Issue:October 2023

BIOLOGICS DEVELOPMENT - Five Steps to a Robust Cell Line Development Process


Biologic production is accelerating, with an increasing num­ber of these essential treatments reaching the market as therapies for a myriad of diseases, including Crohn’s disease, hemophilia, diabetes, and cancer.1 This increasing demand is reflected in the biologics market, which is predicted to expand at a compound annual growth rate of 8.1% to reach $535.5 billion by 2028.2 As the demand for biologics rises, ensuring robust cell line develop­ment is essential.

Working to construct a robust cell line that will lead to effi­cient, reliable, and reproducible production is the foundation of biologic manufacturing. Cell lines often have distinct associated characteristics that may be beneficial for producing one product but detrimental to another. When deciding between cell lines, each must be carefully considered for the desired biologic to en­sure a suitable choice.

Cell line development is the use of various techniques to op­timize a cell line for the production of a stable target biothera­peutic in high yield. Not taking the time to implement a robust cell line development process can lead to difficulties down the manufacturing pipeline, which wastes time and increases costs, and could even halt the timeline, as the process may need to be repeated and optimized until the challenges are overcome.

But what steps can be taken to help ease these challenges? The following discusses the critical steps for designing and im­plementing a robust cell line development process to help over­come the obstacles that often hinder biologic production. By taking the time to understand the cell line, the biologic, and by using advanced synthetic biology techniques to tailor the method to the desired biologic, a proactive approach for robust CLD can be devised.


Cell line engineering can be challenging, and issues can arise throughout manufacturing. Examples include cell line insta­bility, low yield, low product purity, problems with reproducibility and scalability, or regulatory compliance issues. Fortunately, a number of methods can be adopted to overcome these issues, such as testing various cell lines, generating stable pools, com­pleting extensive and high-throughput screening for clone selec­tion, and using synthetic biology to introduce beneficial characteristics into a cell line. These five techniques, used to gen­erate a robust cell line, are explored in depth further.

1: Choosing the Appropriate Cell Line

Deciding upon the cell line at the earliest possible point is key, as it will impact all subsequent development and manufac­turing steps. The conditions for cell growth, expression, and prod­uct isolation depend on the selected cell line. As well as impacting factors, such as product quality, quantity, reproducibility, and efficiency, a robust cell line development platform must also offer scalability. There­fore, it forms the foundation of biologic development and manufacturing.

Many cell line options have been suc­cessfully used for biologic development projects. Chinese hamster ovary (CHO) cells are a logical choice, with more than 70% of recombinant proteins produced using this line.3 With a clear history that sets the basis of documented traceability and ICH compliance, CHO-K-derived cell lines are a safe and easy-to-use option with a track record for gaining regulatory approval. A number of advantages make CHO lines a common choice, including the following:3

  • Protein folding and post-translational modification processing, such as glyco­sylation, are closely conserved in nature with human proteins and so are more likely to be accepted by the human sys­tem.
  • The lines are uniquely tolerant to changes in pH, oxygen levels, pressure, or temperature throughout manufac­ture, so they can be tailored to the pro­tein at hand.
  • The lines are able to grow in serum-free suspension cultures with high cell con­centrations, which eases scale-up as it is preferred for large-scale bioreactors.
  • There is previous understanding of CHO cell lines with extensive literature available and a regulatory track record, which can help accelerate timelines for approval.

As listed, CHO cells offer several ad­vantages, making them an apt choice. The CHO cell line, however, does have limita­tions, such as restricted growth and low productivity (when expressing complex ar­tificial molecules), but through genetic en­gineering, it can be tailored for the desired application.4

2: Productivity Improvement Techniques for Genetic Engineering & Non-ge­netic Optimization of the Cell Line

Biologics are complicated molecules, and past research has shown the CHO cell line may not have the capabilities to pro­duce a particular compound in high yields and at high quality without first being en­gineered to enhance the expression of the gene of interest to overcome low produc­tivity issue.5

Alterations to the core DNA must be completed to impart specific characteristics to the cell line. This is done using genome editing techniques, such as CRISPR Cas, Zinc Finger Nucleases (ZFNs), or Tran­scription Activator-Like Effector Nucleases (TALENs), to implement specific character­istics.6 Genetic engineering of the CHO line has been shown to fundamentally alter the function of the molecules ex­pressed.

Biologic expression becomes inher­ently more difficult as complexity grows. The biopharmaceutical industry is moving toward more intricate molecules, such as next-generation antibodies and protein-based drugs like fusion and multi-specific proteins. However, these revolutionary therapeutics often have lower expression levels in CHO cells as they are artificially designed. While effective for their de­signed purpose, how these proteins are devised can lead to issues with decreasing solubility and increased aggregation. En­gineering can be used to address these is­sues by introducing the genes for chaperone production (additional proteins that are produced during expression and aid protein folding) into the cell line to help with protein solubility and, in turn, increase product yield. Alternatively, engineering cells to improve the volume of recombi­nant protein production at low tempera­tures can also benefit CHO-based expression systems.7

Non-genetic cell culture optimization of an existing product-expressing CHO cell line can also improve productivity, for example, by using a low temperature, pH control, media selection, or a feeding strategy. These cell culture process opti­mizations are synergic to maximize the ex­pression potential of native CHO production cell lines. Different clones have different responses toward cell culture process changes, and therefore, design of experiment (DoE) is necessary to evaluate multiple CHO cell lines. Developing a scale-down cell culture model representa­tive of at-scale production forms the basis of the DoE.

