Bioanalysis最新文献

Immunogenicity undermines the safety and effectiveness of biologics and emerging modalities. Routine workflows are constrained by drug/target interference in binding assays, matrix and dilution biases in functional assays, fragile cellular/innate readouts, and the difficulty of translating titers into patient-level risk. Advances in artificial intelligence (AI) and real-world evidence (RWE) offer practical remedies. This narrative review searched five search engines and databases and included evidence from published original research, reviews, and regulatory data, and reports between 2022 and 2025. It identifies common failure locations and links them to practical AI and RWE solutions. We outline the regulatory and data underpinnings for decision-grade RWE, provide a closed-loop blueprint, and summarize recent developments in T- and B-cell epitope prediction, neoantigen prioritization, and pharmacovigilance/NLP pipelines for case deduplication and normalization: Define laboratory panels and computable phenotypes, map multi-source data to a common model, deduplicate, train transparent internally validated models, externally validate across distributed networks, route outputs to clinic/lab actions, and continuously monitor for drift with auditable updates. AI-augmented RWE can replace assay-centric snapshots in immunogenicity assessment with a learning system that focusses on clinically significant ADA/NAb effects, targets limited laboratory resources, speeds up signal verification, and enhances post-market vigilance.

Accurate quantification of oxidative mitochondrial DNA (mtDNA) lesions remains technically challenging due to the limitations of existing assays, which often require large sample inputs, multi-day workflows, and offer limited sensitivity. Here we introduce FALCON-qPCR (Fpg-assisted Long-PCR), a streamlined, high-sensitivity method for quantifying oxidative damage in mtDNA. FALCON-qPCR couples digestion with formamidopyrimidine [fapy]-DNA glycosylase (Fpg) to long-range PCR and qPCR-based normalization, enabling precise lesion quantification from as few as 10,000 cells (~300 ng total DNA) within a single day. The assay provides a robust dynamic range and reproducibility across diverse biological systems, including human cell lines, hepatocellular carcinoma biopsies, and Caenorhabditis elegans. Compared with established methods, FALCON-qPCR exhibits markedly higher sensitivity in detecting mtDNA damage induced by hydrogen peroxide, antimycin A, and rotenone. Its performance was further demonstrated in assessing mitochondrial toxicity of ruthenium-based compounds, highlighting its potential for pharmacological screening. By integrating enzymatic lesion recognition with quantitative amplification in a unified workflow, FALCON-qPCR eliminates the need for mitochondrial isolation. This methodological advance provides a rapid, accurate, and scalable platform for studying oxidative DNA damage, with broad applicability in mitochondrial research and translational toxicology.

Background: Reliable methods for measuring plasma concentrations of cannabidiol (CBD) and its metabolites are essential for pharmacokinetic studies. Several methods have been published, some involve complex procedures. This study aimed to develop a simple yet robust method for quantitating CBD, 7-OH-CBD and 7-COOH-CBD in human plasma.

Research design and methods: Sample preparation was evaluated using various solvent systems. Analytical parameters were examined and optimized. The method was validated for sensitivity, accuracy, precision, selectivity, specificity, carryover, dilution integrity, long-term stability, freeze-thaw stability and recovery.

Results: A simple protein precipitation method using acetonitrile provided optimal sample preparation. All analytes were quantified in negative ionization mode, with complete separation achieved on a C₁₈ column within 3.5 minutes using an isocratic mobile phase. The method met all validation criteria. Analytes remained stable under long-term storage at -30°C, through freeze-thaw cycles and at refrigerated temperatures, although 7-COOH-CBD showed a slight decrease after two months at refrigerated temperatures.

Conclusion: This study presents a simple, rapid and robust bioanalytical method for the simultaneous quantification of CBD and its major metabolites across clinically relevant plasma concentrations. The method's reliability and efficiency make it highly suitable for routine clinical use and pharmacokinetic applications.

