Development of a mertansine-specific DNA aptamer and novel high-throughput sandwich enzyme-linked oligonucleotide assay for quantification and characterization of trastuzumab emtansine.

We developed a novel DNA aptamer, D8#24S1, which specifically recognizes mertansine (DM1), the cytotoxic payload of the antibody-drug conjugate (ADC) trastuzumab emtansine (T-DM1), and applied it for T-DM1 analysis. D8#24S1 was obtained through SELEX and was shown to specifically recognize DM1 with high affinity (dissociation constant, K = 84.2 nM).

Simple and fast one-step FRET assay of therapeutic mAb bevacizumab using anti-idiotype DNA aptamer for process analytical technology.

We developed an aptamer-based fluorescence resonance energy transfer (FRET) assay capable of recognizing therapeutic monoclonal antibody bevacizumab and rapidly quantifying its concentration with just one mixing step. In this assay, two fluorescent dyes (fluorescein and tetramethylrhodamine) labeled aptamers bind to two Fab regions on bevacizumab, and FRET fluorescence is observed when both dyes come into close proximity. We optimized this assay in three different formats, catering to a wide range of analytical needs.

Development of an on-site therapeutic drug monitoring method using a portable spectrometer.

We report on the development of an on-site therapeutic drug monitoring (TDM) method for vancomycin (VCM) utilizing a portable spectrometer and commercially available immunoturbidimetric assay reagents designed for automated clinical chemistry analyzers. The method enables the quantification of VCM in plasma samples within 10 min, with a good correlation between the measured values and the theoretical values (r = 0.995).

Precolumn derivatization LC/MS method for observation of efficient hydrogen sulfide supply to the kidney via d-cysteine degradation pathway.

d-Cysteine (d-Cys) is metabolized to hydrogen sulfide (HS) by d-amino acid oxidase (DAO)/3-mercaptopyruvate sulfurtransferase pathway. The pathway is required for HS supplementation that ameliorates acute kidney injury after the oral administration of d-Cys in mice. However, whether the rate-limiting activity of DAO regulates the tissue-selectivity or the extent of d-Cys degradation and HS supplementation remains unclear.

Development of a DNA aptamer that binds to the complementarity-determining region of therapeutic monoclonal antibody and affinity improvement induced by pH-change for sensitive detection.

Therapeutic monoclonal antibodies (mAbs) are successful biomedicines; however, evaluation of their pharmacokinetics and pharmacodynamics demands highly specific discrimination from human immunoglobulin G naturally present in the blood. Here, we developed a novel anti-idiotype aptamer (termed A14#1) with extraordinary specificity against the anti-vascular endothelial growth factor therapeutic mAb, bevacizumab. Structural analysis of the antibody-aptamer complex showed that several bases of A14#1 recognized only the complementarity determining region (CDR) of bevacizumab, thereby contributing to its extraordinary specificity.

Rapid chiral discrimination of oncometabolite dl-2-hydroxyglutaric acid using derivatization and field asymmetric waveform ion mobility spectrometry/mass spectrometry.

2-Hydroxyglutaric acid is a chiral metabolite whose enantiomers specifically accumulate in different diseases. An enantiomeric excess of the d-form in biological specimens reflects the existence of various pathogenic mutations in cancer patients, however, conventional methods using gas or liquid chromatography and capillary electrophoresis had not been used for large clinical studies because they require multiple analytical instruments and a long run time to separate the enantiomers. Here, we present a rapid separation method for dl-2-hydroxyglutaric acid using a chiral derivatizing reagent and field asymmetric waveform ion mobility spectrometry/mass spectrometry, which requires a single analytical instrument and <1 s for the separation.

Machine learning guided prediction of liquid chromatography-mass spectrometry ionization efficiency for genotoxic impurities in pharmaceutical products.

The limitation and control of genotoxic impurities (GTIs) has continued to receive attention from pharmaceutical companies and authorities for several decades. Because GTIs have the ability to damage deoxyribonucleic acid (DNA) and the potential to cause cancer, low-level quantitation is required to protect patients. A quick and easy method of determining the liquid chromatography-mass spectrometry (LC/MS) conditions for high-sensitivity analysis of GTIs may prospectively accelerate pharmaceutical development.

Development of a selective and sensitive analytical method to detect isomerized aspartic acid residues in crystallin using a combination of derivatization and liquid chromatography mass spectrometry.

