Analysis of dynamic changes in the proteome of a Bcl‐XL overexpressing Chinese hamster ovary cell culture during exponential and stationary phases

Abstract

Mammalian cell cultures used for biopharmaceutical production undergo various dynamic biological changes over time, including the transition of cells from an exponential growth phase to a stationary phase during cell culture. To better understand the dynamic aspects of cell culture, a quantitative proteomics approach was used to identify dynamic trends in protein expression over the course of a Chinese hamster ovary (CHO) cell culture for the production of a recombinant monoclonal antibody and overexpressing the antiapoptotic gene Bcl-xl. Samples were analyzed using a method incorporating iTRAQ labeling, two-dimensional LC/MS, and linear regression calculations to identify significant dynamic trends in protein abundance. Using this approach, 59 proteins were identified with significant temporal changes in expression. Pathway analysis tools were used to identify a putative network of proteins associated with cell growth and apoptosis. Among the differentially expressed proteins were molecular chaperones and isomerases, such as GRP78 and PDI, and reported cell growth markers MCM2 and MCM5. In addition, two proteins with growth-regulating properties, transglutaminase-2 and clusterin, were identified. These proteins are associated with tumor proliferation and apoptosis and were observed to be expressed at relatively high levels during stationary phase, which was confirmed by western blotting. The proteomic methodology described here provides a dynamic view of protein expression throughout a CHO fed-batch cell culture, which may be useful for further elucidating the biological processes driving mammalian cell culture performance.

Recovery of Chinese hamster ovary host cell proteins for proteomic analysis

“…Although the optimal organic solvent concentration is less than typically used for CHO lysate precipitation [25, 26], this finding agrees with previous reports that 50% acetone is sufficient to precipitate most proteins greater than 20 kDa [21]. Conversely, Thongboonkerd et al have shown that a final organic solvent concentration of 90% was required to optimally precipitate urinary proteins [23].…”

“…Although research has previously demonstrated high recovery with less than 1 hr organic solvent incubation [21], incubation times for organic solvent precipitation typically range from several hours [25] to overnight [12, 24]. Decreasing precipitation incubation time can increase throughput without sacrificing protein recovery.…”

5.2 Functional -Omics for Cell Lines and Processes: The -Omics Technologies on the Example of CHO Cells

“…Today's typical fed-batch bioprocess runs over 14 days, covering a variety of cellular states and environmental conditions, with obvious changes in growth rate, productivity, and, most importantly, product quality. The real power of -omics analysis is the ability to monitor these changes [78,79] and to generate an understanding of the needs of cells that enable them to generate high quality and value products [80].…”

Overexpression of Serpinb1 in Chinese hamster ovary cells increases recombinant IgG productivity

“…Unfolded Protein Response (UPR) related genes such as X-box binding protein 1 (Xbp-1) (Cain et al, 2013), GADD34 and ATF4 have also been overexpressed and resulted increase in recombinant protein productivity. In order to reduce apoptosis, Bcl-XL, Mcl-1, MEM and Akt have all been overexpressed and improved culture longevity in various recombinant CHO cell lines (Arden et al, 2007;Carlage et al, 2012;Hwang and Lee, 2009;Majors et al, 2009). Additionally, some researchers successfully overexpressed genes that led to improved CHO cell growth or protein expression.…”