Bioanalysis最新文献

Ultrasmall and highly fluorescent gold nanoclusters (Au NCs) have been widely used for the construction of sensing and imaging platforms. Specifically, through a combination of surface functionalization and spectral analysis and/or imaging techniques, effective intracellular detection and imaging are realized. In this review, we summarize the recently adopted intracellular analysis and imaging events with Au NCs-based probes. The synthesis of Au NCs is briefly introduced based on stabilizer selection. The principles and applications of fluorometric intracellular detection systems toward different analytes, including small molecules and biomacromolecules, are presented by turnoff, turn-on, and ratiometric tactics. The cell imaging events are summarized based on conventional imaging and high-resolution imaging techniques, respectively. In the end, this review highlights the challenges of intracellular applications with Au NCs.

Background: Most oligonucleotide bioanalytical assays currently only quantify the pharmacologically-active antisense strand, though there have been recent efforts to simultaneously quantify the sense strand using hybridization ELISA or solid phase extraction LC-MS. Hybrid LC-MS, which offers both high sensitivity and specificity unlike the currently used platforms, has not been applied to quantify both siRNA strands simultaneously.

Materials & methods: A hybrid LC-MS assay utilizing LNA capture probes was developed and applied to quantify both strands of a 21-mer lipid-conjugated siRNA (SIR-3) using tandem mass spectrometry (MS/MS). A similar approach using high-resolution mass spectrometry (HRMS) was also evaluated.

Results: The final LC-MS/MS method was capable of quantifying both strands of SIR-3 at concentrations between 0.600 and 1000 ng/mL in cynomolgus monkey tissue homogenates with acceptable accuracy and precision. The LC-HRMS assay demonstrated similar sensitivity and assay performance as the LC-MS/MS assay.

Conclusions: Overall, this manuscript presents orthogonal methods to existing siRNA bioanalytical workflows that with high sensitivity and specificity can provide greater information about the concentration and biotransformation of an siRNA analyte.

This article examines the transformative potential of blockchain technology and its integration with artificial intelligence (AI) in clinical trials, focusing on their combined ability to enhance integrity, operational efficiency, and transparency in the data governance. Through an in-depth analysis of recent advancements, the article highlights how blockchain and AI address critical challenges, including patient data privacy, regulatory compliance, and security. The article also identifies key barriers to adoption in the mentioned integration, such as scalability limitations, association with existing healthcare systems, and high implementation costs. By presenting a comprehensive overview of the current research and proposing strategic directions, this work emphasizes how the synergy between blockchain and AI can revolutionize clinical trials through process automation, improved stakeholder trust, and robust transparency.

The 18th Workshop on Recent Issues in Bioanalysis (18th WRIB) took place in San Antonio, TX, USA on May 6-10, 2024. Over 1100 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 18th 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 "IVDR Implementation in EU & Changes for LDT in the US" and on "Harmonization of Vaccine Clinical Assays Validation" were the special features of the 18th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority 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 2024 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 2024 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, LBA and Cell-Based Assays and in Part 2B 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 17 of Bioanalysis, issues 5 and 3 (2025), respectively.

The 18^th Workshop on Recent Issues in Bioanalysis (18^th WRIB) took place in San Antonio, TX, USA on May 6-10, 2024. Over 1100 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 18^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 "IVDR Implementation in EU & Changes for LDT in the US" and on "Harmonization of Vaccine Clinical Assays Validation" were the special features of the 18^th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and Regulatory Agencies 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 2024 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 2024 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication (Part 3) covers in the Part 3A the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity and in Part 3B the Regulatory Inputs on these topics. Part 1 (Mass Spectrometry Assays and Regulated Bioanalysis/BMV) and Part 2 (Biomarkers/BAV, IVD/CDx, LBA and Cell-Based Assays) are published in volume 17 of Bioanalysis, issues 4 and 5 (2025), respectively.

Undruggable targets account for roughly 85% of human disease-related targets and represent a category of therapeutic targets that are difficult to tackle with traditional methods, but their considerable clinical importance. These targets are generally defined by planar functional interfaces and the absence of efficient ligand-binding pockets, making them unattainable for conventional pharmaceutical strategies. The advent of oligonucleotide-based proteolysis-targeting chimeras (PROTACs) has instilled renewed optimism in addressing these challenges. These PROTACs facilitate the targeted degradation of undruggable entities, including transcription factors (TFs) and RNA-binding proteins (RBPs), via proteasome-dependent mechanisms, thereby presenting novel therapeutic approaches for diseases linked to these targets. This review offers an in-depth examination of recent progress in the integration of PROTAC technology with oligonucleotides to target traditionally undruggable proteins, emphasizing the design principles and mechanisms of action of these innovative PROTACs.

