The host cell system is a critical determinant of recombinant protein quality and yield, with direct influence on protein folding and post-translational modification factors that shape the pharmacokinetic and pharmacodynamic profiles of the final product. Although Chinese Hamster Ovary (CHO) cell lines share a common origin, individual parental clones exhibit unique growth characteristics and require tailored culture conditions to perform optimally. Furthermore, established CHO lines are often specialized for distinct production strategies, making cell line selection a pivotal step in process development. CHO cells continue to be the industry standard for recombinant protein production due to their adaptability, reliability, and scalability. While advances in cell culture, transfection, and purification have significantly improved yields, large-scale bioreactor implementation remains resource intensive. Therefore, optimizing upstream conditions and workflows is essential to reduce costs and ensure consistent, high-quality output.
Recombinant protein production with CHO cells
The production of recombinant proteins, including monoclonal antibodies (mAbs), relies on proper protein folding and post-translational modifications, most notably glycosylation. Typical workflows for the production of recombinant proteins using CHO cells include:
- Delivery of recombinant DNA into host cells - Introduction into the host genome can be achieved by retroviral transfection, lipofection, electroportation, or calcium phosphate precipitation.
- Selection of transfected cells - Achieved by using enzyme-deficient parental clones that are dependent on exogenous nutrients. Co-transfection of recombinant DNA with an enzyme (e.g., Dihydrofolate Reductase [DHFR]) enables selection of positive clones by removal of these nutrients.
- Expansion and evaluation of selected clones - Clones are evaluated based on their functional characteristics, including growth rate, productivity, and product quality.
Choosing an optimal CHO cell line
Common CHO cell lines include CHO-K1, CHO-S, and CHO-DG44. Research has shown that CHO-K1 cells are well-suited for the production of specific mAbs, while CHO-S cells produce proteins with lower levels of glycosylation but a higher growth rate, making them better suited for biomass formation.
Optimizing cell culture conditions
Fed-batch culture is the most common method for growing CHO cells for protein production and involves semi-continuous addition of nutrients to growth media. Fed-batch culture does not remove waste products from growing cultures, meaning their accumulation must be closely monitored.
The productive capacity of fed-batch culture is highly dependent on several parameters, including:
- Temperature: 36-37°C
- PH: 6.8 - 7.2
- Dissolved oxygen (DO): 30-60% saturation
Some protocols utilize a shift to a lower temperature at later stages of culture to promote cell growth and protein production.
A shift from conditions of 37°C and pH 7.05, to 30°C and pH 6.75, can improve CHO production of a recombinant Epo-Fc fusion protein, as measured by size exclusion chromatography. You can read more in the handbook on designing experiments in protein production from Cytiva.
Figure 1. Shows gel filtration analyses after the protein A capture showing different Epo-Fc conformations depending on cultivation conditions: (A) 30°C, pH 6.75 and (B) 37°C, pH 7.05. From Figure 5.1.1 in Cytiva Handbook: Design of experiments in protein production and purification
Protein glycosylation is also dependent on environmental conditions, and glycan profiles of CHO products can impact the immunogenicity, efficacy, and circulatory half-life of mAbs. Techniques have been developed to introduce factors to culture media to influence glycan profiles, including manganese, galactose, and uridine.
Optimizing transfection of recombinant DNA
Recombinant gene expression is dependent on the site of integration into the chromosome; recombinant DNA is only expressed when integrated at a transcriptionally active site. Optimizing transfection can reduce downstream inefficiencies when selecting for productive clones and promote amplification of the recombinant gene.
Chromatin opening elements in the vector DNA, such as scaffold/matrix attachment regions (S/MARs), can promote stable transfection efficiency and increase the proportion of positive CHO clones. Bacterial Artificial Chromosomes (BACs) are viral vectors that incorporate the entire locus, including regulatory elements that control gene expression. As such, BACs are not susceptible to the positional effects of random integration and can drive stable and efficient gene expression.
Read the guide from Cytiva to learn more about protein production workflows. Contact Cytiva for more scientific support.
