Cell-Line Development and Engineering

Therapeutic antibodies remain a rapidly growing class of medical treatments accounting for, by 2018, eight of the ten best-selling drugs worldwide.¹ Important for treating diseases ranging from rheumatoid arthritis to cancer, almost all of today’s antibody therapies are manufactured using mammalian cells.²

Mammalian cells are adaptable, can produce large volumes of therapeutic proteins, and manufacture proteins similarly to human cells, reducing the risk of immune rejection.³ It remains time consuming, however, to develop a homogeneous population of cells⁴ that consistently give a large yield of high-quality product.⁵

Cell-line development thus remains a bottleneck in drug development, especially now as companies are increasingly developing therapies based on molecules that don’t exist in nature. Bispecific and multi-specific antibodies, for example, are more complex than traditional monoclonal antibody-based therapies and can be harder to express.⁵

Optimizing the cell-line development workflow

The cell-line development process typically has five stages, according to Helge Schnerr, Ph.D, Global Campaign Manager at Sartorius. The first consists of gene cloning and the selection of a population of cloned cells. Once these are identified, the next step is analytics to ensure the cell line is producing what is expected. Following that, work begins to optimize the cell cultivation conditions to maximize the yield from the cell line, along with characterization and evaluation of its properties. Finally, companies bank cells in case the manufacturing process needs to be started afresh.

Companies have traditionally sought to improve cell-line development by accelerating and simplifying each stage in this process.⁶ Dr. Schnerr, for example, explained that Sartorius now offers an automated cell selection and picking system to speed up clone selection using nano-well plates, rather than 96-well plates, to screen 100,000s of clones at once. The company also offers Ambr^® bioreactors to “bridge the gap” between clone selection and the biomanufacturing process at a commercial scale.

“Traditionally, people start with micro-flasks in an incubator to see how the cell line acted and survived at larger scales,” he says, explaining that Sartorius is among a few companies to provide a fully automated system that allows drug developers to experiment with different culture conditions in multiple, parallel mini reactors at the same time.

However, as Soléne Chartier, Director of Cell Line Engineering, Revvity's Horizon Discovery, writes, “when handling relatively few cell-line engineering projects […] the upfront expense and need to adapt existing processes can mean that a primarily manual approach is preferred.”

Other traditional approaches to maximizing the productivity of cell lines include process control, data analytics, and single-use bioreactors,⁶ which have a pouch containing the cell culture. According to Dr. Schnerr, the benefit of single-use systems is that “once you’ve finished your experiments, you just clean out the pouch rather than having to clean the whole equipment to prevent any contamination.”

Engineering efficient cell lines

Improvements in the cell-line development workflow haven’t removed the bottleneck in drug discovery. As a result, companies and researchers have increasingly turned to altering the cells themselves.

Some of the simpler approaches use evolution through multiple generations to select for cells better able to resist sources of cellular stress, such as exposure to hydrogen peroxide. Such techniques have the advantage of being less complex than modern gene-editing approaches, which—even when successfully executed—can fail to produce desired results due to the intricate interplay of genes, proteins, and signaling pathways within each cell.^6,7

Genetic engineering can be used to improve product yields by, for example, reducing the rate of cell death under high stress conditions. This could involve, for example, co-transfecting genes that regulate cell death alongside those for the desired antibody product to improve the lifespan of the cell culture and—thus—antibody yield.^3,8 Other examples of improving product yield include engineering cells to survive at lower temperatures³ and with improved lipid metabolism.⁹

Editing with CRISPR-Cas9

Applications of gene editing can include using CRISPR-Cas9 to knock-out genes that lead to cell lines generating unwanted proteins, which are hard to remove in downstream processing or generate harmful immune responses in patients.^10,11

Using CRISPR-Cas9 for gene editing of cells is simple in concept, as it merely involves a guide RNA (gRNA) to direct the Cas9 enzyme to the section of DNA to be edited.¹² However, there are several methods for transfecting cells during cell-line development. These can include, for example, delivering the gRNA and Cas9 as plasmid DNA¹³ or infecting the cells with a lentivirus.

