Enhancing drug development and clinical studies with patient-centric sampling using microsampling techniques: Opportunities, challenges, and insights into liquid chromatography-mass spectrometry strategies.

Microsampling has revolutionized pharmaceutical drug development and clinical research by reducing sample volume requirements, allowing sample collection at home or nontraditional sites, minimizing animal and patient burden, and enabling more flexible study designs. This perspective paper discusses the transformative impact of microsampling and patient-centric sampling (PCS) techniques, emphasizing their advantages in drug development and clinical trials. We highlight the integration of liquid chromatography-mass spectrometry (LC-MS) strategies for analyzing PCS samples, focusing on our research experience and a review of current literatures.

Meeting Report on the 3rd Chinese American Society for Mass Spectrometry Conference-Advancing Biological and Pharmaceutical Mass Spectrometry.

Following the highly successful Chinese American Society for Mass Spectrometry (CASMS) conferences in the previous 2 years, the 3rd CASMS Conference was held virtually on August 28-31, 2023, using the Gather.Town platform to bring together scientists in the MS field. The conference offered a 4-day agenda with a scientific program consisting of two plenary lectures, and 14 parallel symposia in which a total of 70 speakers presented technological innovations and their applications in proteomics and biological MS and metabo-lipidomics and pharmaceutical MS.

Meeting Report on the 2nd Chinese American Society for Mass Spectrometry Conference: Advancing Biological and Pharmaceutical Mass Spectrometry.

The 2nd CASMS conference was held virtually through Gather. Town platform from October 17 to 21, 2022, with a total of 363 registrants including an outstanding and diverse group of scientists at the forefront of their research fields from both academia and industry worldwide, especially in the United States and China. The conference offered a 5-day agenda with an exciting scientific program consisting of two plenary lectures, 14 parallel symposia, and 4 special sessions in which a total of 97 invited speakers presented technological innovations and their applications in proteomics & biological mass spectrometry and metabo-lipidomics & pharmaceutical mass spectrometry.

Measurement of protein in vivo turnover rate with metabolic labeling using LC-MS.

Understanding the protein dynamics of a drug target is important for pharmaceutical research because it provides insight into drug design, target engagement, pharmacodynamics and drug efficacy. Nonradioactive isotope labeling has been the method of choice for protein turnover measurement thanks to the advancement of high-resolution mass spectrometry. While the changes in proteome in cell cultures can be monitored precisely, as the culture media can be completely replaced with H-, N- or C-labeled essential amino acids, quantifying rates of protein synthesis in vivo is more challenging.

Bifunctionalized isotopologs as calibrants for standard-free quantitation of drug metabolites in plasma by liquid chromatography coupled with radiometry and mass spectrometry.

The reported method involves a novel workflow that eliminates the need for authentic reference standards for the quantitation of drug metabolites in biological samples using a single multi-isotopically labeled compound bearing both radio and stable isotopes. The resulting radio and stable bifunctionalized isotopolog (RADSTIL) of the parent drug is employed as a substrate for in vitro biotransformation to targeted RADSTILs of metabolites as calibrants. Inclusion of a radio label enables both radiometric and mass spectrometric detection.

LC-MS bioanalysis of intact proteins and peptides.

Bioanalysis assays that reliably quantify biotherapeutics and biomarkers in biological samples play pivotal roles in drug discovery and development. Liquid chromatography coupled with mass spectrometry (LC-MS), owing to its superior specificity, faster method development and multiplex capability, has evolved as one of the most important platforms for bioanalysis of biotherapeutics, particularly new scaffolds such as half-life extension platforms for proteins and peptides, as well as antibody drug conjugates. Intact LC-MS analysis is orthogonal to bottom-up surrogate peptide approach by providing whole molecule quantitation and high-level sequence and structure information.

2018 White Paper on Recent Issues in Bioanalysis: focus on immunogenicity assays by hybrid LBA/LCMS and regulatory feedback (Part 2 - PK, PD & ADA assays by hybrid LBA/LCMS & regulatory agencies' inputs on bioanalysis, biomarkers and immunogenicity).

The 2018 12 Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches.

Bioanalytical workflow for novel scaffold protein-drug conjugates: quantitation of total Centyrin protein, conjugated Centyrin and free payload for Centyrin-drug conjugate in plasma and tissue samples using liquid chromatography-tandem mass spectrometry.

Aim: Alternative scaffold proteins have emerged as novel platforms for development of therapeutic applications. One such application is in protein-drug conjugates (PDCs), which are analogous to antibody-drug conjugates.

