2022 White Paper on Recent Issues in Bioanalysis: ICH M10 BMV Guideline & Global Harmonization; Hybrid Assays; Oligonucleotides & ADC; Non-Liquid & Rare Matrices; Regulatory Inputs (Part 1A - Recommendations on Mass Spectrometry, Chromatography and Sample Preparation, Novel Technologies, Novel Modalities, and Novel Challenges, ICH M10 BMV Guideline & Global Harmonization Part 1B - Regulatory Agencies' Inputs on Regulated Bioanalysis/BMV, Biomarkers/CDx/BAV, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 16th Workshop on Recent Issues in Bioanalysis (16th WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on the ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Mass Spectrometry and ICH M10. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (LBA, Biomarkers/CDx and Cytometry) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 15 and 14 (2023), respectively.

Chemical derivatization as a novel strategy for selective and sensitive determination of intracellular di-and triphosphate anabolites in peripheral blood mononuclear cells.

Quantitation of intracellular tri-phosphate anabolite, GSK1-TP, was required to understand drug efficacy of a proprietary molecule, GSK1; while quantitation of the di-phosphate, GSK1-DP, provided an indicator of potential GSK1-TP instability during sample processing and storage. A novel derivatization approach with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and hexylamine was developed to mitigate the challenges associated with the analysis of GSK1-DP and GSK1-TP, rendering them more amendable to reversed-phase LC-MS/MS detection. Extensive effort was spent to minimize the analyte loss and cell counts variability during peripheral blood mononuclear cells (PBMC) collection and best practices were thoroughly discussed. A solution of 30/70/2 (v/v/v) RPMI-1640/methanol/trichloroacetic acid proved to be an effective method of analyte stabilization and recovery. Methods were developed for simultaneous analysis of GSK1-DP and GSK1-TP with a limit of quantitation of 2.0 ng/mL in dog and rat PBMC lysate, with a subsequent improvement to 0.1 ng/mL for the analysis of GSK1-TP in human PBMC lysate. All three assays were found to be robust over the PBMC concentration range of 1-25 × 106 lysed cells/mL. This novel methodology could alleviate some challenges associated with the bioanalysis of intracellular phosphorylated anabolites such as PBMC collection variability, analyte instability, poor chromatographic retention, high carryover, matrix effect and insufficient assay sensitivity.

Strategies for effective development of ultra-sensitive LC-MS/MS assays: application to a novel STING agonist.

Background: The continual need for the development and validation of ultra-sensitive (low pg/ml) LC-MS/MS assays in the pharmaceutical industry is largely driven by the ultra-low analyte exposure or very low sample volume. Methodology: Strategies and systematic approaches for sensitivity enhancement are provided which cover all aspects of a LC-MS/MS bioanalysis. A case study where such strategies were applied for the validation of a 5.0 pg/ml assay for a STING agonist is discussed. Conclusion: Analytical protocols were developed to extract analytes from large volume of plasma samples (600 and 400 µl) with high throughput. The guidance provided in this publication can serve as a resource to influence LC-MS/MS method development activities.

Drug monitoring by volumetric absorptive microsampling: method development considerations to mitigate hematocrit effects.

AIM: GSKA is a compound that was in development in clinical trials. A bioanalysis method to quantify GSKA using volumetric absorptive microsampling (VAMS) was developed and hematocrit (HCT) related assay bias was investigated. METHODOLOGY: After accurate sampling of 10 µl blood, VAMS tips were air dried approximately 18 h and desorbed by an aqueous solution containing internal standard. The recovered blood underwent liquid-liquid extraction in ethyl acetate to minimize matrix suppression. Assay accuracy, precision, linearity, carryover, selectivity, recovery, matrix effects, HCT effects and long-term quality control stability were evaluated. CONCLUSION: HCT-related assay bias was minimized in 30-60% blood HCT range, and all validation parameters met acceptance criteria. The method is suitable for quantitative analysis of GSKA in human blood.

Camphanic acid chloride: a powerful derivatization reagent for stereoisomeric separation and its DMPK applications.

BACKGROUND: Camphanic acid chloride has proven to be an efficient chiral derivatization reagent for determination of stereoisomers. RESULTS: The utility of chemical derivatization of various stereoisomers containing hydroxy functional groups with camphanic acid chloride in the presence or absence of a base is highlighted. This procedure is shown to be relatively simple, fast and a cost-effective method of separating racemic drugs and stereoisomeric metabolites in biological matrices. Camphanic derivatives contain two additional chirogenic centers, which are found to enhance stereoisomeric separation on both traditional and chiral stationary phases. CONCLUSION: Four methodologies described herein for separation of multiple stereoisomers in biological samples confirm camphanic acid chloride to be a powerful chiral reagent for stereoisomeric resolution for drug metabolism and PK applications.