Approved biological products are required to be accompanied by analytical tests to demonstrate safety, purity, and potency. Manufacturing and testing processes for approved products are validated and performed under current Good Manufacturing Processes (cGMP).
Potency is defined as “the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner intended, to effect a given result.”1
In vivo models used during proof-of-concept and efficacy studies provide an early readout of potency by measuring a desired physiological response in animals. For product approval, in vitro assays must be developed providing a quantifiable readout that can be validated. The in vitro approach of introducing a gene into a cell line and then demonstrating its expression and functional activity has been used for over a decade in the evaluation of small molecule drug-drug interaction properties. When a small molecule drug is developed, the interaction potential of the molecule as a substrate, inhibitor, or inducer of specific drug transporters is evaluated. The U.S. Food and Drug Administration (FDA) issued the first drug transporter concept paper in 2004, and it issued a draft guidance in 2006. This draft guidance was most recently updated in 2017.2
Principal of potency assay development for cell and gene therapy products
Although in vitro potency assays have been used in small molecule and monoclonal antibody drug development for more than a decade, applications in cell and gene therapy (CGT) product development are more recent. CGT products present specific challenges for developing in vitro potency assays due to:
- complex and/or unclear mechanisms of action
- complicated manufacturing processes
- variable critical quality attributes (CQAs)
- reduced preclinical and early-stage clinical data with which to inform development
The development of in vitro potency assays for CGT products includes at least two steps. First: It must be demonstrated that the vector can transfer genetic material into a cell. Second: It must be demonstrated that the transferred genetic material has the desired biological effect in the transduced cells.
Due to the complexity of some CGT products’ mechanisms of action, more than one assay may be required to characterize different aspects of potency. The challenges related to developing in vitro potency assays for CGT products extend to later-stage considerations for validation and routine testing such as supply chain, assay automation, and the development of reference standards. As more products come to market, the demand to overcome these challenges and improve efficiencies will grow.
Developing in vitro potency assays for cGMP settings
Validating biologically relevant cell lines as in vitro models of therapeutic potency is an approach that is recognized by regulatory authorities. Since 2000, the FDA has accepted in vitro epithelial cell culture systems to waive clinical trials. The FDA Biopharmaceutics Classification System (BCS)-based biowaiver guidance outlines the cell line characteristics and requirements for demonstrating suitability of a test system.3
The development of an in vitro potency assay to accompany an approved CGT product involves a series of activities that can be divided into four key stages.
Stage 1: Establish optimum assay profile
It is important to establish an assay profile and feasibility criteria early in the development process. This includes scientific considerations for the gene of interest, mechanisms of action, relevant biological functions, and whether multiple assays are required to demonstrate these activities. The assay design should account for limited understanding of the final manufacturing processes, as well as for potential differences between drug substance and drug product. It should also limit the amount of testing material required.
Key questions include:
- Should expression and functional activity both be considered within a single assay?
- Will the assay be used for characterizing both the drug substance and drug product?
- Are there preexisting assays that can meet some of these objectives?
- What considerations for the capsid, gene, enzyme, or protein of interest need to be made?
- What requirements for time, cost, and compliance occur at each stage of assay development?
- What level of tolerance for assay sensitivity, specificity, and variability will be acceptable?
A common approach is to employ a relative potency assay to combat inherent assay variability. The response of a test sample is compared to that of a designated reference standard tested in parallel. The potency is reported as relative to the reference standard, rather than an absolute response. Identifying a biologically active reference standard is critical to the final assay design. Once the ideal profile is established, it can be used to guide assay development and ensure that the appropriate controls, replicates, and statistics are established to demonstrate all required assay parameters.
Stage 2: Selection of a test system
One of the key reagents of the potency assay is the biorelevant test system (target cell) that can accept the gene and support the desired mechanism of action. Identifying the target cell profile for the potency assay is an important step, and considerations include:
- general cell culture considerations such as availability, growth, and morphology
- product-specific characteristics such as compatibility with the capsid, promoter, and any interacting enzymes or proteins
- characterization of the cell line to facilitate expression and functional assays
Several cell lines may need to be screened to determine the optimum test system. The creation of custom stably transfected cell lines may be required to achieve the desired profile.
