Challenges to Cell Expansion for Allogenic Cell Therapies

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Allogenic cell therapies produced in large batches from healthy donor cells have the potential to address many of the logistics and manufacturing challenges associated with autologous cell therapies. They face their own production hurdles, however, including the need to generate large homogeneous cell populations while maintaining functionality and avoiding phenotypic variability. Several strategies are being explored to address these issues while also ensuring the development of cost-effective processes and streamlined development and manufacturing timelines.

Several general hurdles

All allogeneic cell therapies, regardless of the cell type and whether they are genetically modified or not, face some fundamental challenges. These challenges include sourcing of sufficient, suitable cells, access to fit-for-purpose reagents and manufacturing systems, and scalability of upstream, downstream, fill/finish, and cryopreservation processes.

Unlike autologous therapies, which use cells from the patient being treated, allogeneic therapies are manufactured in large batches from unrelated healthy donor tissues such as bone marrow or lymphocytes, including natural killer (NK) cells and T cells. “This approach makes allogeneic therapies more attractive, but introduces new complexities related to sourcing and handling of cells from different donors​ that can trigger immune responses in the recipient’s body,” notes Nikhil Tyagi, director of cell therapy process development at Center for Breakthrough Medicines. He adds that cells from different donors will have different propensities for expansion and therefore yields.

Cell-therapy developers also must work with technologies for large-scale manufacturing that are not yet fully optimized for the development and manufacturing of allogeneic products, notes Tatiana Nanda, head of process development–cell therapy, drug product and process analytics at Center for Breakthrough Medicines. “Innovations in areas such as cell culture media and manufacturing equipment are required to increase the efficiency, safety, and yield of the process,” she contends. In addition, Nanda believes that optimization of culture conditions, culture media, growth factors, and implementation of automated manufacturing processes are necessary to address this challenge.

Scaling up cell production, particularly for adherent cells, presents another challenge. “Current expansion methods often rely on labor-intensive and manual processes that are not easily scalable. Developing robust and efficient manufacturing processes that can produce sufficient quantities of cells is a critical requirement in today’s world,” Tyagi says.

Even in cases where large-scale allo cell expansion is achievable, there are still limitations with respect to efficient harvest of high numbers of cells, according to Nanda. She also notes the need for ultra-fast fill/finish, labeling, and visual inspection processes which frequently are limited to a 30–180-minute time window. “High-throughput fill/finish and automated visual inspection capabilities can resolve these bottlenecks,” she says. Custom solutions for large-scale, robotics-enabled cryopreservation also help with cryopreserving large batches of allo drug products.

Donor material properties crucial

The quality and composition/variability of the donated cellular starting material are of paramount importance in allogeneic cell therapy. “These factors can significantly impact the success or failure of large-scale cell expansion,” states Tyagi.

The quality of donated cells directly impacts the safety and efficacy of allogeneic cell therapies, as poor-quality cells may not function as intended, leading to suboptimal therapeutic outcomes. In addition, Tyagi observes that improperly screened cells could potentially transmit infectious diseases.

The composition of the cellular material, meanwhile, can significantly influence the therapeutic effect, because different cell types have different roles and functions. “For instance,” Tyagi says, “the presence or absence of certain cell types, such as T cells in hematopoietic stem cell transplantation, can contribute to complications or beneficial effects.” Inter- and intra-donor variability, meanwhile, can affect the consistency of allogenic cell therapies.

The methods used to process cells (isolate, purify, and store) after donation can also affect their quality and function, including their viability and therapeutic potential, according to Tyagi. He stresses that standardized, validated cell-processing methods are essential to ensure the consistency and effectiveness of allogeneic cell therapies.

Finally, Tyagi notes that the impact of the quality and composition/variability of the starting material can vary depending on the cell type, the need for genetic modification, and the ultimate application of the cell therapy. “Certain cell types may be more sensitive to the quality of the starting material or more prone to variability. Similarly, the need for genetic modification can introduce additional complexity and potential variability,” he explains.

Finally, the ultimate application of the cell therapy can also influence the importance of the quality and composition/variability of the starting material, according to Tyagi. For example, therapies intended for life-threatening conditions may have stricter requirements for the quality and consistency of the starting material.

