Mutić T, Mijajlović A, Stanković V
… +3 more, Djurdjić S, Vlahović F, Stanković D
Bioanalysis
· 2025 Jul · PMID 40791142
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Adrenaline, a critical biomarker of stress and autonomic nervous system function, plays a central role in diagnosing and monitoring several medical conditions such as cardiovascular ailments, endocrine illnesses, and neu...Adrenaline, a critical biomarker of stress and autonomic nervous system function, plays a central role in diagnosing and monitoring several medical conditions such as cardiovascular ailments, endocrine illnesses, and neurodegenerative syndromes. Traditional detection methods, including gas chromatography, high-performance liquid chromatography, mass spectrometry, and spectrophotometry, offer high sensitivity and specificity but are limited by complex instrumentation, high cost, and the need for specialized personnel. These constraints hinder their use in rapid, point-of-care, or decentralized settings. This review provides a thorough summary of conventional and emerging adrenaline detection technologies, focusing on recent biosensing platform advancements. Electrochemical and optical sensors enhanced by nanomaterial-based electrode modifications and advanced molecular recognition strategies have improved sensitivity, selectivity, and portability.
Bioanalysis
· 2025 Jul · PMID 40791022
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BACKGROUND: Cytokines are critical biomarkers, but their accurate measurement is susceptible to pre-analytical variables. This study investigated the effect of delayed blood processing on the quantitation of eight cytoki...BACKGROUND: Cytokines are critical biomarkers, but their accurate measurement is susceptible to pre-analytical variables. This study investigated the effect of delayed blood processing on the quantitation of eight cytokines (CCL2, CXCL10, IFN-γ, IL-2Ra, IL-6, IL-7, IL-18, TNFα) in matched serum and plasma from healthy volunteers. Methods: Blood was processed immediately or after 3, 6, 24, and 48 hours, stored at room temperature or 4°C. Cytokines were quantified using a multiplexed Ella assay. RESULTS: Our data showed only IL-2Ra remained stable across all conditions. The remaining cytokine concentrations were significantly impacted by processing delay and storage temperature, with changes evident as early as 3 hours and more pronounced at 24 and 48 hours ( < 0.05). Storing blood at 4°C generally mitigated changes compared to room temperature, but analyte-specific and matrix-dependent variations persisted. CONCLUSION: These findings emphasize delayed processing significantly alters cytokine levels, highlighting the importance of prompt, standardized processing and careful consideration of pre-analytical factors for reliable cytokine quantitation in clinical studies.
Hughes R, Jordan G, Katterle Y
… +10 more, Jaitner B, Britten C, Gerritsen A, Bandi DR, Haslberger T, van Boekel T, Sigh J, Golob M, Cowan KJ, Timmerman P
Bioanalysis
· 2025 Jul · PMID 40782029
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The use of blank authentic matrix for bioanalytical method validation and sample analysis has long been standard practice within the bioanalytical community and is considered a requirement under current guidelines for bi...The use of blank authentic matrix for bioanalytical method validation and sample analysis has long been standard practice within the bioanalytical community and is considered a requirement under current guidelines for bioanalytical method validation. However, this practice has been increasingly challenged as there are scientifically valid alternatives to replace blank authentic matrices with surrogate matrices. The use of authentic matrices from preclinical animal species conflicts with the ethical 3Rs principles (Replacement, Reduction, and Refinement), a concern that the European Bioanalysis Forum has been addressing for years through data-driven advocacy. In this manuscript, the EBF presents experimental data supporting the use of surrogate matrices in preclinical assays for immunoassay formats. This approach significantly reduces the reliance on authentic matrices, while preserving the quality and integrity of bioanalytical data. The findings advocate for revisiting or clarifying regulatory guidelines, such as ICH M10, to more widely accept the use of surrogate matrices, ensuring alignment with ethical standards without compromising scientific rigor.
