The translation of a research-grade cell culturing process into a scalable, clinical-grade manufacturing protocol is not a simple one. There are many attributes of the culturing process that are crucial for manufacturing, yet are often overlooked by inexperienced Cell Therapy developers. These include, but are not limited to, cell characterization, feed strategy/media optimization, and closed system processing. Here, topics are discussed that should be considered when converting a promising preclinical finding into a scalable process that could be used for the commercial scale manufacture of cell-based products. The topics being discussed are applicable for both autologous and allogeneic cell-based products.
An understanding of the biology of the cells is critical in the development of a successful manufacturing campaign. One should understand the biological attributes of their cell type in order to evaluate any changes introduced to the manufacturing process. Cell Characterization is an activity by which developers determine the critical quality attributes (CQAs) of their cells. The CQAs for cell-based products, as described by the FDA 1, and similarly described by most international regulatory agencies, are as follows:
Identity – what a cell type is (examples: gene expression; cell surface marker expression/FACS)
Potency – what a cell does (examples: secretion of cytokines; evaluation of cell-to-cell interactions)
Purity – product is free of unwanted cell types or impurities (examples: FACS; test for process residuals)
Safety – product is not harmful, free of microbial contaminants (examples: karyotype, compendial sterility)
Cell characterization is the process by which a Cell Therapy developer will chose the tests that will be used to assess the CQAs of a given cell-based product. For a more thorough summary of the different CQAs and unique considerations for each, please see 2. Safety and purity indications can be fairly straightforward; the more difficult tests to choose are those that measure identity and potency. The tests chosen as indicators of identity and potency are product-specific, serving as the characteristics that will define the product. These indicators have evoked fear in Cell Therapy developers, though they should not be so troubling. By and large, experiments performed during preclinical studies should elucidate unique attributes of the cells. For instance, scientists will typically observe unique gene expression, surface marker identifiers, or protein secretions that can be reproducibly measured from the cells and, ideally, point to a potential mechanism of action. These findings can typically be distilled down into well controlled, highly reproducible assays. Once developed, these assays can be used to help ensure that each batch of cells manufactured is consistent with established product parameters. For further reading on potency assay development please see: 3, 4.
Feed Strategy/Media Optimization
While cell characterization is important for establishing the quality parameters of a cell-based product, optimization of media and feed strategy are important considerations for reducing the cost of goods during product development. Media often represents the most expensive raw material in the manufacture of cell-based products. A reasonable approach to reducing media costs is to design experiments that will facilitate the reduction or elimination of expensive media additives (eg recombinant growth factors). Additionally, experiments designed to optimize the feed strategy, to minimize the number of media exchanges required during the manufacturing process, will also be helpful for reducing production costs.
Efforts geared toward reducing or eliminating animal-derived ingredients may not necessarily be economically beneficial in the near term, however moving toward xeno-free or chemically-defined cell culture media can have positive safety and supply-chain implications. For obvious reasons, it is desirable to manufacture products intended for human use in animal origin-free reagents. The employment of xeno-free reagents greatly reduces the chances of contamination of the cultured cells, particularly by mycoplasma 1. The more pressing matter, for the field of Cell Therapy, is that of the peak serum use. There is a limit to the amount of bovine serum that can be produced for clinical-grade cell culturing 5. Cell Therapy developers would be wise to reduce or eliminate (if possible) their dependence on bovine serum in the manufacture of their cell-based products in order to decrease the chances of supply chain issues in the future.
One of the most challenging aspects in Cell Therapy manufacturing is the conversion from a research process to a manufacturing campaign. This is most readily achieved via converting to a closed-system process. Most cell biologists are familiar with the culturing of cells using open-system processing. The scientist works in a biological safety cabinet (BSC) for primary cell isolation, media exchange, cell harvest, and volume reduction/washing by centrifugation. The cells are often grown in culture vessels such as cell culture flasks or well plates. In order to manipulate the cells, the flask or plate must be opened in the BSC and media transferred from the media bottle to the cells via pipette. This is why this type of cell processing is referred to as “open-system”, the cells and the media must be “opened” in the BSC. The challenge with open-system processing for Cell Therapy manufacturing is two-fold. First, each instance in which the cell culture vessel or media is opened represents a safety/sterility risk. Secondly, open-system processing is not easily amenable to large-scale manufacturing.
