The assay validation process
Assay validation ensures the assay produces accurate, reliable, and reproducible results. Although validation is an investment requiring time and resources, it ultimately saves costs by reducing the risk of assay failure, false results, and the need for repeat experiments or additional testing.
PCR is highly sensitive, but understanding the underlying processes is essential to produce reliable data. First, nucleic acids are exposed to nucleases and can degrade quickly if not handled properly. Second, PCR technology’s sensitivity necessitates thorough assay validation for each specific target, sample type, and matrix. This ensures that extraction recovery, nucleic acid degradation, PCR efficiency, and assay specificity are properly controlled, resulting in accurate and reproducible concentration measurements.
The complexities of extraction and analysis arise from the fact that each drug item behaves differently, even between similar ones, such as two different siRNAs or two different messenger RNAs, due to variations in secondary structures, modifications, and other properties. Another challenge is the diverse sample types analyzed in biodistribution and shedding studies, which may introduce different matrix effects impacting PCR efficiency, and present recovery challenges. The validation ensures that the analysis meets stringent regulatory requirements and performs as expected throughout the preclinical and clinical stages.
Method development
With a thoroughly developed method, the validation process becomes straightforward. The time-consuming aspect is the method development, which can be significantly expedited by leveraging expert knowledge and experience. By using established workflows, best practices for design, and carefully selecting kits and instruments, the optimization time can be greatly reduced.
Method development encompasses every step, from the raw organ specimen to the final result.
Biopsy sampling: Considerations on how the dissection is performed, what surrounding tissue may be included, the composition of cell types or organ pieces, whether the sample should be subdivided into different parts of the organ or tumor, and how it is frozen. The type of anticoagulant and tube used are essential for biofluids to simplify downstream extraction.
Tissue processing: Homogenization or lysis, depending on the specific tissue type.
Nucleic acid extraction: This process depends on whether RNA or DNA is being extracted, any modifications, the length of the nucleic acids, and other relevant features. Ideally, the extraction recovery should be checked with the actual test item, as control spikes may show more uniform performance across different kits.
Automation: The extraction process is automated using one of our liquid handlers for standardization. We have established workflows that yield high-quality RNA and DNA from common tissues and biofluids, such as RNA extraction from blood using PAX tubes. We also perform manual extractions when it is the best option for the sample.
cDNA synthesis: Not all RT enzymes perform equally well with the same target and matrix. Target availability is also crucial, as secondary structures may interfere with primer binding. The choice of cDNA primer, whether gene-specific priming, oligo(dT) primers, or random hexamers, can significantly influence cDNA synthesis and, consequently, the quantification accuracy.
Primer and probe design: Optimized to enhance PCR efficiency, specificity, and selectivity, with strategies employed to discriminate between similar sequences.
Data normalization: The samples are normalized against mass/volume, cell number, total RNA/DNA amount, alien spike, or ribosomal RNA, depending on the assay circumstances. When using a reference gene, we evaluate and select the one that demonstrates the most stability across experimental conditions and has an expression level similar to the target gene.
Quality control (QC): All non-automated steps are QC-tested, including data transfer steps and calculations.
The analytical performance characteristics are determined during method development and later confirmed during validation. These characteristics include primer and probe set evaluation, assessing qPCR efficiency, and positive/negative separation in digital PCR (dPCR). Additionally, PCR optimization, calibration curve optimization (for qPCR), extraction optimization, recovery, extraction efficiency, precision, accuracy, specificity, selectivity, and sensitivity of the assay are thoroughly evaluated.
Assay validation
Assay validation involves validating the entire process, from tissue sample preparation to target concentration measurement.
The performance characteristics tested during method validation include the assay’s precision, accuracy, PCR efficiency, dilutional linearity, and co-linearity. The sensitivity of the assay is confirmed by determining the limit of blank (LOB), the limit of detection (LOD), and the lower limit of quantification (LLOQ). Specificity and selectivity are tested using matrices without the test items. Sample stability and extraction efficiency are also assessed. The robustness and ruggedness of the assay are evaluated by running the assay multiple times, with different analysts, on separate days and using different instruments.
The validation is performed on spiked blank samples, also used as assay performance controls during sample analysis (QC samples) and for the calibration curve (qPCR only). A matrix is a sample devoid of the analyte and should ideally match the biological sample. The reference material should, if possible, be the drug product. Still, it could also be plasmid DNA for biodistribution studies, whether the DNA is free in the cytosol or encapsulated DNA for shedding assays to ensure that DNA is released during extraction. QC samples and calibrators should be derived from different stock solutions.
