April 17, 2024

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Bioreactor system for clinical scale expansion of T cells

Bioreactor system for clinical scale expansion of T cells



Bioreactor system for clinical scale expansion of T cells. The expansion procedures developed in the past few decades have brought excellent results for clinical trial development research. In particular, the use of bioreactors is a promising method for in vitro culture of adoptively transferred T cells.

A bioreactor is defined as a container containing cells, other organisms, and/or biologically active substances in a controlled environment: carefully monitor temperature, nutrients, waste, pH, mechanical requirements, and flow rates to achieve a variety of biotechnological processes ( figure 1).

Bioreactor system for clinical scale expansion of T cells

Figure 1. The main concept of the bioreactor chamber including the most common components.

This article first introduces the general considerations for T cell culture and treatment, including a brief overview of the steps other than expansion. Then the specific T cell culture requirements are discussed to emphasize the main goals and methods that guide the development of the new expansion process. Finally, the current gold standards, namely culture flasks and mobile culture bags, are reported and compared with the main bioreactor designs, namely hollow fiber, bag bioreactors and improved flask systems, as well as original and recent recommendations . We summarize the ongoing challenges and technical issues that are still under investigation in order to promote ACT to a wider range of clinical applications.



1. T cells: general considerations

There are a series of methods that can be used to produce powerful therapeutic effects after adoptive transfer of T cells. The production of TIL for cell therapy usually requires standard in vitro expansion steps: TILs are separated from tumor biopsies, washed and concentrated, and then expanded to a clinically relevant population size. However, TIL does not always exist naturally and can only be produced from certain patients. Therefore, other strategies based on T cell receptor (TCR) or chimeric antigen receptor (CAR) have been developed. In this way, normal T cells from patients with other cancers are isolated and transduced by apheresis to produce TCR or CAR T cells (Figure 3).

Bioreactor system for clinical scale expansion of T cells

Figure 3. Tumor-targeted T cells are generated by autologous TIL collection or CAR and TCR process.

An activation procedure must be performed to allow the transduction and proliferation of manufactured T cells. In particular, interleukin 2 receptor (IL-2R), a surface protein expressed on T cells, can be upregulated in vitro. Since pioneering work in the early 1990s, interleukin 2 (IL-2) has been used as the main growth factor that triggers primary culture and implantation proliferation. The complete environment for T cell activation and proliferation includes anti-CD3 antibodies and exogenous IL-2, as well as peripheral blood mononuclear cells as feeder cells. This combination was reported as a standard “rapid expansion protocol” (REP), and the use of co-cultivation with another cell lineage as a pre-stimulation step was also reported. Today is still studying how to improve and perfect REP, such as the use of additional medium supplements (IL-7, IL-15, anti-CD28 antibody), but also pre-stimulation with peptide mixtures focused on pp65 protein, resulting in higher identity Proliferation rate in the culture vessel. Recent alternatives include, for example, scaffolds that mimic T cell activation physiological signals, DNA-based platforms to present anti-CD3 and anti-CD28 antibodies or bispecific antibodies.

Memory T cells can achieve the best results in terms of rapid transplantation, anti-tumor activity and long-term protection. In addition, some people believe that CD4+ T cells are more effective in the body: CD4+ T cells have the same cytotoxic activity as CD8+ T cells; however, the CD4+ subset is more durable after TCR is involved. The most effective T cell subset for treatment has not yet been determined. This unknown is further complicated by how the bioreactor affects T cell subpopulations. For example, compared to CD4+ cells, CD8+ cells grow faster in co-culture in a stirred tank bioreactor.




2. Expansion of T cell activation in a bioreactor environment

Under classical culture conditions, the activation of T cells by CD3 and CD28 antibodies leads to strong activation. The addition of CD28 specifically promotes: pro-survival factors, such as increased IL-2 production; increased respiration of T cells in mitochondria; and increased activated T cells Increased glucose uptake and glycolysis to further meet the energy and biosynthetic needs of the expanding population. However, it has been reported that other T cell activation methods have produced different beneficial effects. The presentation of these clues plays an important role: it is known that placing CD3 and CD28 antibodies on scaffolds of different sizes and materials affects the response of T cells in subsequent activation and expansion.

In addition, the antibodies used also have a great influence on the quality of treatment of the T cells produced. Zhang et al. showed that compared with CD28 costimulation, cell-based artificial antigen presenting cells expressing 4-1BB increased the expansion of CD8+ T cells.

In addition, Bashour et al. showed that CD3 and CD28 immobilized on the patterned surface allowed them to recognize the force that T cells might exert when they bind to these antibodies. Since T cell activation involves many factors, it is obvious that any bioreactor involved in the activation step should be evaluated for its impact on the interaction between T cells and antigen presentation. For example, stirring to improve mass transfer in the bioreactor may significantly affect T cell activation. Although the development of antigen presentation methods may initially be considered beyond the scope of cell expansion bioreactors, it is clear that the method used for activation has a strong impact on the expansion of the produced material and the quality of the treatment.