In closed-system processing, plastic adherent cells are grown in multilayer cell culture vessels, allowing for the culture of large quantities of cells all in one vessel; a smaller footprint which means more cells/vessels can be cultured in a given incubator. Non-adherent cells are often grown in large, gas-permeable cell culture bags. Bags of media are often attached to the culture vessel via sterile, weldable tubing. Process steps such as media exchange, passaging, and harvesting of cells are achieved via the welding of bagged media or trypsin onto the culture vessel and, when necessary, the harvesting of cell suspension into a collection bag. The bagged cells can be sterility sampled for cell count and subsequently used to seed further culture vessels or be subjected to downstream processing. Downstream processing includes large-scale, closed-system volume reduction and washing followed by fill and finish activities that lead to the establishment of individual product doses 6.
The major benefit of closed-system processing is that most cell processing steps can be performed out in the open, in the cleanroom. For closed-system processing, the BSC is only necessary for the initial cell isolation and/or the thawing and seeding of the initial culture vessel. Additionally, closed-system processing is scalable; one operator can manipulate several multilayer vessels at once as opposed to an open-system process in which a single operator can manipulate a small handful of tissue culture flasks, or one or two multilayer vessels at once. This enhanced efficiency in cell culturing significantly reduces the total processing time for the cells, which can have significant implications for product quality, especially cell viability and biological function.
When designing experiments to transition from open to closed-system processing, the CQAs (described above) will be employed to demonstrate product comparability through process development 2. This is why it is recommended that cell characterization activities are initiated early in clinical development. Once candidate measures of identity, purity, and potency are identified, they can be used to ensure that process changes such as serum reduction or closed-system processing do not alter the integrity nor the biological activity of the cells. A strategic approach to translational medicine is ideal, with a targeted focus on quality and cost of goods driving product and assay development decisions. Many describe science as an art, and indeed it can be, but the translation to scientific findings into clinical-grade products is facilitated by switching one’s mindset to manufacturing as early in the process as possible.
For 30 years Lonza has been helping emerging and established pharmaceutical and biotech companies improve production processes, navigate the development and regulatory process, lower the cost of goods, and advance to market faster. Lonza offers world class technology platforms in the areas of GMP cell culture and viral-based therapeutic manufacturing, custom biotherapeutic culture media, a large selection of primary and stem cells and a full line of custom bioassays. Our extensive experience in Cell Therapy process optimization and scale-up innovation helps clients to safely and effectively advance their products through all phases of the commercial pipeline and maximize their return on investment. Our Viral-based Therapeutics group provides viral vaccine manufacturing as well as viral vector mediated gene therapies for the treatment of infectious diseases, neurological disorders, cancer and cardiovascular diseases. From early development through commercial launch and mature market supply our staff can design, develop, and implement a manufacturing process that meets your autologous or allogeneic therapeutic applications.
1. Guidance for FDA Reviewers and Sponsors: Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Somatic Cell Therapy Investigational New Drug Applications (INDs). (ed. FDA) 1-39 (Office of Communication, Training, and Manufacturers Assistance (HFM-40), Rockville, MD, 2008).
2. armen, J., Burger, S.R., McCaman, M. & Rowley, J.A. Developing assays to address identity, potency, purity and safety: cell characterization in Cell Therapy process development. Regen Med 7, 85-100 (2012).
3. FDA Final Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products. (ed. FDA) 1-19 (Rockville, MD, 2011).
4. Bravery, C.A., et al. Potency assay development for cellular therapy products: an ISCT review of the requirements and experiences in the industry. Cytotherapy 15, 9-19 e19 (2013).
5. Brindley, D.A., et al. Peak serum: implications of serum supply for Cell Therapy manufacturing. Regen Med 7, 7-13 (2012).
6. Rowley, J. Developing Cell Biomanufacturing Processes. Chemical Engineering Progress SBE Supplement, 6 (2010).