Advances in CHO Cell Line Development for Biotherapeutics
The landscape of biotherapeutic production is rapidly evolving, driven by an increasing demand for more complex recombinant proteins and a shift toward personalized medicine.
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A diverse array of biological medicines – including therapeutic proteins, hormones, monoclonal antibodies, cytokines, enzymes and vaccines – are now used to prevent and treat a wide range of illnesses. While some of these biotherapeutics were originally obtained from natural origins, such as donated blood, many are now synthesized using advanced biotechnology techniques, providing a safer and more reproducible source.
Building upon the earliest protein-based therapies, such as insulin, the field has continued to make remarkable progress. Nowadays, the development of much larger and increasingly complex proteins, such as bispecific or trispecific antibodies, is ushering a new era of advanced biotherapeutics. Engineered mammalian cell lines have emerged as the preferred system for manufacturing these products – due to their capacity to facilitate appropriate protein folding, assembly and post-translational modifications (PTMs).
In recent decades, mammalian cell line development has undergone profound enhancements, significantly boosting production efficiencies. Achieving titers exceeding 10 grams of protein per liter of culture has become possible through a combination of optimizing host cells, expression vectors, screening methods, media and innovations in process development.
A brief history
High-performing, stable and robust cell lines are the fundamental building blocks for large-scale manufacturing of biotherapeutic proteins.
Chinese hamster ovary (CHO) cells are by far the most commonly used hosts for commercial therapeutic protein production. Their significance dates back to 1986 when the approval of CHO-derived human tissue plasminogen activator revolutionized medicine – opening the door for using mammalian cell culture for manufacturing therapeutic proteins.
In addition to their historical usage, CHO cells offer several advantages – including their capacity to thrive in large-scale suspension culture, their ability to produce high yields of recombinant protein and their compatibility for growth in serum-free, chemically defined media. This last feature ensures both consistency across different batches and enhanced safety by avoiding media containing human or animal-derived additives that could harbor potential contaminants, such as viruses.
However, other legacy systems such as Sp2/0 and NS0 mouse myelomas and baby hamster kidney (BHK) cells are also still in use – and more recently, human cell lines have also been developed, offering reduced immunogenicity and improved PTM.
From gene to medicine
The cell line development process typically begins with cloning the gene of interest into an appropriate vector, such as a plasmid or viral vector. This construct is then introduced into a suitable host cell line, giving rise to a mixed population of cells. Subsequently, single cells carrying the vector are isolated and proliferated – with thousands of monocultures then subjected to a rigorous screening process to select the most suitable clones for large-scale production.
The next step is to confirm the stability of the clone over many generations. Long-term culture brings the risk of spontaneous mutations – and if any of these changes confer a selective advantage, this could lead to unwanted variations in the end product. “For reasons we still do not understand, some cell lines are more likely to create truncation variants,” explained Susan Sharfstein, professor of nanoscale science and engineering, University at Albany.
The Sharfstein laboratory investigates the role of cell physiology and culture conditions in the production of therapeutic proteins and carbohydrates, with the aim of refining production systems.
Optimizing cell growth conditions – such as media composition, temperature and pH – is a critical step for maximizing cell viability and output.
When developing cell lines there are a number of essential issues to consider, including stability and demonstrated clonality, noted Sharfstein, but “growth and specific productivity are the most critical.”
Accelerating cell line development
The cell line development process is a time and labor-intensive process, typically requiring six to twelve months. To help accelerate these timelines, researchers are investigating a variety of vector and cell engineering strategies in efforts to generate high-producing mammalian cells.
In recent studies, Sharfstein and her team have been exploring the impact that epigenetics has on CHO cell productivity, an area of growing interest. Epigenetic silencing has previously been implicated in loss of productivity. However, supplementation of short-chain fatty acids such as butyrate can enhance cell-specific productivities in CHO cell lines. Sharfstein attests to this: “It is well known that addition of sodium butyrate may increase productivity, and this is probably due to an epigenetic mechanism, in which histone deacetylation is inhibited.”
Building on previous work that had demonstrated the impact of transcription factor–transgene interactions on productivity, Sharfstein recently investigated the role of DNA CpG methylation in CHO cells. In a paper published in Antibody Therapeutics, high-producing clones were found to show increased methylation of the CMV promoter region. Increased yield and improved specific productivity were seen in cells treated with the hypomethylation agent azacytidine. “Our recent studies comparing clones with different productivities seem to implicate an epigenetic effect in which some clones have differential methylation in CpG sites in the CMV promoter,” said Sharfstein.
Methylation of cytosines in the promoter region is implicated in reduced transcription, one mechanism for epigenetic silencing. Sharfstein elaborated: “What we saw, which was particularly interesting, was that the differential methylation appeared to block a particular transcription factor binding site in one group of clones, which may lead to differential transcription factor binding in different clones.”
In the last few years, there have been several other notable advances in cell line development. Sharfstein highlighted that “there is increasing interest in targeted integration to increase stability and to put transgenes into hot spots,” with a growing number of different transposase technologies becoming available.
The past decade has also witnessed a rapid increase in automation across all areas of cell line development, leading to significant improvements in efficiency. Furthermore, recent strides in high throughput ‘omics-based technologies – such as genomics, epigenomics, transcriptomics, proteomics and metabolomics – hold immense promise in expediting cell line development timelines.
“The rise of gene-editing technologies, particularly CRISPR is poised to make a substantial impact on the field as gene editing becomes more straightforward and reproducible (once the IP issues are finally resolved)," added Sharfstein. Through the targeted knockout of specific genes, valuable insights can be gained into how to improve the growth and productivity parameters of cell lines. The technology also offers new opportunities for engineering cell lines that can produce proteins with specific and desirable characteristics – heralding a shift toward customized biopharmaceutical production.
In the Sharfstein laboratory, CRISPR is currently being used in work to try to identify which transcription factors bind to the CMV promoter. “We previously explored transcription factor binding by using chromatin immunoprecipitation (ChIP) in which you use antibodies against a particular transcription factor to precipitate or pull down the transcription factor and the DNA associated with it. But what we really want to do is the reverse, pull down a specific DNA sequence (in our case the CMV promoter) and see what proteins come down with it. There are some relatively recent papers describing this “reverse ChIP”, but no one to our knowledge has used it for this application to understand transcriptional regulation of the CMV promoter in CHO cells,” explained Sharfstein.
Benefiting more patients
The future of cell line development holds the promise of early clone identification and closed, automated processes, leading to faster and more cost-effective production. The industry aims to achieve a seamless pipeline from cell selection to product release, all within a controlled and monitored environment, marking a significant evolution in biopharmaceutical manufacturing.
Driven by the commercial success of biotherapeutics, the demand for complex recombinant proteins derived from mammalian cells continues to grow – driving the need for increased efficiency and productivity.
“The biggest challenge and opportunity is in the production of novel moieties – molecules that nature never intended, like bispecifics,” Sharfstein said. “Understanding how these molecules interact with cell physiology is going to provide information that we hope will improve productivity.”
Increased use of computational approaches is also likely to have a significant impact expects Sharfstein, and she plans to use artificial intelligence and machine learning in her future work.
Continued advances in technologies and approaches in this field will help to make advanced therapies more affordable and accessible to a wider range of patients.
About the interviewee
Susan Sharfstein is a professor of nanoscale science and engineering at the University at Albany. Her research focuses on the role of culture conditions and cell physiology on the use of living systems for industrially relevant processes.