Despite the emergence of such standardized procedures, more complex environments based on dedicated culture systems are still being studied to obtain optimal proliferation, thereby generating enough TIL or TCR/CAR cells for treatment. In order to go further, it is necessary to understand the pedigree’s training requirements, ongoing challenges, and specific goals for bioreactor expansion.




3. The purpose of bioreactor amplification

3.1. Number of cells

As mentioned earlier, the main goal of developing a bioreactor system for culturing T cells is to further increase the in vitro proliferation rate compared with static flask culture, beyond the REP and activation steps. The number of isolated cells is still too small to ensure effective autoimmune therapy. In pediatric and young adult CAR-T trials, a complex algorithm and method was developed to achieve the expected 3.24 billion CD3+ T cell apheresis.

Ceppi et al. also described how patients missed promising immunotherapy because they failed to make CAR T therapy, which they believe was partly due to obtaining enough cells from apheresis. For the final therapeutic product, most studies report that a patient needs 100 to 100 billion cells, an average of at least 10 billion. Although it is possible to obtain such cell populations through multiple vessels and heavy labor, patients will benefit from a faster and more reliable process to ensure that the cells are ready on time and as soon as possible. Therefore, regardless of the specific design and geometry, REP is usually used in conjunction with a bioreactor system, even if only partially (for example, only IL-2 or anti-CD3 antibodies are added instead of a complete factor-rich environment).

In this article, the maximum cell density and maximum yield that can be achieved in a specific type of equipment will be discussed. However, it must be noted that the design and functional differences between systems, such as hollow fiber bioreactors and stirred tank systems, make it difficult or almost impossible to strictly compare the maximum cell density without considering all other characteristics and parameters. . Therefore, it should not be the only parameter to be considered when evaluating the potential of a bioreactor for T cell applications.


3.2. Reduce cost and quantity

The increase in the proliferation rate will help reduce the amount of solution and labor time, thereby reducing the cost of cell therapy. In fact, T cell expansion usually lasts for 14 days under classical conditions and due to the low cell density, tens of liters of culture medium may be required to obtain the required number. It is reported that the volume is as high as 50 L to achieve the desired cell population in an open system. Therefore, it is important to optimize the bioreactor design to reduce the volume and improve the T cell culture feed schedule to find a balance between nutrient support, waste removal, and media cost. In addition, it has been shown that partial medium changes can improve cell proliferation, because complete changes lead to the removal of all beneficial autocrine factors, such as interferon-γ, IL-1β and tumor necrosis factor-α, resulting in reduced proliferation (especially when stirring System). However, on the contrary, the development of complex bioreactor systems may increase procurement and startup costs, especially for small academic structures involved in the design and optimization of early steps. Therefore, this balance needs to be carefully considered when evaluating the economic benefits of bioreactors.


3.3. Promote agreements that comply with Good Manufacturing Practices (GMP)

Due to the high labor intensity of current T cell expansion protocols, it is very important to promote the process of ensuring GMP. As mentioned earlier, the reduction of volume and cultivation steps, as well as the automation of routine procedures and the use of closed systems, have made a positive contribution to such methods. The design of the bioreactor should avoid multiple exposures of the cell population to the external environment during the expansion period for feeding, sampling, and transfer from one culture system to another, eliminating the need for large facilities such as clean rooms. GMP is also used in initial and auxiliary steps, such as cryopreservation or transduction of TCR/CAR cells, thereby facilitating the translation of research into clinical trials.

As discussed in the next section, the bioreactor system can incorporate procedures that are performed on either side of the cell expansion phase, further reducing the number of open processing steps, thereby achieving GMP-compliant processing in one unit.




4. Constant or increase cell density: two different methods

Although T cells can be attached to antibody-functionalized surfaces, they are more often cultured under free expansion and non-adherent conditions. Therefore, the cell density is expressed as the number of cells per volume unit. When culturing T cells over time, the optimal density of 1–3 million cells per milliliter should be maintained to benefit from the balance between cell-to-cell communication and available nutrients. Regardless of the culture vessel, this value is compatible with ATCC. The recommendations of T cell lines such as Jurkat cells are the same (https://www.lgcstandards-atcc.org/products/all/TIB-152.aspx#culturemethod).

Proliferation is the ultimate goal. The first method is to continuously increase the available medium volume as the cells proliferate to allow them to expand further, while maintaining the optimal concentration and stability when the culture system is properly designed. Once the limit of 1 x 106 cells/mL is reached, the culture volume is doubled to reduce the cell density to 5 x 105 cells/mL. As a direct result, it is necessary to avoid removing the medium to prevent unnecessary changes in cell concentration or the disposal of part of the cell population; in addition, for the latest expansion phase, removing waste by replacing the medium requires a large amount of fresh medium every day. When the required number of cells is reached, or when the cell concentration no longer increases with time, the culture and volume expansion will stop.

