Table 1.
Optimization strategies to improve recombinant protein transient expression in mammalian cells
| Strategy | Cell lines | VCD (fold) | Yield (fold) | qP (fold) | References | |
|---|---|---|---|---|---|---|
| Cell line modification | ||||||
| Knockout Bax and Bak gene | CHO-K1 | 0.93–1.6 | 3–4 | - | Macaraeg et al. (2013) | |
| Overexpression Bcl-xL gene | CHO-DG44 | 1.15–3 | 0.7–2.7 | 1.5–4.8 | Majors et et al. (2008) | |
| Using Expi CHO-S™ | CHO-S | – | 2.5–6 | 0.99–2.25 | Zhong et al. (2019) | |
| Using HEK293T and HEK 293-6E | HEK 293 | – | 3 –4 | – | Jäger et al. (2013) | |
| Overexpression XBP-1S and ERO1-La | CHO-S, CHO-K1 | – | 5.3–6.2 | – | Cain et al. (2013) | |
| Vector engineering | ||||||
| Promoter | CMV | CHO-K1 | – | 1.4–1.9 | 2.59–4.89 | Bayat et al. (2018) |
| CMV | CHO-S | 1.4–2 | 1.05 | – | Gupta et al. (2017) | |
| CR5, CMV5 | CHOBRI/rcTA | – | 3–4 | – | Poulain et al. (2017) | |
| CMV (NF-κB, CRE,YY1) |
CHO-S CHO-K1 |
– | 1.5 | – | Brown et al. (2015) | |
| Cis-acting element | UCOE | CHO-DG44 | – | 1.5 | – | Nematpour et al. (2017) |
| TM, WPRE, Intron A, SP163 | CHO-K1, HEK293 | – | 1.7–10.5 | – | Mariati et al. (2012) | |
| Signal peptides | human albumin and azurocidin | CHO-K1, CHO-DG44 | – | 1.5–2 | 0.15–1.7 | Kober et al. (2013) |
| Culture medium optimization | ||||||
| Peptone | CHO-DG44 | – | 1.37 | – | Davami et al. (2014) | |
| Wheat peptone | CHO-K1 | 1.3 | 1.6 | – | Burteau et al. (2003) | |
| DMSO + LiAc + fed-batch | CHO-K1 | – | 2–3 | – | Ye et al. (2009) | |
| 10% DMSO, 5 min | HEK293 | – | ~ 1.6 | – | Lynch et al. (2021) | |
| Culture process optimization | ||||||
| Mild hypothermia (32 °C) + sodium butyrate | CHO-S,CHO-K1 | – | 5.3–6.2 | – | Cain et al. (2013) | |
| Mild hypothermia (32 °C) | CHO-T | 2.23–3.45 | – | 1.76 | Sou et al. (2018) | |
| Mild hypothermia (33 °C) | HEK-293 S | – | ~ 1.5 | – | Lin et al. (2015) | |
| Light-activated | CHO-DG44 | – | 4 | – | Minami and Shah (2021) | |
| Transfection conditions screening | ||||||
| Transfection reagent (25 kDa linear PEI) | CHO-S, CHO-K1 | – | 2 | – | Daramola et al. (2014) | |
| Transfection reagent (PEI, PEIpro™) | HEK293-6E, CHO-3E7 | – | 1.1–2.3 | – | Delafosse et al. (2016) | |
| Flow electroporation | HEK293, CHO, NSO | – | 0.9–6.8 | – | Parham et al. (1998) | |
| Transfection ratio (PEI to DNA = 5:1) | Bax Bak DKO CHO-K1 | 0.93–1.6 | 3–4 | – | Macaraeg et al. (2013) | |
| Transfection ratio (3:1) + cell densities (5 × 105) | CHO-DG44 | – | – | 1.37 | Davami et al. (2014) | |
| Co-expressed help gene | ||||||
| DMA + XBP-1S | CHO-k1 | – | 1.5–2.5 | – | Rajendra et al. (2015) | |
| miR-17 + miR-92a | CHO | – | 3 | 2 | Jadhav et al. (2014) | |
| Bcl-xL | CHO-S | – | ~ 2 | – | Zustiak et al. (2014) | |
| NDPK-A + EBNA-1 | CHO-K1 | ~ 1.75–2.5 | ~ 1.5–3.5 | Budge et al. (2021) | ||
| EBNA-1 + PyLT | CHO-DG44 | – | – | 22.9 | Lee et al. (2018) | |
| EBNA-1 + GS | CHO | – | 2 | – | Daramola et al. (2014) | |
| eIF3i | CHO-K1, HEK293,NIH3T3 | – | ~ 1.1– ~ 1.4 | – | Roobol et al. (2020) | |
| Sp35 Fc to CypB = 5:1 | CHO-S | – | 6 | – | Johari et al. (2015) | |
VCD viable cell density; qP specific production; XBP-1S X-box binding protein 1 s; ERO1-La endoplasmic oxidoreductin 1 like protein; UCOE ubiquitous chromatin opening element; CR5 cumate gene switch promoter; YY1 yin yang; NF-κB nuclear factor kappa B; CRE cyclic adenosine; CHOBRI/rcTA cell line: stably expresses the cymene repressor (CymR) and the cumate reverse transactivator; SP163 163-bp long splice variant; TM triple leader sequence; WPRE woodchuck hepatitis virus post-transcriptional regulation element; EBNA Epstein-Barr virus nuclear antigen-1; DMSO dimethyl sulfoxide; LiAc lithium acetate; PEI polyethylenimine; DMA N, N-dimethyl acetamide; NDPK-A nucleoside diphosphate kinase; PyLT polyoma virus large T antigen; eIF3 eukaryotic initiation factor 3; CypB cyclophilin B