Cell Line Development in Drug Discovery

Cell line development is a critical step in drug discovery and development. These cells—which are mostly mammalian but can also be bacterial, yeast, algae, or even plant in origin^1-4 —produce therapeutic proteins and antibodies, and are vital for drug screening, toxicity testing, and studying gene function.^5,6 Since the products they produce enter human bodies, their safety and quality are paramount. Recent advancements are addressing industry challenges and ensuring high-quality, productive cell lines.

A product-first approach

Many leaders in the cell line development space agree that the most pressing challenges faced by the industry center around cell line quality, stability, reproducibility, and predictability—which ultimately impact the final product.

“Only cell lines with these stable characteristics will yield reliable results,” explains Dr. Robin Alexander Krüger, Vice President of Arralyze. “Failing to achieve this can have detrimental consequences on the efficacy of the medication for the patient and increase manufacturing costs due to the need for countermeasures to meet required product quality.”

Richard Hammond, CTO of Sphere Fluidics, believes that achieving reliable results is more likely to succeed if the final product is considered starting at the very beginning of the cell line development process, envisioning the cell as a factory intimately tied to its product. “You can’t split factory quality and product quality,” he says. “Instead, we need to bring them together and ask what is the true definition of quality for that system, which we can answer by thinking about how to screen both sides of the problem simultaneously and then building the tools and techniques to do so.”

A product-first approach like this can also go a long way toward heading off other common challenges, such as scalability, regulatory compliance, and cost of production.

Key characteristics of quality cell lines

So, what should researchers be looking for in a high-quality cellular factory? Initial considerations center around cell viability, density, and titer, according to Ildiko Lissberger, LifeScience Research Segment Marketing Manager at Sartorius. “It’s also essential to define your target parameters, how to evaluate them, and decide which are most important to your bioprocess so that you can evaluate whether your clones can recapitulate all of the required features of your biologic.”

There are a few characteristics of cell lines capable of producing the exact product you need, says Krüger. “This starts with good susceptibility to receiving genetic material through electroporation, viruses, and other techniques, as well as stable and high expression rates for the desired proteins. The host cells should also maintain genetic stability over multiple passages and have low mutation rates to preserve the cell line's integrity.”

Correct and consistent post-translational modifications are also important, as well as fast proliferation, resistance to stress, and general robustness. It’s also important that cell lines are able to adapt to different culture conditions, says Krüger, which will facilitate scale-up efforts by allowing cells to be used in different sized bioreactors and under different media conditions while yielding comparable, predictable product. This includes adaptability to serum-free media, according to David Apiyo, Senior Manager, Applications at Sartorius, reducing variability and risk of contamination from animal-derived media components.

Additionally, the right selection of labware, media, and other consumables is vital to avoiding contamination, invalid results, and other costly setbacks, Apiyo says. And, of course, the cell line should be non-tumorigenic and not cause other toxic side effects to ensure safety along with efficacy.

Another critical challenge is technological limitations that remain with gene editing, cell culture, and high-throughput screening techniques that have been developed to help ensure cell lines meet all of the requirements outlined above.

“Fortunately, screening tools and genetic toolboxes are becoming more robust, which will increase the likelihood of success,” says Erik Nordwald, Associate Director of Process Technology and Innovation at KBI Biopharma. Solutions in automation are also playing a key role, especially because they “provide robust documentation capabilities, ensuring traceability of each step, from clone selection to final cell banking,” adds Apiyo.

Tackling cell line development challenges with advanced technologies

Some of the most promising solutions include platforms and instruments that facilitate single-cell isolation and analysis, microreactors, improved genetic engineering and delivery techniques, and automated, high-throughput instruments and platforms.

Single-cell technologies

“The progress in developing various new single-cell sorting and analysis methods is a significant improvement for cell line development,” says Krüger “Unlike traditional methods, such as limiting dilution, these new techniques allow faster iteration cycles and provide functional information on the cells at an early stage of clone selection. This streamlines the cell line development process, making it more cost-effective and improving the quality of the resulting products.”

The CellShepherd^® workstation from Arralyze is a state-of-the-art single-cell screening platform. It gently dispenses cells to increase viability, and leverages real-time imaging to immediately verify clonality and confirm protein expression simultaneously. Minimal sample amounts are required, which is important for groups working with precious samples or rare cells. The CellCelector Automated Cell Selection and Retrieval Platform from Sartorius is another example of a high-throughput single-cell analysis platform. It also uses real-time imaging to verify clonality and viability.

