Stable Cell Lines: Stepping Stones For Continuous Viral Vector Manufacturing

By Chrysanthi Sitmalidou, scientist II, Orchard Therapeutics

As more therapies reach the clinics and as larger indications meet the clinical criteria with more patients, the need for larger volumes of therapeutic molecules, proteins, and viral vectors is constantly growing. Amid the increasing need, a promising technology started to develop — the stable cell line.

Stable cell line technology is a powerful tool used in biotechnology and pharmaceutical research – an exciting technology with vast potential for advancing scientific research and improving healthcare. It involves the introduction of a specific gene or DNA sequence into a cell, allowing the cell to produce a desired viral vector, protein, or molecule consistently over time. The process typically begins by introducing the gene of interest into the cells using techniques such as transfection or electroporation. The cells are then subjected to a selection process, where only those cells that have successfully incorporated the gene are allowed to survive. This can be achieved by using antibiotic resistance markers.

Once the stable cell line is established, it can be maintained for an extended period of time, ensuring a consistent supply of the desired vector, protein, or molecule. This makes stable cell lines useful for various applications, including drug discovery, protein and viral vector production, and studying gene function. They are quite established as far as it concerns antibody production with CHO cells, but what about when it comes to viral vector production? The truth is, over the last few years many bioprocesses related to the production of viral vectors have been shifting production from transient to stable cell lines due to the advantages the technology offers, such as:

• lack of need for GMP supply of transfection reagents and plasmid stocks,
• batch-to-batch consistency,
• scalability, and
• process intensification.

Where Are We Now?

The current landscape of stable cell lines is dynamic, vibrant, and continuously evolving, driven by advancements in gene editing technologies, increased demand for biopharmaceutical production, and emerging applications in drug discovery. These developments continue to expand the possibilities and potential impact of stable cell line technology in various fields. The demand for stable cell lines has grown in biopharma due to the rising need for large-scale, consistent, and cost-effective production of complex biological molecules.

Stable cell lines undeniably play a crucial role in improving manufacturing processes and production for large quantities of viral vectors.

A Brief Recap Of Stable Cell Line Technology’s Benefits

To begin with, they can deliver consistent, reliable, and scalable production of viral vectors over time. This consistency is vital for manufacturing processes, as it allows for predictable and reproducible production of high-quality products. In addition to this, stable cell lines enable biopharmaceutical companies to scale up production efficiently to meet market demands. Their use also increases productivity by optimizing gene expression levels or modifying cellular pathways. This leads to higher yields of the desired viral vectors, and the increased productivity improves manufacturing efficiency, reduces production costs, and allows for a more sustainable production process.

Another positive aspect is the reduction in batch-to-batch variability in manufacturing. By using a stable cell line with consistent gene expression, variability arising from cell line fluctuations is significantly reduced. This ensures a more predictable and reliable manufacturing process, leading to greater product consistency and quality.

Once a stable cell line is established, it can be maintained for an extended period with reasonable cost and effort. This cost and time efficiency reduces the need for repeated experimentation and saves time compared to transient expression methods, where the introduced gene expression is only temporary. An established stable cell line can also provide a stable and well-characterized platform for process development, which serves as a foundation for developing manufacturing processes and optimizing parameters such as media composition, culture conditions, and purification procedures.

This streamlines process development, accelerates time to market, and reduces costs associated with process optimization.

Finally, stable cell lines that have been thoroughly characterized and validated can simplify regulatory compliance. By providing detailed documentation, including information on cell line identity, stability, and performance, biopharma companies can demonstrate control and traceability throughout the production process. This aids in meeting regulatory compliance requirements and expediting regulatory approvals.

A Typical Workflow For Generating Stable Cell Lines

For stable cell lines producing viral vectors, a typical workflow starts with the stable transfection of the viral components into a host cell line. Common practice is to generate a packaging cell line first that contains the three viral components (Gag pol, Rev, and VSVG) and then stably transfect the transgene with the gene of interest to create a producer cell line. A two-week selection process follows where polyclonal populations are cultured with selection agents to assure the survival of cells with stable integration of vector components.

