Yuk Inn H, Nishihara Julie, Walker Donald, Huang Eric, Gunawan Feny, Subramanian Jayashree, Pynn Abigail F J, Yu X Christopher, Zhu-Shimoni Judith, Vanderlaan Martin, Krawitz Denise C
Early Stage Cell Culture, Genentech, 1 DNA Way, South San Francisco, California, 94080.
Protein Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, California, 94080.
Biotechnol Bioeng. 2015 Oct;112(10):2068-83. doi: 10.1002/bit.25615. Epub 2015 May 12.
To understand the diversity in the cell culture harvest (i.e., feedstock) provided for downstream processing, we compared host cell protein (HCP) profiles using three Chinese Hamster Ovary (CHO) cell lines in null runs which did not generate any recombinant product. Despite differences in CHO lineage, upstream process, and culture performance, the cell lines yielded similar cell-specific productivities for immunogenic HCPs. To compare the dynamics of HCP production, we searched for correlations between the time-course profiles of HCP (as measured by multi-analyte ELISA) and those of two intracellular HCP species, phospholipase B-like 2 (PLBL2) and lactate dehydrogenase (LDH). Across the cell lines, proteins in the day 14 supernatants analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) showed different spot patterns. However, subsequent analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) indicated otherwise: the total number of peptides and proteins identified were comparable, and 80% of the top 1,000 proteins identified were common to all three lines. Finally, to assess the impact of culture viability on extracellular HCP profiles, we analyzed supernatants from a cell line whose viability dropped after day 10. The amounts of HCP and PLBL2 (quantified by their respective ELISAs) as well as the numbers and major populations of HCPs (identified by LC-MS/MS) were similar across days 10, 14, and 17, during which viabilities declined from ∼80% to <20% and extracellular LDH levels increased several-fold. Our findings indicate that the CHO-derived HCPs in the feedstock for downstream processing may not be as diverse across cell lines and upstream processes, or change as dramatically upon viability decline as originally expected. In addition, our findings show that high density CHO cultures (>10(7) cells/mL)-operated in fed-batch mode and exhibiting high viabilities (>70%) throughout the culture duration-can accumulate a considerable amount of immunogenic HCP (∼1-2 g/L) in the extracellular environment at the time of harvest (day 14). This work also demonstrates the potential of using LC-MS/MS to overcome the limitations associated with ELISA and 2D-PAGE for HCP analysis.
为了解提供给下游加工的细胞培养收获物（即原料）中的多样性，我们在未产生任何重组产物的空运行中，使用三种中国仓鼠卵巢（CHO）细胞系比较了宿主细胞蛋白（HCP）谱。尽管CHO谱系、上游工艺和培养性能存在差异，但这些细胞系产生的免疫原性HCP的细胞特异性生产率相似。为了比较HCP产生的动态变化，我们寻找了HCP（通过多分析物ELISA测量）的时间进程谱与两种细胞内HCP种类，即磷脂酶B样2（PLBL2）和乳酸脱氢酶（LDH）的时间进程谱之间的相关性。在所有细胞系中，通过二维聚丙烯酰胺凝胶电泳（2D-PAGE）分析的第14天上清液中的蛋白质显示出不同的斑点模式。然而，随后的液相色谱-串联质谱（LC-MS/MS）分析却显示：鉴定出的肽和蛋白质总数相当，并且鉴定出的前1000种蛋白质中有80%在所有三个细胞系中是相同的。最后，为了评估培养活力对细胞外HCP谱的影响，我们分析了一种在第10天后活力下降的细胞系的上清液。在第10天、14天和17天期间，HCP和PLBL2的量（通过各自的ELISA定量）以及HCP的数量和主要群体（通过LC-MS/MS鉴定）相似，在此期间活力从约80%下降到<20%，细胞外LDH水平增加了几倍。我们的研究结果表明，下游加工原料中源自CHO的HCP在不同细胞系和上游工艺之间可能没有那么多样，或者在活力下降时变化没有最初预期的那么显著。此外，我们的研究结果表明，以补料分批模式运行且在整个培养过程中具有高活力（>70%）的高密度CHO培养物（>10⁷细胞/mL），在收获时（第14天）可在细胞外环境中积累相当数量的免疫原性HCP（约1-2 g/L）。这项工作还证明了使用LC-MS/MS克服与ELISA和2D-PAGE相关的HCP分析局限性的潜力。