Timmerman P, Barfield M, Bartlet JG
… +11 more, Blattmann P, Boehm N, Christodoulou L, Kjelgaard CFD, Iles T, Iltzsche F, Klinge M, Henderson L, Monk L, Nelson R, Liu Y
Bioanalysis
· 2025 Jul · PMID 40757964
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The European Bioanalysis Forum has observed increasing misclassification of pharmacokinetic, anti-drug antibody, and biomarker research assays not used for patient management under the European Union's In Vitro Diagnosti...The European Bioanalysis Forum has observed increasing misclassification of pharmacokinetic, anti-drug antibody, and biomarker research assays not used for patient management under the European Union's In Vitro Diagnostic Regulation, despite their non-diagnostic intent in early clinical development. This misinterpretation, fueled by ambiguous protocol language, limited cross-functional awareness, and inconsistent national implementation, is leading to unnecessary delays in clinical trials and increased and non-added value regulatory burden. Through a structured evaluation involving a focus workshop and regional roadshows, the European Bioanalysis Forum identified some manageable origins of the issue and its operational consequences. This recommendation paper outlines these observations and wants to propose a pragmatic path forward. This includes clearer regulatory guidance to exempt noncommercial, non-diagnostic assays from In Vitro Diagnostic Regulation when not developed or intended as registered diagnostics. We also highlight the importance of stakeholder education and coordinated regulatory dialogue. These steps aim to preserve the regulator's intent of patient protection while enabling timely and efficient clinical research.
Yu Y, Liu D, Zhao H
… +4 more, Dai M, Hu Y, Luo K, Zhang H
Bioanalysis
· 2025 Jul · PMID 40735826
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BACKGROUND: Current HPLC-based methods for doxofylline analysis lack speed and precision. A rapid, specific, and sensitive UPLC-MS/MS method was developed for the determination of doxofylline in this study. RESEARCH DESI...BACKGROUND: Current HPLC-based methods for doxofylline analysis lack speed and precision. A rapid, specific, and sensitive UPLC-MS/MS method was developed for the determination of doxofylline in this study. RESEARCH DESIGN AND METHODS: This method was fully validated and doxophylline-d4 was used as an internal standard. A Kinetex-C18 column (EVO 100Å, 50 × 2.1 mm, 5 μm) was used for the separation procedure, with mobile phases consisting of 0.3% formic acid (A) and 90% acetonitrile solution with 0.3% formic acid (B). The total runtime of the gradient elution procedure was 2.6 minutes. The mass spectrometry analysis was carried out employing a multiple reaction monitoring model and using the transitions of m/z 267.000→181.000 for doxofylline and m/z 271.200→181.100 for the internal standard. RESULTS: The linear range of detection for doxophylline was between 20.0 to 16,000 ng/mL. The intra-batch accuracy deviations of each concentration level ranged from -8.0% to 2.5%, while the intra-batch precisions ranged from 1.3% to 9.0%. And the inter-batch accuracy deviations were -5.8% ~0.8%, while the inter-batch precisions were 2.2% ~7.0%. CONCLUSIONS: This method was applied to pharmacokinetic clinical trials of single oral administration of doxophylline tablets successfully. CLINICAL TRIAL REGISTRATION: www.clinicaltrials.gov identifier is CTR20240006 and CTR20233665.
Timmerman P, Zeiser K, Walker C
… +3 more, Nelson R, Golob M, Barfield M
Bioanalysis
· 2025 Jul · PMID 40728094
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As artificial intelligence enters bioanalysis, traditional validation frameworks, designed for static, deterministic systems are proving unfit for purpose. This paper, developed from the 2025 European Bioanalysis Forum S...As artificial intelligence enters bioanalysis, traditional validation frameworks, designed for static, deterministic systems are proving unfit for purpose. This paper, developed from the 2025 European Bioanalysis Forum Spring Focus Workshop, challenges the assumption that applications using artificial intelligence should be validated like conventional tools. We propose a shift toward adaptive qualification: an approach rooted in scientific oversight, contextual relevance and earned trust. Reframing artificial intelligence as a learning system, more trainee than tool, we explore how oversight must evolve beyond compliance to ensure transparency, robustness and fitness for purpose. Above all, we argue that scientists must remain at the helm. Not to preserve legacy processes, but to guide this evolving landscape with clarity, collaboration and responsibility, keeping innovation sharp and the patient in focus.