Single-cell isolation and colony formation is the next step under close imaging observation. Expansion of the clones and full characterization will result in identifying the final leads and the highest producer clone. An important part of CLD workflow is the stability studies at the end of the development, where the stable production and stable genetic profile of the producer clone are confirmed.

The studies include copy number, titer stability, viability and cell growth, expression without selection, and further characterization like quality testing for GMP applications.

Optimization and further work are required to lock each step of the workflow. Established best practices for generating and maintaining stable cell lines include choosing a host cell line and a selection marker. Stable cell line technologies primarily utilize mammalian cell lines (such as Chinese hamster ovary cells or HEK293 cells), but  the nature of the culture and, to an extent, the whole platform based either in adherent or suspension conditions, will prevent the impact of suspension adaptation to serum-free culture later on, which can potentially change the ranking of the high producer clones. For that reason, it is always advisable to know your top producer clones from the beginning.

When establishing a stable cell line, it is crucial to choose an appropriate selection marker, such as an antibiotic resistant gene, that allows for efficient and reliable selection of cells that have successfully incorporated the gene of interest. The choice of selection marker will depend on the specific cell line and experimental requirements.

Optimizing the transfection methods is an important step to maximize transfection efficiency while minimizing cell toxicity. This may involve testing different transfection reagents and optimizing transfection conditions (such as cell density, DNA concentration, and transfection reagent ratio). After transfection, cells need to be subjected to a proper selection process to isolate those that have stably incorporated the gene of interest. This typically involves using selective media containing the appropriate antibiotics or other selection agents. It is important to carefully monitor and maintain the selected clones to ensure the stability of gene expression and prevent genetic drift or loss of the introduced gene.

Thorough characterization and validation of stable cell lines is essential. This includes confirming the integration and expression of the gene of interest, assessing viral vector production levels, and evaluating stability over time. Techniques such as PCR, flow cytometry, and functional assays may be used for characterization and validation. Detailed documentation and record-keeping of the entire process, including transfection protocols, selection methods, maintenance procedures, and characterization data, are also critical. This ensures reproducibility, allows for troubleshooting if issues arise, and facilitates knowledge sharing with regulatory authorities.

Advanced Technology Accelerating Stable Cell Line Development

Technology has had a significant influence on the development and application of stable cell lines. It has shaped stable cell line technology with the use of high-throughput systems for single-cell isolation and screening platforms. This combination allows for a more efficient and reassuring way of isolating single cells that will eventually grow into stable clones. At the same time, it allows for rapid and automated screening of a large number of clones, enabling identification of the best potential producer candidates and optimizing viral vector production processes more efficiently than ever before.

Moreover, technology has delivered advanced high-throughput imaging and monitoring instruments, which come in handy when the need for monoclonality proof and tracking and monitoring of the stable clone growth comes.

Single-cell high-throughput imaging allows real-time observation of single-cell isolation, monoclonality assurance, and tracking the clonal growth. Technological advancements in automation and robotics have streamlined the process of generating and maintaining stable cell lines. Automated systems can handle tasks such as cell culture, transfection, selection, and monitoring, reducing human error, increasing efficiency, and allowing researchers to work with larger numbers of cell lines simultaneously.

Last but not least, omics technologies, including genomics, proteomics, and transcriptomics, have had a profound impact on stable cell line research. These technologies allow for comprehensive characterization of stable cell lines at the genetic, protein, and cellular levels, providing insights into gene expression patterns and metabolic profiles, and such data guide the optimization and understanding of stable cell lines.

From The Bench To GMP Production

Assuming a stable cell line is to be used to manufacture viral vectors, which implies a GMP environment, certain requirements should be considered during the development of a stable cell line  to be able to transfer it later to GMP production.