The intra- and inter-assay precision should be ≤ 30% coefficient of variation (%CV) for QCs and ≤ 50% CV for LOQs, whether using interpolated qPCR results or absolute dPCR copy number results.
Intra- and inter-assay accuracy for qPCR should range from –50% to +100% relative error (RE) on interpolated copies. This is because the doubling nature of qPCR means that a difference of just 1 Cq can result in the interpolated result being either half or twice the nominal concentration. For dPCR, the inter-assay accuracy for absolute copy numbers should have a relative error (%RE) of ≤ 30% for QCs and ≤ 50% for LOQs.
A PCR efficiency of 100% means that all target sequences are doubled in each cycle. PCR efficiencies between 90% and 110% are acceptable, as demonstrated by a slope between −3.1 and −3.6 in the standard curve. The linear regression should have an R² value of ≥ 0.98.
Dilution linearity demonstrates that the slope remains linear even when the sample is diluted. Co-linearity refers to the scenario where multiple matrices show similar linearity within the assay range, allowing one matrix to be used as a surrogate for the others.
The limit of blank (LOB) is determined as the concentration at which the sample is negative with 95% confidence, while the limit of detection (LOD) is the concentration at which the sample is positive with 95% confidence.
Specificity and selectivity are confirmed when 100% of the unspiked matrices show results below the LOD, and at least 8 out of 10 spiked samples meet the precision and accuracy criteria for the LLOQ.
Stability is confirmed using QC samples that undergo the exact handling as the actual samples, including long-term stability testing. The extraction efficiency is validated by spiking the sample and then measuring the extraction efficiency and recovery.
Sample analysis
A sample analysis run includes QC samples prepared in sets for each plate at different concentration levels, calibrators for qPCR, and blank matrices as negative controls. RT-NTC (Reverse Transcription No-Template Control) can be included to check for DNA contamination in the cDNA.
The run is accepted if all the QC samples and the calibration curve meet the defined precision and accuracy criteria. The R² value of the calibration curve must also meet the required threshold. Additionally, all negative controls must be negative, and the analyzed sample must fall within the assay’s range.
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TATAA Biocenter is accredited by the Swedish Board of Accreditation and Conformity Assessment (SWEDAC) for complying with Good Laboratory Practice (GLP) standards. We also adhere to Good Clinical Laboratory Practice (GCLP) guidelines, which are international quality standards governing the analysis of samples from clinical trials using GLP.
GLP and GCLP validation is mandatory in preclinical and clinical assays to demonstrate accuracy, reliability, and consistency. These standards cover every aspect of study planning, execution, monitoring, documentation, archiving, and reporting. This rigorous process assures both the sponsors, when submitting data for new drug approvals, and the regulatory authorities, when reviewing the data, that it is generated in a regulated manner.
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Committed to quality, innovation, and fostering strong client relationships, we serve as your trusted partner to accelerate drug development. As a contract research organization, our mission is to push the boundaries of science and technology to generate accurate and reproducible data that shortens time-to-market.
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We are GLP accredited for qPCR, dPCR, and molecular biology, GCLP compliant, and ISO/IEC 17025 accredited. Our facility features a strict sample management process, a fully integrated LIMS system, backup for all vital systems, temperature and humidity control, and robust IT security.
Purpose-built PCR laboratory
Our purpose-built laboratory in Gothenburg, Sweden, is specifically designed for PCR. It has controlled air pressure, temporal separation, and biosafety cabinets to minimize contamination risk. This setup enables us to achieve the highest sensitivity and robustness required for validated assays. The lab is equipped with market-leading PCR and NGS instruments and advanced liquid handling systems.
Pioneers in assay development and validation
We are a team of 45 employees, with scientists at the forefront of assay development, optimization, and validation. Our team has co-authored key publications in the field, including Recommendations for Method Development and Validation of qPCR and dPCR Assays in Support of Cell and Gene Therapy Drug Development (AAPS J. 2024) and The MIQE Guidelines for qPCR and dPCR.
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We work closely with our clients to find tailored solutions, offering flexibility in sample types, test items, sample volume, and scalability. We prioritize transparency and proactive communication throughout each project, ensuring our clients are continuously updated on the progress.
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