi: 10.1016/j.jchromb.2010.01.044. Epub 2010 Feb 4.
Polyamine precipitation conditions for removing host cell protein impurities from the cell culture fluid containing monoclonal antibody were studied. We examined the impact of polyamine concentration, size, structure, cell culture fluid pH and ionic strength. A 96-well microtiter plate based high throughput screening method was developed and used for evaluating different polyamines. Polyallylamine, polyvinylamine, branched polyethyleneimine and poly(dimethylamine-co-epichlorohydrin-ethylenediamine) were identified as efficient precipitants in removing host cell protein impurities. Leveraging from the screening results, we incorporated a polyamine precipitation step into a monoclonal antibody purification process to replace the Protein A chromatography step. The optimization of the overall purification process was performed by taking the mechanisms of both precipitation and chromatographic separation into account. The precipitation-containing process removed a similar amount of process-related impurities, including host cell proteins, DNA, insulin and gentamicin and maintained similar product quality in respect of size and charge variants to chromatography based purification. Overall recovery yield was comparable to the typical Protein A affinity chromatography based antibody purification process.
研究了聚胺沉淀条件，以去除含单克隆抗体的细胞培养液中的宿主细胞蛋白杂质。我们考察了聚胺浓度、大小、结构、细胞培养液 pH 值和离子强度的影响。建立了基于 96 孔微量滴定板的高通量筛选方法，并用于评估不同的聚胺。聚烯丙基胺、聚乙烯亚胺、支化聚乙烯亚胺和聚（二甲胺-co-表氯醇-乙二胺）被鉴定为去除宿主细胞蛋白杂质的有效沉淀剂。根据筛选结果，我们在单克隆抗体纯化过程中加入聚胺沉淀步骤，以替代蛋白 A 层析步骤。通过考虑沉淀和层析分离的机制，对整个纯化过程进行了优化。含有沉淀的工艺去除了相似数量的与工艺相关的杂质，包括宿主细胞蛋白、DNA、胰岛素和庆大霉素，并在大小和电荷变异方面保持与基于层析的纯化相似的产品质量。总体回收率与典型的基于蛋白 A 亲和层析的抗体纯化过程相当。
