ISO/PWI 26125-2

Biotechnology Tumorigenicity Testing — Part 2: Nucleic Acid Detection for Undifferentiated Pluripotent Stem Cells in cell and gene therapy products

Scope

This document specifies the general requirements for detecting residual pluripotent stem cells in cell therapy products using reverse transcription quantitative polymerase chain reaction (RT-qPCR) or reverse transcription digital polymerase chain reaction (RT-dPCR) assays, with messenger RNA (mRNA), long none cording RNA (lncRNA) or microRNA (miRNA) as target analytes. It provides guidance on:
 — Sample representativity, including the considerations in sample size；sample replicates；nucleic acid quality control;
— Assay selection and design , including selection of fit-for-purpose test methods; control setup; target gene selection, reference gene selection, normalization strategies, Primer, and Probe Design; and Amplification Strategy
— Method validation, covering general requirements, determination of the limit of detection (LOD), and revalidation;
— Data analysis and reporting, covering quality control for assay data; normalization of data; result interpretation and reporting.
This document is applicable to the development and validation of RT-qPCR or RT-dPCR as methods intended for the detection of residual pluripotent stem cells in cell therapy products.