This white paper (Part 2 of a series on vaccine immunogenicity assay validation) addresses the development of Ligand Binding Assays (LBAs) to support vaccine immunogenicity studies. Developed through a collaborative effort by a working group of experts from 16 global vaccine manufacturers, national regulatory authorities, and a nonprofit global-health foundation convened under the Workshop on Recent Issues in Bioanalysis (WRIB), this manuscript provides practical guidance to complement existing regulatory frameworks for bioanalytical method validation.Part 2 details the iterative process of LBA development, with emphasis on defining the Analytical Target Profile (ATP) as a foundational tool for guiding decision-making across the assay life cycle. Key topics include assay purpose and analyte specificity, regulatory expectations, the use of statistical Design of Experiments (DOE) to systematically optimize assay conditions, and the selection of appropriate reagents, controls, and sample matrices. The paper also addresses the unique challenges posed by the structural diversity of vaccine antigens and the need for representative reference materials.The manuscript further highlights the importance of ongoing performance assessment and the adaptability of assays as they transition from early exploratory phases to pivotal clinical studies. Collectively, the recommendations presented aim to supplement existing regulatory guidance with vaccine-specific considerations, ultimately supporting the development of safe and effective vaccines. This paper is complemented by Part 1 (LBA Validation) and Part 3 (Assay Lifecycle Management, including Maintenance, Monitoring, and Transfers).
Biosimilars are biological medicines developed once exclusivity for the reference product has lapsed and are regulated through a stringent approval pathway, demonstrating the same efficacy, similar safety, and immunogenicity as their reference products. After two decades of clinical experience and scientific progress, regulatory agencies worldwide are transitioning toward a streamlined (also named tailored) biosimilar development that primarily relies on analytical and pharmacokinetic (PK) similarity, without routinely requiring comparative efficacy studies (CES). This narrative review discusses how comparable immunogenicity is demonstrated within streamlined development. Comparable analytical data provide the pivotal evidence for concluding comparable immunogenicity of a biosimilar candidate. Scientific evidence shows that PK similarity studies provide immunogenicity outcomes highly concordant with those observed in CES. Well-designed PK studies, particularly in healthy volunteers, provide sensitive and clinically informative immunogenicity assessments while minimizing confounding factors associated with disease and concomitant treatments. These studies can adequately characterize the onset, magnitude, and evolution of humoral immune responses without underestimating comparative immunogenicity. In the future, in silico and in vitro tools may enable additional risk-based assessment of immunogenicity differences between biosimilar and reference product. In conclusion, extensive scientific and regulatory evidence supports that streamlined biosimilar development provides a scientifically rigorous framework for demonstrating comparable immunogenicity.
A sensitive and validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of the anti-COVID-19 agents molnupiravir (MPV), its active metabolite N-hydroxycytidine (NHC), and baricitinib (BARI) in human plasma. As MPV is rapidly converted to NHC in vivo, whereas BARI is predominantly eliminated unchanged, NHC was generated in vitro by alkaline hydrolysis to enable accurate determination of the active metabolite. Chromatographic separation was achieved on an Agilent Poroshell 120 EC-C18 column using methanol and 0.1% formic acid in water (90:10, v/v). Escitalopram was selected as the internal standard owing to its consistent extraction recovery, and satisfactory chromatographic performance. Plasma samples were prepared by liquid-liquid extraction with ethyl acetate, yielding recoveries of 87.5-91.6% for MPV, 87.5-89.4% for NHC, and 87.9-94.9% for BARI. The method was validated according to FDA bioanalytical guidelines over concentration ranges of 20-500, 20-10.000, and 20-500 ng/mL for MPV, NHC, and BARI, respectively. Accuracy, precision, stability, recovery, carryover, and matrix effect met the acceptance criteria. Normalized matrix factors for MPV, NHC, and BARI ranged from 0.948-0.984, 0.900-0.902, and 0.912-1.014, respectively, with CV values below 1%, indicating negligible matrix interference. The proposed method provides a reliable analytical tool for future pharmacokinetic and therapeutic drug monitoring studies.
