Novel Cryopreservation For Cell And Gene Therapies
By Gamid Abatchev, X-Therma
The concept of cooling organic materials in order to slow their decay is as old as civilization itself. For the preservation of food, it is as simple as freezing with ice to prevent bacterial or fungal growth; at worst, what occurs at both the tissue and cellular levels results in texture and consistency changes, altering the taste of what was frozen but not impacting its safety.
Yet what occurs at the cellular level to cause this shift is ultimately highly destructive – the formation of ice crystals which can rupture and tear cells apart. This ice damage, while less concerning in food, is catastrophic for cell therapies. This requires freezing solutions to prevent ice formation, allowing for the retention of cells’ therapeutic functionality. For these modalities, as well as any tissue preserved for medical use, the need to preserve them in ways which allow them to revert to healthy cells after preservation has created complexity for the drug developers pursuing these therapies to treat rare and often intractable diseases.
The industry standard for decades has been the cryopreservation agent dimethyl sulfoxide (DMSO), a solvent with a demonstrated ability to curb ice formation. Yet the use of DMSO has been a calculated risk – toxicity and a propensity for inducing chromosomal damage at the cellular level, combined with suboptimal pre-freeze and post-thaw incubation times, has been linked to its usage. This has resulted in a delicate balancing act, with drug developers working to incorporate enough DMSO to prevent ice formation, while simultaneously minimizing its use to avoid the detrimental off target effects it presents.
As “living treatments” continue to serve an increasingly significant role in medicine, finding cryopreservation approaches that offer an alternative to toxic, suboptimal solvents is critical to improving their commercial and therapeutic potential. Non-toxic alternatives to DMSO have shown promise in the lab, particularly products that mimic the natural antifreeze proteins (AFPs) found in certain species dodging the effects of freezing temperatures.
DMSO Alternatives Inspired by Nature
The long incumbency of DMSO in the advanced therapy space, despite its dangers, can be chalked up to a lack of long-ranging studies on its detrimental effects, as well as the rapid pace at which cell therapies requiring preservation have been developed. As the number of advanced therapies in the pipeline continues to increase, the burning need for safer cryoprotectants that can be used in manufacturing and larger quantities has begun to spur innovation. Though DMSO is an effective cryoprotectant, the extent of its toxicity and genotoxicity is still not fully understood, and finding alternatives that better protect human health will only serve to improve the safety profile of the drugs entering today’s market.
To address the issues surrounding the use of DMSO, some have begun investigating the potential of AFPs as a cryoprotectant for advanced therapies. These proteins, which are found in several species adapted for cold weather conditions, work by binding to ice crystals, arresting their growth and preventing the formation of the shapes that can tear through a cell’s membranes and organelles. While AFPs have been adapted for use in the laboratory with relative success at smaller scales, a primary challenge inherent to furthering AFPs is scalability. For both naturally simulating and biomimetic peptides, the amount of biomass required to produce enough protein necessitates an economically infeasible, low-yield process. Protein-based products, much like serums, are also highly complex and difficult to chemically define, resulting in a “black box” solution that can be difficult to replicate, particularly to regulatory standards.
Additionally, while synthesizing a chemical process in the lab to produce AFPs may be relatively straightforward, scaling is more complex, as the stability of an AFP’s individual components is likely to decrease. Finally, the organic nature of AFPs is likely to induce immunogenicity in patients, creating an unwanted immune response for the final product.
The Promise of Peptoids for Cryopreservation
The challenges inherent to furthering AFPs has resulted in the pursuit of peptide-mimetics that replicate their antifreeze properties while retaining scalability and reproducibility. These “peptoids” possess a highly similar chemical structure to AFPs, with a few key differences. The central one is a chemical shift of the location of the functional group on the backbone of the peptoid when compared to its natural counterpart. This chemical change plays a major role in improving the peptoid’s favorability with respect to scalability, stability, and biocompatibility.
This also likely plays a role in a peptoid’s decreased immunogenicity, with the altered molecular structure and shape rendering it less recognizable to the body as a foreign agent. The shift in chemical structure also makes peptoids less prone to protease digestion once inside the body, further improving its biocompatibility. The molecular structure of these peptoids permits greater versatility, allowing them to cross cell membranes without any observable toxicity or changes to the cell or tissue. Products such as XT-Thrive®, a bio-inspired, chemically defined, peptoid-based cryoprotectant, have demonstrated this lack of toxicity and simplicity of use, with similar recovery of cryopreserved stem cells observed when compared to DMSO.
The potential detrimental effects of DMSO become more apparent and significant for drug and cell therapy developers concerned with its impact on a final product, making alternatives such as XT-Thrive very appealing and likely to gain greater traction in the market. Importantly, XT-Thrive can be integrated seamlessly into every phase of scale-up, without the same timing constraints that accompany the use of DMSO. Furthermore, its lack of toxicity makes it an integral alternative for applications such as skin grafts, for which it has been demonstrated as an effective cryoprotectant. Peptoid-based cryoprotectants also hold potential for larger tissue preservation – for applications such as IVF, for example, the capacity to cryopreserve eggs or zygotes with additives that are far less likely to induce cellular mutations is a crucial consideration.
Conclusion
Ultimately, the concerns surrounding the use of DMSO as a cryoprotectant for advanced therapies are many. They often include the potential for post-thaw toxicity, in-process toxicity during manufacturing, adverse patient side effects, poor translation from R&D to biomedical applications of procedures involving cryopreservation, mutations that impact the target effects of a therapy, and resulting bottlenecks during upscale in manufacturing. Larger, more comprehensive retrospective and prospective trials are needed to more fully understand the impacts of DMSO for patients; in particular, the burden of tracking the genotoxicity of DMSO has meant that its impact on health at the genetic level is only sparsely documented. Historically, the benefits of DMSO have outweighed the known risks, but that paradigm is changing rapidly, as the number of cell therapies going into human patients increases alongside concerns of exposing sick patients to potentially toxic DMSO.
Peptoids, in formulation and with various freezing processes, have been demonstrated to inhibit intracellular ice crystal formation in freezing temperatures, down to roughly -200° C. This efficacy, coupled with a lack of toxicity and immunogenicity, position peptoid-based products such as XT-Thrive to offer drug developers a safer alternative to DMSO for fragile, crucial advanced therapies. Cheaper and more reliable than extracting and scaling up naturally occurring AFPs, and safer and potentially more efficacious than DMSO, these products may serve to solve challenges at every step of the cryopreservation process.
About the Author
Gamid Abatchev, Ph.D., Research Scientist at X-Therma Inc, focusing on formulation research and development. Currently working at X-Therma to optimize formulations for cryopreservation and subfreezing platforms applicable to cell, tissue, and organ biopreservation. As a Ph.D researcher, defended dissertation in the Biomolecular Sciences program at Boise State University in the lab of Dr. Daniel Fologea, analyzing liposome formulation for controlled drug release, delivery, and novel methods for their production. Undergraduate studies were pursued in biochemistry and molecular biology at the University of California, Davis.