Latest Breakthroughs in the Development of CHO Cell Lines for Producing Recombinant Proteins

Recombinant proteins used in biomedical research, diagnostics and different therapies are mostly produced in Chinese hamster ovary cells (CHO) in the pharmaceutical industry. These biotherapeutics, monoclonal antibodies in particular, have shown remarkable market growth in the past few decades. The increasing demand for high amounts of biologics requires continuous optimization and improvement of production technologies. All these requirements boost the development of more efficient expression optimization systems and high-throughput screening platforms to facilitate the design of product-specific cell line engineering and production strategies. In this minireview, we provide an overview on recent advances in CHO cell line development, targeted genome manipulation techniques, selection systems and screening methods currently used in recombinant protein production.

The CHO Expression Platform for Pharmaceutical Recombinant Protein Expression

Mammalian expression-based systems involve various cell lines of different origins, from hamster (CHO, BHK), human (HEK293, HT-1080, PER.C6, CAP, HKB-11, Huh-7) and mouse (NS0, Sp2/0). However, 70% of biologics, and almost all mAbs, are produced in Chinese hamster ovary (CHO) cells, as the most commonly used and preferred hosts for biopharmaceutical protein production.

Marketed drugs produced using CHO cells include:

Drug	Diseases	Biosimilar Stable Cell Lines	Price
Rituximab	Non-Hodgkin's lymphoma, chronic lymphocytic leukemia, rheumatoid arthritis, etc.	Rituximab Stable Cell Line - CHO-K1	Inquiry
Trastuzumab	Breast and stomach cancers	Trastuzumab Stable Cell Line - CHO-K1	Inquiry
Cetuximab	Bowel cancer, etc.	Cetuximab Stable Cell Line - CHO-K1	Inquiry
Adalimumab	Autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, etc.	Adalimumab Stable Cell Line - CHO-K1	Inquiry
Infliximab	Rheumatoid arthritis, Crohn's disease, sigmoiditis and other diseases	Infliximab Stable Cell Line - CHO-K1	Inquiry
Pembrolizumab	Melanoma, non-small cell lung cancer, etc.	Pembrolizumab Stable Cell Line - CHO-K1	Inquiry
Nivolumab	Melanoma, non-small cell lung cancer, etc.	Nivolumab Stable Cell Line - CHO-K1	Inquiry
Avelumab	Urothelial carcinoma, non-small cell lung cancer, etc.
Bevacizumab	Colorectal cancer, non-small cell lung cancer, breast cancer, etc.

Their popularity lies in the facts that they are capable of high productivity (0.1-1 g/L in batch and 1-10 g/L in fed-batch cultures), exhibit consistently good growth phenotypes, are suitable for large-scale industrial culturing, can easily be adapted to various chemically defined media, are less susceptible to infections by human viruses, and they are able to perform human compatible glycosylation.

Despite sharing the same common ancestor, different CHO lineages exhibit substantial genetic heterogeneity as a result of extensive mutagenesis and clonal selection. The most widely used CHO cell lines include CHO-K1, CHO DXB11, CHO-S, and CHO DG44. Comparative studies from different groups provide large amount of data on cell-specific growth and product formation of different recombinant CHO cell lines for various biologics. CHO-K1 cells favoured cell-specific productivity (7-16 pg/cell/day), wheres CHO-S had a preference for biomass production, but lower mAb expression (2–6 pg/cell/day), which was similar in CHO DG44 cells ((2-4 pg/cell/day) in the same experimental setup.

New Focus Areas in Industrial Cell Line Development

The focus of industrial cell line development has shifted towards the issues of host cell line stability for ensuring stable long-term production, the use of targeted integration techniques for expression optimization, especially for complex engineered recombinant therapeutics, and the development of selection and screening systems for faster and more effective clone selection.

Fig. 1 Current focus areas of innovative research in industrial cell line development.

CHO cell line instability

CHO host cell lines, as all other rapidly growing immortalized cells, are genomically unstable and intraclonally heterogenous, which gives the system robustness and flexibility, but causes problems with production stability. An important issue during the development of stable recombinant cell lines is to generate and identify high producing clones, which do not lose their expression capability over time. The rapidly accumulating transcriptomic data available on CHO cell lines open the way for functional analysis to identify regulators and biomarkers of high production and clonal stability.

Targeted genome manipulation techniques

In recombinant cell line generation, the most intensive research line focuses on the use of targeted genome integration techniques for expression optimization. For the establishment of stable and clonal (isogenic) producer cell lines, transgenes need to be integrated into the host genome. Recombinant CHO pools generated in only 2-3 weeks using transposon-mediated gene integration have been reported to be able to achieve extraordinarily high product titers (>7 g/L) and hence can be used for rapid production of large amounts of proteins. In addition, targeted genome manipulation techniques enable the knock-in of recombinant protein coding genes into well-defined and transcriptionally active genomic sites. Targeted genetic modification of cells has been made possible by the emergence of target-specific genome-editing tools, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) RNA guided nucleases. Among these, the CRISPR/Cas9 system became the most popular for its ease of use (as compared to ZFNs and TALENs), high editing efficiency and low cost. The main applications of targeted genome editing tools involve the knock-out or knock-in of genes of interest in the genome of cells or organisms.

Recent advances in the development of high-throughput selection and screening systems

Many improvement efforts focus on monocloning, as the most time-consuming process during producer cell line establishment. Several new techniques are now available for more efficient single cell isolation replacing the classical method of "limiting dilution". Modern cloning approaches employ specialized instruments to ensure that a single cell is seeded into microtiter plate wells. Fluorescence-activated cell sorting (FACS) has been successively used for sorting single cells with specific cellular attributes indicative of high productivity. Many technology developments and modern instruments providing possibility for high-throughput automated processes have already been successfully introduced in the pharmaceutical industry and are now applied in industrial recombinant protein production.

Future Perspectives

Systems biology approach has opened a new dimension for enhancing CHO-based bioproduction.' Omics technologies provide new opportunities to better characterize and understand complex cellular functions, thus create a basis for the possibility of mechanism-driven cell line optimization, instead of manipulating a few key genes. How this knowledge can be adapted and integrated into industrial bioprocess optimization and development regarding recombinant protein production still remains a challenge and needs to be addressed in the future.

Reference:

Tihanyi, B.; et al. Recent advances in CHO cell line development for recombinant protein production. Drug Discovery Today: Technologies. 2020, 38: 25-34.

* For research use only. Not intended for any clinical use.

Quick Inquiry

Latest Breakthroughs in the Development of CHO Cell Lines for Producing Recombinant Proteins

The CHO Expression Platform for Pharmaceutical Recombinant Protein Expression

New Focus Areas in Industrial Cell Line Development

Future Perspectives

Related Products and Services at Creative Biogene