In contrast, the second method focuses on maintaining a constant volume while increasing the cell density until it reaches a plateau, up to 30 million cells per milliliter. Closed system bioreactors are related to GMP but do not allow medium addition and volume expansion, so they can benefit from this method, especially hollow fiber bioreactors. However, cell viability and function must be carefully characterized after expansion to confirm that they are not affected by waste accumulation and cell concentration, and they must be higher than recommended. For example, most of the research conducted by Ou et al. in stirred tank bioreactors focused on evaluating the function, viability, and quality of the T cells produced. In particular, the assessment of a wide range of phenotypes and functions through anti-tumor activity is actually critical to validating expansion procedures, because in some cases, increased proliferation rates have been shown to cause T cell depletion. In the following sections on different bioreactor systems, when the phenotype is clearly discussed in the study, phenotypic considerations will be reported.

In both cases, an important parameter to consider is the initial cell density. Although a minimum concentration is required to ensure cell-to-cell communication and, in turn, rapid cell proliferation, the recommended density of about 1 million cells per milliliter is usually not required on day 0 of culture and lower seeding densities have been shown to not affect the proliferation rate. As studied by Sadeghi et al., the same problem applies to the initial volume of cell growth. Who shows that a small initial volume will lead to the best results.

In summary, a balance must be found between cell density over time, available volume, waste removal, concentration of beneficial autocrine factors, closed system characteristics, GMP, and cost. Depending on the culture system used for T cell expansion, some of these parameters may not be easy to modify. A bioreactor system is proposed to meet this challenge to provide a more reliable and faster proliferation rate than the current gold standard. Therefore, the following introduces traditional systems and new developments.




5. Gold Standards and Restrictions

Standard plastic culture flasks (although sometimes kept in an unusual vertical position to increase usable volume) and semi-permeable culture bags are known as the gold standard for in vitro T cell expansion. Thanks to the larger air-permeable surface and dedicated ports, the bag can achieve better oxygen exchange and easy sampling. However, flasks are generally cheaper, they can provide a large surface (175 cm²), and according to some studies, even higher proliferation rates can be achieved. They can also be used in combination (flask first, then bag).

In any case, using multiple repetitions of this type of system in combination with REP can produce a large number of cells, but at the expense of increased labor time, unevenness, and high consumable costs due to the low overall cell density in a single product. Therefore, frequent media replacement and cell transfer increase the risk of contamination and interrupt optimal cell contact conditions, which are critical to treatment efficiency. They are indeed open systems, so they are not GMP compliant in nature. In addition, in addition to temperature and CO2 concentration, culture conditions are usually not controlled, resulting in experiments and culture plans based on personal judgment and batch-to-batch differences that are almost impossible to repeat.




6. Commercially available bioreactor system


6.1. Bioreactor based on hollow fiber membrane: Terumo Quantum

The first hollow fiber filter cartridge was developed for a dialysis system more than 40 years ago (Figure 4A), and was later proposed for cell culture and further applications and eventually became a bioreactor. In the early years, some research on T cells in hollow fibers did not focus on cell expansion, but on the production of cytokines and other biomolecules by cells inserted into the system. As the cells grow on one side of the small hollow fibers (200 μm in diameter), the medium is perfused on the other side (Fig. 4B).

The fibers are made of membranes that are permeable to specific target components (biomolecules, gases, nutrients, waste), thus allowing controlled exchange between cell compartments and medium perfusion. The basic principle of applying this type of system to T cell culture is due to the separation geometry, which avoids the shear stress caused by flow perfusion, while keeping the culture medium in motion, such as in shaker bags and stirred tanks, which has the opportunity to re-oxygen And adjust the pH of the medium before sending it back to the cells. In addition, the closed loop of the culture medium in the cell compartment and the concentration of nutrients help reduce the volume required for feeding. The method for cell density here is to increase the concentration in a fixed volume (cell compartment), but the flow rate can increase with the number of cells.

Bioreactor system for clinical scale expansion of T cells

Figure 4. Schematic diagram of hollow fiber used as a cell culture and bioproduction bioreactor

A potential problem with such systems is the size of the molecules to be provided or removed, especially as the cell density increases. The growth inhibitors produced by T cells must be easily removed to ensure continued proliferation and expansion, which can be achieved through fiber membranes. In a similar way, growth factors should be able to reach the cell area from perfusion in turn. Therefore, the molecular weight of the T cell-specific activator (ie, IL-2 and antibody recommended to follow REP) required for expansion will guide the choice of membrane to be used. For example, Trickett et al. Note that polypropylene-based hollow fibers (0.3-0.5μm membrane pore size listed by the manufacturer) are more suitable for IL-2 diffusion than 4 kDa cellulose filters.

A commercial example of a hollow fiber-centric bioreactor system is Terumo Quantum®. The system can culture T cells to a therapeutic dose in a single-use kit in the system; after inoculating 1×108 lymphocytes, it can increase the number of cells by 90-500 times. Therefore, in experiments using the Quantum® system, the number of starting cells is very high, and the quality of the starting material may be a challenge for adoptive T cell transfer. It may be necessary to further study the adaptability of the system to changes in starting material parameters (such as cell number) to confirm its full potential.

