Process automation in manufacturing of mesenchymal stromal cells

The remarkable progress over the past decade in mesenchymal stromal cell (MSC) translational and clinical research has vastly increased the understanding of the cell processing techniques necessary to deliver a safe cellular therapeutic product that is well characterized and potent. Marrow aspirates have proven to be the most widely utilized source of MSCs in current clinical trials. The cellular manufacturing unit operations that define marrow-derived MSC processing, whether autologous or allogeneic, typically includes separation of the mononuclear cell (MNC) fraction from the bulk marrow aspirate, ex vivo cell culture of the MSCs present in the MNC fraction, harvest of the MSCs from culture, washing and concentration of the harvested MSCs, and fill and finish of the washed and concentrated cells into the final drug product packaging, which may be destined for fresh infusion or cryopreservation. The majority of MSCs manufactured for current clinical trials is are obtained by manual methods. This typically includes density gradient separation of marrow aspirates via Ficoll density gradient separation and centrifugation to produce a MNC fraction, which is then placed into static 2D cell culture in plastic tissue culture flasks or cell factories. The MSCs are plastic adherent and proliferate in culture, while the other cellular constituents of the MNCs are nonadherent and will therefore not attach to the plastic cell culture substrate and subsequently may be removed from the culture vessel by a medium exchange, leaving the adherent MSCs to persist. Several passages of the MSCs from one flask or cell factory to another are required to achieve adequate cell yields for clinical doses. MSC doses are typically administered to patients in the 1 × 106 to 10 × 106 cells/kg range. Hanley and colleagues1 have reported that expansion of 200 × 106 MSCs from 25 mL of marrow aspirate in the good manufacturing practices facility at Baylor College of Medicine (Houston, TX) over a 30-day period requires four cell culture passages, the use of 340 T-175-cm2 tissue culture flasks, and 2040 pipettes, with 54,400 open events (manual manipulations) that must be performed in a Class 100 (ISO 5) biosafety cabinet. The washing and concentration of harvested MSCs are generally carried out by multiple centrifugation steps utilizing centrifuge tubes to remove the cell harvest medium, to exchange with wash fluid, and to resuspend the concentrated cell pellet up to the desired cellular concentration in either infusion solution or cryoprotectant. Finally, the resuspended MSCs at the desired final formulation concentration are filled into either cryovials or cryobags for controlled-rate freezing or else placed in other containment for fresh infusion, such as syringes, plastic or glass vials, or IV infusion bags. Due to the exceedingly labor-intensive nature of current MSC production, the risks of product contamination and product variability due to human error involved with open system manipulations, there is much interest in automating MSC manufacturing. Unfortunately, there are currently only a very limited number of automation devices commercially available that are specifically designed for MSC production. Many automated systems in use today for MSC production are either repurposed from technologies designed for established cellular therapies such as marrow transplantation or else from the biologics drug manufacturing industry. However, as MSCs as a therapeutic progress further from early-stage clinical trials to late-stage trials and ultimately commercialization, and as the need to both scale-up and scale-out manufacturing increases, it is anticipated that medical device, laboratory device, and bioprocessing equipment manufacturers will begin to design and commercialize automated systems expressly for MSC production. The majority of automated systems currently in use for MSC manufacturing utilize closed-system processing with single-use plastic disposables. This is to ensure the sterility of the cellular product as well as to potentially limit the need for processing within higher-grade good manufacturing practices cleanrooms, and the concomitant expense of operating and maintaining these facilities. In terms of the first common unit operation in MSC production, isolation of the MNC fraction in whole marrow aspirates, there is currently a dearth of fully automated systems utilizing single use disposables. The Sepax 2 system (Biosafe SA, Eysins) is the primary system on the market today that is commonly utilized for this manufacturing step in MSC production since it is capable of processing the relatively small volumes of iliac crest marrow aspirate utilized to seed MSC ex vivo cell cultures (10-100 mL aspirates) via a functionally closed single-use disposable for density gradient separation. Unlike for marrow aspirate processing, the options for automated MSC expansion are more numerous, and several platforms are available based on a variety of underlying core technologies. These include a hollow-fiber continuous perfusion device, the Quantum cell expansion system (Terumo BCT Inc., Lakewood, CO), a 2D multiplate bioreactor with