BACKGROUND:Quantitating antibody drug conjugates (ADCs) with traditional ligand binding assays (LBAs) requires cytotoxic payload targeting reagents, which may be needed for ADC capture/detection. LBAs are drug antibody ratio insensitive (DAR insensitive) and prone to under- or over-estimating DAR species. METHODS:This hybrid LBA LC MS/MS assay measures concentrations for a novel ADC drug (BMS-X) with an engineered, site‑specific, non-cleavable linker, and a unique payload. Recombinant target protein-extracellular domain fused with mouse IgG-Fc was used for ADC pull down in human serum. Pepsin digestion released unique toxin linker peptide complexes for two conjugation sites. RESULTS AND CONCLUSIONS:This novel, validated DAR-sensitive hybrid-LCMS PK assay measured payload-conjugated BMS-X levels in human serum, quantitated surrogate analytes for both unique conjugation sites, and was used to support a first-in-human clinical trial.
The 17th Japan Bioanalysis Forum Symposium was held in Himeji, Japan, in March 2026. Under the theme "Unlock Scientific Potential: Future of Bioanalytical Expert," the symposium provided a comprehensive forum to share and discuss the latest scientific, technological, and regulatory developments in bioanalysis essential for drug development.The scientific program comprised 12 sessions including 35 oral presentations and 2 panel discussions, 38 general poster presentations, and 12 discussion group poster sessions. The topics spanned a broad range of disciplines, including implementation of ICH M10 and data integrity requirements, bioanalysis of oligonucleotides, gene and cell therapies, antibody-drug conjugates, immunogenicity assessment, biomarker analysis, and advances in LC-MS/MS and ligand‑binding assays. In addition, emerging trends such as artificial intelligence and automation in bioanalytical workflows were actively discussed. The newly introduced sessions for junior bioanalysts and Casual Follow-Up Sessions fostered open dialogue and received particularly strong engagement from the participants. The poster sessions by the individuals and by the discussion groups facilitated lively scientific exchange, further enhanced by the record number of company exhibits. Overall, the symposium successfully highlighted the current challenges and future directions in bioanalysis, reinforcing the critical role of bioanalytical experts in advancing drug development and regulatory science.
BACKGROUND:Therapeutic peptide-induced anti-drug antibodies (ADA) can have various effects, including adverse events and drug-neutralizing activity which can lead to loss of efficacy. This has led regulatory agencies to require nonclinical and clinical testing of biotherapeutics to assess immunogenicity risk. RESEARCH DESIGN AND METHODS:An electrochemiluminescence bridging assay was developed to detect antibodies against parathyroid hormone receptor inverse agonist (PTH-IA), a novel synthetic peptide that is being developed to treat Jansen's Metaphyseal Chondrodysplasia. Biotinylated PTH-IA and ruthenylated PTH-IA reagents were synthesized via amine-coupling chemistry, and rat polyclonal anti-PTH-IA positive control antibody was generated by immunizing rats with PTH-IA conjugated to keyhole limpet hemocyanin. The reagents were utilized to determine cut points for the screening and confirmatory assays and assay sensitivity in drug-naïve human plasma. RESULTS:Sensitivity of the screening assay was determined to be 3.61 ng/mL at cut point. The cut point for the confirmatory assay was determined to be 9.34% inhibition, and the cut point factor used to establish the floating plate cut point from the negative control signal was 1.055. The assay was found to be selective and reproducible. CONCLUSION:The assay's performance characteristics demonstrated its suitability for detecting anti-PTH-IA antibodies in human plasma.