Co-cultivation of T cells with polymer particles called Dynabeads is often used for activation steps in the form of hollow fiber, wave bag, and stirred tank bioreactors. For the hollow fiber form, the CD4+:CD8+ ratio produced by dynabead activation of T cells and Quantum® expansion is highly dependent on the donor material. Most samples tend to promote the proliferation of CD8+ cells; however, for a donor, the ratio is close to 1:1. To improve the initial results, the optimization scheme in the Quantum® system found that among other parameter changes, increasing the number of dynabeads per T cell would result in a higher multiple expansion of the bioreactor. The study did not report an effect on the phenotype of T cells. The conclusion regarding the T cell subpopulation produced by the bioreactor is that it may depend on factors unrelated to the bioreactor, but for example, factors related to donor variability, although these studies lack control experiments that use the above-mentioned gold standard method related data.

One unique feature of Quantum® and hollow fiber bioreactors is that they can cultivate adherent cells, so they can also produce lentiviral vectors for transfection of CAR T therapy.

As mentioned earlier, T cell expansion usually requires specific media components, based on REP, but will vary depending on the target application, bioreactor geometry, and protocol. The use of hollow fiber bioreactors will require additional optimization steps, as two different media can be used in the cell compartment (extracapillary space) and the perfusion compartment (inside the fiber). The cultivation plan should also be adjusted and compared with the static system. Large-scale hollow fiber systems and commercial modules are not always suitable for this optimization, but due to the good correlation, smaller bioreactors can easily adjust the composition before scale-up. Dedicated micro hollow fiber bioreactors have been shown to help optimization at a lower cost than full-scale systems and better reliability than flasks. These preliminary steps will greatly increase the yield of the expansion culture, resulting in a proliferation rate much higher than that of the T bottle and similar to a semi-permeable bag, while reducing cost and space required. In addition, since the hollow fiber system has been used in cell culture to produce cell secretion products, there is a good industry infrastructure to support the system, while acknowledging that the requirement to harvest cells at the end of the culture may pose some challenges.


6.2. Improved flask: G-Rex® system

As mentioned earlier, although there are many restrictions, the advantages of conventional culture T-flasks are that they are easy to use and reasonable in price. Therefore, one way to develop a more relevant but easy-to-monitor culture environment is to design “improved flasks” with additional functions to increase the proliferation rate. Although the nature of bioreactors of such devices can be discussed for granted, according to the strict definition of bioreactors, they can indeed be regarded as containers with a micro-controlled environment, especially through gas exchange.

The G-Rex® system for rapid expansion of gas permeability was developed by Wilson Wolf Manufacturing about 10 years ago as an alternative to T-flasks, rotating flasks and semi-permeable bags. The main feature of the cell expansion chamber is that the semi-permeable membrane at the bottom is in contact with the main cell culture surface area (Figure 5). Compared with conventional T-flasks, this container can be filled with a very large volume of culture medium, which is limited to 1 mL/cm2 to avoid cell hypoxia. The device has been tested in suspension cells and can be used in conjunction with REP, including adding feeder cells. Therefore, it is considered for the expansion of T cells. According to the manufacturer’s information, a larger initial volume and no specific requirements for cell passaging and medium changes mean increasing cell density in a constant volume for T cell expansion.

Figure 5. The geometry of G-Rex (breathable rapid expansion) flask models of different sizes, with 5-cm2, 100-cm2, and 500-cm2 culture surfaces, respectively. Gas exchange (O2, CO2) occurs through the bottom semi-permeable membrane.

G-Rex® flasks have been studied using different protocols as semi-open or closed systems. After activating REP in a regular culture flask, regulatory T cells first continuously expand and transfer in the size/number of G-Rex® models in a manner similar to the passage of adherent cells. These studies confirmed that despite using the same protocol, the proliferation rate was higher than that of the T bottle. Then optimize G-Rex® for use as a completely closed system (except for the regular addition of IL-2) or as a method for increasing the volume to achieve a higher T cell proliferation rate. Ultimately, the goal is to obtain a closed system for each patient that can provide enough cells for treatment while drastically reducing space, labor, and costs. Bajgain et al. successfully reported such results. After 10 days, a 1.5×109 cell population was achieved with primary CAR cells in a single 1L G-Rex® bottle. According to this study, this is achieved through the combination of oxygen exchange and an almost unlimited nutrient reserve provided by a large amount of culture medium. With more than 10 ml of culture medium per square centimeter of the gas exchange membrane, the additional available volume has no effect on cell behavior. This high expansion potential can be explained by a decrease in cell death time rather than an increase in proliferation rate, so fewer divisions (and shorter incubation times) are required to achieve the desired cell number. This interesting method, that is to improve cell survival rate instead of just focusing on the proliferation rate, can also be studied and optimized in other bioreactor designs.

In addition to cell number and viability, regulatory T cells and TCR against human papillomavirus were used to evaluate the function of T cells expanded in the G-Rex® system. After expansion, they succeeded in obtaining cells that are potent and specific in killing targeted tumor cells. In addition, Forget et al. showed that the phenotype of T cells expanded in G-Rex® flasks and semi-permeable bags ensures better viability and can improve tumor control after implantation compared with conventional T flasks. Thanks to the better oxygen consumption during growth confirmed by mitochondrial respiration. They also demonstrated the maintenance of the high polyclonal diversity of TIL expanded in this system.

