Bioanalytical method transfer is a critical and often vulnerable step in drug development, yet many challenges arise not from technical limitations but from misalignment, communication gaps and loss of scientific context...Bioanalytical method transfer is a critical and often vulnerable step in drug development, yet many challenges arise not from technical limitations but from misalignment, communication gaps and loss of scientific context as work moves between organizations. The European Bioanalysis Forum conducted a multi-table Pharma/CRO workshop and community survey to identify the behaviors that most influence transfer success. The discussions revealed consistent themes: early SME engagement, shared understanding of scientific intent, proactive communication, joint risk management and a clear responsibility split. This paper summarizes these insights and provides practical, interaction-focused recommendations to strengthen collaboration across method development, transfer and validation. Rather than prescribing templates or checklists, the European Bioanalysis Forum outlines behaviors and principles that promote clarity, predictability and scientific convergence. Reliable method transfer emerges from partnership mind-set and deliberate interaction, not procedural formality.
Bispecific antibodies (bsAbs) represent a rapidly expanding class of biotherapeutics that enable novel mechanisms of action beyond the conventional monoclonal antibodies (mAbs). While their clinical success continues to...Bispecific antibodies (bsAbs) represent a rapidly expanding class of biotherapeutics that enable novel mechanisms of action beyond the conventional monoclonal antibodies (mAbs). While their clinical success continues to grow, the increased structural complexity, non-native architecture, and dual-targeting mechanisms introduce unique immunogenicity risks and sophisticated bioanalytical challenges. In this review, we examine the clinical immunogenicity profiles of approved and selected investigational bsAbs, highlighting key determinants that drive immunogenicity risk. We also discuss critical hurdles, considerations and emerging solutions in immunogenicity evaluation for bsAbs, including limitations of widely used anti-drug antibody (ADA) assay format, matrix interference, domain-specific characterization, and functional neutralization assessment. Finally, we propose an integrated immunogenicity assessment framework that combines predictive tools, clinical bioanalytical data, and reverse-translational investigations to refine therapeutic platforms and reduce immunogenicity risk in next-generation bsAb development. To support this evolution, we advocate enhanced industry transparency and the publication of immunogenicity results in detail, especially for programs terminated due to high immunogenicity. Together, these approaches aim to support more informed decision-making and enable the safe and effective advancement of complex bispecific antibody therapeutics.
AIM: During clinical sample analysis unforeseen problems arose using a previously validated assay for the quantification of diclofenac in the concentration range from 0.0500 to 50.0 ng/mL. Here, we describe the troublesh...AIM: During clinical sample analysis unforeseen problems arose using a previously validated assay for the quantification of diclofenac in the concentration range from 0.0500 to 50.0 ng/mL. Here, we describe the troubleshooting process leading to the identification of the root cause and resulting changes in assay development strategy for robust supported liquid extraction (SLE)-based assays for clinical application. METHODS: Individual steps of the sample preparation workflow and subsequent analysis with liquid chromatography and mass spectrometry (LCMS) were examined. Troubleshooting results were tested using stored validation samples and pooled clinical samples for confirmation. RESULTS: The root cause for altered assay performance could be identified as the SLE plate batch effect attributable to variability in the sorbent material (diatomaceous earth). Reducing the sample loading volume applied to the SLE plates yielded purer extracts and consistent chromatographic performance. Quantitative recovery and signal consistency were restored after the modification. CONCLUSION: Assay robustness was improved by underloading SLE plates. After adjusting the method and re-validation, clinical samples were successfully analyzed. The internal method development strategy was adjusted to avoid full capacity loading of SLE plate with natural sorbents.
Tian N, Chen M, Zhang S
… +8 more, Wang J, Huang B, Kao S, Zheng T, Qin Y, Zhao X, Zhang Z, Zhou X
Bioanalysis
· 2026 Mar · PMID 42163838
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OBJECTIVE: This study aimed to develop a rapid, high-sensitive, and accurate amplified luminescent proximity homogeneous assay (AlphaLISA) for measuring plasma concentrations of growth differentiation factor 15 (GDF15) a...OBJECTIVE: This study aimed to develop a rapid, high-sensitive, and accurate amplified luminescent proximity homogeneous assay (AlphaLISA) for measuring plasma concentrations of growth differentiation factor 15 (GDF15) and assessing its application value in the diagnosis of cardiovascular disease (CVD). METHODS: Based on the AlphaLISA method, plasma GDF15 levels were quantified in healthy controls (HCs), heart failure (HF), and myocardial infarction (MI) patients using a double-antibody sandwich format. The diagnostic efficiency of GDF15 for HF and MI was assessed by ROC curve analysis. RESULTS: The established GDF15-AlphaLISA took only 15 minutes with good linearity (R >0.99) and wide detection range (0.072 ~ 200 ng/mL). The intra- and inter-analytical precision were within 10%, and the recovery rate was 96.06% ~109.41%. There was no significant cross-reactivity with CVD biomarkers (Galectin-3, ST2, cTnI) or with other TGF-β superfamily members (TGF-β1, GDF8, BMP2, BMP6). Results showed that GDF15 could significantly distinguish HCs and patients ( < 0.0001), and ROC curve showed that GDF15 had a high diagnostic efficiency for HF and MI (AUC >0.95). CONCLUSION: We have successfully established a sensitive and rapid GDF15-AlphaLISA method, which can promote the timely diagnosis of HF and MI, and it is a good auxiliary means for the initial diagnosis of HF and MI.
