Quantitative polymerase chain reaction in the bioanalytical laboratory and technical and scientific considerations for nonclinical and clinical assay characterization, validation and sample analysis

Abstract

In this manuscript, the European Bioanalysis Forum reports back on their discussions on practical and scientific considerations related to bioanalytical applications of quantitative polymerase chain reaction. This publication follows an earlier publication in which the European Bioanalysis Forum recommends to consider principles of context of use when defining assay acceptance criteria for method validation criteria and sample analysis.

Drug Regulatory-Compliant Validation of a qPCR Assay for Bioanalysis Studies of a Cell Therapy Product with a Special Focus on Matrix Interferences in a Wide Range of Organ Tissues

“…Although qPCR-based assays have emerged as an important bioanalytical method for assessing the pharmacokinetics of human-cell-based medicinal products [ 4 , 5 , 16 , 21 ], the regulatory guidelines on the validation of bioanalytical methods released by the EMA, FDA, and ICH [ 24 , 25 , 26 , 27 ] focus on methods suitable for small- and large-molecule drugs such as chromatographic and ligand-binding assays. While the basic concepts and parameters of method validation described in these guidelines can be adapted to cell quantification via qPCR, in the absence of specific regulatory recommendations including definitive acceptance criteria for a validated qPCR assay, researchers must rely on published evidence from bioanalytical scientists, as well as recently issued best practice recommendations [ 15 , 20 , 42 ] and white papers from scientific networks [ 21 , 22 , 23 , 43 ].…”

“…The validation presented here followed the validation parameters set out in the EMA Guideline on Bioanalytical Method Validation [ 26 ], recently superseded by the ICH Guideline M10 on Bioanalytical Method Validation and Study Sample Analysis [ 24 ], using predefined acceptance criteria ( Table 1 ). The acceptance criteria for accuracy and precision, which were set to within ±40% and ≤40%, respectively, are within the range of those recently recommended by the European Bioanalysis Forum [ 22 ]. The data obtained show that human MSCs can be detected and quantified with acceptable linearity, accuracy, and precision within the range of 125 (LLOQ) to 20,000 (ULOQ) cells/200 µL lysate ( Table 1 ).…”

“…The yield and quality of extracted genomic DNA can vary widely depending on the physical and biochemical nature of each tissue [ 16 , 31 ], and the tissue from which the DNA was extracted can have a significant effect on the efficiency, accuracy, and precision of the qPCR assay [ 34 ]. Therefore, current regulatory guidelines [ 24 , 25 ] and best practice recommendations [ 15 , 20 , 21 , 22 , 43 ] advise researchers to assess potential matrix effects by determining the recovery of the target DNA spiked into each tissue of interest during assay validation, whereby recovery rates in a wide range between 30% and 100% are considered to be expected [ 15 , 20 ].…”

“…Therefore, any qPCR method must be validated to ensure the reliability of the data that will be obtained. Remarkably, however, despite the growing number of qPCR applications in regulated bioanalysis, and even though qPCR is one of the officially recognized methods for determining the pharmacokinetics of cell therapy products [ 11 ], regulatory guidance on the use of qPCR is limited [ 13 , 20 , 21 , 22 , 23 ]. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Guideline M10 on Bioanalytical Method Validation and Study Sample Analysis [ 24 , 25 ], which recently superseded the guidelines on bioanalytical method validation released by the European Medicines Agency (EMA) [ 26 ] and the US Food and Drug Administration (FDA) [ 27 ], focuses on pharmacokinetic methods that are suitable for classical small-molecule drugs and large-molecule biologics, such as chromatographic methods and ligand-binding assays, but do not address the specific requirements for the proper validation of PCR assays [ 13 , 20 , 21 ].…”

Recommendations for Method Development and Validation of qPCR and dPCR Assays in Support of Cell and Gene Therapy Drug Development

“…These recommendations had the intention of raising the standard for publishing reproducible and repeatable data in scientific journals (13), and later, MIQE guidelines were released for dPCR (14). Following the increase in cell and gene therapy drug development, several manuscripts and white papers from various authors in the industry have been published with recommendations and best practices for developing and validating bioanalytical PCR assays (15)(16)(17)(18)(19)(20)(21)(22). International Organization for Standardization (ISO) standards have also been published and available (23).…”