Kollapareddy A, Sayyad K, Kowtharapu LP
… +4 more, Konduru N, Mondal T, Varkolu M, Gundekari S
Bioanalysis
· 2025 Jul · PMID 40728075
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AIMS: This study aims to address the clinical need for managing secondary hyperparathyroidism (SHPT) in chronic kidney disease (CKD) patients on dialysis and hypercalcemia in individuals with parathyroid carcinoma or pri...AIMS: This study aims to address the clinical need for managing secondary hyperparathyroidism (SHPT) in chronic kidney disease (CKD) patients on dialysis and hypercalcemia in individuals with parathyroid carcinoma or primary hyperparathyroidism (PHPT) ineligible for parathyroidectomy. Cinacalcet, a key calcimimetic agent, targets the calcium-sensing receptor (CaSR) in the parathyroid gland to lower elevated parathyroid hormone (PTH) levels. The study focuses on developing a robust high-performance liquid chromatography tandem mass spectrometry method for cinacalcet quantification to support therapeutic monitoring and pharmacokinetics. METHODS: A bioanalytical method using cinacalcet-D3 (CCT-D3) as an internal standard and tandem mass spectrometry in positive ion mode for accurate quantification employing Triple Quad Mass spectrometer in normal mode and a Zorbax Eclipse XDB-C18 column. RESULTS: The method was comprehensively validated, demonstrating reliability through high precision, accuracy, linearity within the range of 0.300-150.00 ng/mL, and stability, all in compliance with established bioanalytical guidelines. Furthermore, its environmental sustainability was assessed using modern green chemistry evaluation metrics. CONCLUSION: This study presents a robust, selective LC-MS/MS assay for quantifying cinacalcet in human plasma. The assay demonstrates excellent linearity, precision, and recovery, making it suitable for pharmacokinetic studies, therapeutic drug monitoring, and bioequivalence or bioavailability assessments.
Sanford EJ, Chen J, Tran J
… +2 more, Korboukh I, Zhang G
Bioanalysis
· 2025 Jul · PMID 40705657
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BACKGROUND: Oligonucleotide therapeutics (ONTs) are a rapidly growing class of drug, with 20+ approved drugs on the market and more undergoing preclinical and clinical investigation for various indications. Many groups i...BACKGROUND: Oligonucleotide therapeutics (ONTs) are a rapidly growing class of drug, with 20+ approved drugs on the market and more undergoing preclinical and clinical investigation for various indications. Many groups in the field are appending chemical modifications to modulate tissue specificity. Conjugation of long-chain fatty acids to siRNA molecules increases the hydrophobicity of the analyte and poses analytical challenges for extraction and LC-MS. RESULTS: We report the development and optimization of an SPE extraction method for a lipid-conjugated siRNA. To improve assay quantitation by LC-MS, a stable isotope label internal standard (SILIS) was evaluated that enabled robust quantitation with high accuracy and precision (±5% in most cases). CONCLUSION: We demonstrate the performance of the assay in mouse plasma and tissue homogenates and apply the assay to the determination of tissue exposure and plasma PK profile for a novel lipid-conjugated siRNA molecule and suggest that a SILIS quantitation approach should be standard practice in siRNA bioanalysis.