BACKGROUND:Relative accuracy (RA) and linearity are distinct validation characteristics in bioassay validation, yet quantitative relationships between their acceptance criteria are rarely defined. This study investigated how predefined RA tolerances determine admissible linearity acceptance ranges. RESEARCH DESIGN AND METHODS:A theoretical framework for assessing dilutional linearity was established using a b-fold serial dilution design. Relative accuracy was defined as the ratio of observed to expected geometric means, evaluated against a tolerance threshold (θ). Linearity was determined through linear regression of logb(GMobserved) against logb(Dilution). Analytical expressions were derived for acceptable slope intervals based on θ and the number of valid dilution levels. A simulated bioassay dataset illustrated its application. RESULTS:Analytical derivations showed that admissible slope intervals depend exclusively on θ and the number of valid dilution levels. The resulting limits were invariant with respect to both the initial geometric mean and the dilution fold factor. In the illustrative example, the proposed framework supported acceptance of linearity while retaining all valid dilution levels. CONCLUSIONS:This study provides a mathematically consistent method for deriving linearity acceptance criteria directly from predefined RA requirements. Although illustrated using simulated data, it offers a tool for dilution-based bioassay validation and may support more coherent validation decisions.
Monitoring anti-drug antibody (ADA) responses is critical for evaluating the safety and efficacy of protein therapeutics. While traditional three-tiered testing (screening, confirmation, titration) reliably identifies ADA incidence, onset and magnitude, modern, complex biologics necessitate adapting our assessment strategies. Consequently, advanced assay designs are required to capture the full biological and clinical impact of these immune responses. Structured to guide bioanalytical scientists through this paradigm shift, this review first explores the transition toward integrated functional assessments, detailing how "active" pharmacokinetic (PK) and pharmacodynamic (PD) assays, neutralizing antibody (nAb) testing, and domain-specific assays help align immunogenicity characterization with a drug's mechanism of action (MoA). Next, we highlight Model-Informed Assay Development (MIAD) as a transformative tool for optimizing drug tolerance and estimating ADA-Reagent-Drug complex (ARC) formation. We then outline a risk-based, fit-for-purpose (FFP) framework for developing these advanced assays. Furthermore, we provide practical considerations for reporting advanced characterization data in regulatory filings. Finally, we conclude by differentiating benign ADA release from active inflammatory responses driven by the interaction of complex biologics with antigen-presenting cells, and explore future perspectives, specifically how Systems Immunogenicity and Artificial Intelligence (AI) will transition the field from retrospective monitoring to predictive immunogenicity profiling.
BACKGROUND:HTL0039732 (HTL) is a novel E-type prostanoid receptor 4 (EP4) antagonist for the treatment of advanced solid tumors. An analytical method was developed to facilitate the analysis of patient samples as part of an ongoing Phase I/IIa clinical trial. METHODS AND RESULTS:A LC-MS/MS method for quantifying HTL in human plasma was developed and validated per regulatory guidelines. The assay was selective, with a 2 ng/mL detection limit observed. It demonstrated good precision (CV ≤11.9%) and accuracy (86-101%), with linearity from 10 to 2000 ng/mL. Recovery was consistent, and no significant matrix, anticoagulant, or carryover effects were observed. CONCLUSIONS:A sensitive and selective method for measuring HTL in human plasma was successfully developed and applied to clinical samples, generating first‑in‑human pharmacokinetic data for HTL. The assay is now being utilized in an early‑phase clinical trial setting. CLINICAL TRIAL REGISTRATION:https://clinicaltrials.gov/study/NCT05944237.
Oligonucleotide therapeutics (ONTs) represent a rapidly expanding drug modality driven by advances in chemical modification, sequence design, and delivery technologies. These innovations have improved pharmacokinetics, tissue targeting, and clinical efficacy, but they may also introduce modality-specific challenges for immunogenicity risk assessment. Although ONTs are regulated as small molecules and routine immunogenicity testing is not universally required, anti-drug antibody (ADA) responses have been reported and may impact pharmacokinetics and safety in certain contexts. Compared with protein therapeutics, regulatory guidance, and practical experience for immunogenicity assessment of ONTs remain limited. This review summarizes the diverse oligonucleotide types, chemical modifications, and carrier modalities used in approved and emerging ONTs and examines how these attributes influence immunogenic risk and ADA assay development. Key challenges include potential nonspecific protein binding, reagent generation, immunodominance of conjugated components, and the need for specialized assay formats. Building on established principles from protein biologics while recognizing their limitations for oligonucleotides, this review highlights risk-informed strategies for ADA assay design to support immunogenicity assessment as ONT platforms continue to evolve.