Regarding the disadvantages, it was mentioned that compared with horizontal culture flasks, cells cannot be observed in G-Rex® during culture. In addition, some of the analyzed controls are T cells cultured in 24-well plates, and this method is no longer considered the gold standard. Finally, it must be mentioned that most studies reporting the results of G-Rex® disclose conflicts of interest with manufacturers.


6.3. Improved culture bag: wave bioreactor and GE Xuri system

The Wave bioreactor was originally developed by Singh et al. as an alternative bioreactor, which can be easily scaled up compared to previous systems. It is proposed for various applications, such as adherent and non-adherent cell culture and adenovirus production. Then recently I specifically studied the expansion of T cells.

The system uses a sterile disposable culture bag as the main container on the platform, ensuring heating and rocking motion (variable angle, amplitude and speed) to induce “waves” in the culture medium (Figure 6). Even at small volumes and high cell densities (Donia et al., 2014), these specific exercises can provide better oxygen exchange, uniformity, and easy access to nutrients, while reducing the impact on cells compared with agitating systems. pressure. The culture medium can be perfused through the bag, and the standard tube port allows sampling and inoculation. The bioreactor has been successfully used in Class I and Class II clinical trials. Therefore, compared with static and uniaxial vibrating bags, the main innovative feature of this system is the combination of complex wave circuits and flow perfusion in a closed system. This combination provides better homogeneity even at the cell scale because it avoids the gradient of the highest concentration of beneficial secreted factors in the closed environment of the production cell. Therefore, from a technical point of view, it is a more complex system than the current gold standard, but in fact it will not increase labor time because monitoring and sampling are simple or even automated. It is shown that adding IL-2 by inoculating the bag daily or as a single initial load in the perfusion reservoir leads to the same results regarding T cell expansion: the REP program can be adjusted in a simpler way using the Wave bioreactor, which also Comply with GMP. Hollyman et al. paid special attention to biosafety inspections and respect for FDA and NIH guidelines when using the Wave system. In addition, all steps of the REP process can be executed directly in the system, and only the later stages of REP are suitable for operation in other designs, such as bioreactors based on hollow fiber membranes. In addition to increasing the proliferation rate, this method can also increase the standard yield by shortening the culture process by making it easier to handle and monitor. The use of transparent culture bags is an advantage in itself because they are disposable, can be sterilized, are optically transparent, and allow microscopic observation and non-invasive optical measurement through the culture medium.

Figure 6. Geometry of Wave bioreactor with perfusion system


Because of these characteristics, the Wave bioreactor has been used for T cell expansion, using a constant volume and increasing cell density method, combined with flow perfusion, and the method is similar to the hollow fiber bioreactor. However, due to a separate rocking motion provides Mixing and oxygen exchange, so it is possible to study a mixing method to adapt the parameters to the cell density: Sadeghi et al. use a constant volume, but only start to perfuse the medium after reaching a certain cell density. More importantly, they showed that when the process starts with a smaller volume, the results of cell expansion are better. Therefore, another method can be developed based on cell density or nutrient levels (glutamine and glucose) in combination with increased volume and flow perfusion.

Interestingly, except for the available volume, all parameters can be adjusted over time during cell proliferation, such as rocking rate, rocking angle, perfusion flow rate, etc. In general, the proliferation rate obtained by the Wave bioreactor is similar to or significantly higher than the static bag method, and requires a smaller volume. It has been shown in other gas permeable systems that high oxygen levels in the culture medium can play an important role in the increase in T cell expansion. Since the Wave bioreactor is expected to increase oxygen exchange and entry, even in a closed environment of cells, the environment is more stable than the breathable static bag that needs to be updated regularly, which can explain the higher proliferation rate. It should be noted that the control group is mostly static bags, not non-wave shake bags, nor other forms of dynamic bioreactors. These additional groups can be included to highlight the specific effect of the rocking exercise/perfusion combination on the proliferation rate. When the goal is to verify large-scale expansion with maximum biosafety, sometimes no other system is even used for control.

In addition to the number of expanded cells, the T cell population in the Wave bioreactor was analyzed based on the phenotype, focusing on the balance between CD4+ and CD8+ cells. Some people claim that the number of CD4+ cells in the Wave culture population is too high compared to static bags; or that the two groups are similar; while others emphasize how the Wave system can help monitor balance. Therefore, further research is needed, but it is important to note that compared with other systems, Wave Bioreactor has made specific efforts to analyze this aspect (although phenotypic analysis can be monitored in other studies, especially using G-Rex® flask or stirred tank bioreactor.

In addition to the direct impact of bioreactor technology on cell phenotype, the impact of exogenous reagents used in the system to provide the required components for the cell culture must also be considered. In some studies, Dynabeads were used as part of the activation process before and after inoculation in the Wave bioreactor bag. This highlights how the system can benefit from the use of culture bags while being combined with other methods and supplementary equipment. The Dynabeads used in these studies activate T cells through the combination of CD3 and TCR and CD28 receptor co-stimulation. It is known that this method will produce different ratios of CD4+ and CD8+ T cells depending on the bead dose used. Obviously, in order to further understand the cell expansion process, the effect of T cell activation needs to be separated from the effect of the bioreactor, which has many of the effects discussed earlier.

