It’s Time for CDMOs to Embrace the CRISPR Advantage

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The biotech and pharmaceutical pipeline is packed with experimental biological therapeutics such as antibodies, recombinant proteins, enzymes, and vaccines, which hold transformative potential for many health conditions. Taking these promising biotherapeutics from bench to market hinges on developing, characterizing, and optimizing specialized cell lines for research and manufacture. Fueled by the pressing need for improved biotherapeutics, the cell line development market is seeing double-digit growth and is expected to hit $1.7 billion by 2028 (1), much of it driven by contract development and manufacturing organizations (CDMOs).

There is much to be gained by improving and accelerating the development and optimization of cell lines. Discovered by Emmanuelle Charpentier and Jennifer Doudna, clustered regularly interspaced short palindromic repeats genome editing, also known as CRISPR, offers multiple benefits for CDMOs when it comes to the development of cell lines for biotherapeutics and viral vectors, realizing the promise of the biologics revolution.

Advancing cell line development to propel biotherapeutic innovation

CDMOs provide essential support that underpins innovation in biological therapeutics at all stages of the pipeline, from development and clinical trials through to commercial manufacture. They aim to ensure cell line stability and proliferative capacity, maintaining a good, high-quality yield even for hard-to-express proteins or viruses.

Right now, cell line development is cumbersome and relies on a series of laborious and time-consuming manual processes. This creates a bottleneck in biotherapeutic development and manufacturing that is throttling the pace of progress.

Consequently, CDMOs are actively exploring and leveraging both new and existing technologies to streamline the cell line development process at every step. By integrating these advances, CDMOs can deliver exceptional services and value to their customers in bioproduction, research, and diagnostics and maintain a competitive edge.

Challenges in developing, characterizing, and optimizing cell lines for biotherapeutics

Developing effective cell lines that are stable and optimal for maintaining a good yield is challenging, even for experienced teams. Many of the normal biological functions of the cell can interfere with biotherapeutic production, including the unwanted expression or aggregation of particular proteins, challenges in achieving correct post-translational modifications, and triggering apoptosis.

Optimizing and characterizing existing cell lines or developing new ones using genome engineering technologies can help circumvent unwanted biological processes and overcome bottlenecks in biotherapeutic development. These techniques can also help CDMOs generate value by developing their own proprietary cell lines for preclinical research in a wide range of disease indications.

For example, CDMOs are increasingly employing sophisticated workflows and technologies to optimize and analyze cell culture conditions. CRISPR/Cas9– the most widely-used gene editing technology—is a fast, efficient way to achieve desired characteristics within cell lines, and it can be automated and performed at scale. One such approach is so-called directed evolution (2), where cells go through multiple rounds of editing and selection to produce a cell line exhibiting desirable attributes for biotherapeutic production.

Harnessing CRISPR gene editing to develop next-generation cell lines

CRISPR can help at all stages of drug and cell line development. It can improve accuracy and expedite the development of bioproducts during the drug target identification and validation, screening and hit confirmation, and lead optimization stages. Moreover, it can also decrease late-stage attrition of biotherapeutics in development.

Leveraging CRISPR technology, CDMO teams can alter, add, or delete sequences in the genome with high precision, speed, and efficiency. This can help overcome hurdles, such as protein aggregation or the triggering of apoptosis, that reduce the effectiveness of cell lines for biotherapeutic development (3).

With just a few carefully selected genetic tweaks, CRISPR can modify complex biological pathways to modulate gene expression, avoid epigenetic silencing effects, enhance post-translational modification of proteins, or eliminate aggregation. Gene editing also widens the scope for bioprocessing cell lines grown in cheaper media or at reduced temperatures.

Using CRISPR, CDMOs can also create knockouts or inducible knock-in/overexpression models for specific genes to develop more accurate, disease-relevant cell lines for pre-clinical trials. CRISPR makes it possible for research teams to identify underlying mechanisms that can be modulated to enable high-level, quality protein production, expand product diversity, and control and improve product quality and yield, regardless of the cell type used.

CRISPR can also be used to improve and simplify downstream processing of biotherapeutics, particularly antibodies. After antibodies are expressed in a cell line, they must be purified away from unwanted cellular components and other proteins. However, in some cases, certain proteins may co-purify with the antibody, requiring additional processing steps, each of which can be very costly and technically challenging.

If any such issues are identified prior to production, CRISPR can be used to remove or modify any interfering proteins within the manufacturing cell lines, alleviating the need for additional and expensive purification steps.

Another advantage of CRISPR is that its versatility extends far beyond the usual ‘workhorses’ of bioproduction such as Escherichia coli and Chinese hamster ovary (CHO) cells. As well as mammalian and bacterial cells, it can also be applied to yeast and plant cells, both of which are growing in prominence as biomanufacturing platforms (4,5).

