Cell Line Development: A Story of Innovation in Biologics and Cell-Based Therapies

Everything started back in 1982, when the US Food and Drug Administration (FDA) approved the first recombinant insulin, marking the birth of the modern biologics industry.

Previously, insulin and other biologics were made using animal cells that naturally produced biomolecules, a process that involved slow production and complex isolation and purification.

The aforementioned recombinant insulin was produced by DNA-engineered Escherichia coli. Over time, biologics were produced in other recombinant bacteria and yeasts. As biologics increased in complexity, it became clear that bacterial and fungal cells lacked the machinery needed for human-like therapeutics.

Enter mammalian cells: the real game changers in biomanufacturing.

The first biologic produced in engineered mammalian cells—Chinese Hamster Ovary (CHO) cells—was tissue plasminogen activator (tPA), a thrombolytic medication approved by the FDA in 1987. Since then, improvements in mammalian cell lines have powered the development of novel biologics, such as monoclonal antibodies.

This article will dive into the history of cell line development and how advances have supported the production of biologics and cell-based therapies.

Why CHO and HEK293 cells stand out

CHO cells quickly established themselves as the dominant production platform and now account for ~70% of biologics production. To date, most monoclonal antibodies, which dominate the biopharmaceutical industry, are still produced in CHO cells.

A combination of traits makes CHO cells well-suited as protein factories: they grow robustly in suspension, adapt well to large-scale bioreactors, and can be engineered for stable, high-level expression over long production runs.

In parallel, the rapid rise of gene and cell therapies created demand for a different type of production platform. These therapies rely on viral vectors, most commonly adeno-associated viruses (AAVs) and lentiviruses, to deliver genetic material into cells.

In cell therapies, such as chimeric antigen receptor (CAR) T-cell therapy, viral vectors are used ex vivo to genetically modify cells before reinfusion. In gene therapies, the vectors themselves contain the therapeutic gene, which is administered directly to patients.

For these applications, Human Embryonic Kidney (HEK)293 cells have become a dominant manufacturing platform. They are highly amenable to transient transfection with multiple plasmids and provide the human cellular machinery needed for efficient viral assembly and genome packaging.

In essence, CHO cells are optimized for stable, long-term recombinant protein production, while HEK293 cells are suited to complex workflows required for viral vector manufacturing.

Gene editing techniques that support cell line development 

Early cell line development has relied—and in many cases still relies—on the random integration of transgenes into host genomes. In these workflows, scientists transfect cells with plasmids and then screen large numbers of clones to identify those that produce high levels of the desired protein.

Although well established, this approach can lead to considerable variability, since productivity and stability depend on where the gene integrates within the genome. Therefore, strategies that provide greater control over transgene insertion are increasingly adopted.

The field was shaped by the introduction of the Sleeping Beauty transposon system, first reported in 1997, which enabled stable gene insertion in mammalian cells with a preference for open chromatin, for which it is considered a “semi-targeted integration system.” A decade later, the PiggyBac transposon system offered higher cargo capacity and the ability to excise DNA without leaving a genomic footprint—valuable features for flexible engineering in cell line development.

Although these systems are used today, Lekan Daramola, biologics research and development consultant, noted that: “Random integration is still widely used for stable integration of the gene of interest into the desired host cell line.”

“If a company has already established a platform with random integration, they would need a very good reason to move [to a different system],” he continued.

Daramola added that biomanufacturers may adopt transposase systems due to the stability profile of the cell lines.

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The defining tool of modern gene editing emerged in 2012 with the CRISPR-Cas9 system, which enables targeted DNA modification. Although highly specific to gene editing, he highlighted that patent barriers may hinder use by biologic manufacturers.

“Aside from the freedom-to-operate huddle for commercial use, CRISPR can be used to target and insert landing pads [predefined DNA insertion sites] at specific genomic loci when developing a targeted integration platform.”

Daramola believes the adoption of targeted integration platforms will continue to grow: “It’s becoming more common to see CRISPR and similar technologies being harnessed to design host cells by specifically knocking out genes in a host cell line to improve productivity or to reduce process impurities and further enable the expression of increasingly complex modalities.”