Stage 3: Assay optimization
During this stage, each independent step of the assay is optimized with respect to the overall potency assay. Process optimization may include:
- fine-tuning of cell culture conditions such as the cell density, plating format, and time for transduction
- production, handling, and stability of transduced cell lysate to form a functional reagent
- functional assay parameters including substrate concentration, solvent, and stability; incubation time and temperature; performance of reference standard; and specificity of positive and negative controls
- detection method used to quantify the output from the functional assay (extraction, linearity, specificity, accuracy, and precision for the analyte of interest)
At this stage, it is recommended to use statistics to guide assay design using multifactorial design of experiments and an appropriate replication strategy based on known or expected sources of variability. Statistical analysis should be used throughout assay optimization to determine acceptance criteria for control treatments, reference standard and test samples; data distribution and transformation to fit the appropriate statistical model; and procedures for identifying and managing outliers.
Each step of the assay should have its own system suitability and acceptance criteria to ensure the inclusion of reliable data. There are several guidelines available for the design and validation of bioassays for general application, and those specific to potency assays for CGT products have been promulgated by several authorities:
- The FDA
- The European Medicines Agency4
- The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (validation guidelines)5
- United States Pharmacopeia (chapters for bioassay design and validation)6–9
The guidelines agree on the relevant parameters required for assay validation, such as accuracy, precision, linearity, and specificity. However, the assessment of those parameters is specific to each product type. This is due to the interdependency of many methods and reagents commonly used in assay development.
Stage 4: Long-term program management
The potency assay development process follows the life cycle of the CGT product. At different stages of product development, the approach, design, and refinement of the assay may change. From an analytical testing perspective, the labs performing these complex assays must be able to operate within a GMP-compliant facility while satisfying CFR Part 11 and Annex 11 guidelines for data integrity.
Critical reagents must be characterized for grade and qualification criteria with a well-defined supply chain and built-in redundancy. Redundancy is also necessary for trained personnel and specialized equipment for successful implementation in a GMP setting. Statistical design and analysis approaches may be customized to adapt traditional United States Pharmacopeia guidelines to the complex nature of CGT products. Finally, from a commercial standpoint, the intended lot release schedule and product availability may shape the final potency assay design, along with any regulatory feedback on the potency assay approach. It is important to have discussions with the FDA early and often to communicate the intended potency assay approach.
1. U.S. FDA Guidance for Industry—Potency Tests for Cellular and Gene Therapy Products, CDER January 2011.
2. U.S. FDA Draft Guidance—In Vitro Metabolism- and Transporter-Mediated Drug-Drug Interaction Studies, CDER October 2017.
3. U.S. FDA Guidance for Industry—Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System, CDER December 2017.
4. EMA Guideline on the quality, nonclinical, and clinical aspects of gene therapy medicinal products, March 2018.
5. ICH Q2(R1) Validation of Analytical Procedures.
6. U.S. Pharmacopeia (2012) Chapters: <1032> Biological Assay Development, <1033> Bioassay Validation, <1034> Biological Assay Analysis.
7. Li J et al. Use of Transporter Knockdown Caco-2 Cells to Investigate the In Vitro Efflux of Statin Drugs. Drug Metab. Dispos. 2011; 39: 1196–1202.
8. Zhang W et al. Silencing the Breast Cancer Resistance Protein Expression and Function in Caco-2 Cells Using Lentiviral Vector-Based Short Hairpin RNA. Drug Metab. Dispos. 2009; 37: 737–744.
9. Darnell M et al. Investigation of the Involvement of P-Glycoprotein and Multidrug Resistance-Associated Protein 2 in the Efflux of Ximelagatran and Its Metabolites by Using Short Hairpin RNA Knockdown in Caco-2 Cells. Drug Metab. Dispos. 2009; 38: 491–497.