Cell Lines: Current Challenges in Cell Line Development for Therapeutics
Delve into the evolving role of cell lines in therapeutics, from bioprocessing to new genetic tools addressing stability and quality issues.
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Cell lines are a crucial tool in scientific research; without them, many experiments would be impossible.
Cell lines are “a culture of mammalian cells that can grow indefinitely in certain cultivation conditions in a laboratory setting, and retain a distinct phenotype and function, and can be stable over many population doublings,” explains Dr. Paula Meleady, associate professor in the School of Biotechnology at Dublin City University.
Numerous cell lines have been derived over the past 70 years and are used extensively in biomedical research to help scientists understand many fundamental biological processes. “For example, a cancer cell line is derived from cancer cells from a specific tissue (e.g., lung, breast) and is used in research to study the biology of cancer and to evaluate drugs used in cancer treatments,” Meleady adds.
This article explores some of the challenges in developing cell lines for therapeutics, such as stability of the cell line, and how they are being addressed.
A vital part of the drug development process
Cell lines are essential to drug development, allowing researchers to test the efficacy of their therapeutics in vitro before moving onto in vivo studies. Cell lines are used to test drug metabolism and cytotoxicity, study gene function and to generate a wide range of biological medicines, from proteins such as antibodies, recombinant protein products like insulin, to vaccines.1
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Cell lines derive from a single original cell and are genetically identical, so they “offer a more uniform product, meaning that every molecule in a therapy is the same; or as close as can be,” explains Dr. James Budge, a postdoctoral research fellow with a focus on Chinese Hamster Ovary (CHO) cell lines at the University of Kent. “The biology of each individual cell can have an impact on the product’s biotherapeutic properties such as the glycan profile, which refers to sugars added to the molecule in the cell which work to prevent recognition by a patient’s immune system and improve its stability and half-life post administration.”
“Over the past 30 years, the CHO cell line has been used for the production of biotherapeutics, particularly monoclonal antibodies,” says Meleady. “CHO cells are the most commonly used host cell line for biotherapeutic production, with over 70% of biopharmaceuticals produced from these cells.”
“CHO cells were initially used to study molecular cell genetics; the reduced number of chromosomes compared to human cells was an appealing property for these studies,” adds Budge. These cells can be cultured for long periods and showed robust genetic stability, while also allowing for genetic manipulation. “It is this robustness, ease of culture and the ability to produce complex protein-based materials with human-like post-translational modifications which have seen these cells become the dominant host for production of biotherapeutic materials,” he adds.
Stability of cell lines
The production of high amounts of biotherapeutics relies on the generation of stable cell lines. “Cell line development begins with the transfection of a suitable host cell line with the gene of interest, leading to the random integration of DNA from the gene of interest into the host genome,” explains Meleady. “The engineered cell line will start to grow and produce the product of interest, e.g., monoclonal antibody biotherapeutic, usually under selection pressure.”
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“Stability of cell lines presents a major challenge,” says Budge. Cell lines need to display and maintain functional features as similar as possible to primary cells, but those which have undergone many passages may harbor spontaneous mutations that affect their phenotype, native functions and responsiveness to stimuli, which could lead to unwanted variations in the end product.1
“Phenotypic instability is a very common challenge in biopharmaceutical production and is due to the ‘plasticity’ of CHO cells, which have a high propensity for genomic rearrangements, such as deletions or translocations,” explains Meleady. “This can be a source of cell line instability during the bioproduction process, resulting in the cells growing slower, a reduction in viability and a fall-off in productivity of the biotherapeutic of interest.”
There is a lot of research focusing on trying to find solutions to this problem, Meleady adds. “This is required to ensure stable, long-term production of the biotherapeutic of interest, to ensure bioprocess consistency and to assure acceptable product quality for the regulatory authorities so that the product is of the highest quality and safe for patient administration.”
Targeted integration techniques, including transpon-based technologies, which “enable the knock-in of recombinant protein coding genes into well-defined and transcriptionally active genomic sites is an active area of research at the moment,” adds Meleady.
Genetic engineering may minimize the risk of instability by knocking in or out specific genes. “Gene editing techniques could be used to modify the genetic landscape of the host cell to ensure stable and consistent expression of the desired genes, for example, through the use of CRISPR-based genome editing tools,” explains Meleady. “This technology offers new opportunities to engineer the host cell line to produce proteins with specific and desirable characteristics, such as high cell densities, high viability, high productivity and highest product quality.”
“For example, knocking out apoptosis-related genes has the potential to increase cell survival and culture longevity,” Meleady continues. “Knocking out host cell proteins has the potential to eliminate the production of proteins naturally produced by the host cell line that could co-purify with the desired product. By knocking out genes encoding these host cell proteins, the risk of contamination can be reduced, simplifying downstream processing and improving product purity.”
Single cell cloning and monoclonality
“One challenge surrounding the development of cell lines is cloning out individual cells and selecting the one with the desired properties,” says Budge. “Scientists need to ensure that the cell line is generating high titers but also producing molecules with the best product quality to meet its therapeutic potential.”
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Prior to cloning, cells are incubated individually and screened for the desired characteristics. Clones can be isolated using limiting dilution cloning, which is straightforward but time-consuming, with months spent obtaining results.2 Automation can speed up clone development and colony screening, reduces the chances of contamination and can be a useful in ensuring stability and product quality.
“Another challenge is that regulatory authorities require substantial documentation of the methods used during cell line development, to ensure clonality which is crucial for product quality and safety,” says Meleady. “A monoclonal cell line originates from one parent cell, to try to eliminate the risk of genetic heterogeneity that could affect the characteristics of the protein being produced.”