Bioanalysis
· 2025 Jul · PMID 40689874
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BACKGROUND: Background: An oligonucleotide drug named nusinersen sodium is used to treat Spinal Muscular Atrophy (SMA), requires accurate detection for therapeutic research. There are no published reports on liquid chrom...BACKGROUND: Background: An oligonucleotide drug named nusinersen sodium is used to treat Spinal Muscular Atrophy (SMA), requires accurate detection for therapeutic research. There are no published reports on liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for detecting nusinersen in rat cerebrospinal fluid (CSF). METHODS: An LC-MS/MS method has been created and verified to detect nusinersen in Sprague-Dawley (SD) rat CSF. The method employed solid-phase extraction for post-extraction analysis and used dT20 as an internal standard. Negative ion multiple reaction monitoring (MRM) mode scanning and the electrospray ionization (ESI) source were used. The method was validated over a concentration range of 5-2000 ng/mL with a Lower Limit of Quantification (LLOQ) of for nusinersen at 5 ng/mL. RESULTS AND CONCLUSIONS: The method achieves extremely high accuracy and precision, good linearity, high extraction recovery, and provides a useful approach for evaluating the pharmacokinetics of nusinersen in rats.
Rajapaksha RD, van Kesteren F, Kuehl PJ
… +1 more, Farmer JT
Bioanalysis
· 2025 Jul · PMID 40685990
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BACKGROUND: The use of duplicate analysis in biomarker assays is a standard practice. While it does not inherently increase variability or contribute to assay failure, it can reveal the high variability associated with m...BACKGROUND: The use of duplicate analysis in biomarker assays is a standard practice. While it does not inherently increase variability or contribute to assay failure, it can reveal the high variability associated with manual pipetting, thereby highlighting potential issues within the assy. RESEARCH DESIGN AND METHODS: This study evaluated the reliability of singlicate analysis compared to duplicate analysis for interferon-gamma (IFN-γ) assays using ELISA in mouse and non-human primate (NHP) serum samples. Assay performance was assessed across 50 plates, with results analyzed for minimum required dilution (MRD), standard curve linearity, surrogate sample accuracy, recovery, precision, and variability between analysts, including both an experienced and a novice analyst. RESULTS: There were minimal differences in relative accuracy, and precision between singlicate and duplicate analysis, with CVs less than 5% for both methods. Singlicate and duplicate-generated standard curves were strongly correlated (NHP R = 0.9995, mouse R = 1.0000). Analyst variability had less impact on singlicate analysis, with lower inter-analyst CVs (1.0-6.5%) compared to duplicate analysis (5.0-7.5%). CONCLUSIONS: These findings underscore the robustness of singlicate analysis. A workflow for singlicate assay validation is proposed, demonstrating its potential to streamline biomarker assay development while ensuring regulatory compliance. This study supports the adoption of singlicate analysis as a viable alternative to conventional duplicate methods for biomarker assays.
Bioanalysis
· 2025 Jun · PMID 40660492
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BACKGROUND/AIM: The three-tiered testing strategy for an anti-drug antibody (ADA) assay is a common practice for assessing the immunogenicity to therapeutic products. Efforts to streamline the ADA testing process led to...BACKGROUND/AIM: The three-tiered testing strategy for an anti-drug antibody (ADA) assay is a common practice for assessing the immunogenicity to therapeutic products. Efforts to streamline the ADA testing process led to proposals of using signal to noise () as a substitute for titer when determining ADA magnitude. This study aims to identify the critical factors that may influence the correlation of and titer. METHOD/RESULT: Experimental and clinical ADA datasets were examined to assess how drug concentration and the assay drug tolerance affect the measurement and correlation of and titer. Under various experimental conditions the influence of drug on titer was minimal across a range of drug concentrations. However, drug presence affected the , particularly when drug concentrations approached the assay drug tolerance. Clinical ADA datasets showed a moderate to strong correlation between and titer, demonstrating similar patterns of drug impact on both measurements, as observed in the experimental data. CONCLUSION: The presence of drug in clinical samples and the drug tolerance of the ADA assay simultaneously influence the measurement and correlation of and titer. The fold difference between drug tolerance and the maximum drug concentration in samples is a key factor in determining this correlation.
