Cut point determination is a critical component in the development and validation of antidrug antibody (ADA) assays, directly influencing the assessment of immunogenic responses. Removal of analytical and biological outliers from employing Tukey's outlier test may lead to reduced variability and consequently low or ultra-low cut points (ULCPs, defined here as signal-to-noise ratios ≤1.10). Such ULCPs raise concerns about assay reliability and the potential for elevated false-positive rates. To evaluate this, we analyzed ADA studies from two contract research organizations employing ULCPs. Given the heightened assay sensitivity from modern platforms and reagents, our findings indicate that ULCPs do not inherently compromise ADA assay performance or reliability. These results support the continued use of ULCPs when appropriately validated and statistically justified.

The 19th Workshop on Recent Issues in Bioanalysis (19th WRIB) took place in New Orleans, LA, USA on April 7-11, 2025. Over 1200 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 19th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on "Implementation Practice for the Newest ELN/LIMS Systems" and on "Vaccine Cell-Based/Functional & Molecular Assays as part of the harmonization of vaccine clinical assays global initiative" were the special features of the 19th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and Regulatory Agency 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 2025 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 2025 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1) covers in the Part 1A the recommendations on Mass Spectrometry Assays and Regulated Bioanalysis/BMV and in Part 1B the Regulatory Inputs on these topics. Part 2 (Biomarkers/BAV, IVD/CDx, Ligand-Binding Assays and Cell-Based Assays) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 18 of Bioanalysis, issues 1 and 2 (2026), respectively.

SB225002 was a selective non-peptide antagonist of CXC Chemokine Receptor 2 (CXCR2), but its detection method in mass spectrometry and pharmacokinetic (PK) parameters in mouse were not reported. Following methanol-induced protein precipitation and centrifugation, the analyte was separated using an Agilent ZORBAX SB-C18 column (2.1 mm × 100 mm, 3.5 μm, Agilent, MA, USA) with an isocratic mobile phase comprising 25% methanol (phase A) and 75% water containing 0.3% formic acid (phase B). The mobile phase was delivered at a flow rate of 0.3 mL/min. The PK of SB225002 were assessed in a mouse model. SB225002 demonstrated a strong linear relationship in range of 20.00 to 4000.00 ng/mL, with recovery between 96.9% and 99.6%, and matrix effects ranging from 98.9% to 104.6%. SB225002 demonstrated acceptable short-term, long-term, and freeze-thaw stability, with all other items meeting the requirements. The method was utilized to assess PK of SB225002 in mouse, revealing rapid absorption (tmax: 0.4 ± 0.1 h, mean±SD) and metabolism (t1/2: 3.4 2.6 h), along with significant inter-individual variability. This study successfully developed and validated a UHPLC-MS/MS method to determine SB225002 in mouse plasma and first reported its pharmacokinetic parameters in mouse, potentially facilitating further clinical research.

Advances in analytical techniques now enable biomarker detection from sample volumes as small as one microliter, reducing the need for large-volume specimen collection. This capability has supported the development of low-volume blood collection devices that allow frequent, longitudinal sampling outside traditional healthcare facilities, promoting decentralized and patient-centered sampling (PCS). The development of user-friendly, at-home sampling devices with reduced invasiveness and minimized sampling discomfort further enhances participant recruitment and retention in long-term longitudinal studies. These longitudinal studies are essential for tracking disease progression, uncovering molecular mechanisms underlying disease, and gaining insights into how external stimuli, such as diet, medication and physical activity, affect physiological responses. In this review, we highlight current low-volume sampling technologies and their role in longitudinal PCS environments, alongside biomarker detection assays for small-volume specimens.

