More similar than different: Host cell protein production using three null CHO cell lines

Autor: Feny Gunawan, Julie C. Nishihara, Donald E. Walker, X. Christopher Yu, Eric Huang, Martin Vanderlaan, Denise C. Krawitz, Judith Zhu-Shimoni, Inn H. Yuk, Pynn Abigail Friederike Joyce, Jayashree Subramanian
Rok vydání: 2015
Předmět:
Zdroj: Biotechnology and Bioengineering. 112:2068-2083
ISSN: 0006-3592
DOI: 10.1002/bit.25615
Popis: 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 107 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. Biotechnol. Bioeng. 2015;112: 2068–2083. © 2015 Wiley Periodicals, Inc.
Databáze: OpenAIRE
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