We determined above that the wave system complies with GMP, but it does not fully automate the extension procedure. In the protocol released in 2020, the addition of culture medium follows a manual procedure, samples are taken from the bioreactor and an offline cell count is performed, and then the cell count is used in conjunction with the reference table to set the culture medium volume and perfusion rate. The same reference book states that the Wave system does have built-in pH and dissolved oxygen sensors. These types of soft sensors are often used in larger-scale bioreactors for culturing cell types other than mammalian cells.

In any case, there are some limitations in using this system. Compared with the static small-scale process, many optimization steps are required, involving basic culture parameters and different technical parameters unique to the Wave bioreactor. Such optimizations can be difficult because they have various effects: for example, the sway rate changes the shear stress, but it also affects aeration and thus oxygen exchange. Compared with other systems, the initial purchase investment may be larger, but this may be different because conventional culture will be cheaper due to the reduced amount of medium (up to half of the consumption of other designs). Since the system is a combination of multiple motions and perfusions, the risk of electrical and mechanical failure to change cell behavior is more important, so the setup requires strong redundancy.


6.4. Single-system multi-step bioreactor: Miltenyi Biotec Clinimacs Prodigy

So far, we have discussed bioreactors that specifically focus on the cell expansion stage of the self-adoptive transfer of T cell products. However, many processes for developing these products are multi-stage linear processes. Before cell expansion, peripheral blood mononuclear cells need to be extracted from blood separation products; then T cells are specifically selected; then cell activation, followed by possible genetic modification. The task after expansion is to formulate the cells into a suitable buffer for biological preservation or infusion of the product, provided that it passes quality control measures (Wang and Rivière, 2016). Given that a bioreactor is a general term for systems that transform substances in a biological process, a complete bioreactor system may be able to perform multiple steps in an advanced treatment production line.

One such device is the MiltenyiBiotec Clinimacs Prodigy system. The system can perform cell selection, activation, transduction, expansion, cell harvesting and preparation in a piece of hardware and its associated disposable tube set. In the study of Mock et al., the results obtained by Prodigy are comparable to the process used in the GMP facility: Prodigy’s cell number, viability, and sterility results are neither improved than the results obtained from the comparison, and more importantly, Deteriorated process (including culture bag and Wave bioreactor). This shows that the benefit of the system lies in the automation of multiple steps in a system. Certain parts of the system are not only automated by pre-programming when performing operations, but also controlled to some extent: pressure and liquid sensors can detect when new tasks are completed; cameras can monitor the progress of cell separation during the centrifugation step ; Microscope can be used to assess the progress of cell culture. Multiple steps enclosed in one device may reduce clean room space and allow production at satellite locations, which helps realize the benefits of decentralized manufacturing processes.

The table drawn by Mock et al. shows that many of the steps that Prodigy performs still require operator interaction, so it is not a fully automated system. In addition, providing multiple functions in a single system hardware may have drawbacks. Regardless of the culture system, cell expansion is the longest stage in the production of T cell-based immunotherapy, assuming that lentiviral vectors can be effectively provided. The longer cell expansion step means that while Prodigy is running during the cell expansion phase, the centrifugation and cell separation functions remain idle. This is not ideal, considering that these therapies are currently autologous, so the device can be used to process materials for individual patients. Allowing bottlenecks in each step of advanced therapy manufacturing, single-system multi-step bioreactors may limit the throughput of the bioprocessing chain due to insufficient hardware availability. This will encourage future multi-step bioreactors based on throughput analysis of all processing chains, thereby minimizing the impact of bottlenecks.

Another significant disadvantage of this system is its limited capacity, which may limit its use in the production of multi-dose therapy and in the production of allogeneic therapy. The culture vessel is the same as the unit used for centrifugation, which suggests that this multiple use may not be the best design for cell culture.


6.5. Mixing system and self-made design

Another type of bioreactor and kinetic vessel is the rotating flask and further stirring system (Figure 7). Through mechanical or magnetic stirring, their main advantages are to provide a simple geometry to provide uniform culture conditions and excellent gas exchange, and easy to sample. There are different models on the market, and can be easily scaled up by increasing the volume of the storage tank. Perfusion can increase the cell density achieved in the stirred tank and reduce the volume required to expand cell production. However, the perfusion studied by Kropp et al. requires additional hardware to retain the pure cell stem cell aggregates in the bioreactor. The stirred tank also allows the use of REP during the culture process and is mainly used for a constant culture volume.

Figure 7. Standard design of a rotating flask with mechanical stirring

In order to optimize the controlled environment for cultivation in such systems, one of the main parameters that need to be adjusted is the rate of the stirring mechanism. The shear stress generated by the high stirring rate does endanger the beneficial effects of medium mixing on cell proliferation. For example, Foster et al. have studied the effect of rotational speed on T cell culture and Carswell and Papoutsakis, the latter combined with jet-based exposure. The speed range of up to 120 RPM in a rotating flask or 180 RPM in a large-scale (2 L) stirred bioreactor was successfully used without changing cell viability and proliferation.

