Initial research into CAR T technology relied on manual, open systems for development. As the technology matures, CAR T cell manufacturers are continually looking for process improvements that can decrease overall costs. They are also looking for improvements that will decrease contamination, improve batch-to-batch consistency, and allow for monitoring and capture of critical information, which will help with the ever-changing regulatory environment. To this end, CAR T cell therapy manufacturers are turning to automated, closed systems with integrated software controls to achieve lower manufacturing costs, maintain product consistency, and meet regulatory requirements. 

Closed versus open systems for manufacturing CAR T cell therapies

Numerous steps in the methods for isolation and expansion of CAR T cell therapies have options that can be performed using an open or closed system. For example, density gradient centrifugation for the isolation of PBMCs is typically performed using an open system, and T flasks commonly used for research-scale cell growth are an open system. However, open systems can expose the cell therapy product to a room’s environment and require increased user interaction, such as working under a laminar air flow hood [1]. These open processes tend to be more labor intensive and can take up larger footprints, especially when trying to reach a larger manufacturing scale (Table 1). Another consideration in using open systems is the need to utilize a grade A or grade B manufacturing facility, whereas a closed system can be implemented in a grade C facility. The difference in manufacturing conditions required for a closed system can considerably decrease costs, labor, and space requirements. All of these issues can significantly increase manufacturing costs. In research settings, manual open processes may be the route chosen. However, these methods should be avoided in clinical applications and final cell therapy manufacturing because of the increased risk of contamination and batch-to-batch product variability, which can hinder regulatory approval and possibly result in product failure.

Table 1. Comparison of cell culture options.

OptionsTypical working volumeClinical or RUO*AdvantagesDisadvantages
Static bags5 mL–3 LRUO or ClinicalClosed systemLimited gas transfer and working volume
T-flasks/static plates100 µL–370 mLRUOEconomical; great for screening multiple conditionsOpen system;
small scale
Rocking motion bioreactor300 mL–50 LClinicalClosed; automated; scalable; Gas, liquid, DO, and pH control and sensing; perfusion capable; digital integrationLarge footprint; high cost
G-Rex8 mL–5 LRUO or ClinicalSupports scaling; automated DO and pH control; supports higher oxygen mass transfer coefficient (kLa) values compared to static cultureDifficult to close system; requires training; high cost
Stirred tank bioreactor250 mL–2,000 LClinical

Closed; automated, scalable; gas, liquid, DO, and pH control and sensing; perfusion capable;
digital integration; supports higher oxygen mass transfer coefficient (kLa) values

Large footprint;
high cost

*RUO = research use only.

According to the European Medicines Agency (EMA), a closed system is “A process system designed and operated so as to avoid exposure of the product or material to the room environment. Materials may be introduced to a closed system, but the addition must be done in such a way so as to avoid exposure of the product to the room environment (e.g., by means of sterile connectors or fusion systems)”  [2]. Systems can be closed using a variety of devices and techniques (e.g., sterile barrier filters, disposable sterile doc connectors). The use of single-use technologies (SUTs) further assists closure of the system by providing enhanced protection outside of a clean room or biosafety cabinet. SUTs include plastic single-use bags, bioreactors, tubing, filter capsules and connectors, making them compatible with closed system techniques[3]. An important consideration when using SUTs is compatibility of all parts, particularly the connections between bags and tubing. If tubing sizes and bags are mismatched, connections can still be made, but will require aseptic joining in a laminar flow hood. 

The importance of closed processes and SUT

Contamination in bioprocessing is costly. It results not only in product loss, but can also lead to facility shutdown for cleaning and validation. According to the FDA, for aseptic processing of cell therapy products, “Cellular therapy… represent a subset of the products that cannot be filter-sterilized… Where possible, closed systems should be used during manufacturing[2]. Closing the system significantly reduces risk of contamination by viral, bacterial, or other adventitious agents. In addition, closed systems may be placed in a controlled but non-classified environment [4], which could improve manufacturing flexibility (e.g., reducing the facility footprint or reducing the amount of segregation in a facility). In addition, equipment and personnel can be moved around more easily to meet production needs. These simpler designs enable manufacturing suites that are easily duplicated at multiple sites. Combined with SUT, closed systems can greatly reduce processing time including cleaning, setup, and batch turnaround times. Moreover, cost of goods will be significantly lower due to reduced operating costs, which can include labor costs, energy costs for environmental monitoring and air handling, costs to grow the material, as well as facility costs [5]

The importance of automation in GMP manufacturing

To further improve manufacturing costs, cell therapy manufacturers will look into automating most of the manual steps in the process. Automation is not restricted to production; it can also be expanded to include analytical steps such as offline or inline process analytical technology (PAT).
Implementation of automation is critical for large-scale cGMP manufacturing [6]. The EMA suggests that “The use of automated equipment may ease compliance with certain GMP requirements and may also bring certain advantages in respect to product’s quality[2]. Automation would improve operator safety, reduce human errors, and help enable processing robustness and reproducibility. Automated systems can simplify operations overall. Manual procedures that have multiple steps and/or require multiple operators can be combined within a single machine with a single operator, reducing the product turnover time and the number of personnel required in the operation space. As a result, facility production capacity will increase. The overall cost of goods for similar quantities of cell therapy products will significantly decrease. 

