Bo Y, Lu J, Jiang M
… +6 more, Liu X, Le L, Liu X, Lan H, Gong J, Yang F
Bioanalysis
· 2026 Jan · PMID 41665416
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BACKGROUND: BPI-43487 is a novel irreversible fibroblast growth factor receptor 4 inhibitor. RESEARCH DESIGN AND METHODS: A simple and reliable liquid chromatography-tandem mass spectrometry method was developed and vali...BACKGROUND: BPI-43487 is a novel irreversible fibroblast growth factor receptor 4 inhibitor. RESEARCH DESIGN AND METHODS: 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 C 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. RESULTS: Calibration curves demonstrated good linearity ( ≥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. CONCLUSIONS: This method was further successfully applied to a pharmacokinetic study of BPI-43487 capsules in Chinese patients with advanced solid tumors. CHINA DRUG TRIALS: http://www.chinadrugtrials.org.cn is CTR20210565).
Balińska N, Lemska A, Mazurkiewicz-Bełdzińska M
… +1 more, Studzińska S
Bioanalysis
· 2026 Jan · PMID 41640372
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BACKGROUND: Risdiplam has been used to treat spinal muscular atrophy for 3 years. There are limited number of papers devoted to its analytics. Until now, risdiplam and its metabolites have only been analyzed using a C18...BACKGROUND: Risdiplam has been used to treat spinal muscular atrophy for 3 years. There are limited number of papers devoted to its analytics. Until now, risdiplam and its metabolites have only been analyzed using a C18 column, while the sample preparation method involved protein precipitation. RESEARCH DESIGN AND METHODS: Risdiplam was analyzed using reversed-phase UHPLC. The experiment was designed to compare the retention of risdiplam on five columns using various mobile phases. The protein precipitation was used as the sample preparation method. RESULTS: Risdiplam shows greater retention on phenyl columns, where π-π interactions take part in retention. The increase of mobile phase pH caused increased risdiplam retention, while salt concentration had no significant effect. An octadecyl column with pentafluorophenyl groups was selected with a mobile phase containing 10 mM ammonium formate (pH 4) and acetonitrile. The method was characterized by good linearity, repeatability, and short analysis time. It was applied to risdiplam analysis in serum samples after protein precipitation with different solvents. Finally, proteins were effectively precipitated using 10% TFA solution, providing 90% recovery. CONCLUSIONS: The developed procedure of extraction and determination of risdiplam is simple, fast and reliable. It may find application for routine monitoring of risdiplam or for quality control.
Yang X, Qin H, Ling J
… +4 more, Dong L, Jiang Y, Zou S, Hu N
Bioanalysis
· 2026 Jan · PMID 41636591
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OBJECTIVES: Disruption of bile acid homeostasis is implicated in the pathogenesis and progress of several liver diseases, including drug-induced liver injury (DILI). A rapid liquid chromatography-tandem mass spectrometry...OBJECTIVES: Disruption of bile acid homeostasis is implicated in the pathogenesis and progress of several liver diseases, including drug-induced liver injury (DILI). A rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying 30 bile acids in human serum was developed and applied to analyze serum bile acid profile in voriconazole administered patients with invasive fungal infections. METHODS: The stable isotope-coded bile acids were used as internal standards. The analytes in serum samples (50 μL) were extracted by protein precipitation and isolated by a Kinetex EVO C column (50 × 2.1 mm, 2.6 μm). The detection was performed using multiple reaction monitoring (MRM) in electrospray negative ionization mode. RESULTS: All of the 30 bile acids were sufficiently separated within 10 min with good linearity ( > 0.99) over tested calibration ranges. The accuracy and precision values were in the range of 85.42-114.3% and 1.8-13.8%, respectively. The matrix effect, recovery, stability, and dilution integrity all met the acceptance criteria. Serum bile acid profile changed in patients with supratherapeutic voriconazole concentration (>10 μg/mL). CONCLUSIONS: This LC-MS/MS method for the quantification of 30 bile acids in human serum might apply a new analytical approach for liver function evaluation.
