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Buffers, stabilizers, and cryoprotectants play major roles in cell therapy formulations.
While many stem-cell therapies remain at the preclinical stage, several are progressing through the clinic toward late-stage trials. Developing successful treatments based on induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) in part depends on creating effective formulations in which cell viability is maintained during cryopreservation, storage and handling, shipment, and reconstitution prior to delivery to the patient. Excipients, therefore, play an important role in bringing novel cell therapies to the market.
As with most biologic drug products, the predominant excipient in cell therapy formulations is the buffered saline solution. In this case, the saline solution is an osmotically balanced water solution designed to keep the cells from drying out and dying, according to Ryan Guest, senior CMC translation consultant for eXmoor pharma.
In addition to physiological saline, Carole Nicco, CSO at BioSenic notes that other isotonic solutions containing chemical substances present in blood, such as Ringer’s lactate, are also used to help maintain the electrolyte balance and keep the cells stable, sterile, and viable with proliferative capacity until their application. Furthermore, these isotonic solutions offer chemical easy systemic and local application, according to Nicco.
Classically, most formulations will also have protein to balance the solution as a buffer or reservoir for smaller molecules and enable transport via active protein transporters on cell membranes, Guest notes. The standard is an albumin (0.5–2.5%), such as human serum albumin. Albumin constitutes 50–75% of the colloidal osmotic pressure of blood (1–3) and performs a very similar role in MSC and iPSC formulations, Guest says. More specifically, albumin is a useful component in cell therapy formulations for balancing water availability during product hold times, cryopreservation, and thawing prior to product administration.
In fact, Guest observes that the presence of an equivalent protein in cell therapy formulations is predicted to have a significant impact on cell survival. In MSCs, for instance, the absence of an appropriate protein can reduce viable recovery by 20–40%, he comments.
The other important excipients for cell therapy products are cryoprotectants, given that the majority of these treatments are cropreserved. The cryopreservation of MSC- and iPSC-based therapies using 2–10% dimethyl sulfoxide (DMSO) in solutions containing a high content of serum is a common procedure, Nicco observes. She adds that while DMSO has been used to reduce the formation of ice in cells stored in liquid nitrogen since 1959, it is toxic, resulting in undesired clinical and biological side effects. Serum also introduces variation and safety risks.
“There is, consequently,” Nicco says, “a move toward solutions containing bio-preservation media free of DMSO, serum, and other proteins, optimized for the preservation and distribution of these products at low temperatures, either in cold (2–8 °C) or cryopreserved conditions (-70 °C to -196 °C).” The new excipients, she explains, eliminate toxicity issues as well as the need for human product excipients (serums, proteins) that can induce a risk of contamination to the drug product. Complex formulations involving dextran-40, lactobionate, sucrose, mannitol, glucose, adenosine, and/or glutathione are examples, according to Guest.
The type of stem cell therapy generally does not impact the choice of excipients, although Guest does note that some iPSCs can be more acutely sensitive to the final formulation, hold times, and routes of administration. For example, anucleated products, specifically designed to deliver payloads, have a reduced capability to produce proteins or perform cellular repair. Nicco adds that iPSCs and cells differentiated from them are commonly multicellular systems, which she says also makes them more sensitive to the stresses of freezing and thawing than single cells.
Overall, however, similar types of excipients are used for the preservation of cell-based therapies, regardless of the cell type or method of manipulation, according to Nicco. “It is the route of administration, the need for transport, and the storage temperature that influence the choice of excipients,” she states.
Special applications, for instance, such as therapies for artificial skins and wound healing may have specific properties or additional ingredients for sealing the wounds, Guest notes. Hence, the excipients or scaffolds are part of these therapies. Dosage size is another important factor, he adds. “Larger doses need to take into account any toxic side effects of the excipients, such as DMSO, and may place maximal limits upon the total volume of excipients to be administered,” he observes.
One of the most critical issues for cell therapy manufacturers is the maintenance of the cells in an appropriate medium/excipient from the end of the culture until the time of administration to the patient. “Not only should the medium keep the cells viable with their properties intact, but the route of administration must also be taken into account,” Nicco states. She adds that a good excipient “is solvent-free and suitable for fresh or frozen suspensions of living cells formulated in a proprietary formulation adapted to the route of administration, such as intravenous, intra-articular, or intracranial.”
In addition to understanding the delivery mechanism, Guest stresses the importance of considering the container and dose when choosing excipients. The next important factor, he says, is whether the cell therapy product will be supplied frozen, as that will require selection of a cryoprotectant. Formulation development experiments should then be designed carefully to optimize potency while taking into consideration hold periods and freeze/thaw steps and eliminating or otherwise minimizing the use of diluents and wash steps. In addition, Guest recommends evaluating existing administration formulations, containers, equipment, and protocols to minimize the need for new or changes to clinical practices.
