A Primer on Potency Assays for Release of Biologics

A key criterion in determining the quality of a biologic product is whether it has maintained its biological activity or “potency.” As a critical quality attribute, potency is assessed during characterization of Good Manufacturing Practice (GMP), manufactured products for use in clinical development, and is included as a specification for lot release and stability testing of drug substance and drug product. Given the importance of potency as a quality characteristic of biologics and the variety of methods that can be used to measure it, we will present a brief review of the topic here.

1. What is a potency assay? A potency assay is a quantitative analytical procedure used to measure the therapeutic activity of a biologic by comparing its activity to a well-characterized reference standard in a biological test system. Often referred to as a “bioassay,” the potency assay frequently uses a mammalian cell line to measure a biological response but may also be a solid-state binding assay that uses biologic reagents such as antibodies to measure binding activities. It is considered one of the most important of many release assays in the manufacturing process because it verifies the expected therapeutic activity of the biologic to ensure both safety and efficacy.

2. What kinds of assays are used to measure potency in release testing? Potency-release testing is typically done with solid-state binding assays (i.e., Biacore, Octet, ELISA), cell-based assays, or a combination of cell-based and binding assays. The potency assay should measure potency based on a relevant mechanism of action (MOA) of the therapeutic. Furthermore, potency testing should include testing all relevant activities associated with different functional regions of the molecule that can vary from batch to batch (i.e., Fc and variable regions of antibodies, glycosylation sites). Binding assays are often used early in clinical development because of the difficulty of developing a cell-based assay reflective of the drug’s MOA. They are later replaced or used in conjunction with a cell-based assay to assess potency.

3. What are the attributes of a potency assay used for release and stability testing? Prior to use in release and/or stability testing, a potency assay should be validated for the following parameters: specificity, accuracy, precision (repeatability and intermediate precision), detection limit, quantitation limit, range, linearity, and robustness. Validating the potency assay confirms that a specific threshold for each of these parameters is met. During validation, it is also critical to demonstrate that the potency assay is stability-indicating. This is done by measuring the potency of stressed samples that show loss of activity. Stressed samples can be generated through heat, pH, and UV-light treatment.

The method is validated for its intended purpose in a GMP quality-control laboratory using trained analysts and documented controlled practices. The validated method is then performed according to a standard operating procedure (SOP) and is monitored for performance using assay-quality controls.

4. When are potency-release assays required? All manufactured biologics have potency testing on the specification for release of the drug substance and product. Potency testing is also performed to ensure stability of the drug substance and product for the period the drug is to be stored prior to use. Due to the importance of characterizing the drug substance and product, potency tests must be put in place early in the development process. However, because of the challenges in developing quantitative cell-based assays, many companies initially develop solid-state binding assays instead of cell-based assays to release manufactured drugs early in their clinical programs.

Regulatory expectations depend on the stage of clinical development and the type of product (see also #5 below). For example, a monoclonal antibody, for which the MOA may be binding only to antigen, likely will require only a solid-state binding assay for phase 1 with a later commitment to develop a cell-based assay. In contrast, an antibody drug conjugate will likely require both solid-state binding and cell-based assays by phase 1 because its MOA, comprised of both the binding to antigen and subsequent killing of target cells, is integral to drug composition and structure. By phase 2/3, the expectation of regulatory agencies is that a cell-based potency assay is developed, although it is understood that in rare cases, limitations to developing a cell-based assay can be discussed with the FDA.

5. When would more than one potency assay be used for release? A combination of cell-based and ligand-binding assays is sometimes employed to measure overall activity of the therapeutic as well as binding activities of different functional regions of the therapeutic (i.e., variable and Fc regions of antibodies). For example, an antibody that causes antibody-dependent phagocytosis (ADCP) or cellular cytotoxicity (ADCC) by binding to a surface protein on a target cell through its variable region, forming a bridge to an effector cell through its Fc region, has three critical biological activities that should be measured in potency assays: 1) binding of the variable region to the surface protein; 2) binding of the Fc region to the Fc receptor on the effector cell; and 3) induction of a biological signal in the effector cell.

For this molecule, potency based on the binding activities of the variable and Fc regions can be quantitatively measured by solid-state assays, whereas potency based on the signaling to an effector cell can be quantitatively measured using a cell-based assay. The three assays together give a more complete assessment of the molecule’s potency predicated on the different regions contributing to its biological activity. As previously mentioned, the binding assays are typically used for release early in clinical development and are then replaced by a more difficult-to-develop cell-based assay by phase 2/3.

6. What are other uses of potency testing during the manufacturing process? In addition to the importance of potency testing to monitor drug stability and to verify the therapeutic activity of a drug product prior to its release, potency testing is also informative to understanding the impact of changes in the manufacturing process. When the manufacturing process is first being developed, potency testing can facilitate process optimization. This is often done with an early-stage potency test that may not be fully validated.