BioanalysisPub Date : 2026-05-07DOI: 10.1080/17576180.2026.2668687
Anisha Mandal, Swambabu Varanasi
{"title":"Fluorescent carbon dots for therapeutic drug monitoring: advances in sensing methotrexate and 6-mercaptopurine.","authors":"Anisha Mandal, Swambabu Varanasi","doi":"10.1080/17576180.2026.2668687","DOIUrl":"https://doi.org/10.1080/17576180.2026.2668687","url":null,"abstract":"<p><p>Carbon dots (CDs) are an emerging class of nanomaterials distinguished by excitation-dependent photoluminescence, tunable surface chemistry, excellent biocompatibility, and scalable green synthesis. Advances in precursor design and single-step heteroatom doping have produced CDs with enhanced quantum yields and tailored electronic structures, expanding their use in bioanalytical applications, particularly sensing. A key application is therapeutic drug monitoring (TDM) of chemotherapeutics such as methotrexate (MTX) and 6-mercaptopurine (6-MP), which have narrow therapeutic windows and high interpatient pharmacokinetic variability, making precise monitoring essential to prevent toxicity or therapeutic failure. Conventional method, including HPLC, LC-MS, and immunoassays, are robust but costly, complex, and non-portable. CD-based fluorescent sensors, using quenching and enhancement mechanisms, offer low toxicity, facile functionalization, and tunable emission. These features support their potential integration with portable and point-of-care platforms, including smartphone-assisted systems, real-time, personalized TDM. By bridging nanomaterial innovation with clinical diagnostics, CDs provide a promising platform for next-generation, sensitive, and patient-centric TDM.</p>","PeriodicalId":8797,"journal":{"name":"Bioanalysis","volume":" ","pages":"1-13"},"PeriodicalIF":1.8,"publicationDate":"2026-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147832985","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
BioanalysisPub Date : 2026-05-05DOI: 10.1080/17576180.2026.2667854
Devangi Mehta, William Wargin, Stephanie Wallace-Teliz, Lauren F Stevenson, Greg Rigdon
{"title":"Rethinking immunogenicity: an integrated approach reveals the PK/PD impact of pre-existing and drug-sustaining ADA.","authors":"Devangi Mehta, William Wargin, Stephanie Wallace-Teliz, Lauren F Stevenson, Greg Rigdon","doi":"10.1080/17576180.2026.2667854","DOIUrl":"https://doi.org/10.1080/17576180.2026.2667854","url":null,"abstract":"<p><strong>Aims: </strong>The traditional immunogenicity paradigm resulting in anti-drug antibody (ADA) and neutralizing antibody (NAb) positive/negative status and ambiguous or imprecise titers may overlook clinically relevant effects of ADA on pharmacokinetics (PK) and pharmacodynamics (PD). Employing risk-based strategies that integrate PK and PD analyses with ADA magnitude using signal-to-noise (S/N) can provide early insight into potential clinical impact of immunogenicity.</p><p><strong>Materials and methods: </strong>In a Phase 1 evaluation of DLX-2323, a humanized single-chain variable fragment (scFv) antibody that binds human IL-1β, ADA-sensitive PK and PD assays were employed to measure DLX-2323 concentration, PD activity, and assess the impact of ADA on PK and PD in healthy participants.</p><p><strong>Results: </strong>The presence of pre-existing ADA was observed in one-third of participants and resulted in high variability of DLX-2323 exposure and PD activity. Pre-existing ADA magnitude correlated with a drug-sustaining impact on PK and PD, with lower clearance (CL/F) and volume of distribution (Vd/F) of DLX-2323 relative to increasing ADA signal.</p><p><strong>Conclusions: </strong>Based on the immunogenicity risk assessment, the use of ADA-sensitive free PK and PD assays to measure <i>in vivo</i> ADA effects on drug exposure and PD activity provided clinically meaningful information that standalone neutralizing antibody assays could not.</p>","PeriodicalId":8797,"journal":{"name":"Bioanalysis","volume":" ","pages":"1-15"},"PeriodicalIF":1.8,"publicationDate":"2026-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147832987","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
BioanalysisPub Date : 2026-04-27DOI: 10.1080/17576180.2026.2664205
Ting Jiang, Zhaomeng Feng, Long Yuan