Methodology: Liquid chromatography-mass spectrometry methods for quantitation of total protein, conjugate and free payload for a PDC based on Centyrin scaffold were developed.

Quantitation of intact monoclonal antibody in biological samples: comparison of different data processing strategies.

Aim: Sample extraction using immuno-affinity capture coupled with LC-high-resolution mass spectrometer has recently emerged as a novel approach for the determination of concentrations of large molecules at intact level in biological matrix.

Methodology: In the current work, different data processing strategies for intact protein bioanalysis, deconvoluted mass spectra or extracted ion chromatogram, were applied to quantitate monoclonal antibody in biological samples for comparison of assay performance.

Conclusion: Both deconvolution and extracted ion chromatogram strategies showed similar selectivity, sensitivity, accuracy and precision.

Bioanalysis of sulprostone, a prostaglandin E analogue and selective EP agonist, in monkey plasma by liquid chromatography-tandem mass spectrometry.

Sulprostone is a potent prostaglandin E (PGE) analogue and one of the first identified selective G-protein-coupled receptor 3 (EP) agonists. It has been investigated as a potential antiulcer agent and frequently used in the research of EP antagonist. To assist pharmacokinetic and pharmacodynamic studies, a rapid and sensitive LC-MS/MS method was developed and qualified for the quantitation of sulprostone in monkey plasma.

LC/MS/MS Bioanalysis of Protein-Drug Conjugates-The Importance of Incorporating Succinimide Hydrolysis Products.

Bioanalysis of antibody-drug conjugates (ADCs) is challenging due to the complex, heterogeneous nature of their structures and their complicated catabolism. To fully describe the pharmacokinetics (PK) of an ADC, several analytes are commonly quantified, including total antibody, conjugate, and payload. Among them, conjugate is the most challenging to measure, because it requires detection of both small and large molecules as one entity.

Clinical and pharmaceutical success from discovery to regulatory approval: biomarkers, modeling and analytical technologies.

The 8th Annual Shanghai Symposium on Clinical and Pharmaceutical Solutions through Analysis (CPSA): Clinical and Pharmaceutical Success from Discovery to Regulatory Approval: Biomarkers, Modeling and Analytical Technologies (CPSA Shanghai 2017); Renaissance Shanghai Pudong Hotel, Shanghai, China, 12-14 April 2017 was held on 12-14 April 2017 in Shanghai, China. The meeting was featured with highly interactive events including diversified symposia, workshops, roundtable discussions, conference awards and poster sessions. There were over 220 participants with 61 oral presentations and 20 posters presented.

LC-MS/MS quantification of 7α-hydroxy-4-cholesten-3-one (C4) in rat and monkey plasma.

7α-hydroxy-4-cholesten-3-one (C4) is an oxidative enzymatic product of cholesterol metabolism via cholesterol 7α-hydroxylase, an enzyme also known as cholesterol 7-alpha-monooxygenase or cytochrome P450 7A1 (CYP7A1). C4 is a stable intermediate in the rate limiting pathway of bile acid biosynthesis. Previous studies showed that plasma C4 levels correlated with CYP7A1 enzymatic activity and could serve as a biomarker for bile acid synthesis.

Standard-Free Bioanalytical Approach for Absolute Quantitation of Drug Metabolites Utilizing Biosynthesis of Reciprocal Radio and Stable Isotopologues and Its Application.

The following work describes a combined enzymatic and bioanalytical method that permits absolute quantitation of metabolites in biological samples without the requirement for reference metabolite standards. This technique was exemplified using a radio (C) isotopologue and a stable (C) isotopologue of acetaminophen as substrates for in vitro biosynthesis of the corresponding radio and stable isotope labeled metabolites, namely, C- and C-glucuronides and sulfates. By supplanting the use of authentic metabolite standards, traditionally used to calibrate C-metabolites via liquid chromatography-tandem mass spectrometry (LC-MS/MS), C-metabolites were radiocalibrated by their C-isotopologues via liquid chromatography coupled with radioactivity detection and mass spectrometry (LC-RAD/MS).

Simultaneous Catabolite Identification and Quantitation of Large Therapeutic Protein at the Intact Level by Immunoaffinity Capture Liquid Chromatography-High-Resolution Mass Spectrometry.

As therapeutic recombinant fusion proteins become more widely applicable for the treatment of various types of diseases, there is an increased demand for universal methods such as liquid chromatography (LC)-mass spectrometry (MS) for the determination of their pharmacokinetic properties, particularly their catabolism. The most common approach of analyzing proteins by LC-MS is to digest them into peptides, which can serve as surrogates of the protein. Alternatively, we have developed a novel high-resolution mass spectrometry (HRMS) based approach for analyzing large-molecule proteins at the intact level in biological samples without digestion.