BACKGROUND:Semaglutide is a human glucagon-like peptide-1 (GLP-1) peptide analog. To date, the literature does not report a validated HPLC-MS/MS assay using a triple-quadrupole mass spectrometer for the quantitation of semaglutide in human plasma in support of pharmacokinetics. A robust, sensitive HPLC--MS/MS assay supporting this class of compounds, is provided and full validation demonstrated. METHODS AND RESULTS:A triple-quadrupole HPLC-MS/MS method has been developed and fully validated for the quantitation of semaglutide in human plasma, using a stable isotope labeled internal standard and an effective, efficient protein precipitation sample extraction. The method has a sensitive analytical range of 5 ng/mL to 200 ng/mL (and to 1000 ng/mL with a dilution QC) and exhibits excellent accuracy (92.7% to 109.4%) and precision of <10.6%. This highly selective method provided high recovery of the analyte, with minimal matrix effects. CONCLUSION:A robust and simple HPLC-MS/MS method to quantify semaglutide in human plasma has been fully validated in accordance with the 2022 FDA M10 guidance. This method is suitable for implementation in clinical studies within the validated analytical range and stability conditions and can be extended to the bioanalysis of other GLP-1 peptide-based therapeutics with similar structural and physiochemical properties.
BACKGROUND:Accurate determination of salivary fluoride (F) depends on appropriate analytical conditions, particularly the buffering system used before ion-selective electrode measurements. RESEARCH DESIGN AND METHODS:This laboratory-based analytical study addressed the practical challenge of selecting between two total ionic strength adjustment buffers (TISAB II and TISAB III) for salivary F-determination using samples obtained from previously conducted clinical trials. Unstimulated saliva from eight participants was collected at baseline and at 5 min, 30 min, 1 h, 4 h, and 12 h after exposure to three F-treatments: F-varnish, 1,450 ppm F-dentifrice, and 5,000 ppm F-dentifrice. F-concentrations were measured using an F-ion-selective electrode after buffering with either TISAB II or TISAB III. Data were analyzed using paired statistical tests and Pearson correlation analysis (α = 0.05). RESULTS:No significant differences were observed between the buffers across most conditions, although differences were detected at later time points following dentifrice use. A strong positive correlation was found between the two buffering systems (p < 0.001), and no differences were detected in the area under the concentration-time curve. CONCLUSIONS:These findings indicate that both TISAB II and TISAB III provide comparable analytical performance for salivary fluoride determination under the tested conditions, although differences may occur at lower fluoride concentrations.
BACKGROUND:In clinical research, alternatives to venipuncture can reduce participant burden and increase accessibility and sampling opportunities for biomarkers. Residual pancreatic beta-cell function is assessed in clinical research and in diabetes care using fasting and stimulated C-peptide measurements, and changes are assessed over time. Operational, technical, and acceptability considerations of utilizing Tasso+ SST devices for C-peptide collection compared to venipuncture-derived serum samples were investigated. METHODOLOGY:Samples were collected from prospectively enrolled healthy participants via Third Generation Tasso+ SST micro-sampling devices and phlebotomy. Eleven fasted Tasso+ samples collected on-site or remote underwent alternative handling conditions and were evaluated for consistency, quality, and stability. Participants responded to device acceptability questionnaires. RESULTS:The Tasso+ SST micro-sampling device showed excellent concordance with simultaneous venipuncture-derived fasted C-Peptide, with concentration being largely conserved in healthy volunteers despite alternative sample handling. Delayed centrifugation over 72 hours caused no consequential degradation of C-peptide when kept at 4°C. Questionnaire responses indicate acceptable device usability and associated comfort levels. CONCLUSION:Fasting C-peptide levels can be accurately collected using micro-sampling collection techniques. Tasso+ C-peptide stability is seen when kept at 4°C. Micro-sampling devices can be used with shipping mechanisms (without centrifugation) to decrease participant burden, expand trial populations, and aid biomarker measurements.