{"title":"Overcoming inconsistent DNA extraction recovery across tissue types in qPCR assays supporting biodistribution studies.","authors":"Ting Jiang, Zhaomeng Feng, Long Yuan","doi":"10.1080/17576180.2026.2664205","DOIUrl":"https://doi.org/10.1080/17576180.2026.2664205","url":null,"abstract":"<p><strong>Aims: </strong>DNA biodistribution studies are essential in the development of cell and gene therapies. A key step of these studies is the extraction of vector DNA (vDNA) and genomic DNA (gDNA) from complex biological samples. In this work, key DNA extraction parameters were evaluated and optimized to ensure consistent and efficient DNA recovery.</p><p><strong>Methods: </strong>Recovery was evaluated in multiple tissue types by spiking known amounts of vDNA and/or mouse gDNA into surrogate rabbit matrices, performing extraction using a DNA extraction kit under various conditions, and then quantifying the recovered DNA by qPCR and comparing with those spiked post extraction.</p><p><strong>Results: </strong>Parameters including proteinase K incubation, RNase amount, DNA binding beads amount, and tissue input were systematically evaluated and found to play a significant role in recovery. By optimizing these parameters, consistently high recovery was achieved across various tissue types including brain, liver, and spinal cord from mouse, rabbit, and non-human primates.</p><p><strong>Conclusion: </strong>Proteinase K incubation, RNase amount, tissue input, and DNA binding beads volume were identified as key parameters influencing DNA recovery from tissue samples. The findings provide valuable insights for researchers to evaluate and optimize their own extraction protocols, ultimately enhancing the reliability and accuracy in supporting biodistribution studies.</p>","PeriodicalId":8797,"journal":{"name":"Bioanalysis","volume":" ","pages":"1-12"},"PeriodicalIF":1.8,"publicationDate":"2026-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147760856","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
BioanalysisPub Date : 2026-04-20DOI: 10.1080/17576180.2026.2652757
Michael Wright, Richard Hughes, Gareth Satchell, Sofia Adolfsson, Ross Bamford, Riejanne Bax-Seigers, Laura von Berg, Esther Biemans, Tinie van Boekel, Charles Britten, Valeria Castagna, Lode De Cauwer, John Cook, Thomas Krogh-Meibom, James Lawrence, Marcel van der Linden, Deborah McManus, Deepa Reddy Bandi, Kamil Sklodowski, Rebecca Watson, Daniel Wirth, Xichen Zhang, Philip Timmerman
{"title":"An update on the European Bioanalysis Forum recommendation on singlicate analysis for ligand binding assays: biomarker and anti-drug-antibody assays.","authors":"Michael Wright, Richard Hughes, Gareth Satchell, Sofia Adolfsson, Ross Bamford, Riejanne Bax-Seigers, Laura von Berg, Esther Biemans, Tinie van Boekel, Charles Britten, Valeria Castagna, Lode De Cauwer, John Cook, Thomas Krogh-Meibom, James Lawrence, Marcel van der Linden, Deborah McManus, Deepa Reddy Bandi, Kamil Sklodowski, Rebecca Watson, Daniel Wirth, Xichen Zhang, Philip Timmerman","doi":"10.1080/17576180.2026.2652757","DOIUrl":"10.1080/17576180.2026.2652757","url":null,"abstract":"<p><p>Duplicate measurements have long been used in regulated ligand-binding assays, particularly for pharmacokinetic assays. The European Bioanalysis Forum has previously shown that single-well analysis can be suitable for pharmacokinetic endpoints when supported by appropriate method development and validation. This paper extends that evaluation to biomarker and anti-drug antibody assays. A collaborative project involving multiple companies assessed whether single-well measurements provide outcomes comparable to duplicate formats across a wide range of assay types and platforms. The combined data showed strong agreement between both approaches and indicated that duplicate measurement seldom adds scientific value. The European Bioanalysis Forum therefore recommends a data-driven strategy in which replicate number is defined during method development and justified for the intended use of the data. Where performance supports it, single-well analysis should be considered the default approach.</p>","PeriodicalId":8797,"journal":{"name":"Bioanalysis","volume":" ","pages":"1-8"},"PeriodicalIF":1.8,"publicationDate":"2026-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147721560","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
BioanalysisPub Date : 2026-02-01Epub Date: 2026-03-19DOI: 10.1080/17576180.2026.2641928
Kyra J Cowan, Rob Nelson, Annelies Coddens, Karien Bloem, Laura Creed, Hannah Higgins, Jonathan Jimenez Novoa, Christopher Jones, Issa Jyamubandi, Yvonne Katterle, Marco Klinge, Thomas Krogh-Meibom, Ben Leatherdale, Charles Britten, Deepa Reddy Bandi, Theo Rispens, Katherine Sime, Nicoline Videbæk, Michaela Golob, Philip Timmerman
