Bioanalysis最新文献

Fresenius Kabi has developed FKS518, a fully human monoclonal antibody biosimilar to denosumab. The clinical development program included two randomized comparative trials: a PK study in healthy volunteers and a safety and efficacy study in osteoporosis patients. The demonstration of similarity and equivalence between FKS518 and reference denosumab included the quantitation of the serum biomarker C-terminal cross-linking telopeptide of Type 1 collagen (CTx-1), a well-established marker of bone resorption. This paper details the development, validation, and analytical performance of the method for CTx-1 quantitation, emphasizing how the Context of Use (CoU) shaped the validation requirements. Application of the method in samples from the pharmacokinetic (PK) equivalence study allowed demonstration of pharmacodynamic (PD) similarity between FKS518 and Prolia. This publication also addresses the use of endogenous serum control (ESC) samples in monitoring assay performance. A retrospective analysis indicated that applying more flexible ranges and/or omitting ESC-based criteria for run acceptance would not have substantially changed the CTx-1 results. While recognizing the value of ESCs for stability and trending analysis and the importance of rigorous biomarker method development and validation in the assessment of biosimilarity, this paper fosters the discussion whether run acceptance based on tight ESC acceptance limits is always necessary.

Aims: The aim of the work was to develop and validate an anti-drug antibody (ADA) assay for RGB-19, a proposed biosimilar to tocilizumab, capable of detecting anti-tocilizumab antibodies in the presence of high concentrations of soluble IL-6 receptor (sIL-6R), the drug's pharmacological target.

Methods: A bridging electrochemiluminescent immunoassay was applied. To mitigate target interference, sarilumab - a high-affinity anti-sIL-6R antibody - was incorporated into the sample preparation. The assay was validated per EMA and FDA guidelines.

Results: The assay demonstrated high sensitivity, robust precision, and selectivity across normal and diseased matrices. Drug tolerance exceeded 280 µg/mL at regulatory sensitivity thresholds, and target tolerance was confirmed up to 1000 ng/mL sIL-6R. Performance monitoring during Phase I and III clinical sample analysis confirmed stability and acceptable false-positive rates.

Conclusion: The validated ADA assay effectively neutralized sIL-6R interference and provided reliable immunogenicity assessment for RGB-19 clinical trials. Its robustness supports biosimilarity evaluation and ensures compliance with regulatory standards. Validation of this ADA assay is essential for regulatory compliance and patient safety. By ensuring accurate ADA detection under clinically relevant conditions, it supports biosimilarity demonstration and streamlines regulatory review, ultimately facilitating timely access to cost-effective biologic therapies.

Clinical trial registration: https://jrct.mhlw.go.jp.Identifiers are: jRCT2031230029 and jRCT2031220512.

The 19^th Workshop on Recent Issues in Bioanalysis (19^th WRIB) took place in New Orleans, LA, USA on April 7-11, 2025. Over 1200 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 19^th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on "Implementation Practice for the Newest ELN/LIMS Systems" and on "Vaccine Cell-Based/Functional & Molecular Assays as part of the harmonization of vaccine clinical assays global initiative" were the special features of the 19^th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and Regulatory Agency experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.This 2025 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2025 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication (Part 2) covers in the Part 2A the recommendations on Biomarkers/BAV, IVD/CDx, Ligand-Binding Assays and Cell-Based Assays and in Part 3B the Regulatory Inputs on these topics. Part 1 (Mass Spectrometry Assays and Regulated Bioanalysis/BMV) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 18 of Bioanalysis, issues 3 and 1 (2026), respectively.

Aim: To develop and validate a robust LC - MS/MS method for the simultaneous quantification of endogenous deoxycholic acid (DCA), 1β-hydroxydeoxycholic acid (1β-OH-DCA), 1β-hydroxydeoxycholic acid glycine conjugate (1β-OH-G-DCA), and 1β-hydroxydeoxycholic acid taurine conjugate (1β-OH-T-DCA) in human plasma as biomarkers for the assessment of CYP3A-mediated drug-drug interactions (DDI).

Materials and methods: A surrogate matrix was employed for the preparation of calibration standards and quality control samples. Analytical quantification was achieved using protein precipitation extraction coupled with UHPLC - MS/MS analysis.

Results: A multiplex LC - MS/MS assay was successfully developed and validated for the simultaneous quantification of DCA (4-2000 ng/mL), 1β-OH-DCA (0.05-100 ng/mL), 1β-OH-G-DCA (0.05-100 ng/mL), and 1β-OH-T-DCA (0.05-100 ng/mL) in human plasma. The method effectively addressed the analytical challenges associated with developing an integrated assay for endogenous analytes spanning markedly different concentration ranges and was compliant with the ICH M10 bioanalytical method validation guidance.

Conclusion: This is the first report describing the development and validation of a 4-in-1 LC - MS/MS method for the simultaneous quantification of DCA, 1β-OH-DCA, 1β-OH-G-DCA, and 1β-OH-T-DCA in human plasma. The assay provides a robust bioanalytical platform for evaluating CYP3A-mediated DDIs and offers a methodological framework applicable to other multiplexed methods for endogenous biomarkers.

Aim: To develop and validate a rapid, sensitive, and selective LC - MS/MS method for quantifying polmacoxib (POL) in rat plasma and integrating for preclinical pharmacokinetic evaluation.