Aim: Airway mucins in sputum are promising respiratory disease biomarkers, despite posing substantial analytical challenges due to their physicochemical properties and rare and heterogenous nature of the matrix. We aimed to identify a suitable sputum collection and processing method, and qualify a bioanalytical method for MUC5AC and MUC5B quantification in clinical samples.

Method: Mucins were quantified in induced and spontaneous sputum collected from the same COPD patients, following various sample processing procedures. LC-MS/MS method used truncated recombinant mucins as surrogate analytes in surrogate matrix.

Results: Frozen spontaneous sputum was found to be a suitable and convenient matrix for mucin quantification and fit-for-purpose method qualification was performed.

Conclusion: Our methodology provides accurate and reliable MUC5AC and MUC5B quantification and facilitates multi-site clinical sputum collection.

Aim: The purpose of this study is to establish a light-initiated chemiluminescence assay (LiCA) for the quantitative analysis of Humulus scandens pollen-specific IgE (sIgE) antibodies.

Methods: The best chemibeads coupling method in detecting Humulus scandens pollen - sIgE was selected. The working concentrations of the antigen-antibody and the reaction buffer were optimized as components of the reaction system. Then the assay performance was evaluated and the results of LiCA were compared with ImmunoCAP methods.

Results: In the range of 0.23 kU_A/L to 100.51 kU_A/L, LiCA demonstrated good linearity. The coefficients of variation for repeatability and intermediate precision ranged from 5.96% to 8.58% and 7.53% to 11.25%, respectively. The limit of blank (LoB), limit of detection (LoD), and limit of quantitation (LoQ) were determined to be 0.044 kU_A/L, 0.086 kU_A/L, and 0.11 kU_A/L, respectively. Furthermore, LiCA exhibited a statistically significant correlation with ImmunoCAP (r = 0.990).

Conclusion: The established LiCA-based quantitative detection method for Humulus scandens pollen-slgE has good analytical performance and potential clinical application prospects.

Background: Metabolic labeling with heavy water (D₂O) followed by LC-MS has become a powerful tool for studying protein turnover in vivo. Developing a quantitative method to measure partially labeled low-abundance proteins poses many challenges because heavy isotopomers of peptides, especially their changes through deuterium labeling, are difficult to detect.

Methods: A workflow that coupled immunocapture and LC-high-resolution MS to determine the synthesis rate of HSD17β13 protein in mouse liver was presented. Deuterium labeling of tryptic peptides was analyzed, and data were fitted into an exponential rise equation.

Results & conclusion: HSD17β13 protein t_1/2 were calculated to be 31.8, 36.1, and 28.9 hr from 3 different peptides with an average of 32.3 hr. The established workflow can be adapted from hybrid LC-MS protein quantitation assays to assess protein turnover in vivo using D₂O metabolic labeling.

Background: The Bicycle® toxin conjugate (BTC) zelenectide pevedotin, formerly known as BT8009, is a novel bicyclic peptide targeting the Nectin-4 tumor antigen conjugated to the cytotoxin monomethyl auristatin E (MMAE) via a valine-citrulline cleavable linker. Zelenectide pevedotin is currently being investigated in a Phase 1/2 (Duravelo-1, NCT04561362) clinical trial to determine safety and efficacy in patients with tumors associated with Nectin-4 expression. A simple regulated bioanalytical assay was developed to quantify intact zelenectide pevedotin in patient plasma samples.

Methodology: Quantitation of the intact zelenectide pevedotin and its analog internal standard BCY6063 encompassed a routine protein precipitation procedure followed by reverse phase chromatographic separation paired with tandem mass spectrometric detection.

Results: Intact zelenectide pevedotin was quantified over the assay range of 5-2500 ng/mL using 50 µL of human plasma. The method was validated according to local standard operating procedures and has met all US Food and Drug Administration and European Medicines Agency regulatory guidance criteria.

Conclusion: A bioanalytical method for measuring zelenectide pevedotin in plasma samples was developed and successfully applied in evaluating over 3000 samples from patients in a Phase 1/2 clinical study of zelenectide pevedotin in patients with advanced solid tumors.