ABSTRACTBackgroundLC-MS/MS is widely used for the quantification of phosphorodiamidate morpholino oligomers, but its low sensitivity is a problem. In addition, solid-phase extraction, a pretreatment method generally used for LC-MS/MS analysis, results in low recovery when applied to phosphorodiamidate morpholino oligomers. Therefore, a highly sensitive bioanalytical method for the determination of phosphorodiamidate morpholino oligomers using LC-MS/MS with improved pretreatment is desired.ResultsProtein precipitation was applied to LC-MS/MS analysis for viltolarsen, as a typical phosphorodiamidate morpholino oligomer, in plasma, and PPT with MeOH achieved high recoveries (>90%). Reversed-phase liquid chromatography (RPLC) with gradient elution improved ion suppression compared to hydrophilic interaction liquid chromatography. A combination of PPT and RPLC achieved higher sensitivity (lower limit of quantification, 1 ng/mL) than those of approved phosphorodiamidate morpholino oligomers (10 to 20 ng/mL in their clinical reviews), and the between-run accuracy and precision ranged from 97.1% to 109% and 2.56% to 10.5%, respectively. This method was also shown to be applicable to another phosphorodiamidate morpholino oligomer, ψM23D (+07–18).ConclusionA simple and highly sensitive bioanalytical method for the determination of viltolarsen was established by optimizing RPLC and PPT, and the method was successfully applied to a phosphorodiamidate morpholino oligomers with a different sequence.

BPI-43487 is a novel irreversible fibroblast growth factor receptor 4 inhibitor. A simple and reliable liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of BPI-43487 and its active metabolite BPI-43739 in human plasma. BPI-43487B was used as the internal standards (IS). Plasma samples were protein precipitated by acetonitrile and processed samples were chromatographed on an AQUITY UHPLC BEH C18 column (50 × 2.1 mm, i.d. 1.7 μm) with acetonitrile and 10 mM ammonium acetate containing 0.1% formic acid (v/v) as the mobile phase. Calibration curves demonstrated good linearity (R ≥0.99) over the concentration range of 10.0-5000 ng/mL for both analytes. Intra- and inter-batch precisions were ≤9.2% for BPI-43487 and ≤8.7% for BPI-43739. The accuracies were 94.6-104.8% for BPI-43487 and 95.0-108.4% for BPI-43739. This method was further successfully applied to a pharmacokinetic study of BPI-43487 capsules in Chinese patients with advanced solid tumors. http://www.chinadrugtrials.org.cn is CTR20210565).

Immunogenicity assessments are critical for evaluating biological therapeutics. Preexisting immune reactivity can affect clinical safety and efficacy and confound anti-drug antibody (ADA) assay cut point determination, complicating identification of treatment-emergent ADA. In the ALXN-X program, approximately 50% of naïve human serum samples showed preexisting reactivity, with some signals exceeding background by more than 100-fold, challenging ADA method development (cut points, sensitivity, drug tolerance). Competitive inhibition experiments were conducted to map the binding site of the preexisting reactivity and depletion with protein A/G agarose was performed to further characterize the preexisting reactivity. A modified ADA assay with an engineered drug molecule was developed to mitigate its impact on assay performance. This preexisting reactivity binds to the C-terminal conserved framework sequence of the drug molecule. Characterization indicated that the preexisting reactivity is likely attributable to immunoglobulins. The modified ADA assay can effectively mitigate the impact of the preexisting reactivity on the assay performance, while maintaining the ability to detect ADA response specific to the unique epitopes of ALXN-X. Preexisting, immunoglobulin-mediated reactivity targeting ALXN-X's Cterminal framework sequence complicates ADA assay performance. A modified ADA assay mitigates this interference while preserving detection of ALXN-X-specific ADA, supporting more reliable immunogenicity assessment.

ABSTRACTIntroductionRoutine conjugation protocols are typically used by bioanalytical laboratories for production of their assay critical reagents. Novel molecules can pose unique challenges to the production of high-quality conjugated critical reagents required for clinical bioanalytical assays. Using routine conjugation protocols, we observed gross instability of conjugated-drug surrogate material for use in antidrug antibody (ADA) assays in clinical autologous Chimeric Antigen Receptor (CAR)-T cell programs, thus halting assay development.MethodsWe highlight our approach in developing process conditions for CAR-T supporting conjugated critical reagents, where recombinant CAR scFv-hFc is the surrogate drug material to be conjugated. We show that when routine platform process approaches are not appropriate to produce conjugated novel molecule critical reagents, in-silico modeling can be used to determine conditions imparting repeatable generation of stable reagents. Implementing this modeling is advantageous as it decreases laborious efforts to develop stable novel critical reagent production methodologies.ConclusionAs a result of these studies herein, optimal stable conjugated critical reagents were produced which enabled successful development and validation of clinical CAR-T ADA assay. This work provides a framework that can be applied to the production of other bioanalytical assay critical reagents when platform conjugation approaches fail.