Chen J, Zhao H, Gardner C
… +6 more, Wang J, Chen M, Li M, Amancha P, Rajadhyaksha M, Pelto R
Bioanalysis
· 2026 Jan · PMID 41630589
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BACKGROUND: 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 dete...BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
Bioanalysis
· 2025 Dec · PMID 41622783
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Pre-existing anti-drug antibodies (pre-ADA) pose a significant challenge in the development of biotherapeutics. This article underscores the importance of early pre-ADA screening in mitigating adverse immune responses an...Pre-existing anti-drug antibodies (pre-ADA) pose a significant challenge in the development of biotherapeutics. This article underscores the importance of early pre-ADA screening in mitigating adverse immune responses and optimizing therapeutic efficacy. Drawing on collaborative insights from three pharmaceutical companies, the paper presents a comparative analysis of pre-ADA assessment strategies, including timing, assay formats, and risk thresholds. It details how each company integrates pre-ADA data into immunogenicity risk evaluation, emphasizing the shift toward earlier screening during lead candidate selection. The article also explores assay-specific considerations, such as detection sensitivity and isotype coverage, and outlines criteria for ranking molecular candidates based on pre-ADA reactivity. By characterizing positive pre-ADA responses and proposing standardized practices, the paper offers actionable guidance for incorporating pre-ADA screening into preclinical workflows. This work highlights the growing relevance of pre-ADA assessments in an increasingly complex biopharmaceutical landscape and advocates for harmonized methodologies to support the development of safe and effective medicines.
Bioanalysis
· 2025 Dec · PMID 41612967
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In recent years, the study of extracellular vesicles (EVs) has gained increasing attention due to their pivotal role in intercellular communication and their potential as noninvasive biomarkers and innovative therapeutic...In recent years, the study of extracellular vesicles (EVs) has gained increasing attention due to their pivotal role in intercellular communication and their potential as noninvasive biomarkers and innovative therapeutic tools. Their molecular cargo, shaped by the source cell and its microenvironment, offers an accurate mirror of cellular states and pathophysiological dynamics. Characterizing this content provides critical insights into disease mechanisms, while paving the way for novel diagnostic, prognostic and therapeutic strategies. Among their components, proteins and metabolites stand out for their functional relevance and biomarker potential. In this context, proteomics and metabolomics have emerged as key approaches to unravel the molecular complexity of EVs and clarify their biological functions, thereby advancing their translation into clinical applications. This review highlights the contributions of these disciplines, outlines associated analytical workflows, and discusses major technical challenges. A systematic literature search was conducted in PubMed, Web of Science, Scopus, and Google Scholar for studies published between 2012 and 2025, focusing on proteomic and metabolomic analyses of EVs. Emphasis is placed on the need for methodological consensus to standardize protocols, improve inter-laboratory reproducibility, and support the successful clinical application of EV-based strategies.
Zhang Y, Liu Y, Yuan C
… +7 more, Zhuge W, Zou B, Dong L, Wu X, Ouyang Z, Zhang M, Wang H
Bioanalysis
· 2026 Jan · PMID 41612943
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BACKGROUND: As the synthetic abused drug with high addictive potential, methamphetamine (METH) and its major metabolite amphetamine (AMP) are chiral compounds. The -enantiomer of METH is primarily abused because of its p...BACKGROUND: As the synthetic abused drug with high addictive potential, methamphetamine (METH) and its major metabolite amphetamine (AMP) are chiral compounds. The -enantiomer of METH is primarily abused because of its potent psychoactive effects, whereas the R-enantiomer may originate from the metabolism of selegiline, a prescription medication for Parkinson's disease. RESEARCH DESIGN AND METHODS: This research aimed to develop a robust and reliable analytical method to distinguish illicit METH abuse from legal selegiline therapy. A novel, simplified chiral stationary phase liquid chromatography-tandem mass spectrometry (CSP-LC-MS/MS) method was developed and validated for the rapid determination of - and S-enantiomers of METH and AMP in human hair, eliminating the need for derivatization pretreatment. RESULTS: Employing an Agilent Chiral-V column under isocratic conditions, the developed CSP-LC-MS/MS method achieved efficient baseline separation (resolution ≥2) and rapid quantification of the / enantiomers of METH and AMP within 10 min. Analysis of hair samples from three METH abusers revealed a predominance of the -enantiomers. Conversely, only the R-enantiomer was detected in the hair of a selegiline user. CONCLUSIONS: This research enables precise enantiomer differentiation, offering critical insights into drug metabolism and forensic discrimination between illicit METH abuse and legitimate selegiline treatment.
