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Harnessing the human immune system as a foundation for therapeutic technologies capable of recognizing and killing tumor cells has been the central objective of anti-cancer immunotherapy. There has been an increasing interest in improving the effectiveness and accessibility of this technology to make it widely applicable for adoptive cell therapies (ACTs) such as chimeric antigen receptor T (CAR-T) cells, tumor infiltrating lymphocytes (TILs), dendritic cells (DCs), natural killer (NK) cells, and many other. Automated, scalable, cost-effective, and GMP-compliant bioreactors for production of ACTs are urgently needed. The existing and most advanced systems for ACT manufacturing, including cell culture bags, G-Rex flasks etc.
- adoptive cell immunotherapy
- CAR-T
- T cell
- bioreactor
1. Introduction
A wide range of immunotherapeutic products have found extensive use in modern science and medicine. From various cell products to antibodies and cytokines, they are produced worldwide on an industrial scale. Therefore, scaling up of the biotechnological processes to meet the growing needs represents a serious challenge. Multiple optimization approaches exist for scaling up the production of immunotherapeutic products. The main principles are based on closed systems and automation. The more autonomous the process becomes, the cheaper and more efficient it gets for large-scale manufacturing. The use of closed systems can significantly reduce the risk of contamination, thereby increasing the quality of the product and lowering the infrastructural requirements.
The production of most immunological products is based on the patient’s own immune cells. Therefore, any biotechnological process related to cell cultivation, when scaled up and automated, will have to end up with bioreactor systems of some kind. In this regard, given the wide range of practical issues to be addressed, the development of modern bioreactors for industrial production of immunotherapeutics is extremely important. Industrial use of cell culture bioreactors is carried out after careful development and optimization of the protocol, since methodology and used materials can have a significant impact on the efficiency of the whole process [1].
Primary parameters to consider for biotechnology processes include pH, temperature, dissolved oxygen levels that can be controlled by medium oxygenation and replenishment (e.g., by perfusion), mixing of cell suspension to achieve uniform distribution of cells inside the bioreactor, etc. Bioreactor systems may use different basic principles and therefore may vary in performance and specific application. In addition, certain mechanical features may be advantageous for the efficient cultivation of one cell type while leading to low proliferation or even cell death in other cases. Thus, there is no universal solution to enhance throughput and achieve the required product quality, however, with careful selection of the appropriate system, high yields of quality cell products can be achieved. The following contents describe various cultivation systems that are currently used in both academia and industry for the manufacturing of a wide range of cell products (such as CAR-T cells [2], Tregs [2], TILs [3], etc.). Technical solutions for cell product manufacturing can be classified into cultivation systems, bioreactors with a certain level of automation, and semi- and fully-automated systems (Figure 1).
Figure 1. Primary types of cell cultivation systems, bioreactors, and automated systems. Cell cultivation systems are simple, require additional equipment, and involve manual cell processing at all steps. Bioreactors are more mechanized and allow certain steps to be completed in a closed aseptic mode. Semi-automated and automated systems are highly autonomous and require minimal human intervention.
2. Cell Culture Bags
Cell culture bags are made of polymeric materials and may have up to several aseptic ports for media input/output, sampling, and harvest. The main advantage of bags compared to T-flasks is a significantly lower risk of contamination. Bags may be manufactured from a wide range of gas-permeable polymers, such as silicone, ethyl vinyl acetate, polyolefins, etc. However, it should be taken into account that the bag material and its shape can significantly affect the cell proliferation and expansion.
Li et al. cultured human T cells in bags made of various materials such as silicone, polyolefin/ethyl vinyl acetate (EVA), fluorinated ethylene propylene (FEP) and compared results to regular T-flasks [4]. In polyolefin/EVA and FEP bags, cell expansion was nearly twice as slow as control T-flask, while the fold of expansion in silicone-made bags was the same as in the control. The authors assume that the higher gas permeability of silicone is the main reason for this observation.
Zuliani et al. performed the cultivation of tumor infiltrating lymphocytes in polyolefin bags and found that cell expansion was reduced by 9.8% compared to regular plates [3]. The authors then proposed a complex compartmentalized bag shape, which could provide local increase in cell concentration and thus provide better contact between TILs and feeder cells. Thereby, in principle, the bag shape may be re-designed to achieve enhanced cell growth for a given material.
The mixing option may also be implemented in bags by connecting input and output ports by a continuous tubing accompanied with a magnetic pump. Li et al. incubated human T cells in bags at different conditions: static, with periodic activation of magnetic pump, and with continuous pumping [3]. The use of the magnetic pump allowed it to avoid cell damage, unlike centrifugal or peristaltic pumps. The periodic pumping substantially increased T cell numbers, while the continuous pumping resulted in 75% decrease compared to the resting control T-flasks. Such results may be explained by the type of bag material and its effect on proliferation of T cells.
In general, the main advantages of the bags are a high level of aeration and relatively low cost. Bags of various intricate shapes can be designed for a local increase in intercellular interactions, which is important in the cultivation of lymphocytes. However, due to conflicting data for many types of immune cells, careful selection of bag material is required.