The isomerization of amino acids in peptides and proteins induces structural changes and aggregation. The isomerization rate of aspartic acid (Asp) is high and causes various serious diseases including Alzheimer's disease and cataract. Herein, a method for the comprehensive separation and sensitive detection of isomerized crystallin containing Asp (l-α-Asp, l-β-Asp, d-α-Asp, and d-β-Asp) was developed using chiral derivatization and reversed-phase UHPLC separation.

Bioanalytical methods for therapeutic monoclonal antibodies and antibody-drug conjugates: A review of recent advances and future perspectives.

As the use and development of therapeutic monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) increases, the need for accurate and robust bioanalytical methods is also increasing. Up to about a decade ago, bioanalysis of therapeutic mAbs was performed only by ligand-binding assay (LBA), and this was the only available method for absolute quantitation. Recent technological advances in liquid chromatography (LC)-tandem mass spectrometry (LC-MS/MS) and LC-high-resolution mass spectrometry can realize specific, accurate, and multiplex bioanalysis of these with a wide dynamic detection range.

An accurate differential analysis of carboxylic acids in beer using ultra high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry based on chiral derivatization combining three isotopic reagents.

We developed a highly sensitive and accurate differential method for analyzing chiral and achiral carboxylic acids in Japanese commercial beers, using ultra high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS), based on chiral derivatization combining three isotopic chiral reagents: C-DMT-3(S)-Apy, C-DMT-3(S)-Apy, and d-DMT-3(S)-Apy. By combining these reaction solutions and analyzing the resulting mixture, simultaneous comparative analyses of the three samples could be achieved while offsetting the matrix effects on the samples. Using this approach, it was possible to differentiate the beers according to the type (draft, low-malt, and non-alcoholic), manufacturer, and storage conditions (temperature and storage period), based on the concentrations of carboxylic acids in the beers.

DL-Amino Acid Analysis Based on Labeling with Light and Heavy Isotopic Reagents Followed by UPLC-ESI-MS/MS.

L-Pyroglutamic acid succinimidyl ester (L-PGA-OSu) and its isotopic variant (L-PGA[d]-OSu) were synthesized and used as the chiral labeling reagents for the enantioseparation of amino acids by reversed-phase UPLC-ESI-MS/MS. The enantiomers of amino acids were labeled with the reagents at 60 °C for 10 min in an alkaline medium. The resulting diastereomers were well separated by the reversed-phase chromatography using an ODS column, packed with small particles (1.

Introducing an Experimental Design Approach for Efficient Optimization of Chiral Derivatization Conditions for D- and L-Glyceric Acids.

A sensitive analytical method was developed for individual analyses of D- and L-glyceric acids by chiral derivatization - liquid chromatography-tandem mass spectrometry. To elucidate rapid and efficient optimization for derivatization we newly introduced a concept of design of experiments (DOE). The optimization of major 5 factors in the derivatization could be predicted with only 28 measurements.

Chiral Metabolomics Using Triazine-Based Chiral Labeling Reagents by UPLC-ESI-MS/MS.

The determination of enantiomers of biological molecules is an important issue because a significant difference in the activity of the enantiomers is generally observed in biological systems. Chiral separations can be carried out by direct resolution using a chiral stationary column or by indirect resolution based on the derivatization with a chiral reagent. Many chiral-labeling reagents for ultraviolet-visible and fluorescence detections have been developed for various functional groups, such as amine and carboxylic acid.

Isotope Corrected Chiral and Achiral Nontargeted Metabolomics: An Approach for High Accuracy and Precision Metabolomics Based on Derivatization and Its Application to Cerebrospinal Fluid of Patients with Alzheimer's Disease.

We propose a chiral metabolomics approach based on a data-dependent MS/MS analysis (DDA) using high-resolution quadrupole-time-of-flight mass spectrometry (Q-TOFMS) and C-isotope coded derivatization (ICD) reagents, i.e., iDMT-( S)-A and iDMT-( S)-PO.

Anti-Idiotype DNA Aptamer Affinity Purification⁻High-Temperature Reversed-Phase Liquid Chromatography: A Simple, Accurate, and Selective Bioanalysis of Bevacizumab.

This study presents a simple, accurate, and selective bioanalytical method of bevacizumab detection from plasma samples based on aptamer affinity purification⁻high-temperature reversed-phased liquid chromatography (HT-RPLC) with fluorescence detection. Bevacizumab in plasma samples was purified using magnetic beads immobilized with an anti-idiotype DNA aptamer for bevacizumab. The purified bevacizumab was separated with HT-RPLC and detected with its native fluorescence.

High-Throughput Bioanalysis of Bevacizumab in Human Plasma Based on Enzyme-Linked Aptamer Assay Using Anti-Idiotype DNA Aptamer.