BACKGROUND:Cumene is a high-production volume aromatic hydrocarbon recently reclassified as a Category 1B carcinogen, and 2-Phenyl-2-Propanol (2P2P) is its primary urinary metabolite. Highly sensitive analytical methods, required to detect the low-level exposures typical of the non- occupationally exposed general population, are not available for this compound in literature to this day. RESEARCH DESIGN AND METHODS:This research details the development and validation of a fully automated Gas Chromatography-Mass Spectrometry (GC-MS) method using SPME extraction to quantify 2P2P at ng/L levels. RESULTS:The study established reference values by analyzing 242 non-exposed workers, yielding a background range of 31.6-652.7 ng/L (5th-95th percentiles). The literature review encompassed global production trends, environmental ubiquity, and human metabolism. Furthermore, the study utilized biomarkers like cotinine and S-Phenyl Mercapturic Acid (SPMA) to statistically evaluate smoking as a potential confounder. CONCLUSION:This method fills a significant technical gap by providing a noninvasive, highly sensitive tool for health surveillance. Establishing empirical background reference values is essential for accurately distinguishing between environmental and occupational exposure. Critically, statistical analysis confirms that cigarette smoking is not a significant confounding factor for urinary 2P2P levels, reinforcing its reliability as a specific biomarker for cumene.
BACKGROUND:Bruton's tyrosine kinase inhibitors (BTKis, ibrutinib, zanubrutinib, orelabrutinib) are key targeted therapies for B-cell lymphomas, but interindividual variability and adverse reactions limit their use. Metabolized by CYP3A, co-medications or hepatic dysfunction cause plasma fluctuations, necessitating therapeutic drug monitoring (TDM). Cerebrospinal fluid (CSF) distribution is also critical for CNS lymphoma treatment. OBJECTIVE:To develop and validate a UHPLC-MS/MS method for simultaneous quantification of three BTKis and one metabolite in human plasma and CSF to support TDM. METHODS:After protein precipitation with methanol, samples were analyzed on an ACQUITY HSS T3 column with a 3-min gradient at 0.3 mL/min and ESI (+) MRM. Mobile phase: water-acetonitrile-formic acid-ammonium acetate (1 M) (950:50:1:1, A) and (50:950:1:1, B). RESULTS:The method showed excellent selectivity and linearity (r > 0.997) over 1-200 ng/mL (ibrutinib, zanubrutinib, ibrutinib metabolite) and 5-1000 ng/mL (orelabrutinib) in both matrices. Recoveries were 89.4-103.8%, matrix factors 0.93-1.06, and precision CV ≤8.5%. Stability was confirmed. The method was applied to 34 clinical samples (25 plasma, 9 CSF) from 20 patients. CONCLUSION:This method detects four BTK-related analytes in plasma/CSF with a 3-min run time, evaluation of hemolytic/lipemic matrices, and clinical validation, increasing throughput, reducing costs, and broadening applicability, providing preliminary feasibility data for clinical TDM.
BACKGROUND:The identification of anti-drug antibodies (ADA) raised against biologic drugs is important for understanding and ensuring efficacy and safety. Because the immunogenicity of novel biologics is unknown before clinical trials, ADA assays are often developed with positivity thresholds assigned based on statistically determined cut-points defined by testing of samples from treatment-naïve donors. RESEARCH DESIGN AND METHODS:While the standard approaches are based on reasonable theoretical models, we aimed to identify the impact of alternative analytical pipelines on assignment of ADA positivity by analysis of Tier 1 screening and Tier 2 confirmatory ADA bridging assay data for up to 138 mAbs across up to hundreds of serum samples. We evaluate the utility of data augmentation through bootstrapping and the impact of outlier removal approaches on the consistency of ADA status determinations. RESULTS AND CONCLUSION:We find that bootstrapping supports assay development efficiency by improving confidence in threshold setting in the context of limited numbers of test samples and that while outlier exclusion approach led to different apparent levels of ADA positivity, immunogenic drug products were identified by differences in the distribution of sample profiles from naïve and treated participants by each method evaluated. CLINICAL TRIAL REGISTRATION IDENTIFIERS INCLUDE:NCT02716675 https://clinicaltrials.gov/study/NCT02716675NCT02568215NCT02568215 https://clinicaltrials.gov/study/NCT02568215NCT03875209NCT03875209 https://clinicaltrials.gov/study/NCT03875209NCT04173819NCT04173819 https://clinicaltrials.gov/study/NCT04173819.