3. G-Rex Flask
One of the most popular devices for suspension cell culturing is the G-Rex flask, designed and manufactured by Wilson Wolf Manufacturing. G-Rex is a round cylindrical flask with a special gas-permeable silicone membrane at the bottom (Figure 1). The vessel is filled with media and then cells are inoculated. Cells sediment during cultivation and form a thick layer above the membrane. The large media volume provides sufficient quantities of nutrients while the silicone membrane ensures efficient gas exchange, which reduces the risk of oxygen starvation [5]. The media exchange is performed manually inside the laminar flow cabinet. Cells form a dense layer at the flask bottom, which makes it easy to replenish the medium without disturbing the cells. The indisputable advantages of the G-Rex system include low medium consumption and compatibility with standard laboratory equipment, such as laminar flow cabinets and CO2 incubators, which significantly reduces technical requirements for cell culture manipulations in comparison to conventional bags [6]. This significantly lowers the cost of switching from standard T-flasks to bioreactors, which facilitates the scale-up in laboratory, pre-clinical, and clinical settings (Table 1). G-Rex vessels are also more efficient than T-flasks and are better suited for the production of various cell products under GMP-compliant conditions [7][8][9].
Table 1. Main systems for adoptive cell therapy cultivation.
| Culturing Method | Maximum Cell Density | Available Volume/ Size |
Advantages | Disadvantages |
|---|---|---|---|---|
| Stirred Flask | 1.7 × 106 cells/mL | 50 mL–35 L | Availability Scalability High level of aeration |
Controversial efficacy data for the cultivation of some cell products Careful selection of the shape of blades is required |
| G-Rex flask | 10–40 × 106 cells/cm2 | 2 cm2–500 cm2 8 mL–5 L |
High density of cultivation Relatively low cost Can be operated using standard laboratory equipment (laminar flow hood, cell culture incubator) |
Quality control check is required for each flask Additional steps needed for manual processing of the cell product Higher risk of contamination |
| Rocking motion bioreactors | 10 × 106 cells/mL | 250 mL–100 L | Scalable to large volumes Uniform cell mixing High level of media aeration Perfusion capability Sensors for pH, dO, temperature Aseptic media addition and sampling |
Low efficacy for some cell types Additional expensive equipment is required |
| Stirred tank bioreactors | 2 × 106 cells/mL | 250 mL–1 × 104 L | Great scalability Effective mixing with adjustable intensity High level of media aeration Range of sensors for monitoring cell culture Flexibility of control settings (rpm, shape of paddles, etc.) |
Controversial efficacy data for the cultivation of some cell products Additional expensive equipment is required Careful selection of the shape of blades is required |
| Hollow fiber | 1 × 109 cells/mL | area (cm2) to volume (mL) ratio = approx. 100–200 | Capability to culture cells at extremely high density Scalability Versatility (fibril configuration, cartridge size, etc.) Continuous addition of fresh medium and waste removal |
Additional expensive equipment is required Need to carefully select the membrane/fibril properties (porosity, packing density, diameter, etc.) Complicated collection of the cell product |
| CliniMACS Prodigy | 8 × 106 cells/mL | 500 mL | Semi-automated system Multicomponent device (all-in-one) Suitability for clinical application Availability of ready-made kits for various steps of the processes |
High cost of the system and consumables Cell expansion is carried out in a centrifuge chamber (CentriCult Unit, a part of CliniMACS Prodigy system) |
Nevertheless, the G-Rex system has some drawbacks with regards to integration with automated GMP protocols for manufacturing of immunotherapeutic cell products, especially at the stages of cell seeding and quality control. The problems arise mainly due to the difficulty of transferring large volumes in a sealed and sterile manner from the flask to other devices, e.g., for washing and buffer exchange. The issues also include problems with sampling and limited maximal volume of the vessel. For example, for TILs manufacturing it may take up to 30 G-Rex flasks to produce the quantity of clinical grade cell product sufficient to effectively treat a single patient. At the same time, some types of perfusion bioreactors allow one-step production of large amounts of therapeutic cell products due to much higher volume of the expansion vessel. For example, in WAVE (Cytiva, Wilmington, NC, USA) rocking, the motion bioreactor volume of the cultivation bag can reach up to 100 L with a possible cell density of up to 10E7 cells/mL [10]. Eventually, using a large number of G-Rex vessels substantially complicates the quality control, making it necessary to individually analyze samples from each vessel for sterility, cell product identity, and efficacy.
Despite all the drawbacks, G-Rex remains one of the most popular solutions for scaling up biotechnological processes and is widely used in immunotherapy manufacturing. G-Rex is known for particularly efficient cell expansion when scaling up TILs production [6][11][12], which is explained by close interactions of lymphocytes with feeder cells followed by passive proliferation. Comparable efficacy TILs production was also demonstrated by gas-permeable bags (EXP-Pak by Charter Medical, Dublin, Ireland) [13]. For CAR-T cells, G-Rex flasks showed substantial efficiency. Gagliardi et al. demonstrated that the transduction efficiency of T cells in G-Rex compared to retronectin-coated bags was lower (55% versus 73%) [14]. However, the cell expansion and the total number of transduced effector cells was significantly higher in G-Rex than in the bags.
