Profiling of Host Cell Proteins by Two-Dimensional Difference Gel Electrophoresis (2D-DIGE): Implications for Downstream Process Development
Profiling of Host Cell Proteins by Two-Dimensional Difference Gel Electrophoresis (2D-DIGE): Implications for Downstream Process Development
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DOI：
10.1002/bit.22532
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发表时间：
2010-02-01
影响因子：
3.8
通讯作者：
Ghose, Sanchayita
中科院分区：
文献类型：
--
作者：
Jin, Mi;Szapiel, Nicolas;Ghose, Sanchayita
宿主细胞蛋白 (HCP) 是使用细胞培养技术生产的生物药物的主要杂质。 HCP 需要在下游过程中受到密切监控并充分去除。然而，HCP 是一种复杂的蛋白质混合物，具有显着不同的分子和免疫学特性。全面了解 HCP 的组成以及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。本文描述了比较蛋白质组分析方法的使用，即。二维差异凝胶电泳 (2D-DIGE) 用于检查 CHO 细胞培养物收获流中的 HCP 组成。使用该技术检查了上游工艺参数（例如细胞培养基、生物反应器控制策略、补料策略和细胞培养持续时间/细胞活力）对 HCP 谱的影响。在所有研究的参数中，细胞活力对 HCP 谱产生最显着的变化。 2D-DIGE 还用于比较单克隆抗体产生细胞和无效细胞培养物之间的 HCP 差异。发现生产细胞培养物中的 HCP 种类在空细胞培养物中得到了很好的体现，这证实了使用空细胞培养物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。它可以直接比较不同条件下 HCP 组成的变化，并可以揭示单个物种的特性（pI、MW），而免疫测定则可以灵敏地量化给定样品中的 HCP 总量。生物技术。生物工程。 2010；105：306-316。 (C) 2009 年 Wiley 期刊公司。
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. Biotechnol. Bioeng. 2010;105: 306-316. (C) 2009 Wiley Periodicals, Inc.