Sphere Fluidics has developed a single-cell analysis platform called Cyto-Mine^® that brings many of these same advantages to the table. It uses the power of microfluidics to create, effectively, a two-million well plate using water-in-oil emulsion known as “picodroplets.” The assay speed and scale this enables from extremely small input volumes saves researchers time and money, says Hammond—up to 40–50% on time and 70% on cost. “It's such a great way of interrogating these very large numbers of cells to really understand which ones are showing the phenotype and the performance you need,” he adds.

Early data with collaborators shows that the cells selected with the Cyto-Mine^® are also extremely capable of scaling. “Our collaborators selected their cells in the picodroplets and then are able to show substantial production at a titer of 50 liters—so they’ve demonstrated scalability over 10 or 11 orders of magnitude,” explains Hammond.

Microreactors

One of the reasons why Cyto-Mine^® yields such promising results, according to Hammond, is that each picoliter-sized droplet acts as a single reactor for a wide range of experimental interrogation. “Microfermenters” like this are one of the exciting enabling techniques identified by Nordwald, who says they allow assessments to be done in both a high-throughput and scalable manner. Sartorius has also developed a microbioreactor systems called the Ambr^® 15 Cell Culture Generation 2: Automated Bioreactor System. Like the Cyto-Mine^®, it speeds up screening, reduces overall costs, and translates to larger-scale bioreactors.

Improved genetic engineering and delivery

No discussion of cell line development would be complete without genetic engineering and delivery of genetic material to cells, especially when the use of non-mammalian cells is increasing. KBI Biopharma, for example, has leveraged state-of-the-art gene-editing technology to produce a proactively altered E. coli cell line, PUREcoli™, which gives reproducibly high titers and product quality. It can be combined with various PUREplasmids™—a library of elements that can be combinatorially assessed to develop bespoke solutions for customers, says Nordwald.

“Another major advancement is new techniques for delivering and introducing genetic material into host cells in a stable form, says Krüger. “Techniques like CRISPR and lipofection can deliver genetic material to cells without using viruses or harsh transfection conditions.”

These sorts of advances are made possible by lowering costs of DNA synthesis and the use of robots, according to Nordwald, which increase scale and enable researchers to test a variety of possible genetic alterations.

High-throughput instruments and platforms

Robots and other high-throughput instruments are key enabling technologies that underpin all of the solutions discussed above, from KBI Biopharma’s improved E. coli strain to the CellShepherd^®, Ambr^® 15, and Cyto-Mine^®.

“Automated, high-throughput systems accelerate development timelines by enabling faster and more informed decision-making,” explains Apiyo, which says that Sartorius has developed a suite of automated instruments that accelerate cell line development from clone selection to protein harvest to cell banking. “Moreover, an often-overlooked benefit is the enhancement of documentation support, which is especially important in an industry governed by stringent regulatory standards. Automated systems provide robust documentation capabilities, ensuring traceability of each step, from clone selection to final cell banking.”

Krüger, Nordwald, and Hammond all agree that support of GMP-compliance is critical for any tool or platform selected to aid in cell line development—and the technologies and tools offered by their companies were developed with this at the forefront.

“Reliability, stability, and reproducibility of experiments in drug discovery and development are of utmost importance; therefore, GMP compliance is a necessity in this field,” summarizes Krüger.

Conclusions

Cell line development has evolved significantly over the years, although critical challenges remain. Exciting new single-cell, gene-editing, and automation solutions have been developed to address these challenges—saving researchers time and money while enabling them to deliver quality, safe, and efficacious drugs to the patients who need them.

References

1. SECRETERS - European Union’s Horizon 2020 Programme. Microbial protein cell factories fight back? Trends Biotechnol. 2022;40(5):576-590. doi: 10.1016/j.tibtech.2021.10.003

2. Srinivasan P and Smolke CD. Biosynthesis of medicinal tropane alkaloids in yeast. Nature. 2020;585(7826):614-619. doi: 10.1038/s41586-020-2650-9

3. Barolo L, Abbriano RM, Commault AS, et al. Perspectives for Glyco-Engineering of Recombinant Biopharmaceuticals from Microalgae. Cells. 2020;9(3):633. doi: 10.3390/cells9030633

4. Karki U, Fang H, Guo W, et al. Cellular engineering of plant cells for improved therapeutic protein production. Plant Cell Rep. 2021;40(7):1087-1099. doi: 10.1007/s00299-021-02693-6

5. Allen DD, Caviedes R, Cárdenas AM, et al. Cell lines as in vitro models for drug screening and toxicity studies. Drug Dev Ind Pharm. 2005;31(8):757-68. doi: 10.1080/03639040500216246

6. Jin H, Zhang C, Zwahlen M, et al. Systematic transcriptional analysis of human cell lines for gene expression landscape and tumor representation. Nat Commun. 2023;14(1):5417. doi: 10.1038/s41467-023-41132-w