Abstract
Over the past few years, the biopharmaceutical industry has increasingly utilized mammalian cell expression systems for producing biologics. The current state of glycosylation mechanisms in these systems, along with the fact that monoclonal antibodies are most often used as therapeutic proteins, has a big impact on how biologics develop. Therapeutic recombinant glycoproteins, including monoclonal antibodies, exhibit different biological properties due to their varied glycan profiles. Thus, developing cell genetic modification strategies not only enhances cell-specific productivity but also optimizes glycan profile distribution to increase therapeutic activity. Moreover, advancements in “omics” technologies provide new possibilities for improving these aspects, particularly for Chinese Hamster Ovary (CHO) cells.
Cell Engineering
Expression Systems
In the biopharmaceutical industry, glycoprotein production is achieved through either transient or stable gene expression in mammalian cells. Transient expression is preferred for rapid and cost-effective methods as it bypasses the lengthy process of integrating plasmids into the genome, making it significantly faster. However, the production rate depends on various factors, including transfection efficiency, the cytotoxicity of transfection reagents, and the long-term fed-batch culture strategy. Although the protein levels obtained are not as high as those from stable gene expression systems, transient transfection remains suitable for many applications, especially high-throughput screening and early product characterization. To date, only viral vectors for gene therapy have been produced through transient transfection and have been clinically applied.
When large-scale glycoprotein production is required, stable gene expression systems are the preferred method. For these systems, several aspects have been processed and optimized to enhance productivity, process robustness, and reduce cell line generation time. Stable gene expression involves integrating the gene of interest into the host cell genome, where it can be maintained and expressed over many generations of cell division.
Selection Systems
Over the years, numerous selection systems have been developed to enhance protein production rates and selection efficiency. For stable expression, gene markers are typically integrated into the expression plasmid along with the cDNA encoding the target gene. The number of integrated plasmid copies and the integration site in the host genome are crucial factors. Common selection markers in the biopharmaceutical industry include the glutamine synthetase (GS) and dihydrofolate reductase (DHFR) genes. TNS0 and Sp2/0 cell lines lack sufficient endogenous GS expression, allowing for selection by simply removing glutamine from the medium. For CHO cells, combining methionine sulfoximine (MSX, a glutamine analog) with glutamine-free medium inhibits endogenous GS activity, providing sufficient selection pressure. Additionally, recent developments in CHO GS-knockout (KO) cell lines have increased the stringency of the selection system.
Similarly, the DHFR selection system uses CHO cell lines deficient in DHFR. By integrating the recombinant DHFR gene into the plasmid and subjecting the cells to increasing concentrations of methotrexate (which inhibits DHFR enzyme activity) and nucleic acid precursor starvation, the target gene is amplified. Other selection systems, such as the Oscar™ system from the University of Edinburgh, utilize hypoxanthine-guanine phosphoribosyltransferase (HPRT) for purine synthesis, although DHFR and GS systems remain the most widely used for large-scale commercial production.
Gene Expression
Despite the success of these selection systems, they still face a major issue: they rely on random plasmid integration and expression in the host genome. This randomness produces heterogeneous cell populations with varying expression levels across clones. Transgenes may be inserted into heterochromatin regions, leading to very weak gene expression. This necessitates screening numerous clones (typically hundreds to thousands) to find those with plasmids integrated into highly active chromatin regions (“hot spots”). Recently, several molecular and cellular biology tools have been developed for targeted gene integration, reducing the randomness of gene insertion and increasing predictability for high transgene expression.
Recombinase-mediated cassette exchange (RMCE) and site-specific nucleases like Zinc Finger Nucleases, Transcription Activator-Like Effector Nucleases (TALEN), and CRISPR/Cas9 have shown promise in this regard. RMCE utilizes site-specific recombinases to exchange specific sequence cassettes on plasmids with corresponding sequences in the host genome. This technique improves the success rate and reduces the time required to develop stable cell lines expressing monoclonal antibodies. The second generation of tools, including TALEN, and CRISPR/Cas9, induce double-strand breaks at precise locations in the host genome, facilitating targeted integration through non-homologous end joining (NHEJ) or homologous recombination (HDR).
Cell Growth, Proliferation, and Survival
Optimizing cell growth, proliferation, and survival is crucial for maximizing protein yield in mammalian cell expression systems. Process parameters such as pH, temperature, and mixing, along with media formulation, play vital roles in this optimization. Commercial media formulations are often proprietary, making the optimization resource-intensive. Additionally, small molecules from chemical libraries, such as sodium butyrate (NAB) and valproic acid (VPA), can enhance protein yield by maintaining chromatin in an open configuration through histone acetylation.
Optimizing the timing and concentration of these inhibitors is essential to mitigating unwanted effects like cell cycle arrest or apoptosis. Combining these inhibitors with other strategies, such as reduced temperature (30–32 °C) during the production phase, can further improve productivity by slowing down cell cycle progression and shifting cells from a proliferative to a production mode. Anti-apoptotic engineering strategies, including the overexpression of Bcl-2 family genes (Bcl-2, Bcl-xL, and Mcl-1) and downregulation of pro-apoptotic genes (Bax, Bak, Caspase-3, -7, -8, and -9), have been shown to enhance cell survival and protein yield.
Protein Folding and Secretion
Proper folding and secretion of glycoproteins are critical for producing functional therapeutic proteins. As proteins are translated, they undergo glycosylation and folding in the endoplasmic reticulum (ER) and Golgi apparatus before secretion. The levels of ER-associated proteins and molecular chaperones significantly impact protein folding. Overexpression of certain chaperones and folding enzymes, such as protein disulfide isomerase (PDI) and BiP, has been shown to improve productivity in some cases. However, results can be mixed, depending on the host cell line and the specific protein being produced.
ER stress response elements like X-box binding protein 1 (XBP-1) and glucose-regulated protein 78 (GRP78) are also crucial for proper protein folding. Overexpression of XBP-1, a transcription factor involved in the unfolded protein response (UPR), has shown positive effects on protein production in some studies. Another key factor is GADD34, which restores translation by dephosphorylating eukaryotic initiation factor 2 alpha (eIF2α) under ER stress, thereby enhancing protein production levels.
Glycosylation Optimization
Glycosylation is a critical post-translational modification affecting the stability, efficacy, and immunogenicity of therapeutic glycoproteins. CHO cells, the most commonly used mammalian cell line for glycoprotein production, do not replicate all human glycosylation types, such as α-2,6-sialylation and α-1,3/4-fucosylation. Efforts in metabolic engineering and genetic modification aim to address these limitations by introducing human glycosylation pathways into CHO cells. For example, introducing enzymes like sialyltransferases and fucosyltransferases can help achieve more human-like glycosylation patterns.
Conclusion
The production of therapeutic glycoproteins in mammalian cells has seen significant advancements, driven by the need for optimized glycosylation, enhanced cell productivity, and robust expression systems. CHO cells remain the workhorse for glycoprotein production, but ongoing research and development efforts continue to improve their efficiency and the quality of the glycoproteins they produce. Future innovations in cell engineering, process optimization, and glycosylation control will further advance the field, ensuring the availability of high-quality therapeutic proteins to meet growing healthcare demands.
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