Chronic spontaneous urticaria is a common immunological disease that affects over 1% of the population. Recently, the anti-FcεR1α autoantibody has been discovered as a prominent biomarker related to the disease. Therefor...Chronic spontaneous urticaria is a common immunological disease that affects over 1% of the population. Recently, the anti-FcεR1α autoantibody has been discovered as a prominent biomarker related to the disease. Therefore, there is a need for robust, qualified assays to characterize and measure anti-FcεR1α autoantibodies in human serum. However, biomarker assay qualification is a topic of discussion and can sometimes be left open to interpretation. Currently, there is little guidance or best practices outlined for the method qualification of autoantibodies. These molecules are typically endogenous and such assays fall somewhere between quantitative and qualitative.Herein, we develop an electrochemiluminescence assay for the detection of anti-FcεR1α autoantibodies and provide an outline of method qualification. Two assay formats are investigated with one being fully developed and optimized. Other considerations stemming from immunogenicity qualifications are also assessed, such as isotyping, cut-point, and a confirmatory assay.
Bioanalysis
· 2026 Mar · PMID 42152800
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BACKGROUND/AIM: The biotin-drug extraction with acid dissociation (BEAD) assay is used to enhance drug tolerance when detecting anti-drug antibodies (ADAs) during biotherapeutic development. A critical step of it is the...BACKGROUND/AIM: The biotin-drug extraction with acid dissociation (BEAD) assay is used to enhance drug tolerance when detecting anti-drug antibodies (ADAs) during biotherapeutic development. A critical step of it is the acid-induced elution of ADA from the biotin-labeled drug immobilized on streptavidin-coated-beads (SA-beads). Although biotin binds streptavidin extremely tight, the dissociation dynamics between the biotin-labeled drug or the entire ADA-biotin-drug complex and SA-beads remain uncharacterized under acidic conditions. This study characterizes biotin-labeled drug dissociation and develops a systematic approach to optimize BEAD. METHOD/RESULT: Acid-induced dissociation was observed in a modified ADA bridging assay and the Biolayer Interferometry (BLI) analysis. Dissociation magnitude depended on the acid type and pH, with reduced sensitivity to acid concentration and exposure time. Acetic acid at pH 2.65, lactic acid at pH 2.23, and glycine at pH 3.00 produced the greatest dissociation, whereas acids at pH 4 produced the slowest rate. From these findings, we developed an optimization workflow focusing on concentrations of biotin- and ruthenium-labeled drugs in Master-Mix, SA-bead amount, and initial biotin-labeled drug. CONCLUSION: The manuscript proposes a fit-for-purpose optimization framework and provides guidance in acid selection and reagent tuning to improve drug tolerance and assay efficiency across ADA workflows.
Denhoff L, Dernek C, Heins L
… +4 more, Gu C, Renfro A, Pasas-Farmer S, Kernstock R
Bioanalysis
· 2026 Mar · PMID 42142340
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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 outl...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.
Bioanalysis
· 2026 Mar · PMID 42138690
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Immunogenicity is a biological response to pharmacologic therapeutic intervention and therefore fits squarely within established definitions of a biomarker. Yet, unlike most biomarker assays, where analytical performance...Immunogenicity is a biological response to pharmacologic therapeutic intervention and therefore fits squarely within established definitions of a biomarker. Yet, unlike most biomarker assays, where analytical performance is tailored to context of use, anti-drug antibody (ADA) testing has converged on a largely uniform, three-tiered paradigm built around statistically derived cut points and titer reporting. This perspective argues that this approach arose from understandable, risk-averse responses to rare, high-impact safety events, combined with early analogies to the vaccine field, rather than from first principles thinking aligned with drug development needs.As a result, immunogenicity datasets are often reduced to binary classifications that discard biological context, inflate reported incidence, and complicate efforts to relate immune responses to clinically meaningful outcomes such as pharmacokinetics, pharmacodynamics, efficacy, or safety. Titer-based readouts, while appropriate for large vaccine-like responses, are frequently insufficiently granular to capture the full spectrum of response magnitudes relevant to contemporary biotherapeutic modalities.Reframing immunogenicity as a context-of-use-driven biomarker measurement leverages complete, continuous response profiles (e.g. screening-tier signal-to-noise) alongside pharmacokinetic/pharmacodynamic (PK/PD), and clinical outcomes to identify clinically relevant immunogenicity thresholds. This approach preserves nuance, improves interpretability and stakeholder communication, and focuses attention on clinical impact most relevant to patients and regulators.
