Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK.
Biotechnol Bioeng. 2012 Apr;109(4):971-82. doi: 10.1002/bit.24383. Epub 2011 Dec 12.
The characterization of host cell protein (HCP) content during the production of therapeutic recombinant proteins is an important aspect in the drug development process. Despite this, key components of the HCP profile and how this changes with processing has not been fully investigated. Here we have investigated the supernatant HCP profile at different times throughout culture of a null and model GS-CHO monoclonal antibody producing mammalian cell line grown in fed-batch mode. Using 2D-PAGE and LC-MS/MS we identify a number of intracellular proteins (e.g., protein disulfide isomerise; elongation factor 2; calreticulin) that show a significant change in abundance relative to the general increase in HCP concentration observed with progression of culture. Those HCPs that showed a significant change in abundance across the culture above the general increase were dependent on the cell line examined. Further, our data suggests that the majority of HCPs in the supernatant of the cell lines investigated here arise through lysis or breakage of cells, associated with loss in viability, and are not present due to the secretion of protein material from within the cell. SELDI-TOF and principal components analysis were also investigated to enable rapid monitoring of changes in the HCP profile. SELDI-TOF analysis showed the same trends in the HCP profile as observed by 2D-PAGE analysis and highlighted biomarkers that could be used for process monitoring. These data further our understanding of the relationship between the HCP profile and cell viability and may ultimately enable a more directed development of purification strategies and the development of cell lines based upon their HCP profile.
在治疗性重组蛋白生产过程中，宿主细胞蛋白（HCP）含量的特征描述是药物开发过程中的一个重要方面。尽管如此，HCP 谱的关键组成部分及其随加工过程的变化尚未得到充分研究。在这里，我们研究了在 fed-batch 模式下培养无模型 GS-CHO 单克隆抗体产生哺乳动物细胞系的不同时间点的上清液 HCP 谱。使用 2D-PAGE 和 LC-MS/MS，我们鉴定了许多细胞内蛋白质（例如，蛋白质二硫键异构酶；延伸因子 2；钙网蛋白），与培养过程中观察到的 HCP 浓度普遍增加相比，其丰度发生了显著变化。在整个培养过程中，与普遍增加相比，丰度发生显著变化的 HCP 取决于所检查的细胞系。此外，我们的数据表明，这里研究的细胞系上清液中大多数 HCP 是通过细胞裂解或破裂产生的，与细胞活力的丧失有关，而不是由于细胞内蛋白质物质的分泌而存在的。SELDI-TOF 和主成分分析也进行了研究，以实现 HCP 谱快速监测变化。SELDI-TOF 分析显示了与 2D-PAGE 分析观察到的 HCP 谱相同的趋势，并突出了可用于过程监测的生物标志物。这些数据进一步加深了我们对 HCP 谱与细胞活力之间关系的理解，并可能最终使更有针对性地开发纯化策略和基于 HCP 谱的细胞系开发成为可能。
