The FDA defines critical quality attributes (“CQAs”) as “physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality.” The agency advises that limits in a process’ early development may be broader than in the late stage, when investigators are still gathering information about the product.
Despite regulatory oversight, standard guidelines do not yet exist for critical quality attributes submitted with each new cell and gene therapy. Even so, regulators expect critical quality attributes to have associated assays that illustrate a therapy’s performance and safety.
Common CQA categories include safety, identity, sterility, purity and potency.
Safety
Safety is an underlying parameter for all CQAs. It is defined in 21 CFR 600.3 as “relative freedom from a harmful effect to persons affected, directly or indirectly by a product when prudently administered, taking into consideration the character of the product in relation to the condition of the recipient at the time.”
Identity
Identity testing is required to “distinguish one product from another that is produced in the same facility.” Parameters include physical, chemical, cultural testing, and in vitro/in vivo immunological testing. Autologous patient samples pose a challenge because various lots will not differentiate from each other. Therefore, identity tracking mechanisms/protocols are necessary to avoid mix-ups between patient-specific products.
To determine the identity of cell and gene therapies, one uses a quantitative test (phenotype or biochemical assay) to confirm the number of target cells present in the product. During this process, when identifying non-target cells or impurities, reserachers characterize them to understand their role, function, and potential impact.
All CQAs are closely related; in the case of identity and purity, the case is even more so. For the purpose of “identity” in this document, identity strictly refers to the characterization of the target cell population, whereas purity refers to non-target cells and other non-biological impurities.
Sterility
Sterility is defined as the absence of “viable contaminating microorganisms.”
This parameter poses a particular challenge to cell and gene therapy (“CGT”) manufacturers. Traditional forms of sterility testing require a 14-day culture period to determine whether a drug product meets the FDA’s threshold.
The FDA recommends the use of “aseptic manufacturing processes” to maintain sterility as much as possible. This includes closed-system processing and sterile manufacturing equipment and sterilized raw materials when possible. However, testing CGT products for 14 days prior to administration to patients is nearly impossible. This is due to the nature of the therapy itself and a need to maintain a supply chain which minimizes the time from development to administration. Rapid disease progression, rendering the therapeutic no longer viable, presents another obstacle.
Traditional drug products have the option for “terminal sterilization,” which removes adventitious agents from the final product. Cell and gene therapies don’t have the same option, as that process would also kill the viable living cells.
Therefore, FDA guidance is to perform a gram-negative stain prior to patient administration and to have an action plan developed for positive test results after administration to the patient.
For allogeneic therapies, one may confirm sterility by screening donors for various transmissible diseases. This includes screening for HIV type 1 & 2, HBV, HCV, human transmissible spongiform encephalopathy and syphilis.
Purity
Purity is a measure of the “impurities in the final product that come from the manufacturing process.” This may include additives or other products not intended for the final product that may result in a hypersensitivity reaction or other adverse events. Examples of impurities include endotoxins, residual solvent, antibiotics, animal product and unintended cell types (i.e. lymphocyte subsets).
Proof of CGT product purity requires validation of impurity removal or acceptable residual levels.
Potency
“Potency” refers to an ability to effect a given result, as indicated by laboratory tests or by adequately controlled clinical data. Tests used to determine potency must be in vivo or in vitro and designed specifically for that product. Potency testing is a subset of conformance testing, comparability testing, and stability testing.
One measures potency using assays to determine biological activity. Published examples from the FDA include:
a) “Mixed lymphocyte reaction assay to determine if T-cells are active against a target cell or measure cytokine secretion to assess biological activity
b) Indirect assay to correlate cell phenotype with a function – i.e., dendritic cells might up regulate the expression of a co-stimulatory molecule such as CD86 during activation, and high expression of CD86 could be an indirect measure of dendritic cell activation and potency.
c) In a matrix assay, one evaluates a number of different characteristics, and the cumulative assessment of these characteristics provides a measure of potency.
Potency testing is obtained prior to lot-release. The assay should be “fully validated to demonstrate product activity, have a quantitative readout, should be stability indicating and provide a measure of product consistency.”
Karanu et al. suggest that viability may eventually become a standardized measure of potency because it is one of the most common and widely utilized assays in CGT.
It’s important to note that CQAs do not stand alone. Each attribute depends on another. For example, confirming identity might also lead to identifying an impurity. This can affect the product’s sterility and the overall safety profile. Therefore, defining CQAs requires a holistic approach. Each attribute interrelates and contributes towards a measure of an overall safety profile.
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