Overall, G-Rex flasks have proven to be an affordable and efficient solution for scaling up production without requirements for sophisticated additional equipment. In addition, G-Rex flasks consistently demonstrate high efficiency of cell cultivation due to the technology of medium oxygenation through a gas-permeable silicone membrane.
This entry is adapted from the peer-reviewed paper 10.3390/bioengineering9120808
References
- Watanabe, N.; Mo, F.; McKenna, M.K. Impact of Manufacturing Procedures on CAR T Cell Functionality. Front. Immunol. 2022, 13, 876339.
- MacDonald, K.N.; Salim, K.; Levings, M.K. Manufacturing next-generation regulatory T-cell therapies. Curr. Opin. Biotechnol. 2022, 78, 102822.
- Zuliani, T.; David, J.; Bercegeay, S.; Pandolfino, M.-C.; Rodde-Astier, I.; Khammari, A.; Coissac, C.; Delorme, B.; Saïagh, S.; Dréno, B. Value of Large Scale Expansion of Tumor Infiltrating Lymphocytes in a Compartmentalised Gas-Permeable Bag: Interests for Adoptive Immunotherapy. J. Transl. Med. 2011, 9, 63.
- Li, M.; Chin, L.-Y.; Shukor, S.; Tamayo, A.; Maus, M.V.; Parekkadan, B. Closed Loop Bioreactor System for the Ex Vivo Expansion of Human T Cells. Cytotherapy 2019, 21, 76–82.
- Lapteva, N.; Vera, J.F. Optimization Manufacture of Virus- and Tumor-Specific T Cells. Stem. Cells Int. 2011, 2011, 434392.
- Somerville, R.P.; Devillier, L.; Parkhurst, M.R.; Rosenberg, S.A.; Dudley, M.E. Clinical Scale Rapid Expansion of Lymphocytes for Adoptive Cell Transfer Therapy in the WAVE® Bioreactor. J. Transl. Med. 2012, 10, 69.
- Bollard, C.M.; Gottschalk, S.; Huls, M.H.; Leen, A.M.; Gee, A.P.; Rooney, C.M. Good Manufacturing Practice-Grade Cytotoxic T Lymphocytes Specific for Latent Membrane Proteins (LMP)-1 and LMP2 for Patients with Epstein–Barr Virus-Associated Lymphoma. Cytotherapy 2011, 13, 518–522.
- Vera, J.; Brenner, M.; Dotti, G. Immunotherapy of Human Cancers Using Gene Modified T Lymphocytes. Curr. Gene Ther. 2009, 9, 396–408.
- Vera, J.F.; Brenner, L.J.; Gerdemann, U.; Ngo, M.C.; Sili, U.; Liu, H.; Wilson, J.; Dotti, G.; Heslop, H.E.; Leen, A.M.; et al. Accelerated Production of Antigen-Specific T Cells for Preclinical and Clinical Applications Using Gas-Permeable Rapid Expansion Cultureware (G-Rex). J. Immunother. 2010, 33, 305–315.
- Budiyati, A.D.; Pawitan, J.A. Integrating Bioreactor System to Speed Up Tumour Infiltrating Lymphocytes (TILS) Immunotherapy Commercialization. Int. J. Appl. Pharm. 2020, 12, 34–38.
- Bajgain, P.; Mucharla, R.; Wilson, J.; Welch, D.; Anurathapan, U.; Liang, B.; Lu, X.; Ripple, K.; Centanni, J.M.; Hall, C.; et al. Optimizing the Production of Suspension Cells Using the G-Rex “M” Series. Mol. Ther.-Methods Clin. Dev. 2014, 1, 14015.
- Xiao, L.; Chen, C.; Li, Z.; Zhu, S.; Tay, J.C.; Zhang, X.; Zha, S.; Zeng, J.; Tan, W.K.; Liu, X.; et al. Large-Scale Expansion of Vγ9Vδ2 T Cells with Engineered K562 Feeder Cells in G-Rex Vessels and Their Use as Chimeric Antigen Receptor–Modified Effector Cells. Cytotherapy 2018, 20, 420–435.
- Sarnaik, A.A.; Hamid, O.; Khushalani, N.I.; Lewis, K.D.; Medina, T.; Kluger, H.M.; Thomas, S.S.; Domingo-Musibay, E.; Pavlick, A.C.; Whitman, E.D.; et al. Lifileucel, a Tumor-Infiltrating Lymphocyte Therapy, in Metastatic Melanoma. J. Clin. Oncol. 2021, 39, 2656–2666.
- Gagliardi, C.; Khalil, M.; Foster, A.E. Streamlined production of genetically modified T cells with activation, transduction and expansion in closed-system G-Rex bioreactors. Cytotherapy 2019, 12, 1246–1257.
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