Growing cells can be subjected to genetic drifts, mutations or loss of a plasmid. By testing and documenting cells on day zero, scientists can prove the cell line came from a single cell, thus minimizing risk and ensuring a consistent production process and product quality. Emerging techniques include fluorescence-activated cell sorting (FACS), the sorting of cells based on fluorescent characteristics; and microfluidic drop-based single-cell printers with the ability to image newly-plated wells containing single cells. 3,4
Productivity
“The majority of significant improvements over recent years has been the ability to achieve higher cell densities in culture,” explains Budge. “More often than not, more cells mean more product, and improvements to culture media and feeding strategies have been central to this. Advancements in perfusion culture techniques and continuous culturing has also played a key role more recently.”
Budge has had success in improving product titers by manipulating the lipid content of CHO cell membranes; “By overexpressing certain lipid modifying genes, we’ve been able to change cellular properties such as membrane fluidity and/or endoplasmic reticulum size, which improves the capacity of our CHO hosts to produce biotherapeutic material.”
“More recently we have been working on transposon ‘jumping gene’ technology, which is now commonly used in industry to produce cell pools,” says Budge. “These technologies allow you to ‘cut and paste’ DNA cargo into a host cell genome and ultimately can reduce the time taken to generate cell pools (leading to quicker cell line selection) and increase product yields by increasing the frequency of integration events at desired positions in the CHO genome. Furthermore, since the cell pools produced using transposons have higher titers, an increased percentage of ‘high expressers’ are present in a pool. This can reduce the requirement for intense selection processes.”
Quality control
Product quality is of key importance for the production of biotherapeutics, as Meleady explains: “Correct posttranslational modifications (PTMs), especially glycan structures, are crucial for the potency and control of pharmacokinetic and pharmacodynamic properties of biotherapeutics. Heterogeneity of N-glycans can be an issue during cell line development and bioproduction. This heterogeneity, which can arise due to the variability of N-glycan processing (and often linked to the ‘plasticity’ of CHO cells), may compromise the activity and safety of biotherapeutics, particularly monoclonal antibodies.”
Meleady says control measures include the establishment and confirmation of clonality to ensure the stability of the host cell line. “There is also extensive stability testing carried out during cell line development, including monitoring the product for any changes to sequence and PTM modifications (e.g., glycosylation), i.e., using extensive quality control analytics.”
Artificial intelligence (AI) and machine learning will also have an important role going forward in cell line development, Meleady believes. AI tools can automate cell culture experiments from start to finish, while AI-powered software could generate 2D and 3D cellular models and supervise automated feeding and media changing, for example. This could help researchers discover novel drug targets and investigate toxicity more quickly.
Machine learning can make protocol development more efficient, flexible and powerful; their algorithms can prompt decision-making and allow for real time customization. It also allows for fully automated workflows to generate reproducible results across multiple experiments, expediting milestones and improving confidence in results.
Other challenges
Cell lines are cost effective, easy to grow and provide an unlimited supply of material, but they don’t truly represent how cells operate and interact within the body, particularly when used in biomedical research and drug development. The two-dimensional nature of cell culture systems means they lack some aspects of life within an organism, such as contact with tissue surfaces. Three-dimensional biology such as organoids or spheroids grown in a scaffold would better represent the in vivo environment and give more physiologically relevant information, but these cultures can be inconsistent as there is no standardized way to grow them.1,5
Potential contamination is also a concern; some are obvious, like bacterial overgrowth, while others, such as microbial mycoplasma, are less so. Mycoplasma contamination can remain undetected in cell lines for long periods of time, highlighting the importance of scientists testing their cell lines frequently.1
“CHO and human embryonic kidney cells have been the workhorses behind the production of complex, multichain and multidomain molecules such as monoclonal antibodies and the bioprocesses and technologies surrounding them have been exquisitely optimized to produce such molecules,” Budge explains. “Newer molecules such as bispecific antibodies present new challenges, and the tried and tested bioprocesses require some redesign to tackle these.”
These more complex therapeutic proteins can be used to increase the frequency of correct pairings and therefore the yield of the required protein but can be difficult to express in CHO cells.
Cell lines are vital to the drug development process and without them, we wouldn’t be able to test the efficacy of many important medicines. But developing cell lines isn’t without challenges. Over the years, much effort has gone into improving and streamlining the process to improve the stability, purity and quality of cell lines. Technology is increasingly being used to automate processes and improve decision making, saving time and improving productivity – and it’s likely its use will increase, particularly as new molecules are developed.
References
1. Kaur G, Dufour JM. Cell lines. Spermatogenesis. 2012;2(1):1-5. doi: 10.4161/spmg.19885
2. Ye M, Wilhelm M, Gentschev I, Szalay A. a modified limiting dilution method for monoclonal stable cell line selection using a real-time fluorescence imaging system: a practical workflow and advanced applications. Methods Protoc. 2021;4(1):16. doi: 10.3390/mps4010016
3. Yeh CF, Lin CH, Chang HC, Tang CY, Lai PT, Hsu CH. A microfluidic single-cell cloning (SCC) device for the generation of monoclonal cells. Cells. 2020;9(6):1482. doi: 10.3390/cells9061482
4. Liao X, Makris M, Luo XM. Fluorescence-activated cell sorting for purification of plasmacytoid dendritic cells from the mouse bone marrow. J Vis Exp. 2016;(117):54641. doi: 10.3791/54641
5. Cacciamali A, Villa R, Dotti S. 3D cell cultures: Evolution of an ancient tool for new applications. Front Physiol. 2022;13:836480. doi: 10.3389/fphys.2022.836480