The 16th Japan Bioanalysis Forum (JBF) Symposium was held from March 3 to 5, 2025, at the Himeji Culture and Convention Center (Arcrea Himeji). The theme for the symposium was 'Bioanalyst Network: Spread and Circulation of Knowledge.' This theme symbolizes our commitment, based on the principle at the JBF foundation to provide a platform for discussion and interaction among bioanalysts in academia, industry, and government. It aims to restore the interaction among bioanalysts that has diminished during the Coronavirus disease 2019 (COVID-19) pandemic, and to discuss and share knowledge about rapidly evolving scientific technologies and the extensive insights of bioanalysis under regulatory. The symposium discussed a wide range of topics, including the latest mass spectrometric techniques, immunogenicity assessment, antibody - drug conjugate evaluation, biomarkers, ICH-M10, ICH-S12, data integrity, reliability assurance, and the interactive discussion program 'iDG,' inviting domestic and overseas experts. Approximately 400 attendees from various fields, including pharmaceutical companies, contract research organizations (CROs), academia, and regulators, gathered in person.

Objective: This study detailed the troubleshooting and optimization of a challenging neutralizing antibody (NAb) assay using a previously published PEG precipitation, Acid Dissociation, and Biotin-drug as Assay Drug (PABAD) method to overcome high drug interference.

Materials and methods: A strong NAb positive control, PC3, showed poor recovery in the PABAD workflow. A PABAD-compatible PC, PC31 was selected among different anti-idiotype antibodies with varied kinetic profiles. A three-tier Design of Experiment (DOE) approach was applied to optimize assay conditions. Centrifugation-based Bluewasher protocols were also established to automate the handling of PEG pellets.

Results: The reason for the poor performance of PC3 with the PABAD method was identified, and a sensitive NAb assay was successfully developed using PC31.

Conclusions: The poor recovery of PC3 was not due to incompatibility with the PABAD method, but rather to its non-specific interaction with serum factors. Once a specific NAb PC was identified, the NAb assay was successfully developed using PABAD, demonstrating a broad application of the method. DOE-assisted assay development cut the development time by half and meaningfully improved the assay sensitivity. Finally, the implementation of the Bluewasher in the pellet wash further reduced assay variability and enhanced automation of the PABAD method.

Aim: Quantification of drug concentrations in human skin is essential for understanding mechanisms of action in dermatologic therapeutics but remains analytically challenging. The study aimed to develop and validate a standardized tissue homogenization and liquid chromatography - tandem mass spectrometry workflow for quantifying povorcitinib in human skin biopsies.

Materials & methods: Key skin homogenization parameters, including solvent composition, processing volume, and homogenate density, were systematically evaluated. Wet and dry homogenization approaches were compared. Following protein precipitation, samples were analyzed using liquid chromatography - tandem mass spectrometry.

Results: Eighteen solvent systems and various processing volumes were evaluated, with three solvent mixtures producing homogeneous preparations. Limiting homogenization volumes to less than half tube capacity and using 20 mg/mL improved sample consistency. Povorcitinib stability under acidic and thermal conditions was confirmed. Comparable performance was observed between wet and dry homogenization methods, and a dry workflow was validated.

Conclusions: This study provides the first systemic evaluation of human skin homogenization parameters and a direct comparison of wet and dry approaches. The validated dry homogenization method enabled accurate and reproducible quantification of povorcitinib in skin biopsies from a phase I clinical study and offers a robust framework for quantifying a broad range of analytes in human skin tissues.Clinical trial registration: https://www.clinicaltrials.gov/study/NCT06505265 [clinicaltrials.gov] identifier is NCT06505265.

Background: Hybrid LC - MS/MS methodology offers orthogonal ADA quantitation but is limited by endogenous immunoglobulin matrix interference challenges.

Methods: We developed a novel semi-quantitative LC-MS/MS isotyping assay for rabbit IgG/IgM immunogenicity in K₂EDTA plasma using double immunoprecipitation with double acid dissociation.

