Comprehensive assessment of host cell protein expression after extended culture and bioreactor production of CHO cell lines
Comprehensive assessment of host cell protein expression after extended culture and bioreactor production of CHO cell lines
复制标题
CHO细胞系扩展培养和生物反应器生产后宿主细胞蛋白表达的综合评估
DOI：
10.1002/bit.28128
复制
发表时间：
2022
影响因子：
3.8
通讯作者：
Lee, Kelvin H.
中科院分区：
文献类型：
--
作者：
Hamaker, Nathaniel K.;Min, Lie;Lee, Kelvin H.
生物制造业正在朝着连续过程发展，这将涉及更长的培养时间和更老的细胞年龄。由于宿主细胞蛋白（HCP）水平的波动，这些上游趋势可能会给下游纯化带来不可预见的挑战。为了了解中国仓鼠卵巢（CHO）细胞在这些时间尺度上表现出的HCP表达不稳定性程度，一个行业联盟合作开发了一项研究，以表征HCP水平在30、60和90个细胞周期（代表约60天）内的年龄依赖性变化。将生物反应器中批量生产率高达3 g/L的单克隆抗体（mAb）生产细胞系与其亲本CHO-K1宿主平行老化。随后，在自动化生物反应器系统中培养每个年龄的两种细胞类型，以生成用于HCP分析的收获细胞培养液（HCCF）。使用互补蛋白质组学技术、二维电泳（2DE）和液相色谱-串联质谱（LC-MS/MS）对1500多种HCP进行定量。虽然高达13%的蛋白质显示出随年龄变化的表达，但在两种细胞系之间进行比较时观察到更多的变化，其中高达47%的HCP差异表达。一个具有年龄依赖性表达的小子集（50名HCP）先前报告为高风险和/或难以清除的杂质，存在问题;然而，其中绝大多数随年龄下调。我们的研究结果表明，HCP表达在这个时间尺度上的变化可能不会像最初预期的那样剧烈，并对下游加工构成巨大挑战，但对表达有问题的HCP的监测仍然至关重要。
The biomanufacturing industry is advancing toward continuous processes that will involve longer culture durations and older cell ages. These upstream trends may bring unforeseen challenges for downstream purification due to fluctuations in host cell protein (HCP) levels. To understand the extent of HCP expression instability exhibited by Chinese hamster ovary (CHO) cells over these time scales, an industry‐wide consortium collaborated to develop a study to characterize age‐dependent changes in HCP levels across 30, 60, and 90 cell doublings, representing a period of approximately 60 days. A monoclonal antibody (mAb)‐producing cell line with bulk productivity up to 3 g/L in a bioreactor was aged in parallel with its parental CHO‐K1 host. Subsequently, both cell types at each age were cultivated in an automated bioreactor system to generate harvested cell culture fluid (HCCF) for HCP analysis. More than 1500 HCPs were quantified using complementary proteomic techniques, two‐dimensional electrophoresis (2DE) and liquid chromatography coupled with tandem mass spectrometry (LC‐MS/MS). While up to 13% of proteins showed variable expression with age, more changes were observed when comparing between the two cell lines with up to 47% of HCPs differentially expressed. A small subset (50 HCPs) with age‐dependent expression were previously reported to be problematic as high‐risk and/or difficult‐to‐remove impurities; however, the vast majority of these were downregulated with age. Our findings suggest that HCP expression changes over this time scale may not be as dramatic and pose as great of a challenge to downstream processing as originally expected but that monitoring of variably expressed problematic HCPs remains critical.
登录
查看更多内容
影响因子：
3.3
作者：
Marcus K;Lelong C;Rabilloud T
通讯作者：
Rabilloud T
影响因子：
3.9
作者：
Gronemeyer, Petra;Ditz, Reinhard;Strube, Jochen
通讯作者：
Strube, Jochen
生物制造过程中的宿主细胞蛋白质
DOI：
--
发表时间：
2019
期刊：
Current applications of cell culture engineering
影响因子：
--
作者：
Baik, J.Y.;Guo, J.;Lee, K.H.
通讯作者：
Lee, K.H.
影响因子：
4.6
作者：
Park JH;Jin JH;Lim MS;An HJ;Kim JW;Lee GM
通讯作者：
Lee GM
影响因子：
3.8
作者：
Chusainow, Janet;Yang, Yuan Sheng;Yap, Miranda G. S.
通讯作者：
Yap, Miranda G. S.