In addition to speed, if needed after optimization under static conditions, many other parameters such as osmotic pressure, IL-2 concentration, pH, oxygen tension and feed schedule can be monitored or controlled in the stirring system. The effect of shear stress can be reduced by modifying the overall culture program and stirring mechanism (such as cell retention filters or magnetic stirrers). After Bohnenkamp et al. studied different feed strategies in stirred bioreactors. It shows that updating a small part of the medium every day (no more than half of the total volume) will result in a higher proliferation rate than every 2/3 days. The worst method is to completely update and remove waste and paracrine factors. Therefore, one disadvantage of agitated bioreactors may be the need for frequent intervention in media replacement, which limits the potential of a closed system. We can suggest combining a stirred vessel with medium perfusion, but as far as we know, this method has not been extensively studied for T cell expansion, although stem cells for example have been reported recently.

In fact, stirred tank bioreactors have shown very good results in the expansion of other cell lineages, especially induced pluripotent stem cells (iPSC), and immune cells can be derived in vitro. Not only can these methods pave the way for alternative in vitro expansion of immunotherapy, but T cell-based processes can also benefit from the advancement of other cell types. For example, the optimization of feeding strategies described by Kropp et al., and the study of human pluripotent stem cells can help improve the perfusion cycle and process monitoring. Although this review focuses on systems developed primarily for T cell culture, for other bioreactor designs, this cross-analysis between cell types may also be kept in mind. Interestingly, Kropp et al. confirmed the results of Bohnenkamp et al. with stem cells and showed that continuous medium renewal by perfusion produces better results (47% increase in cell yield and stem cell specific parameters) than repeated batch changes.

So far, T cell culture bioreactors have not reached any consensus, and there are many different ways to improve closed and automated systems. Although not widely reported recently, self-made and more primitive methods can be proposed as an alternative to the main categories previously introduced. A compelling proposal by Klapper et al. Use a closed system parallel plate bioreactor, consisting of two disc-shaped chambers. The culture medium and the gas chamber are separated by a gas-permeable but liquid-impermeable membrane (Figure 8), and the two phases are perfused during the culture. The medium is provided by a 7-liter reservoir for the 14-day multiplication step, thus forming a completely closed system. The flow rate is adjusted over time and between batches to keep the lactic acid concentration between 0.5 and 1 mg/mL, slow and laminar, and harmless to the cell suspension. This procedure allows the use of REP, especially IL-2 in a single bolus injection on day 0 (confirmation of closed system potential) and low serum concentrations, which is relevant for further clinical validation. We can mention here that in the past, studies on the effect of serum concentration in a stirred environment on cell development have shown conflicting results. This particular parameter can be further studied. It was found that the limitation of this method was the surface area of ​​the disc chamber, but it was confirmed that it had a higher proliferation rate than the semi-permeable bag used as a control. Novel designs can increase the scalability of bioreactors, although they still have limitations that limit their use in therapeutic manufacturing.

Bioreactor system for clinical scale expansion of T cells

Figure 8. Example of original homemade design. A disc-shaped chamber is composed of two compartments, the culture medium and the gas, separated by a gas permeable but liquid impermeable membrane. The culture medium reservoir can be perfused according to the lactic acid concentration during the entire culture time (14 days) to ensure a complete closed system (Klapper et al., 2009).



7. Discussion and outlook: Automation, especially process control, as the ultimate goal

In summary, as an overall result, most of the studied systems showed better results in terms of T cell proliferation compared to static standards; or, in the case of similar results, improved ease of use and reduced cost and labor time (Table 1). However, as mentioned earlier, the maximum cell density should not be the only parameter to be considered when assessing the progress made by the new system. So far, no consensus has been reached on the best bioreactor, and different designs are still being studied and characterized to achieve clinically relevant cell numbers faster and more reliably. One issue that highlights the best approach is to use various cell sources and controls from one study to another.

There is a lack of direct comparison between different bioreactor systems, and most of the research is only conducted between a specific system and standard, usually under static conditions. In addition, many parameters are involved and the method of calculating the expansion rate is not always clearly reported: whether to report only the proliferation rate of the entire culture process or bioreactor step is not always clear, and sometimes it is difficult to base Cell concentration is used to distinguish the absolute number of cells inoculated. It may be useful to conduct comparative studies between common geometries with the same parameters, especially seeding density, medium composition, cultivation duration, and feeding schedule, although intellectual property rights and proprietary processes and results may make such cross-analysis difficult carried out.

The quality by design (QbD) and design of experiments (DoE) methods proposed in this review will be useful tools for performing these multi-parameter studies to truly understand the design space and operating conditions in the production of autologous cell therapy. These future studies also need to determine how the cell expansion bioreactor and its new functions affect the QbD principle. In addition, since REP conditions are widely used in bioreactor methods, it may be interesting to compare the results with a control group that does not use REP (ie, a basal medium without factors that trigger T cell activation and proliferation) to study the difference between the two. Possible synergies between. REP and specific bioreactor functions. This can even highlight advanced systems where activation steps can be reduced to achieve the same extended range, thereby reducing costs and labor.