Bioanalysis
· 2025 Jun · PMID 40629991
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The accurate quantification of antibiotics in biological matrices is pivotal for supporting individualized medication, optimizing antibiotics therapy and addressing the global crisis of antimicrobial resistance (AMR). LC...The accurate quantification of antibiotics in biological matrices is pivotal for supporting individualized medication, optimizing antibiotics therapy and addressing the global crisis of antimicrobial resistance (AMR). LC-MS/MS has emerged as the gold standard for clinical antibiotic quantification, particularly valued for its accuracy, multiplexing efficiency, and compatibility with complex sample types. This review provides a comprehensive overview of the principles, methodologies, and clinical applications of LC-MS/MS in antibiotic quantification, with a focus on its role in therapeutic drug monitoring (TDM), pharmacokinetics (PK), and epidemiological studies. Key challenges, including sample preparation and matrix effects, are critically discussed. Tailored strategies for analyzing diverse antibiotic classes (e.g. β-lactams, aminoglycosides, colistins, macrolides, and glycopeptides) are highlighted by considering their physicochemical properties, including polarity, acid-base characteristics, and structural complexity. Furthermore, emerging trends in automation, high-resolution mass spectrometry, and quality control frameworks are outlined to guide future advancements. By bridging technological innovation with clinical needs, LC-MS/MS continues to be a cornerstone of precision medicine and a vital tool in global AMR mitigation efforts.
Lawless MJ, Chan N, Patel K
… +2 more, Wang M, Colter DC
Bioanalysis
· 2025 Jun · PMID 40629851
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The extracellular domain of the neurotrophin receptor p75 has been shown to be a prominent biomarker for both disease diagnosis and progression for amyotrophic lateral sclerosis. This urinary analyte may serve as a valua...The extracellular domain of the neurotrophin receptor p75 has been shown to be a prominent biomarker for both disease diagnosis and progression for amyotrophic lateral sclerosis. This urinary analyte may serve as a valuable fluid biomarker which greatly increases the ease of sample collection in both healthy volunteers and patients. In this paper, the method development and validation for an electrochemiluminescence assay is described. This assay completely uses commercially available reagents and can be performed using common lab equipment found in most bioanalytical labs. This method shows good accuracy and precision, high sensitivity as well as good parallelism illustrating the ability of the method to detect and report on urinary concentrations of neurotrophin receptor p75. The assay can quantitate as low as 78 pg/mL of neurotrophin receptor p75 and > 98% of healthy urine samples tested fell within the dynamic range of the assay.
Nishidate M, Yanagisawa C, Irie H
… +3 more, Aida K, Miyayama T, Terao K
Bioanalysis
· 2025 Jun · PMID 40552557
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BACKGROUND: Delta-like protein 3 (DLL3) is considered to inhibit the Notch pathway in the tumorigenesis of small cell lung cancer (SCLC) and other neuroendocrine carcinomas, making it a potential therapeutic target in th...BACKGROUND: Delta-like protein 3 (DLL3) is considered to inhibit the Notch pathway in the tumorigenesis of small cell lung cancer (SCLC) and other neuroendocrine carcinomas, making it a potential therapeutic target in the treatment of cancer. Since the soluble form (sDLL3) is expected to be useful for predicting the status of DLL3 expression on tumors, analytical methods to measure sDLL3 are required. RESEARCH DESIGN AND METHODS: Assay methods using ELISA and the SMCxPRO platform were developed to analyze sDLL3 concentration in human serum. The performance of the ELISA was evaluated and the SMCxPRO assay was fully validated, and the comparability of the 2 assays was assessed. RESULTS: The performance of the ELISA was acceptable, and in the SMCxPRO assay validation, all pre-defined validation acceptance criteria were met. The 2 assays were comparable within the range of quantification. Concentrations ranged from below the limit of quantification (<1.00 pg/mL) to 18.0 pg/mL for healthy volunteers and from 1.27 pg/mL to 519 pg/mL for SCLC patients by SMCxPRO assay. CONCLUSIONS: Two sensitive assay methods to measure sDLL3 in human serum were successfully established. These assays have potential as novel blood-based assays to assess the status of DLL3 expression on tumors in humans.