3: Reducing Variability Between Product Batches

Batch variability leads to unreliable biologic production. Changes in product yield and quality for each batch can result in missed targets and hard-to-predict scales. Variability can be the result of nu­merous circumstances, from varying growth conditions or materials to cell line composition. Therefore, working to con­struct a robust cell line and adhering to strict conditions is essential.

Cell line engineering is only made possible by using techniques for efficient transfection and cell enrichment. These high-throughput methods test the viability of an engineered cell line with the product vector to see if the cell engineering has im­plemented the desired beneficial charac­teristic (such as increased product yield or solubility) for biologic production.

There are a number of different ap­proaches for inserting the product vector into the target cells, including those based on viral, chemical, and physical transfec­tion for integration. Choosing the best transfection technique for both the vector and cell line is essential to ensure robust vector uptake and limit cell variability.7 Cell enrichment is a method of single cell sorting to isolate positive cells, such as using antibody-based cocktails or flow cy­tometry.8 Innovative technologies, such as single-cell printers and high-resolution im­agers, make this high-throughput screen­ing possible to ensure monoclonality and increase clone screening efficiency. The method used depends on the applica­tion, but for biologic production, a method that leads to high purity is essential.

Optimizing transfection techniques is just one method for limiting variability. An optimized cell line development workflow is also essential; furthermore, a robust sin­gle-cell cloning technology minimizes the manufacturing batch-to-batch variability based on the selected clone. Finally, ensur­ing cell culture medium, growth condi­tions, extraction techniques, and other production methodologies are meticu­lously replicated is crucial to reducing vari­ability and ensuring reproducibility.

4: Biologic Analytics Aid Production Viability

Biologic analytics are a powerful tool that can significantly aid the development of stable cell lines. Using intensive biologic analytics early in the cell line development process is essential to fully characterizing and understanding the nature of the bio­logic. With these characteristics in mind, biologics developers can design produc­tion methods knowing the protein will not be harmed by the techniques used during manufacture and that the method is de­signed to impart stability to the molecule. This circumvents the need for extensive and complex formulations post-production to repair the protein.

Aided by high coordination and free-flowing communication between the ana­lytical and biotechnology groups, this communicative relationship helps to en­sure meticulous characterization and top-level visibility, which is especially important for emerging types of molecules (various types of fusion and multi-specific proteins), where less information is known.

5: Scale-up for Market

After extensive optimization and analysis for the reliable production of a bi­ologic, the process then needs to be scaled up to produce enough product for the pa­tient population. Scaling a process up can have unforeseen effects on a biologic and can significantly impact the yield and qual­ity of the product. However, this can be eased by adopting specific intermediary techniques to help aid visibility. For exam­ple, using scale-down models to evaluate clones in 50-mL bioreactor spin tubes mimics the larger-scale process so it can be used to predict how a clone will per­form within a large-scale bioreactor. Any issues or changes identified when using the 50-mL bioreactor can then be ad­dressed before scaling up to the commer­cial scale.

As previously discussed, implement­ing a robust cell line development process helps to reduce batch variability, increase product yield and purity, offer scalability, and reduce production costs. Future risks are also mitigated, and the timeline is less likely to suffer from development and manufacturing delays. However, this can still be very challenging, especially when a highly complex biologic is involved.


Working alongside a supportive part­ner can help ensure a cell line strategy is effective and can aid in maintaining tight timelines. Experienced contract develop­ment and manufacturing organizations (CDMOs) have the expertise to help over­come the challenges often encountered at this early stage. They can help with imple­menting techniques to enhance the stabil­ity and productivity of protein drugs for a desired use.


2022 saw a milestone in biologics development: the number of biologic drugs brought to market matched those of new mo­lecular entities (NMEs) for the first time. This growth will only con­tinue, and it is likely biologics will outpace NMEs in the near future.9 A growing understanding of synthetic biology, enhancing our ability to perform complex genetic engineering, facilitates ac­cess to increasingly complex and hard-to-work-with biologics to tackle complex diseases.

Therefore, working to implement robust, scalable, and re­producible cell lines at speed is the foundation of biologic pro­duction and is essential to keep up with the accelerating biologic market. Investing in new technologies and techniques to stream­line CLD will make success more certain and meet customer needs.


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  4. Fischer, S., Handrick, R., & Otte, K. The art of CHO cell engineering: A comprehensive retrospect and future perspectives. Biotechnology Advances, 33:8, 1878–1896, (2015). https://doi:10.1016/j.biotechadv.2015.10.015.
  5. Noh, S. M., Sathyamurthy, M. & Lee, G. M. Development of recombinant Chinese hamster ovary cell lines for therapeutic protein production. Current Opinion in Chemical Engineering 2, 391–397 (2013).
  6. Dangi, A. K., Sinha, R., Dwivedi, S., Gupta, S. K. & Shukla, P. Cell line techniques and gene editing tools for antibody production: A Review. Frontiers in Pharmacology 9, (2018).
  7. Chong, Z. X., Yeap, S. K. & Ho, W. Y. Transfection types, methods and strategies: A technical review. PeerJ 9, (2021).
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  9. Senior, M. Fresh from the biotech pipeline: fewer approvals, but biologics gain share. Nat Biotechnol 41, 174–182 (2023).

Robert Gustines is Senior Vice President Commercial Operations at Bora Pharmaceuticals. He has more than 18 years of experience in business development and marketing in the biopharma/pharmaceutical industry. During this time, he has consistently driven revenue and commercial capabilities and enhanced the performance of BD teams in high-growth, proactive, and technology-based environments. He has held several senior roles in Business Development.