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Validation of biomarker assays is crucial for effective drug development and clinical applications. Interlaboratory reproducibility is vital for reliable comparison and combination of data from different centers. This review summarizes interlaboratory studies of quantitative LC-MS-based biomarker assays using reference standards for calibration curves. The following points are discussed: trends in reports, reference and internal standards, evaluation of analytical validation parameters, study sample analysis and normalization of biomarker assay data. Full evaluation of these parameters in interlaboratory studies is limited, necessitating further research. Some reports suggest methods to address variations in biomarker assay data among laboratories, facilitating organized studies and data combination. Method validation across laboratories is crucial for reducing interlaboratory differences and reflecting target biomarker responses.

The 15th Japan Bioanalysis Forum (JBF) Symposium was held in Kyoto, Japan, between 5 and 7 February 2024. The conference theme, 'Toward the new world - Science as a universal endeavor' indicated that universal discussion based on science is making a basis of regulatory and analytical sciences, now internationally harmonized. The symposium discussed a wide range of topics, including ICH M10, quantitative PCR, immunogenicity, peptide LC-MS, e-notebooks, artificial intelligence, reliability standards, carrier development, inviting domestic and overseas experts. Approximately 360 attendees from various fields, including pharmaceutical companies, contract research organizations, academia and regulators, gathered in person or online.

Nucleic acid (NA) biomarkers play critical roles in drug development. However, the global regulatory guidelines for assessing quantification methods specific to NA biomarkers are limited. The validation of analytical methods is crucial for the use of biomarkers in clinical and post-marketing evaluations of drug efficacy and adverse reactions. Given that quantitative polymerase chain reaction (qPCR) and reverse transcription qPCR (RT-qPCR) methods are the gold standards for the quantification of NA biomarkers, the Biomarker Analytical Method Validation Study Group in Japan has discussed considerations and made recommendations for the development and validation of qPCR- and RT-qPCR-based analytical methods for endogenous NA biomarkers as drug development tools. This white paper aims to contribute to the global harmonization of NA biomarker assay validation.

Background: Although the fit-for-purpose approach has been proposed for biomarker assay validation, practical data should be compiled to facilitate the predetermination of acceptance criteria. Methods: Immunoaffinity LC-MS was used to analyze glucagon-like peptide-1 as a model biomarker in six laboratories. Calibration curve, carryover, parallelism, precision, relative accuracy and processed sample stability were evaluated, and their robustness among laboratories was assessed. The rat glucagon-like peptide-1 concentrations in four blinded samples were also compared. Results: The obtained results and determined concentrations in the blinded samples at all laboratories were similar, with a few exceptions, and robust, despite the difference in optimization techniques among laboratories. Conclusion: The results provide insights into the predefinition of the acceptance criteria of immunoaffinity LC-MS-based biomarker assays.

Background: Many bioanalytical methods for antisense oligonucleotides (ASOs) using LC-MS have been reported. However, no data have been available on the reproducibility and robustness of a single bioanalytical method for ASOs. As such, in the current study, we evaluated the reproducibility and robustness of LC-MS-based bioanalytical methods for ASOs in multiple laboratories. Methods/Results: Seven independent laboratories were included in this study. Mipomersen was measured by ion-pairing LC-MS (IP-LC-MS) as a model ASO using different LC-MS. The validation results of calibration curve, accuracy, precision and selectivity met the criteria of conventional bioanalytical method validation guidelines using LC/GC-MS for drugs in all laboratories. Meanwhile, carryover (>20%) was detected in three laboratories. Conclusion: We first demonstrated the multicenter-validated IP-LC-MS bioanalytical method for ASOs. Our data showed that the method was sensitive, robust and reproducible. However, the occurrence of carryover should be carefully monitored in its future application.

Biomarkers are an important drug developmental tool. Assessment of quantitative analytical methods of biomarkers is not included in any regulatory documents in Japan. Use of biomarkers in clinical evaluations and supporting the post-marketing evaluation of drug efficacy and/or adverse reactions requires assessment and full validation of analytical methods for these biomarkers. The Biomarker Analytical Method Validation Study Group is a research group in Japan comprising industry and regulatory experts. Group members discussed and prepared this 'points to consider document' covering measurements of endogenous metabolites/peptides/proteins by ligand binding assays and chromatographic methods with or without mass spectrometry. We hope this document contributes to the global harmonization of biomarker assay validation.

Background: Ion-pairing reverse-phase LC coupled with high-resolution mass spectrometry (IP-LC/HRMS) has gained attention in oligonucleotide therapeutic bioanalyses owing to its high sensitivity and selectivity. However, optimization and validation of IP-LC/HRMS-based methods are rare. The objective of this study is the development of a sensitive and reproducible IP-LC/HRMS-based bioanalytical method using clinically approved mipomersen as a model for antisense oligonucleotides. Materials & methods/results: Mipomersen was extracted from rat plasma using Clarity OTX SPE and quantified by IP-LC/HRMS. The calibration range was 0.5-250.0 ng/ml. The developed method met the general regulatory criteria for accuracy, precision, carry-over, selectivity, matrix effect and dilution integrity. Conclusion: A highly sensitive and reliable method for mipomersen measurement with potential antisense oligonucleotide bioanalysis applications has been developed.