In 1957, Puck isolated the original Chinese Hamster Ovary (CHO) cell line. Since then, CHO cells have been widely utilized in the pharmaceutical industry for the production of recombinant proteins required for biomedical research, diagnostics, and various therapeutic applications. These biological therapies, particularly monoclonal antibodies, have witnessed remarkable market growth over the past few decades, encompassing a broad range of products such as monoclonal antibodies (mAbs), vaccines, hormones, and other protein-based biologics.
Since 2002, the U.S. Food and Drug Administration (FDA) has approved more than 300 biological products, and this number continues to rise. Biologics including proteins, nucleic acids, carbohydrates and their complexes are being increasingly applied in diagnostic and therapeutic fields. By 2017, 70% of commercially available biologics were produced using Chinese Hamster Ovary (CHO) cells, while the majority of the remaining approved monoclonal antibodies were manufactured predominantly in myeloma cell lines such as SP2/0 and NS0. As the most commonly used and preferred host for biopharmaceutical protein production, CHO cells feature multiple superior characteristics: high expression yields (0.1–1 g/L in batch culture and 1–10 g/L in fed-batch culture), compatibility with large-scale industrial cultivation, easy adaptability to various chemically defined media, low susceptibility to human viral infection, and the capability to perform human-like post-translational modifications.
Over the decades, research groups worldwide have screened subclones of CHO cells by inducing mutations or adapting cells to different culture conditions, further optimizing the performance of CHO cell lines. The most widely applied variants include CHO-K1, CHO DXB11, CHO-S and CHO DG44. Among them, CHO DXB11 (dhfr+/-) and CHO DG44 (dhfr-/-) are dihydrofolate reductase-deficient cell lines screened via methotrexate (MTX) selection; CHO-GS-/- (CHO-K1SV) relies on the glutamine synthetase-mediated selection system with methionine sulfoximine (MSX). CHO-S and its derivative cell lines have been adapted to suspension culture to achieve higher production capacity by elevating producer cell density.
CHO cells exhibit excellent adaptability to genetic manipulation and variable culture conditions, making them well-suited for large-scale industrial manufacturing. On the downside, such cellular plasticity endows CHO cells with a high tendency for genomic rearrangements (deletions, translocations), which serves as a major source of cell line instability during bioproduction.
Genetic modification of CHO cell lines is aimed at achieving higher productivity and better product titers. Core optimization strategies include upregulating the expression of genes associated with cell proliferation promotion, lifespan extension, stress resistance, apoptosis inhibition, as well as enhanced protein synthesis and secretory capacity.
Numerous studies have demonstrated that transient or stable overexpression of key genes involved in cellular metabolism, protein biosynthesis and glycosylation can respectively improve cell growth rate, production efficiency and product quality. Overexpression of multiple anti-apoptotic transcription factors such as BCL2, XIAP, AVEN and MCL1 also enhances cell viability and culture performance. In contrast to forced overexpression of beneficial genes, alternative cell line optimization approaches focus on eliminating or suppressing unfavorable genes through genomic knockout or RNA-mediated gene silencing. Targeted genomic manipulation enables partial or complete deletion of specific genes, and gene function can also be ablated via mutagenesis or siRNA-mediated silencing. Genes involved in chromatin remodeling (e.g., HDACs) and glycosylation (e.g., FUT8, SLC35C1) have become key targets for knockout research, enabling precise engineering of CHO cell lines to produce recombinant proteins with defined post-translational modification profiles. Host cell engineering allows the generation of optimized CHO derivatives that outperform parental cell lines in growth, stress tolerance and productivity. Engineered production cell lines have been proven to achieve expression titers of 3–7 g/L.
Research on recombinant cell line development has focused on expression optimization via targeted genome integration technology. To establish stable clonal cell lines, transgenes need to be integrated into the host genome. Traditional methods rely on random integration of expression vectors, followed by time-consuming screening of qualified clones from heterogeneous recombinant cell pools with random gene insertion. This approach suffers from two major drawbacks: low integration efficiency and suboptimal integration loci. Various transposon systems (Sleeping Beauty, Leap-in, PiggyBac, Tol2) can effectively boost integration efficiency. Ubiquitous Chromatin Opening Elements (UCOEs) and other regulatory motifs have been adopted to prevent transgene silencing and sustain high-level expression. Bacterial Artificial Chromosomes (BACs) are also applied to ensure locus-independent integration, genetic stability and elevated transgene expression levels post-transfection.
For difficult-to-express (DTE) proteins, Tadauchi et al. conducted systematic research to identify the underlying causes of poor antibody expression. They constructed model cell lines with fixed genomic insertion sites, where integrated transgenes are driven by Tet/Dox-inducible promoters. This system enables the characterization of proteins that are cytotoxic or inherently difficult to express for other reasons. In a representative study, Mathias et al. tracked the intracellular processing of selected recombinant DTE proteins and investigated their distribution in respective organelles of the secretory pathway. The results revealed that protein misfolding acts as the rate-limiting step and a major barrier to secretion in problematic antibodies, as misfolded proteins trigger ER-associated degradation.
In cell line development, apart from recombinant protein expression titer, product quality is also critical for the manufacturing of therapeutic biologics and has always been a core focus of industrial technological development. Accurate post-translational modification of target proteins, especially glycosylation patterns, profoundly impacts the efficacy and pharmacokinetics of these biotherapeutics. Furthermore, the type and abundance of glycosylation moieties such as sialic acid and fucose can influence the biological activity and safety of biological products.
The growing global demand for recombinant proteins has driven continuous innovation in biomanufacturing processes. Systems biology approaches have opened up a new dimension for enhancing CHO-based bioproduction. Multi-omics technologies including genomics, transcriptomics, proteomics, metabolomics, lipidomics and glycomics provide unprecedented opportunities to characterize and decipher complex cellular functions, laying a theoretical foundation for further rational optimization of CHO cell platforms. Translating these multi-omics findings into industrial bioprocess optimization and recombinant protein production development remains a key challenge to be addressed in future research.