Bioanalysis
· 2026 Mar · PMID 42130447
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OBJECTIVE: 2D-LC method and LC-MS/MS self-calibrated internal standard method were used to compared the consistency and correlation of quetiapineserum concentration. METHODS: Quality control samples at low, medium, and h...OBJECTIVE: 2D-LC method and LC-MS/MS self-calibrated internal standard method were used to compared the consistency and correlation of quetiapineserum concentration. METHODS: Quality control samples at low, medium, and high concentrations of quetiapine, as well as serum samples from 138 psychiatric patients taking quetiapine, were analyzed using 2D-LC and LC-MS/MS self-calibrated internal standard method, respectively. Performed a complete validation of both methods. The Bland-Altman plot was used to assess the consistency of the results, while Pearson correlation analysis and Passing-Bablok regression were used to analyze the correlation among the results. OUTCOMES: The linear range for 2D-LC was 60.74-1518.48 ng/ml, and the linear range for LC-MS/MS self-calibrated internal standard method was 60.88-1522.02 ng/ml. For both methods, the relative standard deviation of intraday and daily precision was less than 15%. Calculated using the Bland-Altman plot, the 95.65% LoA was (-11.85, 30.82). The Passing-Bablok regression equation was OLZ = 2.44 + 1.06 × OLZ, and the Pearson correlation coefficient was 0.994 ( < 0.001). CONCLUSION: The 2D-LC and LC-MS/MS self-calibrated internal standard method, exhibit exceptional agreement and correlation in the quantification of quetiapine concentrations in serum. which can be used for conventional therapeutic drug monitoring (TDM).
BACKGROUND: Ibuprofenamine is a novel spray prodrug of ibuprofen designed to reduce gastrointestinal adverse effects through local administration. Its low systemic exposure and extreme susceptibility to rapid ex vivo enz...BACKGROUND: Ibuprofenamine is a novel spray prodrug of ibuprofen designed to reduce gastrointestinal adverse effects through local administration. Its low systemic exposure and extreme susceptibility to rapid ex vivo enzymatic hydrolysis demand a highly sensitive and stabilized bioanalytical method. RESEARCH DESIGN AND METHODS: We developed an LC-MS/MS assay using simple protein precipitation. A rigorous dual-stage stabilization strategy, which employed a high-capacity NaF/citric acid buffer during blood collection and an acidic vehicle prior to plasma extraction-was implemented to completely arrest prodrug degradation. RESULTS: The method achieved a lower limit of quantification (LLOQ) of 40 pg/mL with a linear range of 40-2000 pg/mL. Intra- and inter-batch precision and accuracy were well within ±15%. The stabilization protocol extended sample stability to 120 days at -70°C, effectively eliminating ex vivo hydrolysis artifacts. CONCLUSIONS: This robust, dual-stage stabilized LC-MS/MS method effectively overcomes the bioanalytical challenges of labile ester prodrugs. It was successfully applied to support a clinical pharmacokinetic study of ibuprofenamine spray.
Bioanalysis
· 2026 Mar · PMID 42093462
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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...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.
Mehta D, Wargin W, Wallace-Teliz S
… +2 more, Stevenson LF, Rigdon G
Bioanalysis
· 2026 Mar · PMID 42084489
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AIMS: 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 A...AIMS: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: Based on the immunogenicity risk assessment, the use of ADA-sensitive free PK and PD assays to measure ADA effects on drug exposure and PD activity provided clinically meaningful information that standalone neutralizing antibody assays could not.