Supporting the production of viral vectors

Recombinant viral vectors have been used as vaccines for more than 40 years (6) and came to global prominence in the form of the Oxford/AstraZeneca ChAdOx1 COVID-19 vaccine, based on a modified chimp adenovirus platform. Viruses are also the delivery method of choice for many gene therapy approaches (7).

Demand for large-scale production of viral vectors is therefore increasing each year, with the need for rapid scale-up in the face of further outbreaks of infectious disease. However, because viral vectors can only be produced from cultured mammalian cell lines, manufacturing bottlenecks are holding back progress.

CRISPR gene editing has much to offer CDMOs when it comes to optimizing host cell lines for efficient, safe, and scalable manufacture of viral vectors. Similar to the situation with improving manufacturing of biotherapeutics discussed previously, gene editing of production cell lines can boost viral titers, reduce toxicity of viral components, and iron out issues with downstream processing and purification.

Gene editing can also be used to reduce the risk of recombination events leading to accidental production of pathogenic or replication competent viruses. Typically, viral vector production is enabled by randomly incorporating a harmless version of the full virus genome into the DNA of the host cell, then selecting a production cell line with consistently high viral expression.

Although this is efficient and fast in the short term, it does pose a risk of recombination events occurring. Physically separating the viral components into different areas of the genome essentially removes the risk of recombination but requires two separate integration events which must both be suitably productive for viral gene expression in the same cell.

Traditional methods of random integration require lengthy screening and monitoring to ensure reproducible viral expression, which can add up to a year to vaccine development—an unacceptable delay in a post-COVID world.

With CRISPR, the separate pieces of the viral DNA can be targeted into well-characterized specific genome locations that are known to be stable and support high expression levels yet are sufficiently separated from one another to avoid recombination. This process can be completed in a matter of days and the resulting cell line used in manufacturing within a month or two.

CRISPR genome editing also offers a fast and efficient way to update viral vaccines as the corresponding pathogen antigens adapt and evolve. It can be used to quickly make very specific changes to the protein-coding region of a vector that is integrated into an established manufacturing cell line, taking advantage of the already optimized cell growth, viral titer, and downstream processing.

Similarly, CRISPR can also be used to swiftly update the sequences of therapeutic or diagnostic proteins directly within production cell lines to ensure that we are keeping up the pace in the face of pathogen evolution.

CRISPR is essential for the future of cell line development

CRISPR gene editing unlocks possibilities for developing or optimizing cell lines that were traditionally challenging, with the potential to transform the biotech and pharmaceutical industry. CDMOs stand at a pivotal position to propel the development and commercialization of the next generation of biotherapeutics through the development of better cell lines.

As a patented technology, any commercial use of CRISPR needs to be appropriately licensed in a way that covers not only in-house cell line development and optimization but also the products generated using this technology.

The integration of CRISPR gene editing into cell line development is an essential step towards meeting the ever-increasing demands of the fast-growing global biotherapeutics industry. The time is now for CDMOs to fully embrace this technology or risk being left behind.

References

1. IQ4I Research & Consultancy. Cell Line Development Services Global Market - Forecast To 2030; Research and Markets, December 2022.
2. Griesbeck, O. CRISPR/Cas9-based directed evolution in mammalian cells. Curr. Opin. Struct. Biol. 2021, 69: 35-40. DOI: 10.1016/j.sbi.2021.02.005.
3. Dangi, A.K.et al. Cell Line Techniques and Gene Editing Tools for Antibody Production: A Review. Front. Pharmacol. online, Jun. 12, 2018, 9: 630. DOI: 10.3389/fphar.2018.00630.
4. Kulagina, N. et al. Yeasts as Biopharmaceutical Production Platforms. Front. Fungal. Biol. 22 Sept. 2021, 2. DOI 10.3389/ffunb.2021.733492.
5. Eidenberger, L., Kogelmann, B. and Steinkellner, H. Plant-based biopharmaceutical engineering. Nat. Rev. Bioeng. 2023, 1, 426–439. DOI: 10.1038/s44222-023-00044-6.
6. Travieso, T. et al. The Use of Viral Vectors in Vaccine Development. Vaccines. 4 July 2022, 7, 75. DOI: 10.1038/s41541-022-00503-y.
7. Bulcha, J.T., Wang, Y., Ma, H. et al. Viral vector platforms within the gene therapy landscape. Sig. Transduct. Target. Ther. 8 Feb 2021, 6, 53. DOI: 10.1038/s41392-021-00487-6.

About the author

Eric Rhodes is the CEO of ERS Genomics.