This is a guest post by Ruud Hulspas, Ph.D.
Industry has a different approach to Research & Development than academia. While the academic approach typically focuses on discovery, innovation and proof-of-principle, industry adds feasibility and product (manufacturing) requirements early on to the timelines of R&D projects. Consequently, the tools, technologies, reagents and expertise chosen for academic R&D projects, may be very different from those chosen for industry R&D projects. In contrast to R&D for conventional medicine, R&D for cellular therapy has predominantly taken place in academic environments with little to no consideration for product and manufacturing requirements such as control, efficacy, robustness, economics and sustainability.
Many years of persistent academic work on cellular therapy has recently reached a level of success that persuaded industry to add cell-based products for cellular therapy to its portfolio. Although tools, technologies, reagents and expertise in conventional pharmaceutics may be used to manufacture products that are derived from cells, products for cellular therapy ARE cells. And thus, the current tools, technologies, reagents and expertise available for manufacturing are not necessarily optimized to guarantee robustness and sustainable economics for cell-based products for cellular therapy.
One of the essential tools in cellular therapy is cytometry (i.e. measuring cell characteristics). However, the technologies that enable cytometry continuously evolve as a response to the demand in academic research for the ability to measure previously unknown characteristics of cells and only few technologies in cytometry have been developed with manufacturing of cell-based products in mind. As a result, it is difficult to incorporate cytometry in cell processing workflows for the manufacturing of cell-based products.
Particularly in flow cytometry, much of the instrument setup, measurements and data interpretation is, literally, left in the hands of specialists. In addition, the flow cytometry community has had little success in applying consensus-based methods and it is, therefore, not uncommon that different specialists use different methods. Consequently, flow cytometry may introduce great costs and a significant degree of variability to the process. A recent survey indicated that the causes for variability in flow cytometry reside mostly in data analysis, sample preparation and instrument setup. This is not surprising, as these aspects of flow cytometry are (still) mostly manual processes. Automation can result in a dramatic reduction in variability, while it also reduces the need for specialists and contributes to affordability. Automation is therefore an important development that needs to be completed in order to implement flow cytometry effectively in manufacturing processes of cell-based products for cellular therapy.
The strength of flow cytometry lies within its ability to simultaneously measure a broad range of characteristics on individual cells, while rapidly processing millions of cells. Such multi-parameter, high-speed measurements on individual cells are typically performed at 2,000 – 20,000 cells per second, and have even been successful at rates of up to 240,000 cells per second (personal communications). Subsequent results are recorded in data files for offline detailed analysis and can be archived for quality control purposes. The ability to measure individual cell characteristics by means of relative light intensities allows for the identification of cells by levels of gene expression and/or protein production.
Hence, flow cytometry is also suitable to identify and sort specific cells out of an assortment of cell types. Cell sorting on the basis of flow cytometric technologies is a very powerful tool in cellular therapy as it offers an accurate and controlled way to enrich cell-based products for potency. The problem is that cell sorting on the basis of flow cytometric technologies is currently not suitable for a manufacturing environment. In addition to high costs and lack of robustness, current processing times and sterility and biosafety levels do not meet industrial cell processing requirements. Furthermore, cell sorting by flow cytometry is a highly specialized procedure that needs exceptionally skilled personnel.
If cell sorting on the basis of flow cytometric technologies is to be used in manufacturing of cell-based products for cellular therapy, the typical maximum throughput rate for droplet sorters (approx. 70 million cells per hour for human blood cells) needs to be increased by a minimum of 1.5 fold in order to complete the sorting process within 4 hours. This, of course, also depends on how much the cell suspension has been pre-enriched for the target cell. However, in order to be able to sort sufficient target cells directly from a leukapheresis product (~5E10 cells) in less than one hour, the maximum throughput rate of conventional droplet sorters needs to increase by as much as 1,500-fold. Given the average size of human blood cells and their sensitivity to factors like, pH, viscosity, osmolarity, temperature and shearing forces, a good way to increase the throughput rate is to scale-out the process and have multiple instruments simultaneously sort fractions of the total cell suspension. This approach is currently used in the livestock industry using ‘multi-head’ droplet sorter instruments specifically designed to accommodate lean operation in manufacturing of cell-based products.
While parallel processing may be the answer to how the throughput rate of cell sorting on the basis of flow cytometry should be increased, it does not address the sterility and biohazard issues typically associated with fluidic systems in droplet sorters (i.e. extensive clean-in-place procedures and exposure to biomaterial-containing aerosols, resp.). Fluidic systems in equipment used in manufacturing of cell-based products for cellular therapy should, preferably, be enclosed, disposable and easy to replace.
The ‘Spectrophotometric cell sorter’ described in 1967 by Louis Kamentsky and Myron Melamed, is a cell sorter that sorts on the basis of flow cytometry and focuses on the ability to sort with an enclosed, disposable fluidic system using microfluidic chip technology. The technology is well-suited to be incorporated in a cell sorting apparatus to safely obtain specific cell types under sterile conditions with zero cross-contamination. However, the throughput and sort rate are significantly lower than that of droplet sorters, such that even massive parallel processing cannot provide the rates required in manufacturing processes for cellular therapy. It wasn’t until about 40 years later that fluidic switch technologies allowed for significantly higher cell sort rates fit to be combined with a parallel architecture to reach the throughput rates that can be used in industrial cell processing (Hulspas et al). The ability to safely sort cells on the basis of flow cytometry at more than 2.5 times the throughput rate of droplet sorters while using enclosed, disposable microfluidics is a significant step towards manufacturing high-potency cell-based products.
However, successful incorporation in manufacturing settings requires additional innovation and development in process control. Cell sorting in manufacturing settings needs to be accompanied with systems that not only monitor the process, but also with systems that safely suspend the process when an error occurs without having a negative impact on the product. While these requirements may be obvious to the pharmaceutical industry and regulatory bodies, they are not necessarily obvious to academic research-oriented innovators and business owners. As most of the innovations in flow cytometry have taken place in these academic research-oriented environments, little has been done on incorporating process control systems in cell sorting instruments. The apparent gap in product requirements between academics and industry can be filled when all stakeholders communicate their goals, requirements, experience and opinions effectively. As facilitators and gatekeepers of these communications, professional societies play an essential role in making progress in cellular therapy and reaching the ultimate goal of transforming cellular therapy into a sustainable way to routinely treat patients.