We propose a highly selective, sensitive, accurate, and high-throughput bioanalysis method for bevacizumab utilizing an anti-idiotype DNA aptamer. With this method, bevacizumab in a plasma sample was reacted in a 96-well plate immobilized with the aptamer and further reacted with a protein A-HRP conjugate. The resulting HRP activity was colorimetrically detected using a microplate reader.

Sensitive and Comprehensive LC-MS/MS Analyses of Chiral Pharmaceuticals and Their Hepatic Metabolites Using Ovomucoid Column.

A sensitive analytical method was developed for the simultaneous detection of 11 chiral pharmaceuticals and their hepatic metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an ovomucoid chiral column. After optimization of the LC conditions, all pharmaceuticals examined were enantio-separated with R of >0.82 in LC-MS/MS analysis.

Current Mass Spectrometric Tools for the Bioanalyses of Therapeutic Monoclonal Antibodies and Antibody-Drug Conjugates.

The increase in the use of therapeutic monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) has made the detailed bioanalysis of these drugs essential not only for planning optimal therapeutic programs for clinical practice, but also for evaluating the biological equivalencies in the development of other biosimilars. The ligand binding assays that are widely in use now are being replaced rapidly by the highly accurate, sensitive, and selective analytical method using a mass spectrometer. This review will discuss the progress in and challenges observed during the development of a mass spectrometry-based bioanalytical method for therapeutic mAbs and ADCs.

A novel, simplified strategy of relative quantification N-glycan: Quantitative glycomics using electrospray ionization mass spectrometry through the stable isotopic labeling by transglycosylation reaction of mutant enzyme Endo-M-N175Q.

The lack of a highly sensitive and simple method for the quantitative analysis of glycan has impeded the exploration of protein glycosylation patterns (glycomics), evaluation of antibody drug stability, and screening of disease glycan biomarkers. In this study, we describe a novel and simplified quantitative glycomics strategy. Quantitation by mutant enzyme reaction stable isotope labeling (QMERSIL) to label the N-glycans with either a nondeuterated (d0-) or deuterated (d8-) 4-(2,4-Dinitro-5-piperazin-1-yl-phenyl)-1,1-dimethyl-piperazin-1-ium (MPDPZ)-Boc-asparaginyl-N-acetyl-d-glucosamine (Boc-Asn-GlcNAc) acceptor of a positive charge structure through the glycosynthase (Endo-M-N175Q) transglycosylation reaction with mass spectrometry facilitates comparative glycomics.

Simultaneous optimization of pH and binary organic composition by grid form modeling of the retention behavior in reversed-phase ultra high-performance liquid chromatography.

A novel computer-assisted methodology for the simultaneous optimization of aqueous pH and binary organic eluent composition through a broad range of analytical conditions of reversed-phase ultra high-performance liquid chromatography is proposed. Two of nonlinear prediction models were employed to fit into the retention time (tR) on a linear gradient elution with a predefined slope. One model was derived from Bernoulli-type probability distribution to predict the value of tR against the pH value of the aqueous eluent.

Gas-Phase Stability of Negatively Charged Organophosphate Metabolites Produced by Electrospray Ionization and Matrix-Assisted Laser Desorption/Ionization.

The formation mechanisms of singly and multiply charged organophosphate metabolites by electrospray ionization (ESI) and their gas phase stabilities were investigated. Metabolites containing multiple phosphate groups, such as adenosine 5'-diphosphate (ADP), adenosine 5'-triphosphate (ATP), and D-myo-inositol-1,4,5-triphosphate (IP) were observed as doubly deprotonated ions by negative-ion ESI mass spectrometry. Organophosphates with multiple negative charges were found to be unstable and often underwent loss of PO, although singly deprotonated analytes were stable.

Relationship between dexmedetomidine dose and plasma dexmedetomidine concentration in critically ill infants: a prospective observational cohort study.

Background: Dexmedetomidine is a highly selective central α-agonist used as a sedative in pediatric intensive care unit (PICU). However, little is known about the relationship between dexmedetomidine dose and its plasma concentration during long-term infusion. We have previously demonstrated that the sedative plasma dexmedetomidine concentration is moderately correlated with the administered dose in adults (r = 0.

A novel rapid analysis using mass spectrometry to evaluate downstream refolding of recombinant human insulin-like growth factor-1 (mecasermin).

Rationale: Mecasermin is used to treat elevated blood sugar as well as growth-hormone-resistant Laron-type dwarfism. Mecasermin isolated from inclusion bodies in extracts of E. coli must be refolded to acquire sufficient activity.