Hersey S, McGuire K, Kholmanskikh O
… +74 more, Bivi N, Bandukwala A, Gong B, Irwin C, Ray S, Gunsior M, Avanesov A, Baker B, Bond S, Braun A, Buoninfante A, Dessy F, Fang X, Garofolo F, Gomme E, Hays A, King L, Mayer C, McGregor J, McHenry M, Nowocin A, Rubel C, Sanderink G, Scott B, Scully I, Seyda A, Stoop J, Tang H, Neto JT, Walravens K, Wang K, Wassmer S, Xiao W, Zhu L, Zoghbi J, Brockus C, Petit-Frere C, Coble K, Fischer S, Yearwood G, Cao L, Dysinger M, Grimaldi C, Jiang Y, Joyce A, Kerridge C, Lu K, McCush F, Nolan K, Connor EO, Palmer R, Reese K, Stubenrauch KG, Verch T, Beaver C, Mendez L, Decman V, Bhavaraju K, Huleatt J, Trampont PC, Eck S, Goihberg P, Alcaide EG, Hedrick MN, Jalah R, McGrath S, Ogorman W, Prior S, Sehra S, Shaik S, Standifer N, Stevens C, Stevens E, Sun YS
Bioanalysis
· 2025 Dec · PMID 41609129
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The 19 Workshop on Recent Issues in Bioanalysis (19 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 conv...The 19 Workshop on Recent Issues in Bioanalysis (19 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 19 WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week 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 19 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 2) covers in the Part 2A the recommendations on Biomarkers/BAV, IVD/CDx, Ligand-Binding Assays and Cell-Based Assays and in Part 3B the Regulatory Inputs on these topics. Part 1 (Mass Spectrometry Assays and Regulated Bioanalysis/BMV) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 18 of Bioanalysis, issues 3 and 1 (2026), respectively.
Deshpande K, Hong M, Ware M
… +2 more, Li M, Dysinger M
Bioanalysis
· 2025 Dec · PMID 41566963
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Bioanalytical (BA) data are foundational to clinical trial integrity and regulatory success. Yet, circumstances such as regulatory noncompliance, operational disruptions, resource limitations, or evolving analytical need...Bioanalytical (BA) data are foundational to clinical trial integrity and regulatory success. Yet, circumstances such as regulatory noncompliance, operational disruptions, resource limitations, or evolving analytical needs can necessitate a BA laboratory transition during a clinical trial. Through the lens of a real-world case study and supported by broader industry experience, this manuscript explores the operational, scientific, and regulatory challenges of BA laboratory transitions. We examine four potential scenarios that trigger such changes, the risks to data continuity and compliance, and the significant implications for sponsors. The manuscript outlines actionable, multidisciplinary strategies for managing transitions: from vendor qualification and project planning to Corrective and Preventive Action (CAPA) implementation, method transfer, and ongoing performance assessment. When guided by early risk recognition, transparent communication, and organizational learning, laboratory transitions though challenging, can be successfully navigated to protect trial outcomes and strengthen the resilience of clinical programs.
Rowe BA, Reese KJ, McGrath K
… +3 more, Concannon A, Schwartz AM, Gunn GR
Bioanalysis
· 2025 Dec · PMID 41549967
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AIM: To develop an anti-drug antibody (ADA) assay for a bispecific antibody (bsAb) we will refer to as Drug-1, and to quantify the ADAs against each specific fragment antigen-binding (Fab) site of Drug-1. MATERIALS AND M...AIM: To develop an anti-drug antibody (ADA) assay for a bispecific antibody (bsAb) we will refer to as Drug-1, and to quantify the ADAs against each specific fragment antigen-binding (Fab) site of Drug-1. MATERIALS AND METHODS: Here we use two additional unlabeled monoclonal antibodies (mAbs) Drug-2 & Drug-3 in an ADA confirmation assay format. Drugs-2 & -3 have the same targets as the two Fabs of Drug-1, facilitating our aim to measure and quantify the anti-drug Abs against each specific Fab of Drug-1 by signal inhibition. RESULTS AND CONCLUSION: By using the combination of a screening and a dual confirmation assay format, we were able to accurately quantify the ADAs against each specific Fab of the bsAb Drug-1.
