Recent Advances in Engineering the Unfolded Protein Response in Recombinant Chinese Hamster Ovary Cell Lines

Abstract

Chinese hamster ovary (CHO) cells are the most common protein production platform for glycosylated biopharmaceuticals due to their relatively efficient secretion systems, post-translational modification (PTM) machinery, and quality control mechanisms. However, high productivity and titer demands can overburden these processes. In particular, the endoplasmic reticulum (ER) can become overwhelmed with misfolded proteins, triggering the unfolded protein response (UPR) as evidence of ER stress. The UPR increases the expression of multiple genes/proteins, which are beneficial to protein folding and secretion. However, if the stressed ER cannot return to a state of homeostasis, a prolonged UPR results in apoptosis. Because ER stress poses a substantial bottleneck for secreting protein therapeutics, CHO cells are both selected for and engineered to improve high-quality protein production through optimized UPR and ER stress management. This is vital for optimizing industrial CHO cell fermentation. This review begins with an overview of common ER-stress related markers. Next, the optimal UPR profile of high-producing CHO cells is discussed followed by the context-dependency of a UPR profile for any given recombinant CHO cell line. Recent efforts to control and engineer ER stress-related responses in CHO cell lines through the use of various bioprocess operations and activation/inhibition strategies are elucidated. Finally, this review concludes with a discussion on future directions for engineering the CHO cell UPR.

Keywords: Chinese hamster ovary (CHO) cells; ER stress; biopharmaceuticals; synthetic biology; therapeutic proteins; unfolded protein response (UPR).

Figure 1

Accumulation of unfolded proteins results in the UPR. After translation, proteins that are properly folded are secreted (route 1: Secretion), while misfolded proteins are broken down in order to recycle important amino acids for continued production of other proteins (route 2: ERAD). Stress in the ER occurs when misfolded proteins accumulate. When the chaperone BIP binds misfolded proteins, a downstream transcription cascade (route 3: UPR) is initiated to either relieve burdens on the ER or activate apoptotic pathways if the former cannot be achieved. Italics within the nucleus represent the promoter elements bound by each transcription factor. Previously undefined abbreviations: IRE1-dependent decay (RIDD); site-1 and site-2 proteases (S1P and S2P, respectively). Figure created with BioRender.com.

Figure 2

UPR activation is an optimization problem in CHO cell line development. A moderate amount of ER stress is advantageous for high productivity. If the UPR is only minimally activated, the cell line will exhibit low productivity. Likewise, if the cell line has an overactive UPR, it might exhibit low productivity due to apoptosis. Figure created with BioRender.com.

Figure 3

Effects of reduced temperature (Section 5.4) and chemical treatments (Section 6.1) on UPR activation. The UPR pathways are the same as shown in Figure 1. The PERK pathway is indicated in red, the IRE1 pathway is indicated in green, and the ATF6 pathway is indicated in blue. Activation of a UPR biomarker is indicated by a green arrow. Inhibition of a UPR biomarker is indicated by a blocked red line. The effects of temperature downshift are indicated as TDS. The effects of chemical treatments are shown using their respective abbreviations, which are as follows: 3-methyladenine (3-MA); baicalein (BAI); beta alanine (BALA); beta cyclodextrin (BCD); betaine (BET); BIP inducer X (BIX); thapsigargin (Tg); tunicamycin (Tm); valproic acid (VPA); yeast extract (YEX); copper sulfate (CuSO4); spermidine (SPD); trehalose (TREH); linoleic acid (LA); conjugated linoleic acid (CLA); mannose (MAN); cottonseed hydrolysate (CSH); maltose (MAL); maltodextrin (MD), sucrose (SUC); proteasome inhibitor MG132 (MG132); taurine (TAU), dimethyl sulfoxide (DMSO); hydrogen peroxide (H2O2); sodium butyrate (NaBu); cell cycle inhibitor (CCI); and rosmarinic acid (RA). * The use of TDS causes increases in HERPUD1 for rh-tPA [90] and decreases in HERPUD1 for EPO-Fc [92]. ** Sulaj et al. report downregulation of BIP and PDIA4 in response to Tm [56]. Figure created with BioRender.com.

Figure 4

Positive and negative effects of CHO cell UPR engineering for different products. Based on Table 3. (a) Positive effects include increased titer, yield, and q_p, etc. (b) Negative effects include decreased titer, yield, q_p, etc. UPR targets shown in parentheses are downregulated or knocked out. The total number of positive/negative effects shown on the y-axis for each UPR target includes co-expression studies. The General mAbs category includes ETE Trastuzumab (Tras), DTE Infliximab (Infli), humAb 2F5 IgG, anti-IL-8 IgG, TfR-Ab, DTE Doppelmab, Adalimumab, ETE rituximab, and hTRA-8. The EPO category includes EPO-Fc. The General FcFPs category includes TNFR-Fc and DTE Sp35Fc.