Jiang Z, Smith J, Haskins J
… +4 more, McPhee B, You Z, O'Rourke D, Joyce A
Bioanalysis
· 2026 Feb · PMID 42059819
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BACKGROUND: mRNA-LNP therapeutics have revolutionized drug development across various therapeutic areas, necessitating accurate and robust RNA quantification in bioanalysis. While RT-qPCR is commonly used for RNA quantif...BACKGROUND: mRNA-LNP therapeutics have revolutionized drug development across various therapeutic areas, necessitating accurate and robust RNA quantification in bioanalysis. While RT-qPCR is commonly used for RNA quantification, RT-dPCR has emerged as an alternative, with enhanced precision and robustness. Yet, a systematic comparison of these methodologies for mRNA-LNP bioanalysis remains absent. METHODS: Duplex RT-dPCR and RT-qPCR assays using identical primers and probes were developed to quantify LNP encapsulated IgG heavy chain (HC) and light chain (LC) RNA. Both assays analyzed RNA from 50 rat serum samples dosed with the mRNA-LNP compound, and results were statistically compared for measurement agreement and method reliability. RESULTS: Both assays were qualified, meeting the pre-defined assay acceptance criteria. HC RNA quantification showed strong agreement between the two methods (Lin's CCC = 0.96). However, RT-qPCR significantly underestimated LC RNA levels compared to RT-dPCR, with a dPCR/qPCR ratio mean of 2.83, suggesting gene-dependent variability in quantification accuracy. CONCLUSION: This study provides a systematic evaluation of RT-dPCR and RT-qPCR in RNA quantification, advancing our understanding of their application in mRNA-LNP bioanalysis. These findings underscore the critical need for thorough assay evaluation to ensure reliable mRNA measurements, and prevent potential inaccuracies that could lead to misleading results in drug development and regulatory decision-making.
Bioanalysis
· 2026 Feb · PMID 42057612
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Wider access to biologic medicines has been made possible by biosimilars, which provide clinically equivalent options at a lower cost. However, robust immunogenicity surveillance and reliable switching and interchangeabi...Wider access to biologic medicines has been made possible by biosimilars, which provide clinically equivalent options at a lower cost. However, robust immunogenicity surveillance and reliable switching and interchangeability pathways are still necessary for real-world use. The "totality of evidence" may be translated into standardized, patient-centered switching and interchangeability pathways that maintain efficacy and safety through pharmacy-led stewardship initiatives. This narrative review was carried out using PubMed/MEDLINE, Embase, Scopus, Web of Science Core Collection, and Google Scholar between 1 January 2019, and 1 December 2025. This review shows that biosimilars and reference biologics are clinically equivalent, and switching typically has no appreciable impact on population-level safety or immunogenicity. However, switching should be supported by a pharmacy-led toolkit that includes risk-stratified immunogenicity testing (event-triggered vs. scheduled), a minimum assay set plus drug levels, and an action algorithm to differentiate PK failure, PD failure, and immune-mediated failure because a small subset of patients may experience clinically significant ADA/NAb-mediated loss of response or tolerability issues. The issue is now to "operate switching safely" rather than "prove switching," since regulatory trends encourage interchangeability and simplify evidence requirements.
Bioanalysis
· 2026 Mar · PMID 42037403
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AIMS: 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...AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Wright M, Hughes R, Satchell G
… +20 more, Adolfsson S, Bamford R, Bax-Seigers R, von Berg L, Biemans E, van Boekel T, Britten C, Castagna V, De Cauwer L, Cook J, Krogh-Meibom T, Lawrence J, van der Linden M, McManus D, Bandi DR, Sklodowski K, Watson R, Wirth D, Zhang X, Timmerman P
Bioanalysis
· 2026 Mar · PMID 42007617
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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 ph...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.
Bioanalysis
· 2026 Feb · PMID 41988666
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Bioanalytical method validation is a foundation of drug development, ensuring that pharmacokinetic (PK), toxicokinetic (TK), and biomarker assays produce accurate, precise, reliable, and decision-enabling data. Over the...Bioanalytical method validation is a foundation of drug development, ensuring that pharmacokinetic (PK), toxicokinetic (TK), and biomarker assays produce accurate, precise, reliable, and decision-enabling data. Over the past several decades, advances in analytical technologies including ligand-binding assays (LBAs) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) platforms have transformed the field of bioanalysis. Regulatory guidance from the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), and the International Council for Harmonization (ICH) has evolved to reflect these advancements. This review provides a comprehensive comparison of PK and biomarker assay validation, highlighting critical parameters such as selectivity, sensitivity, calibration, accuracy, precision, dilution/parallelism, specificity, and stability. Best practice recommendations are provided for method development, qualification, and validation, with a focus on fit-for-purpose (FFP) approaches and context-of-use (CoU) considerations. Areas of ambiguity in current regulatory expectations for biomarker validations, common pitfalls encountered during regulatory interactions, and emerging trends in bioanalysis are also reviewed. This review paper aims to guide bioanalytical scientists in developing robust, compliant assays that support regulatory submissions and facilitate informed drug development decisions.