Enhancement of transient gene expression and culture viability using Chinese hamster ovary cells overexpressing Bcl‐x_L

Abstract

Transient gene expression (TGE) provides a method for quickly delivering protein for research using mammalian cells. While high levels of recombinant proteins have been produced in TGE experiments in HEK 293 cells, TGE efforts in the commercially prominent CHO cell line still suffer from inadequate protein yields. Here, we describe a cell-engineering strategy to improve transient production of proteins using CHO cells. CHO-DG44 cells were engineered to overexpress the anti-apoptotic protein Bcl-x(L) and transiently transfected using polyethylenimine (PEI) in serum-free media. Pools and cell lines stably expressing Bcl-x(L) showed enhanced viable cell density and increased production of a glycosylated, therapeutic fusion protein in shake flask TGE studies. The improved cell lines showed fusion protein production levels ranging from 12.6 to 27.0 mg/L in the supernatant compared to the control cultures which produced 6.3-7.3 mg/L, representing a 70-270% increase in yield after 14 days of fed-batch culture. All Bcl-xL-expressing cell lines also exhibited an increase in specific productivity during the first 8 days of culture. In addition to increased production, Bcl-x(L) cell lines maintained viabilities above 90% and less apoptosis compared to the DG44 host which had viabilities below 60% after 14 days. Product quality was comparable between a Bcl-xL-engineered cell line and the CHO host. The work presented here provides the foundation for using anti-apoptosis engineered CHO cell lines for increased production of therapeutic proteins in TGE applications.

Ectopic expression of human mTOR increases viability, robustness, cell size, proliferation, and antibody production of chinese hamster ovary cells

“…Although all of these cell characteristics have been successfully improved by over-expression of different transgenes, our study suggests that ectopic expression of mTOR can simultaneously address several of these bioprocess-relevant bottlenecks. Non-limiting examples for one transgene gene-one impact engineering include Bcl-2 (Astley and Al-Rubeai, 2008), Bcl-x(L) (Majors et al, 2008a), Mcl-1 (Majors et al, 2009), E2F1 (Majors et al, 2008b), which reduce apoptosis and improve viability and robustness of production cell lines under standard or suboptimal culture conditions. Also, controlled proliferation technology, a strategy to arrest production cell lines in the G1-phase of the cell-cycle by targeted over-expression of differentiation factors (e.g., c/ebp-a) or cyclin-dependent kinase inhibitors (e.g., p27 or p21) (Fussenegger et al, 1998;Khoo and Al-Rubeai, 2009) enables cells to devote all of their metabolic energy to the synthesis of product protein instead of cell division and has been shown to dramatically increase specific productivities.…”

High‐level protein expression in scalable CHO transient transfection

“…Figure 4C shows that Mab accumulation continues at similar trends throughout the entire fed-batch process. During preparation of this manuscript, a similar feed strategy in CHO transient transfection has been reported with improved productivities with anti-apoptosis approaches (Majors et al, 2008).…”

“…Very recently, the production yield of monoclonal antibody (Mab) in CHO was reported in the range of 60-80 mg/L by lowering the culture temperature and utilizing the woodchuck hepatitis virus post-transcriptional regulatory element (Wulhfard et al, 2008). Engineered CHO cell lines with overexpression of anti-apoptotic protein Bcl-x L showed enhanced viable cell density and increased productivity (Majors et al, 2008). The glycan profile of the product derived from CHO transient transfection has been shown to be comparable to the product from stably transfected CHO cells (Galbraith et al, 2006;Muller et al, 2007).…”

Improving the secretory capacity of Chinese hamster ovary cells by ectopic expression of effector genes: Lessons learned and future directions

“…Different transfection reagents induce different levels of cytotoxicity in terms of impeded cell growth rate and a drop in viability (our own unpublished observations), as well as the inhibition of protein synthesis (Underhill et al, 2003). Notably, transfection stress can be reduced through process or cell engineering optimization (Johari et al, 2015;Macaraeg et al, 2013;Majors et al, 2008). In addition, variability in transfection efficiency is an inherent problem for TGE (Hansen et al, 2015;Liu et al, 2008); however, optimization and selection of the appropriate method can reduce variability substantially (Davies et al, 2013).…”