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March 05, 2020

Cryopreservation helps cells survive both cooling to extreme temperatures and thawing back to physiological conditions. Our previous post covered the importance and challenges involved in cryopreservation of cellular therapies.

While several factors come into play, here are our top three considerations for developing cryopreservation processes that will ensure the safe and effective manufacturing of cell therapies, with minimal variability and risk to patients.

Consideration 1: Selection of an appropriate container or vessel for cryopreservation

Traditionally, cells are cryopreserved in vials. While vials are easy to handle and commonly used to establish seed banks for large-scale allogeneic cell therapy production, they are not amenable to autologous cell therapy (e.g., CAR-T therapy) workflows, where the final patient doses for such a therapy may exceed billions of cells and may not fit into a vial. Vials are not suitable for administering the cells at a clinical site by the end user, unlike an infusion bag. Also, vials must be processed in a biosafety cabinet and do not include sterile-weldable inputs or outputs, which is the preferred mode of administration. Some manufacturers now offer automated fill-and-finish, closed options using vials.

Cryopreservation of cells in an infusion bag, on the other hand, offers an alternative solution to the challenges associated with using vials. Infusion bags come in various sizes and allow for the storage of higher numbers of cells than in vials. However, infusion bags lack an automated, fill-and-finish solution.

In general, it is important to select a closed, scalable and Good Manufacturing Practices (GMP)-compliant system for cryopreservation that can accommodate patient dosing volumes ranging from tens to hundreds of millilitres of final product.  

Consideration 2: Use of automated unit operations for closed-system workflow

Closed-system workflows help manage contamination risk and operational timelines. Automation simplifies such workflows.

Commercially, liquid Nitrogen-based (e.g., CryoMed™) and liquid Nitrogen-free systems (e.g., VIA Freeze™) are available to enable automation.

In a previous post, we looked at  Sefia™ S-2000, a cell processing system that allows closed and controlled downstream processing, such as volume reduction, wash and final formulation using the FlexCell protocol. This protocol can process up to 10 litres of expanded cell culture. The final product is then formulated into a cryopreservation bag at a constant rate.

To cool the product, the VIA Freeze range of freezers enable controlled-rate cryopreservation. Compared to many other controlled-rate freezers, VIA Freeze bears a smaller footprint and does not require liquid nitrogen to freeze cells, making it easier to incorporate into GMP workflows. It is capable of controlled cooling before and after ice nucleation, which is key to post-thaw cell recovery, as illustrated by this study.

Combined use of such instruments enables closed-system workflows and appropriate storing of cellular products into sterile, weldable cryopreservation bags.

Consideration 3: Choosing an ideal cryoprotectant

Cryoprotectants are specialized solutions that protect cells or tissues from cellular damage during freezing by preventing intracellular ice crystal formation. Dimethyl sulfoxide (DMSO) is the gold standard cryoprotectant and is commercially available at several concentrations that are GMP-compatible and widely used in cellular therapy.

However, DMSO can be toxic to cells for several reasons, such as the changes in osmolarity, long exposure time during formulation and cell membrane damage sustained during room temperature manipulations. Such damage negatively impacts viability post-thaw and recovery of the cellular product. Use of DMSO, a powerful solvent, also presents unique challenges for closed-system processing as it can extract compounds from bags and tubing, thereby leading to impurities in the cellular material.

DMSO-free cryoprotectants are commercially available and efforts are underway to identify alternative cryoprotectants that are less toxic to cells and better suited to closed-system processing.

Along with addressing these considerations, critical process parameters like freezing rate, the composition of the freezing medium used, packaging and storage conditions should be thoroughly tested in early-stage process development.  

Looking for a customized cryopreservation process for your cell or gene therapy product? Contact us now.

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GMP manufacturing good manufacturing practices GMP cryopreservation controlled-rate cooling closed-system processing controlled-rate freezing

Understanding Cryopreservation of Cellular Therapies

Cryopreservation is the process of using ultra-low temperatures to preserve living cells and tissues for a prolonged tim...