Bioassays 2021

Gene therapy potency assays are complex and challenging to implement. Engineered cell lines, including reporter gene systems, can improve cell-based potency assay throughput, precision and costs. For evaluating AAV potency, AAVR overexpressing cells can platform to nearly all serotypes and any transgene to improve permissivity and assay signal.

Engineered cellular therapy products are among the more complex of immunotherapeutic modalities because they are living drugs. Cell-based potency assays are essential to demonstrate that drug product activity is linked to biological critical quality attributes. However, key challenges continue to include an incomplete understanding of CQAs, lack of universal controls, limited lot sizes for use in development, and identifying and mitigating sources of variability in cell-based potency assays. Fortunately, early use of phase-appropriate, risk-based approaches to method development, qualification/validation, and analytical control strategies from clinical stages through to commercialization can ease regulatory burdens.

System suitability assessment is an essential part of bioassay methods. The choice of system suitability parameters and their ranges is critical for ensuring the quality of bioassay results. In this talk, we share a case study of successful bioassay method remediation, which significantly improved a legacy commercial method by replacing inappropriate system suitability criteria with state of the art new criteria. The talk provides an overview of the legacy method investigation, as well as determination, justification and implementation of the new system suitability criteria.

A control strategy, according to ICH Q8, is ‘a planned set of controls, derived from current product and process understanding that ensures process performance and product quality.’

Bioassays are obviously a cornerstone of the control strategy. However, the optimal choice and implementation of a routine bioassay depends on several aspects, for example the Mode of Action(s) of the product; other control strategy elements; and various more practical considerations; which may need to be balanced. For example, MAbs may have various MoAs comprising, among others, direct binding and neutralization, target dimerization, CDC, ADCC, and ADCP; these can all be characterized, but it is not always evident how to translate this into a rational control strategy.

This presentation will discuss some current regulatory perspectives on this topic.

Transcriptional activity within a cell can be used to evaluate cell response to a ligand or promoter activity within a transgene or plasmid within a cell. Catalent has developed a relative potency bioassay using real-time quantitative reverse transcription (RT-qPCR) in a duplex format to assess relative transcription activity in cells treated with ligands or transgenic vectors. The assay utilizes two fluorescent dyes with minimally overlapping emission spectra that allow real-time monitoring of the gene expression of both target and normalizer genes. The assay does not require purification of the mRNA produced by the cells once lysis has occurred. Normalizing the qPCR cycle thresholds (CT) of the target transcript to the reference transcript allows response curve to be generated and compared to a reference standard. The generation of a four-parameter fit curve analysis from raw qPCR cycle threshold data allows for comparison of relative potency and assessment of suitability based on curve parallelism. The assay platform has been used by Catalent to qualify a repeatable, accurate, linear, and specific bioassay for assessing relative potency.

A bioassay case study summarizing the challenges in setting the control system of a combination product of two highly similar monoclonal therapeutic antibodies targeting the same cellular antigen: from similar modes of action and synergistic effect to the control of each molecule bioactivity in vitro.

The pandemic of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) has significantly impacted the lives of the global human population. Many biotech and pharmaceuticals companies are pursuing the development of effective therapeutic and preventive approaches including small molecule drugs and biologics (antibodies, vaccines, etc) at ‘break-neck’ speeds, with some having received emergency-use authorization in the U.S. as well as global marketing authorization approvals. This session will focus on examples of technical challenges encountered in developing appropriate potency assays as well as strategies for accelerated potency assay development for their therapeutic modalities. This session will also focus on a recent round-table discussion and FDA guidance on the development and use of potency assay for biologics targeting SARS-CoV2.