Aug 2026· Analytical and Bioanalytical Chemistry· 0 citations· 37 references
Medicine
TL;DR
This review sets out the analytical validation, standardization, and quality assurance requirements that would allow a research-grade catalytic protease assay to become a laboratory-developed test or an approved in vitro diagnostic and proposes a staged minimum validation-and-reporting framework.
Abstract
Activity-based (catalytic) measurement of proteases in body fluids has emerged as a diagnostic modality distinct from antibody-based concentration measurement, supported by proof-of-concept evidence in oncologic and non-oncologic disease. In analytical chemistry terms, the open question is how to characterize and control a reaction-rate measurement made in a complex biological matrix. This review sets out the analytical validation, standardization, and quality assurance requirements that would allow a research-grade catalytic protease assay to become a laboratory-developed test or an approved in vitro diagnostic. We delimit the assay class across its reporter formats, explain why the metrological framework of clinical chemistry must be adapted for activity measurement in complex matrices, and operationalize the core analytical parameters for a kinetic, matrix-dependent readout: analytical specificity, precision, detection and quantification limits, linearity, trueness, robustness, and interference. We address the unresolved problem of defining and tracing a unit of proteolytic activity, the absence of quality control materials and external quality assessment schemes, and the regulatory pathway under current US and European frameworks. Catalytic protease measurement is analytically plausible and biologically informative but remains research-grade. Its translation depends less on new biology than on analytical validation, harmonization, quality control, and regulatory conformity. We propose a staged minimum validation-and-reporting framework, condensed into a checklist, as a starting point for the field to develop by consensus.
Validation of analytical methods is a mandatory requirement for testing laboratories accredited under ISO/IEC 17025:2017. Immune response to vaccination is inherently heterogeneous, multidimensional, and distributed along continua rather than binary outcomes. ISO/IEC 17025:2017 provides valuable principles and a structured framework to support method validation, but the unique performance characteristics and biological variability of immunoassays often require adapting ISO guidance to assay-specific validation criteria. Its general requirements (impartiality, competence, method validation, measurement uncertainty, and result interpretation) can be meaningfully applied to complex immune-response assays to establish regulatory credibility, but compliance often requires innovation. This study presents a process framework for validating advanced immunological methods, including ELISA cutoff-based assays, multiplex cytokine profiling, functional IFN-γ T cell readouts, and microneutralization assays, in accordance with international guidelines, and provides clearly defined acceptance criteria for each parameter. The validation parameters included evaluation of analytical performance (precision, accuracy, specificity) and uncertainty estimation for the analytical methods we specifically used to assess the immunogenicity of the AVX-COVID-12 anti-SARS-CoV-2 vaccine. Here, we discuss the main challenges encountered, propose methodological strategies, and present validation outcomes to meet stringent accreditation requirements, using robust experimental designs and acceptance criteria informed by the literature. This process framework, based on lessons learned, ensures the reliability and traceability of critical results needed to approve newly developed biotechnological products targeting the immune response in similar conditions.
Andrea Palencia-Reyes, Esmeralda Juárez, Carlos Blancas-Ruiíz et al.· Accreditation and Quality As...· 0 citations
Accurate and comparable quantification of somatic mutations is essential for precision oncology, as clinical decision-making increasingly relies on the quantification of molecular biomarkers. Despite major technological advances, inter-laboratory variability and the lack of metrological traceability remain significant barriers to harmonization and confidence in mutation testing results. Reference Measurement Procedures (RMPs) represent a critical framework to address these challenges by anchoring molecular measurements to common quantitative standards. Here, we describe the development and validation of a candidate RMP for the detection and quantification of the clinically relevant NRAS p.Q61R mutation using digital PCR (dPCR). The assay was systematically optimized to maximize specificity and minimize cross-reactivity between wild-type and mutant alleles. Analytical characterization demonstrated excellent linearity across a broad range of variant allele frequencies (vAF), with a limit of detection of 0.1 %. Precision studies performed on commercially available circulating tumour DNA reference materials (RM) showed good repeatability and intermediate precision, while a full measurement uncertainty budget confirmed the robustness of the approach. Comparison with a commercial dPCR assay provided independent support for assay comparability and consistent vAF estimates across the investigated range. Preliminary inter-laboratory assessment supported transferability of the candidate RMP and comparability of the resulting measurements. Overall, this work establishes a metrologically characterized and transferable dPCR-based RMP for NRAS p.Q61R quantification. Its implementation can support the harmonization of molecular measurements, the value assignment of RM, and the alignment of routine and secondary methods, thereby strengthening the reliability of quantitative biomarker assessment in precision oncology.
