Cell line development is a critical aspect of biotechnology and pharmaceutical research, and plays an important role in Ingenza’s CRDMO services. The process involves establishing and maintaining genetically identical mammalian cells in culture for use in a variety of processes, such as the production of recombinant proteins, including antibodies. This is often a complex journey that encompasses multiple stages, beginning with vector design and gene synthesis, host cell line choice, transfection, clonal selection, characterisation, and expansion, ultimately concluding with a scalable and stable cell line for upstream/downstream manufacturing processes. Each stage demands precision and expertise, and is supported by various screening steps to meticulously select a clone with the desired characteristics, including the highest viability, stability, growth and recombinant target expression.
Host selection for cell line development
Selecting the appropriate cell line for development is the crucial initial stage of the process. The specific criteria for cell line selection will depend heavily on the purpose of the research and the properties of the target product. For example, cell lines used for stable production of recombinant proteins must meet good manufacturing practice (GMP) criteria, have a detailed provenance and be able to adapt to serum-free culture. They should also be suitable for suspension growth conditions, as well as having high growth rates and secretory capacities.1
Chinese hamster ovary (CHO) cells have historically been the go-to cells for pharmaceutical development programmes. In fact, CHO cells are used for the production of more than 70 percent of currently approved recombinant proteins.2 This is largely because the modes of post-translational modification in CHO cells reflect those seen in humans, allowing the production of biologics with complex tertiary and quaternary structures and human-like glycosylation, with the absence of immunogenic α-galactose epitopes. Despite CHO cells still remaining the class favourite, there has been a recent shift to diversify the available mammalian cell lines, and most therapeutic viral vectors are now produced by human embryonic kidney (HEK) 293 cells. Derived directly from human cells, this stable cell line offers high transfection rates, rapid growth and the ability to grow in serum-free, suspension cultures, as well as yielding more human-like post-translational modifications for complex macromolecules.3
Key considerations
Host selection is only the first step, and there are several other challenges to cell line development that must be addressed. Cell line stability is a major issue; even if cell lines are stable at selection, they may show instability after sub-culturing for multiple generations, resulting in wasted time and resources. It is therefore crucial to work with a cell line development expert that can determine the stability profile of a recombinant cell line as early as possible. Cross contamination is another issue when developing mammalian cell lines, as the media is often extremely rich, necessitating stringent cleaning protocols. Mammalian cells are also costly to culture and take on average 18 to 22 hours to double, unlike microbial cells such as Escherichia coli, which can double in just 20 minutes. These challenges make it especially important to assess the viability, identity and purity of cell lines regularly to avoid costly repetition as well as to monitor genetic drift.
Mammalian cell line development for recombinant protein production should also comply with the ICH Q5D regulatory requirement to be derived from a single progenitor cell and, in recent years, the expectations around how to fulfil this requirement have changed. Several rounds of limiting dilution cloning are still employed to achieve a suitably high probability of monoclonality, but this approach is increasingly being paired with the use of equipment that combines cloning and imaging to provide supporting evidence that a cell line originated from a single cell. Fluorescence-activated cell sorters (FACS) and microfluidic devices that combine the ability to perform cloning and cell line screening steps – e.g., Beacon®/Beacon Select™ from Berkeley Lights or Cyto-Mine® from Sphere Fluidics – eliminate the need for multiple rounds of cloning, ultimately saving time and labour to reduce timelines. Given the rapidly increasing demand for the development of complex recombinant proteins, the ability to characterise the purified product early in the cell line development programme is also becoming central to the development of scalable manufacturing processes, with the aim of increasing yield while maintaining consistent product quality.
Cell line development at Ingenza
Ingenza has the in-house expertise and flexibility to work with a range of different hosts to accommodate the requirements of any recombinant protein development programme. For example, we recently delivered a stable CHO cell line capable of producing more than 20 times the amount of human factor VIII previously reported for this host for a project to address chronic unmet needs for haemophilia A treatments in low and middle income territories.
While CHO remains the true workhorse of the biopharmaceutical industry, we also employ other suspension and adherent cell lines – including HEK293 – according to project demands. Our clients rely on our expertise to support a wide range of projects, from assessing short-term transient expression of recombinant targets using alternate promoters and secretion signals for R&D using our inGenius™ CMC platform, to establishing highly productive, stable cell lines for long-term commercial cGMP production.
How can Ingenza help you?
Our stable cell line production process is highly efficient – taking on average just 12-16 weeks – and our expert scientific teams are continually refining and enhancing our capabilities. But it doesn’t stop there – we can advance your programme from cell line development through to preclinical material delivery. Our in-house up- and downstream processing capabilities, supported by advanced analytics and a robust quality management system (QMS), ensure that the final cell line is fully suitable for the target manufacturing process, and the quality of the recombinant product is compliant with regulatory requirements. This includes the development of both tangential flow filtration (TFF) and alternating tangential flow filtration (ATF) perfusion processes, which can be valuable for challenging targets and for reducing the size of bioreactors required. We complement this comprehensive cell line development offering with extensive experience with microbial hosts, ensuring we can provide a solution when mammalian cells are not appropriate or optimal. If you are seeking a trusted cell line development provider, then look no further. Our dedicated team is ready to work with you to meet the individual specifications of your project. Click here to get in touch.
References
- Jagschies, G., Lindskog, E., Lacki, K., & Galliher, P. M. (Eds.). (2018). Biopharmaceutical processing: development, design, and implementation of manufacturing processes. Elsevier. 131-146. https://doi.org/10.1016/B978-0-08-100623-8.00007-4.
- Dumont, J., Euwart, D., Mei, B., Estes, S., & Kshirsagar, R. (2016). Human cell lines for biopharmaceutical manufacturing: history, status, and future perspectives. Critical reviews in biotechnology, 36(6), 1110-1122.
- Tan, E., Chin, C. S. H., Lim, Z. F. S., & Ng, S. K. (2021). HEK293 Cell Line as a Platform to Produce Recombinant Proteins and Viral Vectors. Frontiers in bioengineering and biotechnology, 9, 796991. https://doi.org/10.3389/fbioe.2021.796991