Cryopreservation (II): Cutting edge solutions

• Academic cryopreservation processes are unsuitable for scale-up and can result in quality issues downstream, as evidenced by commercially approved cell therapies. Pluristyx’s cryo-solutions eliminate the variability associated with ‘home-brew’ media and can be used for development at the bench with a path to the bedside.

Introduction to Cryopreservation

Cryopreservation, involving the preservation of cells and maintenance of biological states through suspension in a cryo-media at frozen temperatures, is today the bio-preservation technique of choice for advanced therapy supply chains (Jang et al., 2017, Rafiq et al., 2017). Compared to alternatives such as short term hypothermic preservation, cryopreservation offers extended hold time, logistical flexibility, cost-effectiveness, and high cell recovery rates when the freezing process is properly optimized and employed. The design of the  cryopreservation process is critical to the success of therapeutic candidates, with the speed of freezing and thawing, the composition of the cryo-media and the type of cell being preserved all being key determinants of end cell viability and product efficacy in clinical trials (Cottle et al, 2022). Critical quality attributes, such as cell viability, need to be carefully maintained throughout the process to maximize cell integrity and minimize any loss of product efficacy. Whilst fresh infusion presents logistical challenges that complicate patient administration, properly frozen cell therapy products exhibit comparable performance as fresh cells  (Brezinger-Dayan et al., 2022) and offer significant cost advantages in logistics, storage and transportation.

Kymriah: A case study

Preserving volumes larger than >100 mL of adult stem cells and mammalian tissue culture cells using cryobags is a well-established practice. Cryobags are broadly used to store large batches of cell, gene, and tissue therapies for cryopreservation (Massie et al, 2014). Nonetheless, scaling up to larger volumes poses unique challenges during the development process. The complexities of managing heat transfer issues and controlling ice nucleation become more pronounced in large-scale GMP cryopreservation. Longstanding issues with Novartis’ approved autologous CAR-T cell therapy Kymriah highlights some of the challenges for therapeutic developers in building scaled, sterile cryo-processes. The FDA reported that Novartis had identified approximately 100 possible contamination instances in Kymriah’s manufacturing process. Novartis believes these foreign particulates are most likely due to their use of cryobags (Kansteiner, 2023). This culminated in the FDA handing Novartis a Form 482 for the Morris Plains, New Jersey, manufacturing site, detailing “significant” deviations in Kyrmiah production. Since launch, Kymriah has suffered from a 10% rate of out-of-specification issues or manufacturing failures, bringing the overall cell viability profile down to 70%, 10% below the 80% commercial product specification (Novartis Kymriah® Demonstrates Consistent Efficacy and Safety Outcomes in US Patients When Used in Real-World Setting, 2019).

Challenges with cryomedia

Avoiding out-of-specification incidences, such as those that occur with Kymriah, is the key to realizing the potential of cell therapy products.  One key constraint in the market is the high cost and lack of scalability seen with current commercially available GMP media.  As a result, early-stage therapeutic investigators do not engage in robust cryopreservation optimization activities and approximately 50% opt for home-brew solutions that are ‘Fit-for-Purpose’ to a specific indication.  These ‘Fit-for-Purpose’ solutions are not easily transitioned between products and often yield inconsistent results when products are scaled for commercialization.  As a result, the cell therapy industry is facing a crisis of sub-optimal cryopreservation processes which can result in incidences of out-of-specification, high batch-to-batch variability, and reduced potency out of freeze. It is crucial to optimize the cryopreservation process to reduce cell death and variability (CCRM, 2020). The availability of high-quality, low-cost cryomedia with a path to the clinic would allow groups to engage in robust process development early in commercialization, potentially avoiding out-of-specification occurrences and improving the ability to deliver cell therapies to patients in need. 

A further key constraint in cryo-development is a focus on a ‘one-size-fits all’ solutions, without optimization or sufficient development to tailor workflows for a given therapeutic. Studies have highlighted the impact of varied conditions, including changes in cryoprotectant agent concentration, cell density, incubation time points, delivery methods and cell concentrations, in affecting the post-thaw viability of cells (Chen & Thibeault, 2013). And whilst DMSO-free approaches are currently under investigation as potential options to reduce cell death out of freeze, such solutions are yet to demonstrate comparable potency and viability and DMSO remains the preferred cryoprotectant for commercially approved products (Abazari, 2019).  Further studies have highlighted the need for different cryopreservation approaches for different cell types and configurations, given the different underlying biochemical pathways (Murray & Gibson, 2022). Whilst the creation of completely custom, de-novo solutions can add cost, complexity and delays in early-stage processes, being able to vary at least the choice of cryoprotectant and concentration enables sufficient process flexibility (Siddiqui et al., 2016).

Pluristyx’s solutions:

With rapid growth in the allogeneic cell therapy market, the need for improved cryopreservation solutions that can accommodate large-scale applications is critical for the success of this modality. Pluristyx is pleased to offer our customers PluriPrep™ and PluriFreeze™, innovative cryo-media solutions designed for iPSC and stem cell therapy development. PluriPrep™ provides a versatile base media for cell washing and hypothermic holding, while PluriFreeze™ extends this formulation with the addition of 10% DMSO to ensure high viability and long-term stability of cells post-thaw. These products offer the flexibility to tailor the DMSO concentration to specific product needs, facilitating the development of personalized and cost-effective cryomedia strategies, with custom formulations designed to support cellular metabolism during the freezing process and reduce cell stress. PluriPrep™ and PluriFreeze™ allow flexibility for formulating a final product that can have an increased cryoprotectant that can be customized to the needs of the cell.

In addition to PluriPrep™ and PluriFreeze™,Pluristyx offers end-to-end cryopreservation development services to significantly optimize the freeze-thaw process for cell therapies, ensuring optimum efficacy and functionality for life-saving therapies. Our service offering, based in our dedicated Seattle cryo-suite, was developed with substantial cell therapy development experience together with our therapeutic partners, as well as our internal expertise in cell therapy cryo-development; Pluristyx CTO Dr Brian Hawkins recently received recognition as part of the PDA and Standards Team responsible for drafting a set of cryo-development standards which were recognized by the FDA as a CBER Voluntary Consensus Standard (Research, 2023). The recognition underscores Pluristyx's positioning as a pioneer in cryo-development and improved quality standards in regenerative medicine.

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