The well-known Chinese Hamster Ovary (CHO) cells are widely used in the production of therapeutic proteins such as antibodies and recombinant proteins. To reduce the differences in protein quality attributes between research and development and large-scale CHO production, a growing number of researchers and production personnel opt for the transient transfection of CHO cells to achieve rapid protein expression.

 

The protein expression level of early CHO transient transfection was generally low. According to literatures published between 2004 and 2011, the expression level of CHO transient transfection was mostly in the range of 60–80 mg/L, which was the reason why CHO transient transfection was not widely adopted in the early stage. However, with the continuous optimization of culture media, transfection reagents and transfection methods, the current expression level of CHO transient protein expression has been able to reach several hundred mg/L, and even up to the g/L level in some projects.

 

The operation procedure of transient protein expression involves multiple steps, and many factors can affect the final yield and quality. BioEngine held a live webinar themed Strategies for Enhancing Transient Protein Expression in CHO Cells in May, introducing a variety of process optimization strategies.

 

Common Methods for Improving Transient Transfection Protein Expression Level:

 

Increasing Transfected Cell DensityIn PEI or liposome-mediated transfection, the cell density for CHO transient transfection is usually 0.5–6 × 10⁶ cells/mL, with the high-density transfection range typically at 4–6 × 10⁶ cells/mL. Higher cell density generally results in higher protein yield, especially suitable for projects with a short expression cycle. For example, in a study published by Eli Lilly and Company, cells were cultured to an initial density of 30–35 × 10⁶ cells/mL via perfusion to improve protein expression. Nevertheless, when conducting high-density transfection, it is necessary to take into account the issues of transfection efficiency and medium nutrition.

 

Feed Medium OptimizationHigh cell viability and extended culture cycle directly contribute to high expression levels. The selection of feed medium and its feeding strategy both exert a direct impact on cell status and protein yield.

 

Optimization of PEI and DNA ConcentrationsMany researchers optimize transfection efficiency by adjusting the ratio of PEI to DNA. As shown in the figure below, transfections were performed using different PEI/DNA ratios, and the expression levels were detected on Day 7. The results indicated that different PEI/DNA ratios have an impact on the transient protein expression levels of both CHO cells and 293 cells.

Exploring Parametric and Mechanistic Differences between Expi293FTM and ExpiCHO-STM Cells for Transient Antibody Production Optimization, Antibodies, 2023 

Optimization of Temperature Reduction Strategy

Lowering the culture temperature can slow down cell proliferation, extend the duration of cellular protein production, and ultimately increase protein yield. Studies have shown that when PEI-mediated transfection is applied to CHOK1SV cells with the temperature reduced to 32℃, the monoclonal antibody yield is tripled. In CHO-DG44 cells undergoing PEI-mediated transfection, cooling the culture to 31℃ leads to more than a threefold increase in antibody production.

 

In addition to the above, optimization strategies also include sequence design optimization, transfection reagent optimization, transfection reagent incubation time adjustment, enhancer selection, and feeding strategy refinement. However, since the research and development phase is usually focused on protein screening and preliminary exploration, which involves a large variety of proteins to be expressed and short expression cycles, CHO transient transfection requires not only high protein expression levels but also universality and stability.

 

RapidTrans CHO Kit

BioEngine has optimized a high-efficiency transfection and expression kit for CHO single-protein expression for you - the RapidTrans CHO Kit. This kit is specially designed and optimized for CHO transient transfection and high-efficiency protein expression, containing basal and feed media, as well as the optimal enhancer and PEI transfection reagent. Whether in the research and development phase or large-scale production phase, the RapidTrans CHO Kit can provide you with satisfactory yields and reliable results.

Product Features

Chemically defined, protein/polypeptide-free, and animal-origin-free

Specifically designed for CHO cell transfection

Ready-to-use kit containing basal medium, feed medium and enhancer; PEI available as an optional component

Enables high-efficiency expression of proteins such as antibodies and antigens in basic research and large-scale production

Product Advantages

Supports high-density transient transfection with transfection efficiency > 95%, and protein yield far exceeds that of imported brands

Ideal protein yield can be achieved from Day 5 to Day 9, allowing flexible adjustment of projects

Optimized combination kit eliminates the hassle of component matching and ensures stable transfection results effortlessly

Significantly reduced price, serving as the optimal solution for cost reduction and efficiency improvement

Product Performance

RapidTrans CHO Kit

It features high transfection efficiency, superior viable cell density (VCD) and cell viability, with protein yield significantly higher than that of imported brands.

CHO-S cells were transfected using the RapidTrans CHO Kit at a transfection density of 6×10⁶ cells/mL. The results showed that compared with imported brand media, the RapidTrans CHO Kit achieved higher viable cell density and better maintenance of cell viability, with a transfection efficiency of 99.76% on Day 3.

Data from internal tests and external customer feedback indicate that the expression level can reach over 200 mg/L on Day 5 and nearly 800 mg/L on Day 9.

 

Both Antigen and Antibody Yields Are Significantly Higher Than Those of Imported Brands

The RapidTrans CHO Kit enables high-efficiency expression of antigens and antibodies, meeting diverse application requirements. Results of transient expression for different protein types show that compared with imported brands, the RapidTrans CHO Kit achieves significantly higher antigen and antibody yields on Day 12. The antigen yield can reach more than twice that of imported brands, while the antibody yield can exceed 1.5 times that of imported brands.

 

External Test Results Show

Protein Yield of RapidTrans CHO Kit Is Significantly Higher Than That of Other Brands

The RapidTrans CHO Kit has delivered outstanding performance across various transient expression projects. In the two cases below, CHO-S cells were used to express monoclonal antibodies, with protein harvest conducted on Day 9. The results demonstrated that the antibody yield of the RapidTrans CHO Kit was significantly higher compared with that of imported brands.

 

Reference

[1] Development of a high cell density transient CHO platform yielding mAb titers greater than 2 g/L in only 7 days, Biotechnology Progress ,2020

[2] Exploring Parametric and Mechanistic Differences between Expi293FTM and ExpiCHO-STM Cells for Transient Antibody Production Optimization, Antibodies, 2023

[3] Scalable Transient Protein Expression, Animal Cell Biotechnology: Methods and Protocols, Methods in Molecular Biology, 2014

[4] Mammalian cell transfection: the present and the future, Anal Bioanal Chem, 2010

[5] Mild hypothermia improves transient gene expression yields several fold in Chinese hamster ovary cells, Biotechnol Prog, 2008

[6] Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells, Biotechnol Prog, 2006