integrated perfusion, the Xpansion system (Pall Corporation, Port Washington, NY), and three systems where MSCs may be grown on microcarriers in suspension: the rocking wave bag-based Xuri cell expansion system (GE Healthcare Life Sciences, Marlborough, MA), the fixed-bed iCELLis bioreactor (Pall Corporation), and the stirred tank Mobius system (EMD Millipore, Billerica, MA). A number of systems are also commercially available for cell washing and concentration. A different disposable set configuration allows the Sepax 2 to be utilized to wash and concentrate MSCs, the LOVO cell processing system (Fresenius Kabi, Melrose Park, IL) utilizes spinning membrane technology, the kSep400 (kSep Systems, Morrisville, NC) allows for automated single-use centrifugation, and while not yet commercially available at the time of the writing of this article, GE Healthcare Biosciences will soon launch a cell washing and concentration system based on tangential-flow filtration technology. Unfortunately, there are no automated filling and finishing systems current commercially available for the final formulation and packaging of MSCs. However, Cook Regentec (Indianapolis, IN) is imminently launching an automated cryovial fill and finishing system designed expressly for the cell therapy market, with the capability to perform high-throughput filling of closed 2- and 5-mL cryovials. Recently, two systems have been launched commercially where all or most of the processing unit operations are combined into one single automated device: the CliniMACs Prodigy (Miltenyi Biotec, Bergisch Gladbach, Germany) and the Octane Cocoon (Octane, Kingston, Canada). Although the number of devices which enable the automation of the manufacturing unit operations in MSC manufacturing are increasing as MSC therapy progresses through clinical trials and toward commercialization, a number of technologic and commercial challenges remain as a barrier to the adoption of automation. One of these challenges is scalability, where many current automated technologies have a very limited range of MSC batch sizes and volumes at which they can operate effectively. Devices that are suitable for the manufacture of an autologous product often cannot be used cost-effectively to manufacture the larger lot sizes necessary for allogeneic MSC therapeutics. Another challenge is the current software capabilities of many of these stand-alone devices that address one single-unit operation in MSC manufacturing. Integration of these devices into laboratory and facilities management control systems and the ability to generate electronic batch production records is often difficult or not possible, rendering MSC product tracking and chain of custody throughout the MSC manufacturing process not entirely seamless. In-process control testing of critical quality attributes (CQA) of MSCs during production is also currently far from well integrated into automated platforms, as real-time monitoring and feedback technology for CQAs for cell therapy manufacturing automated platforms is in its technologic infancy with great need for technologic innovation. Sterile tubing connectivity in terms of standardization of disposable single-use tubing material, wall thickness, and diameter has been a challenge for the closed processing of MSC products, whereby oftentimes the product bag processed on one device cannot readily be connected to the input lines of the next device downstream in a manufacturing process. Finally, the current high cost of single-use disposables and their associated electromechanical device platforms, relative to manual tissue culture plasticware also pose a significant challenge to many MSC therapy developers. However, it is anticipated that the costs of both automated devices and single-use disposables will decrease in the coming years as the market for MSC therapeutics increase and the economies of scale and larger commercial volumes allow for the device and single-use disposables manufacturers to lower their cost of goods. Although the number of devices and associated single-use disposable sets currently available to automate MSC manufacturing is small, and MSC manufacturers have had to rely on repurposing equipment designed for the hematopoietic cell transplantation or biologics biomanufacturing industry, a number of devices are starting to be expressly designed and commercialized for the cellular therapy market to automate the manufacturing unit operations associated with MSC production such as marrow separation, ex vivo cell culture, cell washing and concentration, and product filling and packaging. Innovation in device and disposable connectivity, in terms of software to enable effective product tracking and device integration into cell manufacturing facility operating systems, advancements in real-time in-process quality control testing, and standardization of sterile tubing connectivity between manufacturers’ single-use disposable tubing sets, will drive automation forward in the coming years and increase the availability, quality, and lower the cost of MSC for clinical trials and ultimately allow for therapeutic commercialization. The author is an employee of Terumo BCT.

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