As Chartier explains, plasmid delivery of Cas9 can be cost effective but can lead to off-target effects (as the plasmids can remain in the cells for weeks¹³) and the plasmids can become integrated into the cell’s DNA. For difficult-to-transfect cells, she recommends using a lentivirus for delivery of the gRNA and Cas9.

Engineering cells with hairpins

Gene editing to improve yield often involves trying to get the cell line to express the maximum possible amount of antibody, or other protein. That’s according to Nicole Borth, Ph.D., a researcher at BOKU University of Natural Resources and Life Sciences. She and colleagues are among scientists developing the next-generation of techniques for cell-line engineering, which will—hopefully, in the future—enable cell lines to be custom designed from scratch.

Prof. Borth and colleague Johan Rockberg, Ph.D, from the KTH Royal Institute of Technology in Sweden, have developed RNA "hairpins" that act as a volume knob for protein production.¹⁴ The idea is that the level of protein being expressed is sometimes too much for the cell to process and it builds up, rather than being secreted as drug product. By acting as molecular “road-bumps” to the conversion of mRNA into protein, the RNA hairpins allow the level of protein production to be incrementally regulated and tuned to fit the pace of cellular machinery.

According to Prof. Borth, their new technique applies to many different cell lines, and can be used during cell-line development to set the level of protein production that best matches the processing and secretion abilities of the cell.

“People have started to realize that overexpressing a single gene and hoping for more productivity is naïve, at best, as the cellular machinery for protein production is delicately balanced,” she says. “The tools we have developed here will come in handy when people start making designer cells.”

References

1. Lu, R.M., et al (2020) Development of therapeutic antibodies for the treatment of diseases. Journal of Biomedical Science vol. 27 (1) 

2. Frenzel, A., et al (2013) Expression of recombinant antibodies. Frontiers in Immunology, vol. 4 (217) 

3. Dangi, A.K., at al (2018) Cell Line Techniques and Gene Editing Tools for Antibody Production: A Review. Frontiers in Pharmacology, vol. 9 (360) 

4. Li, Z. (2011) In Vitro Micro-Tissue and -Organ Models for Toxicity Testing in Comprehensive Biotechnology (Second Edition), Vol. 4, pp. 551-563

5. Kalia, P. (2021) Mapping the Future of Cell Culture and Cell Line Development. GEN, vol. 41 (8) 

6. Mistry, R. K, et al (2021) A novel hydrogen peroxide evolved CHO host can improve the expression of difficult to express bispecific antibodies. Biotechnology and Bioengineering. Vol. 118 (6), pp. 2326-2337 

7. Cooper, G.M. et al (2000) Pathways of Intracellular Signal Transduction in The Cell: A Molecular Approach, 2nd edition. Sunderland (MA): Sinauer Associates 

8. Zhang, X., et al (2018) Enhanced production of anti-PD1 antibody in CHO cells through transient co-transfection with anti-apoptotic genes Bcl-xL and Mcl-1 Bioprocess and Biosystems Engineering, vol. 41, pp. 633-640

9. Budge, J. D, et al (2020) Engineering of Chinese hamster ovary cell lipid metabolism results in an expanded ER and enhanced recombinant biotherapeutic protein production. Metabolic Engineering, Jan (57), pp. 203-216 

10. Mirasol, F. (2022) Outlining Cell Lines’ Future with Engineering Approaches. BioPharm International, vol. 35 (3), pp.10-13 

11. Unlisted author (2021) How we generate gene-edited cell lines at Abcam, 

12. Synthego (accessed 2023) The Complete Guide to Understanding CRISPR sgRNA in How To Use CRISPR: Your Guide to Successful Genome Engineering 

13. Enzmann, B. (accessed 2023) 5 Reasons Why Ribonucleoproteins Are a Better Alternative to CRISPR Plasmids, Synthego 

14. P. Eisenhut, et al (2020) Systematic use of synthetic 5′-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories. Vol. 48 (20) Nucleic Acids Research