Precision bioanalysis for advanced therapies

Regulatory guidelines for analytical validation in RNA and DNA bioanalysis

The FDA published the ICH M10 Bioanalytical Method Validation Guidance for Industry in 2022, providing a framework for validating bioanalytical methods used to support regulatory submissions. The guidance emphasizes that validation is essential to confirm that a bioanalytical method performs reliably and produces reproducible results. A full validation should assess selectivity and specificity to ensure the method accurately measures the analyte of interest, evaluate matrix effects to understand how biological matrices influence quantification, and include a calibration curve to establish the relationship between analyte concentration and signal response. The lower and upper limits of quantification (LLOQ and ULOQ) must be defined to determine the assay’s quantification range. Additional requirements include evaluating accuracy, precision, carryover, and analyte stability under various conditions.

The ICH M10 guidelines were developed primarily for conventional bioanalytical techniques such as LC-MS/MS and ligand binding assays, which directly quantify the concentration of analytes in biological samples. In contrast, quantitative PCR (qPCR) determines analyte levels indirectly by measuring the cycle threshold (Ct) value and comparing it to a standard curve generated from known concentrations. Within its validated dynamic range, qPCR exhibits a log-linear relationship between Ct and the logarithm of analyte concentration. However, its accuracy and precision depend highly on consistent amplification efficiency and the absence of PCR inhibitors.

Unlike small molecule analytes, nucleic acids are inherently unstable and susceptible to degradation, requiring stringent quality control measures, including internal spike-ins, degradation assessments, and reverse transcription (RT) controls (for RNA targets). Furthermore, defining the lower limit of quantification (LLOQ) in qPCR requires empirical validation, as it is influenced by variability at low template concentrations and assay efficiency.

Specificity in qPCR assays is determined by the primers and probes’ design and the target sequence’s uniqueness, which presents different selectivity challenges compared to LC-MS/MS. These methodological differences highlight the need to adapt bioanalytical validation principles when applying M10-compliant workflows to nucleic acid-based assays.

The ICH S12 guideline on nonclinical biodistribution provides standardized sample timing, group size, and tissue collection recommendations. The guideline highlights the importance of assessing both the presence and quantity of vector DNA, gene expression products, and/or cellular markers in target and non-target tissues. These tissues include but are not limited to the injection site, gonads, adrenal glands, brain and spinal cord, liver, kidneys, lungs, heart, spleen, blood, and other relevant organs depending on the administration route and therapeutic target. qPCR is highlighted in the guideline as a sensitive technique capable of detecting low copy numbers of nucleic acids, making it an ideal tool for measuring the persistence of gene therapy products in biodistribution studies.

The S12 guideline also emphasizes the importance of screening for pre-existing immunity to the viral vector prior to administration, as pre-existing antibodies or immune responses can alter the biodistribution profile by accelerating vector clearance and degrading the therapeutic agent.

High data quality, integrity, and reliability are essential for regulatory acceptance. While studies not conducted under Good Laboratory Practice (GLP) compliance may be acceptable, GLP becomes critical when biodistribution evaluations are integrated into GLP-compliant toxicology studies. In such cases, all in-life evaluations and sample collections must adhere to GLP standards.

The extent of analytical validation required should be based on a risk-based approach, meaning that the level of validation should reflect the potential impact and regulatory importance of the data generated. For example, assays supporting pivotal safety decisions in regulatory filings may require more rigorous validation than those used in exploratory research.

In 2015, the FDA issued guidance on shedding studies for virus- and bacteria-based gene therapies and oncolytic products. Shedding assays are required to determine whether a therapeutic product releases viral particles or genetic material into bodily fluids, a key consideration for environmental risk and potential transmission. Recommended matrices for shedding assessments include urine, saliva, feces, blood, and other relevant body fluids. At least one assay should be quantitative, allowing results to be expressed in terms of genome copies or infectious units. Quantitative PCR (qPCR) is recommended due to its sensitivity, ease of standardization, high throughput, and rapid turnaround time. The FDA requires that such assays be specific, sensitive, reproducible, and accurate, with clearly defined LOD and LOQ and well-characterized specificity to avoid false positives or false negatives. However, the agency does not expect shedding assays to be fully validated. Instead, they should be qualified to meet minimum performance requirements and be fit for their intended purpose.

Overall, the regulatory landscape for nucleic acid-based bioanalytical assays such as qPCR and dPCR is still evolving. While ICH M10, S12, and the shedding guidelines set a foundation, they do not fully capture the unique characteristics of nucleic acid assays. We therefore refer to current best practices, including recommendations published by AAPS in 2024, which were developed by 37 experts from 24 organizations. These recommendations help fill current regulatory gaps. The following section will discuss the specific validation criteria for qPCR and dPCR-based assays.