Bioanalysis
· 2025 Dec · PMID 41527740
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Immunogenicity undermines the safety and effectiveness of biologics and emerging modalities. Routine workflows are constrained by drug/target interference in binding assays, matrix and dilution biases in functional assay...Immunogenicity undermines the safety and effectiveness of biologics and emerging modalities. Routine workflows are constrained by drug/target interference in binding assays, matrix and dilution biases in functional assays, fragile cellular/innate readouts, and the difficulty of translating titers into patient-level risk. Advances in artificial intelligence (AI) and real-world evidence (RWE) offer practical remedies. This narrative review searched five search engines and databases and included evidence from published original research, reviews, and regulatory data, and reports between 2022 and 2025. It identifies common failure locations and links them to practical AI and RWE solutions. We outline the regulatory and data underpinnings for decision-grade RWE, provide a closed-loop blueprint, and summarize recent developments in - and B-cell epitope prediction, neoantigen prioritization, and pharmacovigilance/NLP pipelines for case deduplication and normalization: Define laboratory panels and computable phenotypes, map multi-source data to a common model, deduplicate, train transparent internally validated models, externally validate across distributed networks, route outputs to clinic/lab actions, and continuously monitor for drift with auditable updates. AI-augmented RWE can replace assay-centric snapshots in immunogenicity assessment with a learning system that focusses on clinically significant ADA/NAb effects, targets limited laboratory resources, speeds up signal verification, and enhances post-market vigilance.
Bazzani V, Harding E, Balinski S
… +9 more, Redin ME, Alessi D, Del Mestre P, Baratta W, Cesselli D, Beltrami AP, Turek M, Baccarani U, Vascotto C
Bioanalysis
· 2025 Dec · PMID 41524377
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Accurate quantification of oxidative mitochondrial DNA (mtDNA) lesions remains technically challenging due to the limitations of existing assays, which often require large sample inputs, multi-day workflows, and offer li...Accurate quantification of oxidative mitochondrial DNA (mtDNA) lesions remains technically challenging due to the limitations of existing assays, which often require large sample inputs, multi-day workflows, and offer limited sensitivity. Here we introduce FALCON-qPCR (Fpg-assisted Long-PCR), a streamlined, high-sensitivity method for quantifying oxidative damage in mtDNA. FALCON-qPCR couples digestion with formamidopyrimidine [fapy]-DNA glycosylase (Fpg) to long-range PCR and qPCR-based normalization, enabling precise lesion quantification from as few as 10,000 cells (~300 ng total DNA) within a single day. The assay provides a robust dynamic range and reproducibility across diverse biological systems, including human cell lines, hepatocellular carcinoma biopsies, and . Compared with established methods, FALCON-qPCR exhibits markedly higher sensitivity in detecting mtDNA damage induced by hydrogen peroxide, antimycin A, and rotenone. Its performance was further demonstrated in assessing mitochondrial toxicity of ruthenium-based compounds, highlighting its potential for pharmacological screening. By integrating enzymatic lesion recognition with quantitative amplification in a unified workflow, FALCON-qPCR eliminates the need for mitochondrial isolation. This methodological advance provides a rapid, accurate, and scalable platform for studying oxidative DNA damage, with broad applicability in mitochondrial research and translational toxicology.
Yamada N, Niwa M, Hara H
… +23 more, Ibushi T, Koyama N, Miyayama T, Nagata M, Ohashi Y, Ohyama T, Okuzono T, Shimada E, Shimizu H, Takahashi M, Takamatsu Y, Yamamoto KI, Fukunaga C, Harada H, Ide R, Kawahara T, Kurisu H, Mizuochi M, Nakase Y, Oka T, Otsuka Y, Yamazaki M, Yasui K
Bioanalysis
· 2025 Dec · PMID 41510942
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The 16th Japan Bioanalysis Forum (JBF) Symposium was held from March 3 to 5, 2025, at the Himeji Culture and Convention Center (Arcrea Himeji). The theme for the symposium was 'Bioanalyst Network: Spread and Circulation...The 16th Japan Bioanalysis Forum (JBF) Symposium was held from March 3 to 5, 2025, at the Himeji Culture and Convention Center (Arcrea Himeji). The theme for the symposium was 'Bioanalyst Network: Spread and Circulation of Knowledge.' This theme symbolizes our commitment, based on the principle at the JBF foundation to provide a platform for discussion and interaction among bioanalysts in academia, industry, and government. It aims to restore the interaction among bioanalysts that has diminished during the Coronavirus disease 2019 (COVID-19) pandemic, and to discuss and share knowledge about rapidly evolving scientific technologies and the extensive insights of bioanalysis under regulatory. The symposium discussed a wide range of topics, including the latest mass spectrometric techniques, immunogenicity assessment, antibody - drug conjugate evaluation, biomarkers, ICH-M10, ICH-S12, data integrity, reliability assurance, and the interactive discussion program 'iDG,' inviting domestic and overseas experts. Approximately 400 attendees from various fields, including pharmaceutical companies, contract research organizations (CROs), academia, and regulators, gathered in person.