Ensuring homogenous expanded cell populations

Several strategies are used to ensure that expanded cell populations have an appropriate level of homogeneity. Given the variability in donor cells used as starting materials for cell therapy, cell sorting is widely used because it is an essential technique for isolating specific cell types from a heterogeneous population, according to Tyagi. “By relying on specific cell-surface markers, these techniques, such as fluorescence-activated and magnetic-activated cell sorting (FACS and MACS, respectively), allow for the identification and separation of desired cell populations, ensuring the production of homogeneous cell populations for downstream applications,” he says.

Genetic modification is another approach that enables the engineering of cells to express specific markers or proteins. This technique, Tyagi observes, facilitates the identification and isolation of target cell types, enhancing the homogeneity of the population. Genetic modifications can also improve cell functions and properties for specific applications, further optimizing their suitability for desired therapeutic or research purposes, he adds.

Manufacturing approaches impact the homogeneity of expanded cell populations as well. The composition of the culture medium, the choice of substrate for cell attachment in adherent processes, and physical process parameters during cell culture, such as temperature and dissolved oxygen content, can influence cell behavior and differentiation, according to Tyagi. “By carefully controlling these factors, researchers can create an optimal environment that promotes the growth and differentiation of desired cell types, which helps to maintain the homogeneity of the cell population and ensures consistent and reproducible results,” he contends.

Use of microfluidic devices, meanwhile, provides a means for gaining precise control over cell isolation and culture conditions, Tyagi comments. They can, for instance, isolate specific cell types based on physical properties such as size and deformability. “By culturing cells within microscale environments, these devices provide tight control over culture conditions, enhancing the homogeneity of the cell population,” he says.

It is also worth noting that different combinations of techniques are used to ensure homogeneity after expansion of specific types of cells. For mesenchymal stem cells (MSCs), according to Tyagi, culture conditions play a significant role in maintaining stemness and preventing differentiation. For T cells, genetic modification combined with cell sorting techniques has been a key strategy, particularly for chimeric antigen receptor (CAR) T-cell therapies. Cell sorting is also crucial for isolating hematopoietic stem cells (HSCs). For dendritic cells, a combination of culture condition manipulation and genetic modification is often employed, while for natural killer cells, all three approaches (cell sorting, control of culture conditions, genetic modification) contribute to more homogeneous expanded cell populations.

Maintaining cellular integrity and functionality

Expanded cell populations must not only be homogeneous, but have a high percentage of viable cells with the desired functionality. Many different factors can influence the ability to maintain these attributes during expansion. First, says Tyagi, is the cell source and type. For instance, pluripotent stem cells (PSCs) require a different set of culture conditions compared to somatic cells like T cells or MSCs. “PSCs need a feeder layer or feeder-free conditions with specific growth factors, while T cells and MSCs can be expanded using media supplemented with serum or serum-free conditions,” he explains.

For genetically modified cells, optimization of the mechanism for delivery of the genetic material (e.g., viral vector, electroporation, etc.) is essential, as these methods can impact both cell viability and functionality, according to Nanda. As an example, she points to CAR T-cell therapy production, which involves T-cell activation, genetic modification to express the CAR, and then expansion of the CAR-T cells.

Not surprisingly, the choice of expansion system can also affect cell integrity and functionality. Traditional planar systems, Nanda notes, have limitations in terms of scalability, so suspension culture systems and bioreactors are being explored for large-scale production. She adds that these systems also allow for better control of culture conditions.

As with any drug manufacturing process, quality control (QC) throughout the cell expansion process is a must. “QC measures, including monitoring of cell identity, purity, potency, and safety, are essential to ensure that cells intended for use as cell therapies maintain their desired characteristics,” Nanda says. Advanced techniques like flow cytometry, next-generation sequencing, and functional assays are used for these purposes.

Finally, the use of automation helps to reduce variability and increase reproducibility, while closed systems minimize the risk of contamination. After expansion, appropriate cryopreservation and thawing processes must be established and carefully managed to maintain both cell viability and functionality.

Avoiding differentiated phenotypes

During expansion, it is possible for some cells to undergo changes in their phenotype. For cell therapies, it is essential that all cells in the expanded population have the desired phenotypes. It is therefore necessary to take steps to avoid differentiation. There are several strategies employed to achieve this goal, according to Tyagi, and the specific strategy employed can depend on the cell type, the need for genetic modification, and the ultimate application of the cells.