High-throughput screening and automation techniques to better select your cells

Biologics manufacturing follows a multi-step workflow. After transfection and clone generation, thousands of cells are screened to identify high-producing, stable clones. These are expanded into production cell lines and grown in bioreactors, where they secrete the target protein into the culture medium. The biologic is subsequently harvested, purified, and the final product is isolated through multiple steps.

High-throughput screening and automation have transformed a slow, manual process into a data-driven workflow. These platforms can generate, culture, and screen thousands of candidates in a fraction of the time. This helps explain why traditional random integration approaches remain widely used. “If you're able to screen 1000s and 1000s of clones, ultimately you get the desired one,” said Daramola.

Today, robotic systems handle routine steps while integrated analytical tools assess quality attributes, including productivity, growth kinetics, and product consistency. “Product consistency is uncompromising,” noted Daramola. “Manufacturers might envision higher yields to reduce the cost of goods and improve commercial viability of the drug, however, product consistency must be absolutely established because it’s critical for the patient.”

This shift not only increases scalability and reproducibility but also improves the probability of identifying high-performing clones early in development, shortening manufacturing timelines.

The future is here: improving biologics manufacturing with synthetic gene circuits

Despite advances, there is still room for improvement in both cell line development efficiency and control. This is where synthetic biology may play a central role.

Cell-based therapeutics manufacturing often relies on viral vectors produced in HEK293 cells. In standard workflows, HEK293 cells are transiently transfected with three plasmids to drive viral particle production.

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“Triple transfection is hyper-challenging to begin with, there are just many more variables to consider [compared to simple transfection], and we still arguably haven’t reached optimal points,” said Ioscani Jiménez del Val, associate professor at University College Dublin. His group develops and deploys synthetic biology and engineering strategies to enhance mammalian-cell-derived therapeutic proteins.

Jiménez del Val explained that one potential improvement lies in engineering HEK293 producer lines to reduce variability and increase AAV yields. However, once assembled, viruses are toxic to the cell, leading to the death of viral factories. Developing stable systems in which viral genes are tightly controlled and activated at the appropriate production stage is a promising strategy.

“In our latest manuscript, we proposed creating a stable cell line with a synthetic gene circuit in which the expression of one viral component acts as a regulatory trigger, driving the sequential activation of the remaining genes so that all required viral elements are expressed in a controlled and balanced manner,” he noted.

Another limitation of current inducible gene systems is their reliance on antibiotic-based inducers to control expression, which raises concerns about potential trace residues in the final product. Jiménez del Val highlighted that synthetic gene circuits relying on intrinsic indicators of cellular state could offer one alternative.

Synthetic gene circuits may also address one of the most difficult variables in biologics manufacturing: glycosylation. As Sheryl Li Yan Lim, PhD candidate, explained: “During my thesis project, we tried to modulate two glycosylation genes specifically for fucosylation and galactosylation, because they provide a lot of variability towards the stability and efficacy of a monoclonal antibody.”

This matters because different glycoforms lead to different therapeutic effects. “Reduced fucosylation can enhance pro-inflammatory activity for cancer therapies, while higher fucosylation may be preferable for immune modulation in diseases such as rheumatoid arthritis,” explained Jiménez del Val.

Another research group previously described an on-off switch mechanism to activate or stop glycosylation in the cells, but Jiménez del Val’s team has a different goal: “[We want to] create ‘a dimmer switch version’ to achieve more graded and precise control.”

Both scientists agree that the future of cell line development will rely on precision DNA engineering to enable faster and more efficient production of biologics. Lim noted: “We’re seeing groups investigating techniques such as Prime Editing and the CRISPR-Associated Transposase system, and the efficiencies that are reported are exceptionally high.”

“Broader adoption of synthetic biology will also depend on regulatory frameworks that better incentivize its implementation across the pharmaceutical sector,” added Jiménez del Val.

Reflecting on her own trajectory, Lim said she hopes to work in a manufacturing environment that invests in synthetic biology-driven research and development: “That’s where you grow, and that’s where you’re looking towards the future. Who knows what the next big disease is going to be; if you’re not actively trying to improve current systems and discover new ones, then you’re on the losing end.”