How to Optimize Transient Gene Expression in CHO Cells for Recombinant Antibody

sean.huang

Introduction

Therapeutic antibodies have revolutionized the treatment of various diseases, including cancer, autoimmune disorders, and infectious diseases. As the demand for recombinant antibodies continues to grow, there is an increasing need for rapid and efficient production methods to support drug discovery research.

CHO cells have become the preferred host for recombinant antibody production due to their ability to produce antibodies with human-like glycosylation patterns and their adaptability to serum-free suspension culture. However, optimizing the TGE process in CHO cells can be challenging, as multiple factors influence the efficiency of transfection, cell growth, antibody yield, and quality.

To address these challenges, researchers have been investigating various strategies to enhance TGE in CHO cells. In this week’s Scientific Insight, we will discuss the key factors that affect TGE in CHO cells and provide tips on how to optimize protocols for maximum antibody yield.

Volume of Dilution Buffer for DNAs and Transfection Reagents

The transfection reagent ExpiFectamine is required for transient transfection in the ExpiCHO-S™ cells. Prior to the transfection, both ExpiFectamine and DNA need to be diluted in a buffer, such as Opti-MEM. During the transfection process, it is generally believed that the amount of transfection reagent may affect the expression efficiency. However, research has shown that the volume of the dilution buffer also affects the transfection efficiency of antibodies. In the CHO-S system, as the volume of the dilution buffer increases, the antibody expression level also increases, but beyond a certain point, the antibody expression level gradually decreases. Researchers have also conducted studies using the more cost-effective transfection reagent, PEI, and the trends are consistent with those of ExpiFectamine [1].

Transfection Reagent and DNA Ratio

The ratio of transfection reagent to DNA is a critical factor in the success of a TGE experiment. Too little transfection reagent may result in low transfection efficiency, while too much can be toxic to the cells. The optimal ratio depends on the specific transfection reagent and the amount of DNA used. As a general guideline, it is recommended to start with a ratio of 1:1 to 3:1 (transfection reagent:DNA) and optimize from there.

Incubation Time for Transfection Reagent and DNA

The incubation time for the transfection reagent and DNA complex is another crucial factor. Allowing sufficient time for the complex to form ensures proper delivery of the DNA into the cells. However, prolonged incubation can lead to complex aggregation and reduced transfection efficiency.

Interestingly, the Zhou et al. study found that ExpiCHO-S™ cells preferred a much shorter DNA-PEI complex formation time of 2.5 min compared to 15 min for Expi293F™ cells [1]. Dynamic light scattering analysis showed that the DNA complex size with both PEI and ExpiFectamine™CHO increased over incubation time. This suggests ExpiCHO-S™ cells may uptake smaller DNA complexes more efficiently.

Transfection Ratio

The transfection ratio refers to the number of cells transfected relative to the total number of cells in the culture. A higher transfection ratio generally leads to higher antibody yields, but it also increases the risk of cell toxicity. A transfection ratio of 1:10 to 1:20 (transfected cells:total cells) is a good starting point, but optimization may be necessary for the specific antibody and cell line.

Other Factors to Consider

In addition to the factors mentioned above, several other variables can influence the success of a TGE experiment:

• Cell density: Maintaining an optimal cell density (usually 1-3 × 10⁶ cells/mL) ensures healthy growth and efficient transfection. ExpiCHO-S™ cells are typically transfected at twice the viable cell density of Expi293F™ (6 × 10⁶ vs 3 × 10⁶ cells/mL) [1].
• Plasmid quality: Using high-quality, endotoxin-free plasmid DNA minimizes cell toxicity and maximizes transfection efficiency.
• Media composition: Optimizing the media composition, including the use of serum-free or low-serum media, promotes cell growth and antibody production. Feed media and additives like DMSO can enhance yields, with ExpiCHO-S™ cells responding better to DMSO than Expi293F™ [1].
• Incubation conditions: Maintaining optimal temperature (usually 37°C, with a temperature shift to 32°C for ExpiCHO-S™) and CO2 levels (typically 5-8%) during the transfection and expression phases is crucial.
• Co-transfection of cell cycle regulators: Co-expression of p21 and p27 at a 1:1 ratio enhanced antibody yields in both Expi293F™ and ExpiCHO-S™ cells, with a greater effect in ExpiCHO-S™ [1].

Conclusion

Optimizing transient gene expression in CHO cells for recombinant antibody production is a complex process that involves multiple factors. By understanding the key factors such as transfection reagent to DNA ratio, incubation time, transfection ratio, buffer pH, dilution buffer volume, and other factors like cell density, plasmid quality, media composition, and co-transfection of cell cycle regulators, researchers can develop tailored protocols to maximize antibody yields and quality. The optimized TGE parameters provided by Zhou et al. [1] can serve as an excellent starting point for researchers looking to improve their transient antibody production in CHO cells.

Future Prospects

As the demand for recombinant antibodies continues to grow, the optimization of transient gene expression in CHO cells will remain a critical area of research. Additionally, the integration of high-throughput screening technologies, such as robotic liquid handling systems and miniaturized bioreactors, will allow for the rapid optimization of TGE protocols and the identification of optimal conditions for specific antibody constructs. Furthermore, the application of advanced analytical tools, such as multi-omics approaches and machine learning algorithms, will provide deeper insights into the cellular processes that influence antibody production and quality, enabling the rational design of enhanced TGE systems. As these technologies and strategies continue to evolve, the efficiency and reliability of transient gene expression in CHO cells will improve, accelerating the development of novel antibody therapeutics and advancing the field of drug discovery.

GenScript’s TurboCHO Antibody Production Platform

GenScript has developed the TurboCHO™ antibody production platform, a fully optimized system designed to streamline the antibody production process. The platform leverages extensively researched and tailored protocols for various antibody isotypes, ensuring maximum yields and quality. By utilizing the TurboCHO platform, researchers can save time and resources while obtaining the high-quality recombinant antibodies needed for their drug discovery research.

References:

1. Zhou J, Yan GG, Cluckey D, et al. Exploring Parametric and Mechanistic Differences between Expi293F™ and ExpiCHO-S™ Cells for Transient Antibody Production Optimization. Antibodies. 2023;12(3):53. Published 2023 Aug 10. doi:10.3390/antib12030053