Best Practices in qPCR and dPCR Validation in Regulated Bioanalytical Laboratories

The use of molecular techniques in the bioanalytical laboratory is becoming more common as the number of gene and cell therapy products continues to increase. Currently, there is no bioanalytical regulatory guidance for these techniques, and contract research organizations are dependent on scientific judgment and best practice to execute this work to GxP compliant status for preclinical and clinical studies supporting biodistribution and vector shedding. This manuscript describes the process and rationale for development and validation of quantitative polymerase chain reaction (qPCR) and digital PCR (dPCR) assays as presented in a 2021 AAPS two-day workshop on the harmonization of qPCR. The scope, herein, includes bioanalytical validation parameters and acceptance criteria utilizing these technologies. Additionally, pros and cons of these molecular techniques will be highlighted, and the common pitfalls to avoid will be illustrated. The aim of this manuscript is to provide best-practice, working recommendations, and the facilitation of future regulatory guidance.