Results and conclusions: As compared to single immunoprecipitation, double immunoprecipitation reduced endogenous IgG by ≥90% and IgM by >95% (W1B1) or >55% (W3B2), improved peak-area ratios, and achieved an LLOQ of 100 ng/mL, with acceptable accuracy and precision. Double immunoprecipitation plus acid dissociation mitigates matrix interference and enables robust semi-quantitative hybrid LC-MS/MS ADA isotyping in rabbit plasma.

Major depressive disorder (MDD) is a complex and disabling psychiatric condition, often chronic and recurrent, for which treatment selection remains largely guided by trial-and-error. Objective biomarkers capable of predicting and monitoring therapeutic response are urgently needed to enable personalized interventions and reduce exposure of the patients to ineffective treatments. Mass spectrometry (MS) has emerged as a central analytical technique, providing the sensitivity and specificity required to capture multi-parametric molecular signatures in biofluids such as plasma, serum, cerebrospinal fluid, and urine. In this context, this review summarizes the application of MS-based platforms to characterize molecular changes associated with treatment response in MDD and discusses how integrative multi-omics approaches can generate robust insights into treatment efficacy and resistance. We highlight current applications, discuss opportunities for MS-guided monitoring of therapeutic strategies, and outline how convergent molecular signatures may accelerate the development of clinically informative biomarkers for precision psychiatry in MDD.

Background: Fresenius Kabi developed FKS518, a fully human monoclonal antibody biosimilar to denosumab, that inhibits osteoclast activation by targeting the receptor activator of nuclear factor kappa-Β ligand (RANKL). RANKL exists as a trimeric soluble protein (sRANKL) with high affinity for denosumab. Post-dose, sRANKL levels can rise due to drug-target complex accumulation, creating a risk of interference in bridging anti-drug antibodies (ADA) assays, particularly after acid dissociation.

Methods: We evaluated sRANKL interference in the ADA bridging assay and, to competitively block sRANKL, we introduced a specificity tier by adding osteoprotegerin (OPG). This approach enabled reanalysis of previously ADA-positive samples to confirm whether signals represented true ADA responses or artifacts caused by sRANKL interference.

Results: Acid dissociation significantly exacerbated target interference, resulting in ADA positivity rates of ~96-98% in clinical studies. Introducing a specificity tier corrected incidence to ≤3.9%. OPG incorporation did not change the minimum required dilution (MRD) of the assay and did not affect signals for negative/positive control, confirming assay integrity.

Discussion: These findings underscore the importance of early interference assessment and mitigation. A multi-tiered strategy, encompassing screening, confirmatory, and specificity tiers, provided a robust solution applicable to programs facing similar challenges.

Background: Quantitation of adrenocorticotropic hormone (ACTH) in human plasma by clinical immunoassays is prone to selectivity challenges, whereas quantitation by mass spectrometry assays (MSA) is limited by sensitivity. The use of nano-liquid chromatography (nano-LC) addresses the MSA sensitivity gap but commonly suffers from poor robustness due to LC system backpressure issues, which is mitigated by incorporation of online size exclusion chromatography (SEC).

Research design and methods: An antibody-free, multidimensional nano-LC, high-resolution mass spectrometry method was designed and implemented to measure intact ACTH[1-39] in human plasma. Online SEC separation was performed at relatively high flow rates prior to trap and elute reversed phase separations at sub-µL/min flow rates. ACTH[1-39] was detected using a high-resolution orbitrap mass analyzer.

Results and conclusions: Nanoelectrospray allows measurement of ACTH[1-39] to a clinically relevant concentration of 3-5 pg/mL, with low bias and high precision across the concentration range interrogated. SEC reduces microfluidic backpressure effects commonly observed with the use of nano-LC for bioanalytical applications. Eliminating antibody capture reduces susceptibility to endogenous antibody interferences. This format can be readily adapted for multiplexed measurement of other low abundance peptide hormones in biological samples.