Goodwin L, Faber J, Holmberg KH
… +5 more, McDougall S, Meijer J, Tagliacozzi D, White S, Timmerman P
Bioanalysis
· 2025 Jun · PMID 40537099
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This paper discusses the updated recommendations from the European Bioanalysis Forum on whole blood stability testing in bioanalytical method validation. The recommendations are based on EBF community feedback on the ICH...This paper discusses the updated recommendations from the European Bioanalysis Forum on whole blood stability testing in bioanalytical method validation. The recommendations are based on EBF community feedback on the ICH M10 guidelines and associated training materials. The ICH M10 guidelines highlight the importance of assessing analyte stability throughout the lifecycle of the sample, including in whole blood, immediately after collection, to ensure that the analyte concentration in the tested sample accurately reflects the concentration at the time of collection. Aspects considered in these recommendations are the definition of fresh blood, equilibration times, assay type and how the experiments can be conducted to align with the 3Rs principles.
Bioanalysis
· 2025 Jun · PMID 40525309
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BACKGROUND: Alzheimer's disease (AD) affects over 55.2 million individuals globally with current treatment modalities only providing symptomatic relief. This highlights the importance of combination therapies which targe...BACKGROUND: Alzheimer's disease (AD) affects over 55.2 million individuals globally with current treatment modalities only providing symptomatic relief. This highlights the importance of combination therapies which target multiple pathways simultaneously. Donepezil (DNP), an acetylcholinesterase inhibitor, when combined with Quercetin (QCT), a polyhydroxy flavonoid with neuroprotective properties, might alter progression. However, simultaneous estimation of DNP and QCT for concurrent delivery, calls for robust analytical method that can simultaneously estimate both drugs. RESEARCH DESIGN AND METHODS: A simple, robust, and cost-friendly UHPLC method for simultaneous estimation of DNP and QCT was developed. The analytes were resolved on C18 column (Kromasil C18, 4.6 × 250 mm), with acetonitrile and 0.1% diethyl amine buffer, adjusted to pH 3.5 with glacial acetic acid as mobile phase in ratio 50:50 v/v at 0.6 mL/min, at column oven temperature 40°C in total analysis run time of 10 min. RESULTS: DNP and QCT were quantified by PDA detector at 268 nm and 370 nm, respectively. The developed method was found to be linear, accurate, precise, specific, and robust as per ICH guidelines. CONCLUSION: The developed and validated method efficiently quantified DNP and QCT co-loaded inside liposomes estimating their entrapment efficiency, loading efficiency, and in vivo plasma kinetics.
Yin F, Ye R, Pierce A
… +2 more, Gibbs J, Baratta M
Bioanalysis
· 2025 Jun · PMID 40503733
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AIM: TAK-981 is a highly effective and selective inhibitor of the small ubiquitin-like modifier (SUMO) activating enzyme, and it promotes the expression of type I interferons (IFN-Is). Developing a sensitive bioanalytica...AIM: TAK-981 is a highly effective and selective inhibitor of the small ubiquitin-like modifier (SUMO) activating enzyme, and it promotes the expression of type I interferons (IFN-Is). Developing a sensitive bioanalytical assay for quantitating TAK-981 is essential in the clinical investigations for oncology drug development. MATERIALS & METHODS: TAK-981 and its stable isotope labeled compound (TAK-981C, N) as the internal standard were employed in this LC-MS/MS assay. RESULTS: This assay was successfully validated from 0.1 ng/mL to 100 ng/mL with good accuracy and precision and has been applied to support clinical studies. CONCLUSION: A sensitive and robust LC-MS/MS assay was validated for TAK-981 in human plasma for the first time.
