A relevant and robust potency assay is not only a regulatory requirement, but it is also the basis for producing and delivering a product that works, and is, therefore, one of the most important and challenging issues when developing and testing cell-based therapies.
The potency assay plays a key role in determining thequality of biological products including cellular therapy products (CTPs). As defined in the U.S. Code of Federal Regulations (21 CFR 600.3), the potency the specific ability or capacity of the product to effect a given result. The potency assay is a quantitative test that confirms the therapeutic productprovides a particular response at a certain dose. Since a mechanism of action(MoA) refers to the specific interaction through which the CTP produces its pharmacological effect, it is ideal that the potency assay will represent the product's known or intended MoA.
Throughout the drug discovery and development process, new data is accumulated on the product candidate's MoA. Thus, a potency assay used in the early stages of product development may be deemed non-optimal in later stages of the development, as a deeper understanding of the MoA for a specific therapy is gained. For example, in a proposed anti-inflammatory potency assay for a specific product candidate with what is initially believed to be an anti-inflammatory, MoA may require an alternative, non-anti-inflammatory potency assay as additional data is accumulated from in-vitro and in-Vivo studies, suggesting a new MoA product candidate. As a result, an initial potency assay may be replaced. It is also important to note that during the early product development stages, a potency assay may be developed to target the most critical biological activity of the product candidate known at this time, while in later stages, additional relevant potency assays may be added.
Stages in potencyassay development
The development of a potency assay for cellular therapyproducts entails many steps of innovations and discoveries while keeping inmind the end user and the requirements of the regulatory authorities.
The first step in developing a potency assay for a specificcellular therapy product is to understand the biological basis of thedisease(s) that the product aims to treat, as well as having a good grasp of theproduct's MoA. For example, if a cellular therapy product is hypothesized to elicit a pro-angiogenic response which may be mediated through pro-angiogenic cytokines, the specific pro-angiogenic pathways and relevant factors should be identified and based on those scientific results, a potential potency assay could be developed.
The second step in the assay development should be the convenience of the assay to the end-user. Since potency assays are performed asroutine quality control (QC) tests, the analysis and quantification of thedeveloped essay should be as simple as possible for the person performing theanalysis. In-vivo tests as a potencyassay should be avoided, as these are too complicated, time-consuming andexpensive. It is also recommended for cellular therapy products not to usesingle protein detection for a potency assay, as cells are likely to mediatetheir therapeutic function via multifactorial MoAs. Therefore, potency assay should on one hand reflect the in-vivo process, and on the other hand, should be able to easily and routinely performed by QC personnel.
In the early stages of product development, the potencyassay results may not have defined acceptance criteria, and maybe collectedand documented for information purposes only. However, during clinical development, as part of the final stage in potency assay development, regulatory authorities expect manufacturers to define the potency assay acceptance criteria, particularly before the initiation of pivotal Phase III clinical trials. The acceptance criteria for the assay should be based on accumulated data collected from assays performed during all phases of product development and clinical trials and should be based on a correlation with efficacy as determined in an in-vivo test.
To obtain a biologics license, a validated potency assaywith defined acceptance criteria must be described and justified in the biologicslicense application. Only a validated assay with defined acceptancecriteria can be used, which assures manufacturing consistency. Without suchparameters, there is no certainty that patients receiving the product candidatewill get a consistent potent cellular therapy product.
Potency assay as a tool
In addition to measuring a cellular therapy product's activity, a potency assay is also used as a tool to test the comparability between lots, which were manufactured either following upgrades or as a result of changes to the manufacturing process. Manufacturing process changes may include changes inharvesting procedure, in final product filling or when establishing a newmanufacturing line in a new facility. Such upgrades or changes may occur during product development and clinical trials. Therefore, the potency assay has animportant role in comparing the products manufactured before and after anyupgrades or changes.
A potency assay is also a central tool to test productstability, and therefore should be included in all stability programs. Althoughviability/recovery of the cells may indicate the stability of the product, viable cells may lose their biological activity during storage. Only cells that were shown to be potent post-storage can be used.
Potency assay considerations
When developing potency assays, companies have to considerand address several fundamental issues:
- CTP's MoA is usually multifactorial; was an appropriate assay chosen? Is the assay robust and stable?
For in-vitro cell-based assays, does the heterogeneity of the cells used in the assay undermine the stability of the assay?
What are the proper positive and negative controls?
What is the proper reference sample?
A cell-based potency assay may have several factors that cause assay variability. Therefore, the sources for assay variability should beconsidered and limited as much as possible. To this end, critical reagentsshould be qualified and calibrated, only qualified equipment should be used andonly trained analysts should perform the assay. Elaborated standard operating procedures should be written and be used and controls should be included in theassay.
Since the potency assay is critical in assuring the qualityand consistency of the product but must eliminate inherent variability in thetest system, relative potency and not absolute potency should be specified. The relative potency is calculated by comparing the biological activity of the tested sample to a reference standard. The reference standard should beprepared by using the same manufacturing process as the cell-based product.
Several factors should be taken into account while conducting an in-vitro cell-based assay: the cells used in the assay should be characterized and banked before being used for routine testing by QC. System suitability consists of prespecifiedcriteria by which the validity of the assay is assessed, and should be integrated into each run of the assay to ensure the quality of the assay results. Outliers, which may be random events, should be predefined and omitted beforerelative potency analysis.
Most cell-based assays are performed using a cell culture plate. Complete randomization or at least plate layout of samples is the best approach to minimize plate effects.
Although regulatory authorities do not require a fullyvalidated potency assay until the end of pivotal Phase III clinical trials, itis important that the development of an assay be initiated early during thedevelopment program to have enough time for the validation parameters. It isexpected that accuracy, sensitivity, specificity, precision (repeatability, intermediate precision), linearity and range, system suitability and robustnesswill be established to achieve validated potency assay in the future.