Existing closed automation systems in cell therapy manufacturing

Several steps in patient-specific cell-based therapy development (e.g., CAR T) can be implemented using an automated, closed system: cell isolation, expansion, processing, and formulation. Two categories exist based on the degree of automation [7]:
   1. Integrated closed system 
   2. Modular closed system 
Integrated closed systems are fully automated. They are all-in-one, easy-to-use, and designed as an end-to-end, one-patient-at-a-time solution. Once employed, the integrated closed instruments will be dedicated to producing a specific patient’s cell product for a certain period of time (usually 1-2 weeks). This approach refers to the automation and closure of a single machine for a specific patient or purpose and integrates several steps into a complete workflow.
Modular closed systems are more versatile, with each instrument primarily optimized for a single step. This approach does not restrict bioprocessing companies to a single supplier—they can choose instruments that are best suited for individual steps in the process. More importantly, manufacturers have the flexibility to develop new processes using existing instrumentation as needed. For example, one can use the Rotea instrument (a Thermo Fisher Scientific closed counter-elutriation system) to isolate PBMC/CD3 T cells, and the G-Rex® system to expand the T/CAR T cells (a Wilson Wolf culture expansion system) (see the white paper on this topic). Table 2 summarizes some current cell processing automated systems and their parameters. 

Table 2. Parameters of cell processing automated systems.

Modular systemIntegrated system
Core technologyCounterflow
Electric centrifugation motor and pneumatic
circuitry for piston drive2
Spinning membrane filtration3Acoustic cell processing4Magnetic separation5
Cell recovery>95%70%70%89%85%
Input volume30 mL–20 L30 mL–3 L100 mL–22 L1 L–2L1 L–2 L
Input cell capacity10 x 10910–15 x 1093 x 1091.6 x 1093 x 109
Cell processing time45 min90 min60 min40 minNA

    1.Rotea system; 2. Sepax; 3. Lovo; 4. Ekko; 5. Prodigy

Digital integration of the CAR T cell therapy manufacturing workflow

Software-driven, digital integration plays an essential role to support full automation across the entire cell therapy manufacturing workflow. Digital integration can improve manufacturing productivity and process control by monitoring the entire workflow starting from sourcing of raw materials through product delivery to the clinic. This tracking can ensure data integrity, traceability, and regulatory compliance, plus aid in the scale up of the process. Ideally, a mature manufacturing environment would connect production (hardware and controllers), control layers (e.g., supervisory controls), and manufacturing execution systems (below Figure 1). Software tools can offer the ability to mine and analyze data from upstream and downstream batch records across batches for real-time optimization and troubleshooting.

A pyramid diagram with a red arrow from the base to the point, depicting multiple layers of tiered process control

Figure 1. A comprehensive process control, digital connection and data stream of mature manufacturing for commercialized products. To easily scale up the process and accelerate time to market, it is essential to manage the workflow through a Distributed Controlling System (DCS) to control the process and ensure traceable, reproducible, and secure data connectivity through a Manufacturing Execution System (MES), which can be further integrated into an Enterprise Resource Planning (ERP) system.  
In current digital solutions for cell therapy manufacturing, the workflow is managed by connecting the instrument to a Distributed Control System (DCS). The DCS layer allows for scalable process optimization, workflow management, and data transferring across the entire workflow. Some software systems are more easily configured to DCS and MES than others.


Tremendous efforts have been made to make CAR T cell therapy more effective, safe and persistent in treating patients. Yet, the manufacture of CAR T cells has been prone to errors, lot-to-lot variation, and contamination. These errors commonly result from the use of open processes with manual handling. Using closed automated systems that integrate the complicated, multistep CAR T workflow can easily overcome these challenges. The use of closed integrated systems improves consistency, purity, and safety while helping to lower overall manufacturing costs. These benefits can contribute to making cell therapies more affordable and accessible to patients in the future. 


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  3. Jayaraman, P. et al. Accelerate translational mesenchymal stromal cell therapy through consistent quality GMP manufacturing. 2021. Front Cell Dev Biol 2021 Apr 13;9:648472. doi: 10.3389/fcell.2021.648472.eCollection 2021.
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