Bioreactor system for clinical scale expansion of T cells

For bioreactors that show promising results, as mentioned by Jin et al., it is necessary to quickly confirm that the expansion rate found in cell lines and primary cells from healthy donors can be reproduced from actual patient samples. obtain. In addition, most of the development studies reported here use unmodified T cells (no TCR or CAR-T process). These transduction methods should be combined to check the feasibility and improvement of the proliferation rate. Similarly, how industrial and clinical data are shared or not shared with the scientific community will change how the technology will shift to available and affordable treatment and mass manufacturing platforms.

By using a bioreactor, the parameters in the above-mentioned cell culture process can be better controlled. However, due to the complex T cell biology, the influence of the parameters used in T cell expansion on the resulting cell population is not always clear. Therefore, we recommend adopting a design of experiment (DoE) approach from the initial design and deployment of the bioreactor to the biological process. This method will allow to identify all the parameters that affect the performance of the bioreactor and understand how to change the parameters to affect the output through the effective use of experimental runs. In addition, it is clear that bioreactors can affect many quality attributes of therapeutic cells. This multi-attribute impact requires an understanding of the quality of design (QbD). Considering that cell products will have more than one factor to determine their success as a therapeutic agent, this should be considered from the initial design stage. This approach will expand the bioreactor research to include all the therapeutic quality aspects of the cells produced by the bioreactor, not just the ease with which the reactor produces a large number of cells.

In addition to increasing proliferation, for various bioreactors, the ultimate goal is usually to design a completely closed system to comply with GMP, but it is also moving towards a fully automated system. For example, it can be used as an optional module of the Wave bioreactor to control medium exchange. Due to multiple interventions, automated equipment reduces labor and pollution risks, but it is also the best way to obtain standard results with batch-to-batch reliability. It is indeed recommended to use the standardized conditions of the bioreactor to limit batch variance.

However, the automation of pre-programmed tasks does not allow the system to respond to deviations. Improved systems can be produced by introducing analytical techniques and process control methods. The technical bottleneck is the development of non-invasive and real-time analysis methods for cell culture parameters such as cell concentration, pH, lactic acid concentration, or oxygen consumption. These methods can be embedded in a humid 37°C atmosphere. Al et al. showed that there are many promising methods for online monitoring of the quality attributes of cell therapy, but there are the following challenges: integrating the sensor into the bioreactor, converting the technology to be suitable for one-time use; and some require complex Analysis can only return the method of interest from the surrogate metric.

The measurement will be used to automatically adjust technical parameters such as rocking motion, speed, flow rate, medium renewal or injection in the case of volume increase. This keeps the therapeutic production bioreactor system away from automation. By pre-programming the tasks, it is currently the norm established through the review of the above-mentioned system, turning to closed-loop control, which is a more powerful engineering pillar. Sensors and actuators should not significantly increase costs or reduce safety, thereby jeopardizing the advantages of bioreactors in terms of proliferation rate and GMP compliance.

With a suitable method of detecting quality attributes, there is a challenge to implement a method of providing feedback so that the cost of monitoring equipment can be offset by improved processing. A cost-benefit analysis of online sensing and control feedback in the autologous therapy environment is needed to understand whether these methods can yield valuable improvements on the scale of autologous therapy manufacturing.


When cells form part of the treatment, batch-to-batch variability is a known issue. As can be seen from our comments above, the current generation of bioreactors can stabilize parameters and automatically set the same parameters for each batch of expanded cells. Tasks are performed automatically in the program, regardless of the exact design used. However, many of them lack closed-loop control in response to continuous cell expansion, and the rate of progress between batches may vary greatly, which has been widely used in the bioreactor research cited in this article. Report. The reason for the lack of control is related to the lack of sensors and established methods to perform online sampling of the cell expansion process.

As proposed by Klapper et al., a closed system can be promoted by using reservoir perfusion with sufficient medium throughout the cultivation process. In a homemade design. Perfusion has been used in hollow fiber bioreactors. Although additional steps are required to optimize the flow rate, this can be combined with other systems (such as G-Rex® flasks or stirred tanks) to monitor better media volume and shear stress levels . Interestingly, the lack of research comparing the effects of regular and continuous perfusion in T cell expansion bioreactors can encourage such analysis to obtain the best solution.

In order to move further into the clinic, it is also important to consider additional steps, such as how to change the output and viability of the bioreactor when using cryopreserved cells. Additional recovery periods may have to be included in the training plan. In addition, in order to achieve very high throughput, bioreactors can be used for continuous culture, as proposed by Knazek et al. In the hollow fiber bioreactor. When harvesting expanded T cells, a portion of the cell population will remain in the system for re-expansion from the same cell source. Further research on this method will help develop multi-cycle bioreactors and emergency secondary cells for patients.






the optimal cell density and proliferation rate are not the only goals to ensure the transformation process: automation, process control and a complete closed system are still unachieved final goals, but if you consider from the early steps of development, it can be as before.

The gradual implementation conforms to GMP and REP, and is now widely used.  Proposed process engineering methods to help overcome the challenges of implementing new generation bioreactor technology.



(source:internet, reference only)

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