{"title":"From 3-Tier to adaptive immunogenicity testing strategies: recommendations from the European Bioanalysis Forum.","authors":"Kyra J Cowan, Rob Nelson, Annelies Coddens, Karien Bloem, Laura Creed, Hannah Higgins, Jonathan Jimenez Novoa, Christopher Jones, Issa Jyamubandi, Yvonne Katterle, Marco Klinge, Thomas Krogh-Meibom, Ben Leatherdale, Charles Britten, Deepa Reddy Bandi, Theo Rispens, Katherine Sime, Nicoline Videbæk, Michaela Golob, Philip Timmerman","doi":"10.1080/17576180.2026.2641928","DOIUrl":"10.1080/17576180.2026.2641928","url":null,"abstract":"<p><p>The European Bioanalysis Forum discusses a recommended shift from the traditional 3-Tier immunogenicity testing approach to a more adaptive immunogenicity testing strategy for biotherapeutics, emphasizing clinical relevance, risk-based considerations, assay performance, and collaboration with regulatory authorities to improve the assessment of anti-drug antibodies in therapeutic contexts. The recommendations encourage transitioning toward adaptive 1-Tier or 2-Tier combined approaches tailored to the molecule context-of-use. The new strategy prioritizes the clinical impact of anti-drug antibodies over mere incidence, advocating careful consideration of assay characteristics during assay development. The European Bioanalysis Forum continues to call for published case studies, industry cooperation, and proactive dialogue with regulatory authorities to challenge the traditional 3-Tier approach.</p>","PeriodicalId":8797,"journal":{"name":"Bioanalysis","volume":" ","pages":"217-226"},"PeriodicalIF":1.8,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147479529","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
BioanalysisPub Date : 2026-02-01Epub Date: 2026-04-02DOI: 10.1080/17576180.2026.2653856
Wenyu Li, Jun Chen, Isha Taneja, Wilson Edwards, Wenying Jian, Brian Geist
{"title":"Examining pre-analytical factors for bioanalysis of protein therapeutics crossing the blood-brain barrier through receptor-mediated transcytosis.","authors":"Wenyu Li, Jun Chen, Isha Taneja, Wilson Edwards, Wenying Jian, Brian Geist","doi":"10.1080/17576180.2026.2653856","DOIUrl":"10.1080/17576180.2026.2653856","url":null,"abstract":"<p><strong>Background: </strong>The blood-brain barrier (BBB) poses challenges for delivering large-molecule therapeutics to the brain. While engineered cross-BBB delivering platforms such as antibodies utilizing receptor-mediated transcytosis (RMT) aim to improve drug delivery, accurate quantization of antibody exposure in the brain is critical. This study investigates pre-analytical factors impacting the analysis of BBB-shuttling antibodies in mice and seeks to bring attention to steps that can be optimized to enhance efficiency and throughput.</p><p><strong>Research design and methods: </strong>We evaluated factors such as perfusion, capillary depletion, and freeze-thaw in a humanized mouse model treated with antibodies, with or without a BBB shuttle. Hemoglobin and albumin were investigated as markers for blood contamination.</p><p><strong>Results: </strong>Blood contamination was effectively reduced when cardiac puncture was performed, and perfusion further minimized the contamination. Albumin is a more representative blood contamination marker than hemoglobin for antibody bioanalysis in brain tissue. Capillary depletion did not significantly affect PK analysis under-tested conditions, and no considerable differences were found between frozen and fresh samples.</p><p><strong>Conclusions: </strong>Careful investigation of these pre-analytical procedures should be conducted to understand their impact. This will facilitate the development of an optimized and efficient workflow for discovery screening.</p>","PeriodicalId":8797,"journal":{"name":"Bioanalysis","volume":" ","pages":"245-253"},"PeriodicalIF":1.8,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147589652","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
BioanalysisPub Date : 2026-02-01Epub Date: 2026-03-09DOI: 10.1080/17576180.2026.2641573
Bing Zhang, Lei Zhang, Yanfeng Liu, Weimin Hu, Rui Diao, Lili Xing, Yi Tao, Liang Shen