The three main GMP requirements are based on the Code of Federal Regulations Title 21 from the FDA: monoclonality, proper conditions for GMP transfer, and pre-MCB test.

Monoclonality is clearly stated in the ICH Q5D: “For recombinant products, the cell substrate is the transfected cell containing the desired sequences, which has been cloned from a single cell progenitor.”

Whether using advanced technologies available in the market designed for this purpose or manual methods like limiting dilution cloning, a single cell colony image of the single cell and the entire well is needed for sufficient proof.

Other important aspects of pre-GMP transfer include:

• cell manipulation and generation of research master cell banks (MCB) and working cell banks (WCB) under GxP-like conditions,
• good cell culture practices, and
• provenance of the cell line generation.

Finally, QC testing measures according to USP/EP so that RCB/pre-MCB can be moved to GMP include mycoplasma, sterility, and endotoxin testing data to ensure the integrity and reliability of the stable cell lines.

Fair Warning: Building Stable Cell Lines Is A Huge Undertaking

When developing a stable cell line, there are several limitations and obstacles that can be encountered. The number one obstacle is the resources required and the high cost of this technology. It is fairly considered a big asset by many biotech companies as it is expensive while in development and requires a lot of investment to develop and establish.

In addition, developing a stable cell line can be a time-consuming and labor-intensive process, as it often involves multiple rounds of screening, selection, and optimization, which can require significant investments of time, labor, and materials. There are also cell line-specific issues where different cell lines may present unique challenges during development. For example, a cell line may be difficult to transfect or have specific growth requirements that need to be carefully addressed.

Scale-up challenges may arise when moving a stable cell line from small-scale cultures to large-scale production, which can pose challenges in maintaining stability. Factors such as nutrient availability and shear stress can differ significantly, affecting cell growth and productivity.

Another bottleneck is intellectual property issues. Developing a stable cell line for commercial purposes may involve navigating intellectual property rights and licensing agreements. Ensuring compliance with relevant patents and legal considerations is essential to avoid infringement and legal disputes. These obstacles highlight the complexity and intricacies involved in developing and maintaining a stable cell line.

Overcoming these challenges often requires careful experimental design, monitoring, expertise, adherence to best practices in cell line development, and optimization to achieve a stable and reliable cell line.

Conclusion

Stable cell lines are a powerful tool that contribute to scientific advancements and accelerate research in various fields. They are widely used in the biopharmaceutical industry to produce therapeutic proteins, antibodies, and viral vectors as they allow for consistent and controllable production, aid in drug discovery, facilitate gene function studies, and offer cost and time efficiency. Their applications are vast and have a significant impact on biotechnology and pharmaceutical research.

Overall, stable cell lines improve manufacturing by ensuring consistent and scalable protein production, increasing productivity, streamlining process development, reducing batch-to-batch variability, facilitating regulatory compliance, and expediting time to market. These benefits contribute to efficient and cost-effective manufacturing of biopharmaceuticals, ultimately benefiting patients and healthcare systems.

Despite all the obstacles, stable cell line technology is nowadays considered a highly valued tool in the future picture of the biotech world.

Editor’s note: The contents of this publication represent the views of the presenter and do not necessarily reflect the views of the author’s employer.

About the Author:

Chrysanthi Sitmalidou is a scientist II in vector process development team within Global Technical Development and joined Orchard Therapeutics in September 2020. She is working on the development and optimization of lentiviral vector (LVV) platforms and LVV analytical assay development. She is also responsible for the development of stable cell lines for LVVs and optimization and establishment of a stable cell line platform. Prior to Orchard, she was a senior research associate in Autolus, working on stable cell line development and optimization of cell line development platforms and vector analytical assays for CAR T cell therapies. She holds a M.Sc. in molecular medicine and cancer research and B.Sc. in molecular biology and genetics.