Bioanalysis
· 2025 Jun · PMID 40503728
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Accurate measurement of protein concentration is crucial in biological research, where protein-based reagents play a key role in assay performance and reliability. Traditional methods of protein quantification often meas...Accurate measurement of protein concentration is crucial in biological research, where protein-based reagents play a key role in assay performance and reliability. Traditional methods of protein quantification often measure total protein concentration, failing to account for the active portion capable of binding to its intended target. Calibration-free concentration analysis (CFCA), which uses surface plasmon resonance (SPR) technology, offers a solution by specifically measuring the active protein concentration of the sample. CFCA leverages binding under partially mass-transport limited conditions to directly quantify the functional protein in a sample, overcoming variability associated with recombinant protein production. This article provides a background on CFCA and the case for its more widespread use in protein reagent characterization, as it offers a way to reduce lot-to-lot and vendor-to-vendor variability while improving reproducibility and standardization of assays. This perspective was informed by searching and selecting pertinent articles from PubMed (Nov 2024-March 2025) and by examining the reference lists of key papers.
Bioanalysis
· 2025 Jun · PMID 40501421
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OBJECTIVE: To develop a highly sensitive and specific detection method based on PCR-CRISPR/Cas12a for the detection of () in feces and to evaluate its detection rate in the general population as well as its potential as...OBJECTIVE: To develop a highly sensitive and specific detection method based on PCR-CRISPR/Cas12a for the detection of () in feces and to evaluate its detection rate in the general population as well as its potential as a gastrointestinal tumor marker. MATERIALS AND METHODS: Specific primers and crRNA targeting the 16S rDNA of were designed to construct a PCR-CRISPR/Cas12a detection system. A total of 230 fecal samples were collected from the general population, and bacterial DNA was extracted. The target gene was detected using this system to verify its sensitivity, specificity, and stability. RESULTS: The established detection system demonstrated strong specificity, with stable recognition of , and a minimum detection limit of 10 ng/μL. The detection rate of in fecal samples from the general population was 51.7%. CONCLUSION: The PCR-CRISPR/Cas12a system can efficiently detect in feces, providing a reliable technical tool for exploring its association with gastrointestinal tumors.
Arceo T, Andrews B, Getz J
… +3 more, Haile S, Maia M, Song Y
Bioanalysis
· 2025 Jun · PMID 40501387
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BACKGROUND: Monitoring anti-drug antibodies (ADAs) is a critical component of recombinant protein drug development. Positive controls in ADA assays are essential for evaluating assay quality, yet the influence of their b...BACKGROUND: Monitoring anti-drug antibodies (ADAs) is a critical component of recombinant protein drug development. Positive controls in ADA assays are essential for evaluating assay quality, yet the influence of their binding properties on assay performance is not well understood. RESEARCH DESIGN AND METHODS: We evaluated a panel of surrogate positive controls with varying binding characteristics to investigate how their affinity and kinetic parameters impact ADA assay performance. Binding properties were measured using Bio-Layer Interferometry (BLI), and assays were evaluated for relative sensitivity and drug tolerance. This was a laboratory-based study; no human or animal subjects were involved. RESULTS: We observed a correlation between higher affinity (lower K (equilibrium dissociation constant)) and lower k (off-rate constant) with increased relative assay sensitivity. However, no consistent relationship was found between these binding parameters and drug tolerance. These findings suggest that binding kinetics of the positive control can significantly influence sensitivity but may not predict drug tolerance. CONCLUSIONS: Understanding the relationship between positive control binding properties and ADA assay performance can support the selection of reagents that optimize sensitivity. Limitations include the use of surrogate positive controls, which may not fully replicate the complexity of clinical ADA responses.