{"title":"Development and validation of an ultra-sensitive UPLC-MS/MS method for quantification of monomethyl auristatin E in cynomolgus monkey plasma: application to pharmacokinetic study.","authors":"Bing Zhang, Lei Zhang, Yanfeng Liu, Weimin Hu, Rui Diao, Lili Xing, Yi Tao, Liang Shen","doi":"10.1080/17576180.2026.2641573","DOIUrl":"10.1080/17576180.2026.2641573","url":null,"abstract":"<p><strong>Introduction: </strong>Antibody-drug conjugates (ADCs) represent a cutting-edge approach in cancer therapy, with monomethyl auristatin E (MMAE) frequently used as a payload in ADC development. We have established a novel bioanalytical method characterized by high sensitivity, accuracy, and efficiency for quantifying MMAE in cynomolgus monkey plasma.</p><p><strong>Methodology: </strong>MMAE was extracted using liquid-liquid extraction (LLE) and quantified by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). Results: The method exhibited excellent linearity across a concentration range of 5 to 3000 pg/mL (<i>r</i> ≥ 0.99) in cynomolgus monkey plasma, it fulfilled precision (CV ≤ 15%, 20% for lower limit of quantification (LLOQ)) and accuracy (83.98-112.75%) criteria according to meeting validation requirements per regulatory bioanalytical validation guidelines.</p><p><strong>Conclusion: </strong>This validated method is instrumental in exploring the in vivo pharmacokinetic profile of ADC and elucidating their exposure-response dynamics.</p>","PeriodicalId":8797,"journal":{"name":"Bioanalysis","volume":" ","pages":"227-233"},"PeriodicalIF":1.8,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147376027","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
BioanalysisPub Date : 2026-02-01Epub Date: 2026-04-15DOI: 10.1080/17576180.2026.2658612
Nancy Yu, Justin Low, Frank Macchi, Richard Vandlen, James Zanghi, Benjamin T Andrews
{"title":"Vendor-specific HRP antibody conjugate differences lead to unexpected immunoassay reagent interactions.","authors":"Nancy Yu, Justin Low, Frank Macchi, Richard Vandlen, James Zanghi, Benjamin T Andrews","doi":"10.1080/17576180.2026.2658612","DOIUrl":"10.1080/17576180.2026.2658612","url":null,"abstract":"<p><strong>Aim: </strong>An interaction observed in an anti-drug antibody (ADA) bridging ELISA between the drug and the anti-digoxin (aDIG) conjugated horseradish peroxidase (HRP) led to increased assay background across several commercial aDIG-HRP sources. Understanding the source of this interaction will help inform reagent qualities to be considered in production, evaluation, and selection for bioanalytical assays.</p><p><strong>Methods and materials: </strong>This work evaluates commercial sources of aDIG-HRP through bridging ELISA, binding ELISA, SPR, and chromatography to determine what characteristics of aDIG-HRP lead to the interaction observed in a bridging ELISA.</p><p><strong>Results: </strong>Three of four commercial aDIG-HRP sources evaluated exhibit drug binding and size heterogeneity with large (>250 kDa) species. SE-UHPLC of one source localizes this binding capability to the >6 MDa species. Under typical HRP conjugation conditions, HRP can conjugate to itself, and these larger species can bind to drug.</p><p><strong>Conclusion: </strong>The conjugation of HRP to aDIG may lead to large species linked to HRP self-conjugation that can interfere with bioassays if not carefully controlled. Therefore, it is important to evaluate commercial reagents for consistency, availability, and critical quality when incorporating a new lot/vendor for better understanding of unexpected assay performance and effectively support reagent and assay life cycle management.</p>","PeriodicalId":8797,"journal":{"name":"Bioanalysis","volume":" ","pages":"265-278"},"PeriodicalIF":1.8,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147687897","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
BioanalysisPub Date : 2026-02-01Epub Date: 2026-04-10DOI: 10.1080/17576180.2026.2655299
Xinran Wang, Hongbo Miao, Zhijun Liu, Jianguo Sun, Zhenhui Jiang, Liya Ye, Zhengyan Liang, Shouhong Gao, Zhipeng Wang, Wansheng Chen
{"title":"Quantification and pharmacokinetic assessment of SB225002 in mouse: a selective non-peptide CXCR2 antagonist.","authors":"Xinran Wang, Hongbo Miao, Zhijun Liu, Jianguo Sun, Zhenhui Jiang, Liya Ye, Zhengyan Liang, Shouhong Gao, Zhipeng Wang, Wansheng Chen","doi":"10.1080/17576180.2026.2655299","DOIUrl":"10.1080/17576180.2026.2655299","url":null,"abstract":"<p><strong>Background: </strong>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.</p><p><strong>Methods and materials: </strong>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.</p><p><strong>Results: </strong>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 (t<sub>max</sub>: 0.4 ± 0.1 h, mean±SD) and metabolism (t<sub>1/2</sub>: 3.4 <math><mo>±</mo></math> 2.6 h), along with significant inter-individual variability.</p><p><strong>Conclusions: </strong>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.</p>","PeriodicalId":8797,"journal":{"name":"Bioanalysis","volume":" ","pages":"255-263"},"PeriodicalIF":1.8,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147643759","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}