Bioanalysis
· 2025 Dec · PMID 41508948
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BACKGROUND: Reliable methods for measuring plasma concentrations of cannabidiol (CBD) and its metabolites are essential for pharmacokinetic studies. Several methods have been published, some involve complex procedures. T...BACKGROUND: Reliable methods for measuring plasma concentrations of cannabidiol (CBD) and its metabolites are essential for pharmacokinetic studies. Several methods have been published, some involve complex procedures. This study aimed to develop a simple yet robust method for quantitating CBD, 7-OH-CBD and 7-COOH-CBD in human plasma. RESEARCH DESIGN AND METHODS: Sample preparation was evaluated using various solvent systems. Analytical parameters were examined and optimized. The method was validated for sensitivity, accuracy, precision, selectivity, specificity, carryover, dilution integrity, long-term stability, freeze-thaw stability and recovery. RESULTS: A simple protein precipitation method using acetonitrile provided optimal sample preparation. All analytes were quantified in negative ionization mode, with complete separation achieved on a C column within 3.5 minutes using an isocratic mobile phase. The method met all validation criteria. Analytes remained stable under long-term storage at -30°C, through freeze-thaw cycles and at refrigerated temperatures, although 7-COOH-CBD showed a slight decrease after two months at refrigerated temperatures. CONCLUSION: This study presents a simple, rapid and robust bioanalytical method for the simultaneous quantification of CBD and its major metabolites across clinically relevant plasma concentrations. The method's reliability and efficiency make it highly suitable for routine clinical use and pharmacokinetic applications.
Bioanalysis
· 2025 Dec · PMID 41504465
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AIM: Quantification of drug concentrations in human skin is essential for understanding mechanisms of action in dermatologic therapeutics but remains analytically challenging. The study aimed to develop and validate a st...AIM: Quantification of drug concentrations in human skin is essential for understanding mechanisms of action in dermatologic therapeutics but remains analytically challenging. The study aimed to develop and validate a standardized tissue homogenization and liquid chromatography - tandem mass spectrometry workflow for quantifying povorcitinib in human skin biopsies. MATERIALS & METHODS: Key skin homogenization parameters, including solvent composition, processing volume, and homogenate density, were systematically evaluated. Wet and dry homogenization approaches were compared. Following protein precipitation, samples were analyzed using liquid chromatography - tandem mass spectrometry. RESULTS: Eighteen solvent systems and various processing volumes were evaluated, with three solvent mixtures producing homogeneous preparations. Limiting homogenization volumes to less than half tube capacity and using 20 mg/mL improved sample consistency. Povorcitinib stability under acidic and thermal conditions was confirmed. Comparable performance was observed between wet and dry homogenization methods, and a dry workflow was validated. CONCLUSIONS: This study provides the first systemic evaluation of human skin homogenization parameters and a direct comparison of wet and dry approaches. The validated dry homogenization method enabled accurate and reproducible quantification of povorcitinib in skin biopsies from a phase I clinical study and offers a robust framework for quantifying a broad range of analytes in human skin tissues. https://www.clinicaltrials.gov/study/NCT06505265 [clinicaltrials.gov] identifier is NCT06505265.
Pham KN, Bano N, Rubinstein LJ
… +4 more, Schiller J, Seghezzi W, Vazvaei-Smith F, Xu W
Bioanalysis
· 2025 Dec · PMID 41503629
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OBJECTIVE: This study detailed the troubleshooting and optimization of a challenging neutralizing antibody (NAb) assay using a previously published PEG precipitation, Acid Dissociation, and Biotin-drug as Assay Drug (PAB...OBJECTIVE: This study detailed the troubleshooting and optimization of a challenging neutralizing antibody (NAb) assay using a previously published PEG precipitation, Acid Dissociation, and Biotin-drug as Assay Drug (PABAD) method to overcome high drug interference. MATERIALS AND METHODS: A strong NAb positive control, PC3, showed poor recovery in the PABAD workflow. A PABAD-compatible PC, PC31 was selected among different anti-idiotype antibodies with varied kinetic profiles. A three-tier Design of Experiment (DOE) approach was applied to optimize assay conditions. Centrifugation-based Bluewasher protocols were also established to automate the handling of PEG pellets. RESULTS: The reason for the poor performance of PC3 with the PABAD method was identified, and a sensitive NAb assay was successfully developed using PC31. CONCLUSIONS: The poor recovery of PC3 was not due to incompatibility with the PABAD method, but rather to its non-specific interaction with serum factors. Once a specific NAb PC was identified, the NAb assay was successfully developed using PABAD, demonstrating a broad application of the method. DOE-assisted assay development cut the development time by half and meaningfully improved the assay sensitivity. Finally, the implementation of the Bluewasher in the pellet wash further reduced assay variability and enhanced automation of the PABAD method.
