Analytical methods are critical in the characterization and testing of cell and gene therapy products.  Analytical method development occurs in parallel to product development and is refined as knowledge is accumulated during the development program.  Here we consider approaches for streamlining analytical method development via well-coordinated product and analytical method development efforts. We further discuss the importance of establishing a robust measurement infrastructure including key aspects such as, measurement assurance strategies, implementation of relevant documentary standards and enabling tools and technologies to streamline and integrate various aspects of the measurement process.

Relative potency is a measure obtained from the comparison of a test sample to a standard based on the capacity to produce the expected biological activity. USP <1034> presents several different models and suitability criteria to determine the reliability of the estimate. This talk will cover traditional linear and non-linear bioassay models and case studies showing how to apply the chapter.

Biotherapeutic monoclonal antibodies and their biosimilars open the door to new possibilities for accessible and targeted treatments of oncological, immunological and infectious diseases. Advances in the analytical methods and regulatory processes have provided manufacturers with robust frameworks for their approval and control. Yet, the rapid market expansion and their complexity, whilst better understood, demand additional considerations. International Standards have contributed to the harmonisation of biological medicines for over a century, and now rise to the challenges set by of new medicines like monoclonal antibodies. Understanding their role and correct use is essential to realizing their potential as key tools to support product consistency and traceability over the product life cycle.

The development and verification of test systems for biological assays is a challenging task. Focus of this talk are principle considerations and a practical approach to develop and verify efficient test systems for biological assays using equivalence tests.

Virus-based gene therapies require complex analytical methods to assess key quality attributes during process development, GMP release, and stability monitoring of clinical-grade products. Here we discuss phase-appropriate paradigms for analytical method development, qualification, and implementation into gene therapy programs as they transition from research into clinical stages. In this talk, we will concentrate on a functional potency assay which often requires a multiplex approach to determine the true biological activity of the drug products. We will use one of our case studies as an example to discuss MOA potency assay development strategy, challenges, and method qualification.

The efficacy of gene therapies using recombinant AAV can be limited by an antibody-mediated immunity against the virus. AAV infections, potentially occurring in early childhood, may result in a pre-existing immunity to one or more AAV serotypes. Here we present a novel two-component iLite® reporter cell system for the detection and quantification of neutralizing antibodies against AAV vectors. This assay allows for reliable assessment of anti-AAV antibodies in clinical applications

What does it take to calculate potency and which data do you need? Determination of potency is a critical quality attribute, playing a key role in the development and control of biological products. As there is an increasing need for potency determinations, either a high throughput approach or manning up has to be implemented. In this study we have investigated the use of reduced curves. It will be shown, that by omitting numbers of dilution of the sample investigated, we obtain the same results as if full curves were used, increasing samples analyzed per run.

Increasing complexity of biotherapeutics with novel mechanism of action (MoA) make development of potency assays a challenging task. There is a need for careful assay design for potency (a critical quality attribute), ensuring assays reflect the MoA, while minimizing complexity for a QC release assay. Here we report the design of a cell-free competition bioassay to illustrate reduction of method complexity whilst being MoA reflective and phase appropriate.

The cells are arguably the most critical reagent in any cell-based bioassay. Therefore, understanding the cells is essential to the development of a truly robust bioassay and can be invaluable during technical transfer and troubleshooting activities. This presentation will describe an approach for improving these bioassays by optimizing cell handling methods such as cell counting, culture media supplementation, cell dissociation from flasks, cryopreservation, cell thaw, recovery from thaw and cell banking overall.  A broad overview of these studies followed by a more detailed description of the cryopreservation, cell thaw and cell banking optimizations will be presented.

Bioassays typically have non-constant variance and complex statistical designs; good outlier methods require complex models which yield lower rates for both false and failed outlier detection than simple methods. Multiple statistical detection methods can be helpful. There may be multiple outliers and outliers at different levels (e.g.; well and sample). Automatic outlier removal (usually favored by QA) is strongly discouraged by statistical and regulatory guidelines. Documenting the processes that lead to outlier removal is essential. Analyzing assays with and without suspect observations, then reporting results from the least favorable of these analyses is a good strategy. 

Cell lines are an important element when developing cell-based bioassays. Those cell lines that are used in current Good Manufacturing Practice (cGMP) lot release and stability testing require extensive characterization to understand their growth and performance characteristics. During characterization of the target cell line used in the potency assay for one of Amgen’s Bispecific T cell Engager (BiTE®) molecules, an increase in the cell surface and intracellular expression of the target protein was observed when cells from early passage to later passage were evaluated. The change in cell surface antigen expression had a positive impact on a key method performance parameter and resulted in an increase in the assay signal window. This presentation will describe our findings regarding this cell line in the context of its performance in a T cell dependent cellular cytotoxicity assay.

As a result of recent regulatory guidance, there is increasing demand for diagnostic immunogenicity assays, particularly in gene therapy.  Early planning is essential when designing an assay that may ultimately be used as a diagnostic.  In addition to requirements for documentation and performance verification, diagnostics also have specific needs for critical reagents and controls.  This presentation will describe the regulatory landscape and discuss considerations for design and performance verification of diagnostic NAb bioassays for gene therapies.