Bristol-Myers Squibb Company, Syracuse, New York 13057, USA.
Biotechnol Bioeng. 2010 Feb 1;105(2):306-16. doi: 10.1002/bit.22532.
Host cell proteins (HCPs) constitute a major group of impurities for biologic drugs produced using cell culture technology. HCPs are required to be closely monitored and adequately removed in the downstream process. However, HCPs are a complex mixture of proteins with significantly diverse molecular and immunological properties. An overall understanding of the composition of HCPs and changes in their molecular properties upon changes in upstream and harvest process conditions can greatly facilitate downstream process design. This article describes the use of a comparative proteomic profiling method viz. two-dimensional difference gel electrophoresis (2D-DIGE) to examine HCP composition in the harvest stream of CHO cell culture. The effect of upstream process parameters such as cell culture media, bioreactor control strategy, feeding strategy, and cell culture duration/cell viability on HCP profile was examined using this technique. Among all the parameters studied, cell viability generated the most significant changes on the HCP profile. 2D-DIGE was also used to compare the HCP differences between monoclonal antibody producing and null cell cultures. The HCP species in production cell culture was found to be well represented in null cell culture, which confirms the suitability of using the null cell culture for immunoassay reagent generation. 2D-DIGE is complimentary to the commonly used HCP immunoassay. It provides a direct comparison of the changes in HCP composition under different conditions and can reveal properties (pI, MW) of individual species, whereas the immunoassay sensitively quantifies total HCP amount in a given sample.
宿主细胞蛋白（HCPs）是使用细胞培养技术生产的生物药物的主要杂质组。HCPs 需要在下游工艺中密切监测并充分去除。然而，HCPs 是蛋白质的复杂混合物，具有明显不同的分子和免疫学特性。全面了解 HCP 的组成以及在上游和收获工艺条件变化时其分子特性的变化，可以极大地促进下游工艺设计。本文描述了使用比较蛋白质组学分析方法，即二维差异凝胶电泳（2D-DIGE），来检查 CHO 细胞培养收获液中的 HCP 组成。使用该技术研究了细胞培养培养基、生物反应器控制策略、补料策略和细胞培养时间/细胞活力等上游工艺参数对 HCP 图谱的影响。在所研究的所有参数中，细胞活力对 HCP 图谱的影响最大。2D-DIGE 还用于比较单克隆抗体产生细胞和空细胞培养之间的 HCP 差异。生产细胞培养中的 HCP 物种在空细胞培养中得到了很好的代表，这证实了使用空细胞培养来生成免疫测定试剂的适用性。2D-DIGE 是对常用 HCP 免疫测定的补充。它提供了在不同条件下 HCP 组成变化的直接比较，并可以揭示单个物种的特性（等电点，MW），而免疫测定则可以灵敏地定量给定样品中的总 HCP 量。