BACKGROUND:Quercetin, a dietary flavonoid with emerging therapeutic relevance in myotonic dystrophy type 1 (DM1), has low solubility and poor oral bioavailability. Enzymatically modified isoquercitrin (EMIQ), a water-soluble prodrug, raises systemic quercetin exposure. Pharmacokinetic studies require a sensitive assay that uses minimal sample volume. RESEARCH DESIGN AND METHODS:We developed a single-quadrupole liquid chromatography-mass spectrometry (LC-MS) assay for free quercetin, total quercetin (after enzymatic hydrolysis of glucuronide and sulfate conjugates), and the methylated metabolite isorhamnetin in mouse and human plasma. The method used protein precipitation, 10 µL of plasma, reversed-phase C18 separation, and single-ion recording of [M+H]+ adducts. Validation followed a fit-for-purpose approach consistent with M10 guidelines, and the assay was applied to plasma from EMIQ-treated DM1 and wild-type mice (15 g/L for 6 and 12 weeks). RESULTS:Calibration curves showed r2 > 0.99, with an LLOQ of 0.070 µM for quercetin in both matrices. The assay was successfully validated for quercetin in mouse and human plasma. Total quercetin and isorhamnetin were quantifiable in all treated mice. Exploratory analysis suggested glucuronidation as the major conjugation pathway. CONCLUSIONS:This simple, cost-effective microsampling assay suits preclinical and translational studies of EMIQ in DM1, though the conjugation findings remain exploratory.
The validation of anti-drug antibody (ADA) assays is vital in biologics development, with regulatory bodies like EMA and FDA emphasizing drug tolerance. In patient care, drug tolerance assessments should reflect actual clinical use. We developed an ADA assay for a fully human monoclonal antibody used in oncology, addressing the challenges posed by high circulating drug levels and target biology. Various assay formats were tested using multiple positive controls and drug concentrations that mimic real-world exposure and drug-to-ADA ratios. Assessing different positive controls at various concentrations was key to characterizing sensitivity and drug tolerance. An assay initially showing low drug tolerance with one monoclonal control performed adequately with others. Rather than limiting assessments to a single sensitivity level, we evaluated assay performance using clinically relevant drug and ADA concentrations. This approach ensures the assay's sensitivity and drug tolerance are meaningful for patient management and therapeutic decisions. Early and ongoing collaboration with health authorities supported alignment of clinically relevant performance criteria and interpretation strategies. Ultimately, this patient-oriented strategy guarantees ADA results that inform patient safety and treatment effectiveness for high-dose biologic therapies.
BACKGROUND:PF-07314470 is an antibody that was tested in a first-in-human study to evaluate its safety, pharmacokinetics, and pharmacodynamics. Because PF-07314470 is an interleukin-27 receptor (IL-27 R) agonist, the feasibility of a cell-based neutralizing antibody (NAb) assay was assessed as a tool to characterize anti-drug antibodies (ADA). Identifying a suitable assay endpoint is critical to NAb assay development. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) was chosen, because it reflected activation of IL-27 R and could be measured by different means. METHODS:PF-07314470 was used to activate IL-27 R on engineered Chinese Hamster ovary (CHO) cells, and resultant phospho-STAT3 (pSTAT3) levels were measured using a Meso Scale Discovery (MSD) assay. Experiments were conducted to confirm specificity of the pSTAT3 response and its inhibition by a NAb positive control. Biotin-Drug Extraction and Acid Dissociation (BEAD) was used to enhance drug tolerance. RESULTS:PF-07314470 induced pSTAT3 that was reliably measured by MSD. The pSTAT3 response was specific to PF-07314470 and could only be inhibited by a positive control against PF-07314470. Assay sensitivity and drug tolerance were achieved through implementation of BEAD. CONCLUSIONS:Experiments demonstrated the feasibility of a cell-based NAb assay for PF-07314470. The assay system could be used for other biotherapeutics that modulate pSTAT3.