Jessica Petiti, Sabrina Caria, L. Revel et al.· Methods· 0 citations
Bioanalysis represents a cornerstone discipline within pharmaceutical sciences, dedicated to the qualitative and
quantitative determination of drugs, metabolites, biomarkers, and endogenous compounds across diverse
biological matrices including plasma, serum, urine, saliva, and tissues. The generation of accurate and reliable
bioanalytical data is indispensable for drug discovery, preclinical evaluation, clinical development, therapeutic
drug monitoring, bioavailability, bioequivalence, and regulatory submissions. Over time, bioanalytical
methodologies have undergone significant evolution, progressing from conventional chromatographic techniques
to advanced analytical platforms such as liquid chromatography–tandem mass spectrometry (LC–MS/MS), highresolution mass spectrometry (HRMS), and ligand-binding assays (LBAs). Method development encompasses
systematic optimization of sample preparation, chromatographic separation, detection systems, and analytical
conditions to ensure robust and reproducible performance. Validation of bioanalytical methods is a critical process
that establishes reliability, accuracy, precision, selectivity, sensitivity, recovery, stability, and reproducibility,
thereby confirming suitability for regulatory and scientific applications. International regulatory authorities,
including the United States Food and Drug Administration (FDA), the European Medicines Agency (EMA), the
World Health Organization (WHO), and the International Council for Harmonisation (ICH), have issued
comprehensive guidelines to standardize validation practices, with the ICH M10 guideline providing a globally
harmonized framework. Despite remarkable technological advancements, persistent challenges such as matrix
effects, analyte instability, limited sample volumes, and the increasing complexity of biologics continue to shape
bioanalytical research. Recent innovations—including ultra-performance liquid chromatography (UPLC), micro
sampling technologies, automation, artificial intelligence, and biomarker-driven analysis—have expanded
analytical capabilities, improved efficiency, and broadened the scope of bioanalysis. This review offers a
comprehensive synthesis of bioanalytical method development, validation principles, regulatory frameworks,
prevailing challenges, and emerging trends, underscoring the pivotal role of bioanalysis in contemporary drug
development and personalized medicine
M. S. Jadhav, Dhanashri Mali, S. Amrutkar· International Journal of Dru...· 0 citations
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.
Gregor P Lotz, D. Sickert, R. Staack et al.· Bioanalysis· 0 citations
Introduction / Objective: The selectivity, potency, and stability of antiviral compounds, including Ritonavir (RTV),
require high-precision analytical testing and impurity profiling for confirmation. As a widely used pharmacokinetic
enhancer in COVID-19 therapy, the regulation and clinic validation of impurities and their toxicity are of the utmost
importance. The main goal of the present work was to develop and confirm a simple, accurate, and stability, indicating
HPLC method for determining the Ritonavir content and its related impurity (Impurity, A Free Base) includes theoretical
and experimental toxicity assessments. Methods: Chromatographic separation was performed on a Phenomenex C18
column (250 4.6 mm, 5 m). The mobile phase was 20:80 of water and acetonitrile, flow rate 1.0 mL/min. The method
validation tested different things using ICH Q2(R1) rules, Accuracy, Precision, Specificity, and Linearity. The toxicity
assessment for Impurity A was done by the insilico prediction tools (PROTOX 3.0) and checked with an in vitro study of
toxicity in PBMC cells. Results: The method demonstrated (R = 0.9999) in a range of 1.64 to 29.94 g/mL, with recovery
rates from 92% to 99% and a relative standard deviation below 2%. Ritonavir and its impurity- A with distinct retention
times is 4.3 min and 2.3 min respectively. In silico studies stated that the drug has a medium acute oral toxicity as low
with a LD50 values of more than 2000 mg/kg with mild hepatotoxic and neurotoxic while MTT results showed an IC50
value >500 µg/mL, suggesting low cytotoxicity. Discussion: The validated HPLC method is a reliable, accurate, and
economical way for the routine analysis of Ritonavir and its contaminants. The use of computational and in vitro toxicity
assessments used to identify impurity hazards and comply with quality control standards of antiviral drugs. Conclusion:
A consistent, precise, and cost-effective HPLC method was successfully developed and validated for Ritonavir and its
related impurity. The integration of in silico and in vitro toxicity studies strengthens impurity risk assessment and supports
the safe and effective quality control of antiviral pharmaceutical products.
Balaji Ramakrishnan, J. S, Esakkimuthukumar Mariappan et al.· International Journal of Dru...· 0 citations
BackgroundProstate-specific antigen (PSA) remains the most widely used biomarker for prostate cancer screening, diagnosis, and monitoring. However, despite decades of standardization efforts, significant inter-assay variability persists, with important consequences for clinical interpretation and decision-making.ObjectiveThis review aims to evaluate the impact of PSA calibration and harmonization on reference intervals, clinical thresholds, and population-based screening strategies in contemporary clinical practice.MethodsA literature-based analysis was conducted, examining studies on PSA assay standardization, analytical variability, and evidence from population screening trials, including considerations from a Health Technology Assessment perspective.ResultsThe introduction of the World Health Organization (WHO) International Standard 96/670 improved comparability among PSA assays, yet clinically relevant differences between platforms remain. This variability is driven by differences in calibration, antibody specificity, epitope recognition, and assay design. As a result, PSA values are not directly interchangeable across assays, and assay-specific cut-offs may be necessary to maintain diagnostic performance. Historically established thresholds, such as the 4 μg/L cut-off and the "gray zone," were derived using specific assay systems and are influenced by methodological limitations. PSA-derived metrics, including PSA density, improve specificity but are still affected by inter-assay variability. Evidence from large randomized trials supports a PSA cut-off of 3.0 μg/L for population screening, showing a reduction in prostate cancer mortality when implemented within structured programs; however, this threshold is intrinsically linked to the analytical characteristics of the assays used. PSA also shows relevant intra-individual biological variability beyond analytical variation, with within-subject variation of 6-13%.ConclusionsPSA standardization remains incomplete, and inter-assay variability and biological variability continues to influence clinical interpretation. The implementation of PSA-based screening programs must explicitly consider the assay-specific nature of evidence-derived thresholds, including the 3.0 μg/L cut-off, to ensure consistent, effective, and safe clinical decision-making.
X. Filella· Tumour biology : the journal...· 0 citations