Tounekti O, Shubow S, Wassmer S
… +62 more, Van Tuyl A, Yang L, Gong B, Abhari MR, Kalina W, Myler H, DelCarpini J, Escobar D, Fiscella M, Gorovits B, Gupta S, Irvin SC, Irwin C, Jankowski W, Jawa V, Karauzum H, Kerridge C, Lu Y, Mayer C, McGregor J, Melton A, Murphy R, Nanra J, Nowocin A, Partridge MA, Prior S, Sauna Z, Scott B, Seitzer J, Stern M, Sugimoto H, Taylor M, Wong A, Xiao W, Xu Y, Yang TY, Yuill D, Kubiak RJ, Thway T, Gunn G, Pelto R, Sumner G, Baltrukonis D, Dholakiya SL, Dogmanits S, Garofolo F, Jadhav M, Kramer D, Meyer E, Mohapatra S, Mozaffari R, Peng K, Pine S, Poetzl J, Rasamoelisolo M, Shao W, Staack RF, Swanson S, Taqui A, Vauleon S, Wang Y, Wen Y
Bioanalysis
· 2025 Nov · PMID 41492841
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The 19 Workshop on Recent Issues in Bioanalysis (19 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 conv...The 19 Workshop on Recent Issues in Bioanalysis (19 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 19 WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week 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 19 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 3) covers in the Part 3A the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity and in Part 3B the Regulatory Inputs on these topics. Part 1 (Mass Spectrometry Assays and Regulated Bioanalysis/BMV) and Part 2 (Biomarkers/BAV, IVD/CDx, LBA and Cell-Based Assays) are published in volume 18 of Bioanalysis, issues 3 and 2, respectively.
Hui YG, Wang F, Wu J
… +5 more, Dupree E, Woolf MS, Shah K, Yuan M, Mylott WR
Bioanalysis
· 2025 Dec · PMID 41489311
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BACKGROUND: Hybrid LC - MS/MS methodology offers orthogonal ADA quantitation but is limited by endogenous immunoglobulin matrix interference challenges. METHODS: We developed a novel semi-quantitative LC-MS/MS isotyping...BACKGROUND: Hybrid LC - MS/MS methodology offers orthogonal ADA quantitation but is limited by endogenous immunoglobulin matrix interference challenges. METHODS: We developed a novel semi-quantitative LC-MS/MS isotyping assay for rabbit IgG/IgM immunogenicity in KEDTA plasma using double immunoprecipitation with double acid dissociation. RESULTS AND CONCLUSIONS: As compared to single immunoprecipitation, double immunoprecipitation reduced endogenous IgG by ≥90% and IgM by >95% (W1B1) or >55% (W3B2), improved peak-area ratios, and achieved an LLOQ of 100 ng/mL, with acceptable accuracy and precision. Double immunoprecipitation plus acid dissociation mitigates matrix interference and enables robust semi-quantitative hybrid LC-MS/MS ADA isotyping in rabbit plasma.
Romani SAS, Silva MLD, Ravaglia GB
… +4 more, Ferreira PC, Dos Anjos Carvalho CD, Pereira TFD, Sussulini A
Bioanalysis
· 2025 Dec · PMID 41472447
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Major depressive disorder (MDD) is a complex and disabling psychiatric condition, often chronic and recurrent, for which treatment selection remains largely guided by trial-and-error. Objective biomarkers capable of pred...Major depressive disorder (MDD) is a complex and disabling psychiatric condition, often chronic and recurrent, for which treatment selection remains largely guided by trial-and-error. Objective biomarkers capable of predicting and monitoring therapeutic response are urgently needed to enable personalized interventions and reduce exposure of the patients to ineffective treatments. Mass spectrometry (MS) has emerged as a central analytical technique, providing the sensitivity and specificity required to capture multi-parametric molecular signatures in biofluids such as plasma, serum, cerebrospinal fluid, and urine. In this context, this review summarizes the application of MS-based platforms to characterize molecular changes associated with treatment response in MDD and discusses how integrative multi-omics approaches can generate robust insights into treatment efficacy and resistance. We highlight current applications, discuss opportunities for MS-guided monitoring of therapeutic strategies, and outline how convergent molecular signatures may accelerate the development of clinically informative biomarkers for precision psychiatry in MDD.