An important component of any formulation strategy is consideration of the raw materials.For excipients intended for use in cell therapy products, Guest emphasizes the need to identify available good manufacturing practice (GMP)-suitable sources with acceptable lot-to-lot controls that will enable a reproducible product formulation. It is also critical, he says, to confirm the material is suitable for the manufacturing process and will be available to meet manufacturing demand for the cell therapy throughout its product life cycle.
Equally important, Guest observes, is to ensure the availability of suitable cellular material and drug product for designing formulation experiments. Understanding the patient group and the intended administration route are essential, meanwhile, for identifying potential side effects of any excipients.
Compendial excipients are generally preferred, Guest continues, due to the risk that novel or untested solutions will require significant development to ensure the material does not impact upon the therapeutic benefit of the cellular material. In addition, all materials including excipients must be suitable for the intended route of administration and present minimal risk of inducing toxicity, immune reactions, or the transfer of adventitious agents.
Controlling cellular material from the point of “optimal” donor selection to manufacturing under GMP conditions, commercialization, and application is critical for ensuring the quality, safety, and efficacy of the final drug product. While MSCs used as starting materials for production of advanced therapy medicinal products can only be isolated in authorized centers using globally standardized processes, the optimal conditions for culturing isolated MSCs are not standardized. That poses a major challenge in controlling their quality and therapeutic properties, Nicco contends.
It is, therefore, essential to control the cell sources (e.g., bone marrow, umbilical cord, and adipose tissue), cell density in culture, duration of culture, and cell engineering and composition of culture media and implement in-process quality controls that ensure cell efficacy and safety at all stages of manual and automated manufacturing processes, including cryopreservation, use of cell banks, and transport systems. “Ultimately, the stability and quality of the cell therapy product and the excipients it contains must always be evaluated against reference samples submitted to the same storage facility,” emphasizes Nicco.
Complicating this situation is the fact that it can be difficult to find reliable mode-of-action potency methods for cell-based therapies, according to Guest. “Often both in vitro cell culture and in vivo models are required to confirm the suitability of the formulation,” he says.
In addition, formulation strategy, hold times, cryopreservation, and thawing are intrinsically linked, and it can be difficult to determine the impact of each component of the formulation, Guest observes. “Doing so requires carefully designed experiments and suitable analytical methods that assess not only the impact of formulation changes and freezing and thawing processes, but downstream biological mechanisms such as apoptosis and necrosis, which require time in a suitable cell-culture facility or in vivo models to optimize formulations,” he explains.
“A further difficulty rests with the fact that viability determinations are often tied to the analytical methods, the technicians performing them, and whether biological mechanisms of apoptosis and true cell recoveries are accounted for in the testing strategy,” Guest comments. Therefore, it is important for cell therapy developers relying on external testing laboratories to ensure those outsourcing partners have the proper understanding, expertise, and capabilities needed for appropriate and comprehensive testing.
The cell therapy field is expanding at a rapid rate, and technology is advancing to support the increasing breadth of treatments under development. That includes formulation science and the development of enhanced excipient solutions. Cryoprotectants developed through biomimicry of natural antifreeze proteins to replace DMSO and serum protein-based media are one example highlighted by Nicco.
More work is to be done in this area, however. “There is a clear unmet need for the discovery and development of novel cryoprotectants that can either replace or reduce the required amounts of current gold standards formulated to protect and treat challenging sample types such as MSCs and, even more, iPSC multicellular systems. This multivariate problem is complex, with multiple mechanisms of damage to be addressed and subtle differences between cell types and freezing methods. Combining high-throughput testing with iterative computational algorithms is key to optimizing protocols and excipient formulations to preserve emerging cell-based therapies,” Nicco comments.
Guest, meanwhile, predicts that as the cell therapy field advances, clinical practice will naturally standardize formulation, delivery, and routes of administration through product safety and efficacy data. “That will include the use of animal-free or recombinant excipients and other fit-for-purpose additives developed through the use of artificial intelligence to minimize cytotoxicity while improving stability and hold times,” he states.
Nicco is also confident that the combination of new technologies such as intelligent library design, computational modeling, rapid screening assays, and advances in genomics will lead to a better understanding of the structure-function relationships between drug and excipient. “That greater understanding will lead to more effective and efficient excipients that afford higher-performing cell therapies and ultimately benefit both cell therapy developers and patients,” she concludes.
Cynthia A. Challener, PhD, is a contributing editor to BioPharm International®.
Vol. 37, No. 2
When referring to this article, please cite it as Challener, C.A. Choosing the Right Excipients for MSC and iPSC Therapies. BioPharm International 2024 37 (1).