Materials and methods: POL was quantified using LC - MS/MS on a Varian C8 column with an isocratic mobile phase of methanol and 2 mM ammonium acetate (90:10, v/v; 0.4 mL/min) with run time of 6 min. Detection employed negative ion electrospray and MRM (m/z 360 → 296 for POL; m/z 313 → 257 for rofecoxib as IS). The method was validated as per USFDA guidelines. Male Sprague - Dawley rats received a single oral dose of 10 mg/kg POL, and plasma samples were collected up to 72 h.

Results: The method showed linearity (1.56-800 ng/mL, r²  = 0.9994), LLOQ 1.56 ng/mL, and acceptable accuracy, precision, recovery, and matrix effects. Pharmacokinetics: Cmax 643 ± 32 ng/mL, Tmax 4 h, T1/2 10.4 ± 0.8 h, AUC₀-∞ 5326 ± 106 ng*h/mL.

Conclusions: The validated assay is robust, sensitive, and suitable for pharmacokinetic, bioequivalence, and drug - drug interaction studies of POL.

As outsourcing has become mainstream in the bioanalytical industry, sponsor organizations commonly develop bioanalytical assays in-house before transferring them to contract research organizations (CROs) for validation and sample testing. However, bioanalytical assay transfer processes represent one of the most challenging workflows and can consume substantial resources in terms of personnel, time, and cost. We developed and implemented a systematic approach to enhance bioanalytical assay quality and transfer efficiency, significantly reducing transfer and troubleshooting time while improving overall success rates.

Background: The erythropoiesis stimulating agents (ESAs), erythropoietin (Epo) and darbepoetin (Darbe), are administered clinically to preterm infants to stimulate red cell production and decrease red blood cell transfusions. Darbe is longer acting compared to Epo, allowing for weekly administration; however, Darbe does not have a specific assay to allow for serum measurements.

Research design and methods: We developed a sensitive and specific electrochemiluminescent assay for future reliable measurement of serum and plasma Darbe concentrations in samples from preterm infants participating in the multicenter randomized Phase III Darbepoetin Trial to Improve Red Cell Mass and Neuroprotection in Preterm Infants. A multi-array electrochemiluminescence platform was used to develop an immunoassay to quantitatively measure Darbe concentrations in biological fluids.

Results: Capture and detection antibodies were identified, evaluated, and optimized. Assay sensitivity, intra- and inter-plate reproducibility, and specificity were assessed to verify the utility of the assay for the purpose of accurately measuring Darbe in these samples.

Conclusions: A reliable and accurate assay to measure Darbe concentrations in serum or plasma was developed and validated on a clinically translatable platform. This method will be applied to quantify Darbe serum concentrations in extremely premature infants and to perform population pharmacokinetic analyses.Clinical Trial Registration: https://clinicaltrials.gov/study/NCT03169881. Identifier is NCT03169881.

Aim: Anti-drug antibodies (ADAs) can impact drug efficacy and safety, necessitating sensitive and drug-tolerant assays for accurate detection. The main goal of the study was re-optimization of the Gyrolab immunoassay intended to detect ADAs against MabionCD20 (rituximab biosimilar) in rheumatoid arthritis patient serum. The study focused on eliminating a nonspecific interaction that impacted assay repeatability.

Results: The nonspecific interactions were mitigated by evaluating numerous method modification strategies including additional washing steps, increasing buffer ionic strength, and step-by-step modification of the assay protocol to isolate the one responsible for the observed issue. Reducing molarity and increasing pH of neutralization buffer resulted in elimination of unwanted interactions between assay components and effectively improved assay performance in repeatability and drug tolerance parameters, supporting the assay's suitability for validation.

Conclusion: Careful optimization of assay conditions, particularly buffer composition and pH successfully resolved issues related to the nonspecific interactions and enhanced assay robustness. Optimized Gyrolab-based immunoassay provided a reliable method for ADA detection, supporting immunogenicity assessment in a clinical development program.

Background: Keratan sulfate (KS) is altered in several pathological conditions. We report the capillary electrophoresis-laser induced fluorescence separation and validation of KS-derived disaccharides Galβ(1-4)GlcNAc(6S) and Gal(6S)β(1-4)GlcNAc(6S) in human urine and plasma after keratanase II treatment and AMAC fluorotagging.

Results: The calibration curves of the two disaccharides from 50 to 1000 ng showed an average correlation coefficient greater than 0.9970 and the calculated LOD and LOQ were 3 and 10 ng, respectively. The inter-day precision was ~13% and the inter-day accuracy was ~10% for both disaccharides. The % recovery ranged between 88% and 106% for 200, 500, and 800 ng of each disaccharide added to biofluids. Urine and plasma of Morquio A and B subjects and of patients submitted to ERT treatment were tested. Urinary KS was ~13-16-fold more abundant in MPSIVA and plasmatic KS was significantly ~2-3-fold more than controls. No differences were observed for the disaccharides ratio between Patients and not-affected Subjects.

Conclusion: The capillary electrophoresis separation of the two main KS disaccharides in human biofluids was found specific, sensitive, and accurate and suitable for application to Morquio syndrome early detection and progression as well as for other conditions in which KS is altered in content and structure.