7th Annual Symposium on Clinical and Pharmaceutical Solutions through Analysis.

7th Annual Symposium on Clinical & Pharmaceutical Solutions through Analysis, Renaissance Shanghai Pudong Hotel, Shanghai, China, 20-23 April 2016 The 7th Annual Shanghai Symposium on Innovative Approaches to Reduce Attrition and Predict Clinical Outcomes (CPSA Shanghai 2016) was held on 20-23 April 2016 in Renaissance Shanghai Pudong Hotel, Shanghai, China. The meeting was featured with highly interactive events including diversified symposia, round table discussions, workshops, poster sessions and conference awards. There were over 220 participants from more than ten countries, with 61 oral presentations and 29 posters presented.

A workflow for absolute quantitation of large therapeutic proteins in biological samples at intact level using LC-HRMS.

Aim: The commonly used LC-MS workflow to quantify protein therapeutics in biological samples is 'bottom-up' approach. In this study, the aim is to establish 'top-down' approach for absolute quantitation of therapeutic antibodies or proteins of similar sizes in biological samples at intact level.

Materials & Methods: Using a recombinant human monoclonal antibody as the model molecule, we present a workflow to measure large therapeutic proteins in plasma at intact level based on deconvoluted high-resolution MS (HRMS) peaks.

Recommendations on the Development of a Bioanalytical Assay for 4β-Hydroxycholesterol, an Emerging Endogenous Biomarker of CYP3A Activity.

The availability of reliable assays for measuring 4β-hydroxycholesterol (4β-HC), a CYP3A metabolite of cholesterol, is an important step in qualifying this endogenous moiety as a biomarker of CYP3A activity. Liquid and gas chromatographic methods with mass spectrometric detection have been developed with varying sensitivities, with or without derivatization. Care must be taken to chromatographically resolve 4β-HC from the multiple isobaric cholesterol oxidation products present in plasma, including 4α-hydroxycholesterol (4α-HC).

UPLC-MS/MS assay for the simultaneous determination of ethinyl estradiol, norgestimate and 17-Desacetyl norgestimate at low pg/mL in human plasma.

Previously, because of the difficulty of measuring very low levels (pg/mL) of norgestimate (NGM) due to rapid metabolism to its active and major metabolite, 17-Desacetyl norgestimate (DNGM), only DNGM and the co-administered ethinyl estradiol (EE2) were required to be analyzed in bioequivalence (BE) studies for oral contraceptive NGM/EE2 tablets. Recently, with more sensitive assays available, health authorities have requested that bioequivalence of NGM be also demonstrated in addition to DNGM and EE2. Therefore, it was important to establish a 3-in-1 method for the quantitation of EE2, NGM and DNGM in human plasma.

Bioanalysis of ibrutinib and its active metabolite in human plasma: selectivity issue, impact assessment and resolution.

Ronald de Vries graduated in Organic and Analytical Chemistry at the Free University of Amsterdam, The Netherlands. After working in a Contract Laboratory (CRO) for 7 years, he joined Janssen R&D in 1998. At Janssen R&D, Belgium, Ronald worked in the bioanalytical department that supports both clinical and nonclinical bioanalysis.

Serum apolipoprotein A-1 quantification by LC-MS with a SILAC internal standard reveals reduced levels in smokers.

Background: Absolute quantification of protein biomarkers such as serum apolipoprotein A1 by both immunoassays and LC-MS can provide misleading results.

Results: Recombinant ApoA-1 internal standard was prepared using stable isotope labeling by amino acids in cell culture with [(13)C6(15)N2]-lysine and [(13)C9(15)N1]-tyrosine in human cells. A stable isotope dilution LC-MS method for serum ApoA-1 was validated and levels analyzed for 50 nonsmokers and 50 smokers.

Defining the challenges of the Modern Analytical Laboratory (CPSA USA 2014): the risks and reality of personalized healthcare.

The 17th Annual Symposium on Clinical and Pharmaceutical Solutions through Analysis (CPSA) 29 September-2 October 2014, was held at the Sheraton Bucks County Hotel, Langhorne, PA, USA. The CPSA USA 2014 brought the various analytical fields defining the challenges of the modern analytical laboratory. Ongoing discussions focused on the future application of bioanalysis and other disciplines to support investigational new drugs (INDs) and new drug application (NDA) submissions, clinical diagnostics and pathology laboratory personnel that support patient sample analysis, and the clinical researchers that provide insights into new biomarkers within the context of the modern laboratory and personalized medicine.