Criteria for Selecting a Cell Cryopreservation Solution: What to Check in Cell Therapy Manufacturing

The Role of Cryopreservation Media and DMSO Concentration

A cryopreservation medium is a solution containing a cryoprotectant that reduces cell damage during freezing and thawing. The most widely used cryoprotectant is DMSO (dimethyl sulfoxide); the most common concentration in the literature is 10% (v/v), and a trend toward lowering it to 5% has been reported.[1][2]

DMSO and Infusion Reactions

Gastrointestinal and cardiovascular reactions such as chills, nausea and vomiting, and changes in blood pressure and pulse have been reported in patients infused with cell products containing DMSO.[2][5] A systematic review concluded that these reactions are mostly mild and transient and are related to the dose.[2][3] In hematopoietic stem cell transplantation, 1 g of DMSO per 1 kg of body weight is cited as the generally accepted upper limit, although, depending on the publication, it is stated on a "per infusion" or a "per day" basis.[2][4] Post-thaw washing can reduce DMSO, but it carries a risk of cell loss and damage.[2][5]

Serum and Animal-Derived Components

The U.S. FDA considers that animal-derived materials increase the risk of introducing adventitious agents (microorganisms or viruses that enter unintentionally) and that serum varies widely from batch to batch, and recommends considering non-animal-derived reagents where possible.[6][7] The Korean notification likewise requires data on the origin and source of materials used in the manufacturing process, and proof that animal-derived materials are free of adventitious microorganisms.[8] Because even products labeled serum-free may contain human- or animal-derived proteins such as albumin, claims such as xeno-free or chemically defined should be confirmed from composition data.[7][9]

Ancillary Material or Excipient?

Materials that are used in manufacturing but are not intended to remain in the final product are classed as ancillary materials, and components intended to be part of the final product as excipients; the FDA gives DMSO as an example of an excipient.[6][10] Accordingly, if the cryopreservation medium remains as it is in the final formulation, supporting data from an excipient perspective are needed.[10][11] The relevant reference documents are as follows.[6][8][12][13][14]

Qualification and Regulatory Support Documentation

The FDA recommends listing the grade and source of all materials and having a qualification program that demonstrates suitability for the intended use through certificates of analysis (COA) and other means.[6][7] If there is a Drug Master File (DMF), in which the supplier submits confidential information directly to the FDA, it can be cross-referenced in an investigational new drug application (IND) with a letter of authorization.[6][7]

Freezing and Thawing Process and Evaluation

Slow cooling at about 1 °C per minute is widely used for somatic cell suspensions.[1][11] Programmable controlled-rate freezers are preferred for clinical use, and passive cooling containers placed in a freezer are mainly for research use.[1][15] It has been pointed out that viable cells may decline further between several hours and several days after thawing.[1] The Korean notification requires identity, functional test, viable cell recovery and sterility test data for thawed cells from cell banks.[8]

Containers, Stability and Supply Changes

• Containers: for therapeutic use, use containers that can be hermetically sealed[1], and review compatibility with the formulation (adsorption and leaching).[6]
• Stability: stability studies that specify the storage container, formulation and storage conditions, and data on the frozen shelf life, are needed.[6][8]
• Supply changes: because a change of supplier or process can affect quality, have a system in place for receiving advance written notification of changes.[7][12]

Sources