Optimized culture conditions with cell-specific culture media, growth factors, and cytokines can help maintain the desired cell phenotype during longer expansion periods, Tyagi says. Use of bioreactors, for instance, allows better control of process parameters such as oxygen levels, pH, and nutrient supply, which can influence cell differentiation. Hypoxic conditions, meanwhile, can activate certain signaling pathways that promote stemness and inhibit differentiation. Cells can also be genetically modified to alter specific genes that control cell differentiation to increase the stability of the desired phenotype. Co-culturing stem cells with other types of cells can help maintain their stemness, while 3D culture systems provide a more in vivo-like environment for cells, including favorable cell-cell and cell-matrix interactions.

Assuring aseptic conditions

Maintaining aseptic conditions during longer cell expansion processes poses challenges, but several strategies are being employed to address them. Top of the list, according to Nanda, are the use of automated and closed systems with stringent quality control measures in place. Operator training, facility design, and the use of disposable production systems are also important.

Automated systems, Nanda says, control and monitor culture conditions in real-time, allowing for adjustments in parameters such as temperature, pH, and nutrient supply to ensure optimal growth conditions for the cells. They also reduce manual handling, minimizing the risk of contamination. Closed systems also reduce the potential for contamination by isolating the the cell culture from the external environment, as do stringent quality control measures, including regular testing. Proper QC measures also ensure monitoring and control of relevant process parameters, regular inspections of equipment and materials, and performance of thorough QC and release testing at various stages of the cell expansion process.

Manufacturing technology advanced, but improvements needed

The current state of manufacturing technology for cell therapy expansion is generally well-advanced, but according to Tyagi there are many areas where improvements would be beneficial. Many different bioreactors are available, including stirred-tank, rocking-motion, hollow-fiber, and fixed-bed systems, with each having its own set of advantages and disadvantages. As a result, Tyagi notes the choice of bioreactor can significantly impact the quality and quantity of cells produced. He adds that suppliers are striving to improve bioreactor designs to unlock the full potential of cell therapy applications. Tyagi points to fixed-bed bioreactors as a promising technology for scaling adherent cell therapies with higher yields, but because they are completely closed, direct observation and monitoring are challenges.

Cell harvesting and separation from the growth medium after expansion is typically achieved using centrifugation or filtration methods. The current state of cell harvesting and separation technology is fairly advanced, but Tyagi underscores the existence of challenges related to maintaining cell viability and yield during this process. For instance, while gentle and optimized separation techniques such as density-gradient centrifugation are often used to separate cells from growth medium based on their size and density, process parameters such as rotor speed, separation media, and flow rates typically must be optimized for specific cell types. In addition, while the process is suitable for large cell-culture volumes, high shear forces can damage cells, and the process can be lengthy and require significant energy input, according to Tyagi.

Equipment for cell processing (washing, concentrating, and resuspending) and formulation (addition of additives or excipients) is reasonably advanced, according to Tyagi, but these processes can also create challenges with respect to maintaining cell viability and functionality. For instance, closed and automated cell washing and centrifugation-based technologies can efficiently handle large volumes of cells while maintaining their integrity but can be expensive, require skilled operators, are not suitable for all cell types or very large-scale operations, and there are only a limited number of suppliers offering this type of specialized equipment.

With respect to quality control for allogeneic cell therapy expansion processes, there is a need for more rapid and comprehensive testing methods, according to Nanda. She notes that traditional microscopy and cell counting can be time-consuming and may not capture all relevant information about the cells, while real-time monitoring systems, such as in-line sensors or imaging technologies, can provide valuable data for process optimization and product characterization come with considerations like high cost and technical complexity.

Finally, integration and validation of automated and closed systems for cell-therapy manufacturing can be challenging. In addition, while automated cell culture systems can control all aspects of the cell culture process, from seeding cells to harvesting and processing them, Nanda comments that they can be expensive to implement and maintain, and may require significant customization depending on the specific cell-therapy product being manufactured.

About the author

Cynthia Challener, PhD is a contributing editor for Pharmaceutical Technology Group.

Article details

BioPharm International

Vol. 36, No. 8

August 2023

Pages: 22–25

Citation

When referring to this article, please cite it as Challener, C. Challenges to Cell Expansion for Allogenic Cell Therapies. BioPharm International, 2023, 36 (8) 22–25.