Evolution of Mass Spectrometers for High m/z Biological Ion Formation, Transmission, Analysis and Detection: A Personal Perspective

Journal of the American Society for Mass Spectrometry, Journal Year: 2025, Volume and Issue: unknown

Published: March 5, 2025

Mass spectrometry (MS) has become an essential tool in virtually all academic, pharmaceutical, and biopharmaceutical analytical laboratories. The specialized bespoke area of MS research application high m/z ion (>m/z 6000 mass, >150 kDa) formation, transmission, analysis, detection is a relatively new focus for that seen dramatic acceleration interest over the last two decades. Herein we delve into this exciting aspect MS, discussing how instrumentation been refined evolved native-MS analysis. We cover early groundbreaking experiments showing preservation protein structure gas phase. Additionally, discuss specific instrument optimizations modifications have advanced generation, detection, contributing to known as gas-phase structural biology. Native-MS sample introduction methods, emerging technologies, future perspectives are also examined. Finally, share personal opinions, observations, experiences community or previously unpublished.

Language: Английский

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Novel Native Reversed-Phase Liquid Chromatography (nRPLC)/MS for Antibody–Drug Conjugates (ADCs) Characterization and Drug–Antibody Ratio (DAR) Assessment

Analytical Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: March 31, 2025

Characterization of drug–antibody ratio (DAR) species in antibody–drug conjugates (ADCs) is crucial for assessing the developability/manufacturability and downstream development drug candidates. Although hydrophobic interaction chromatography (HIC) gold standard DAR analysis, elucidating within each HIC peak has historically been challenging. This due to nonvolatility high ionic strength conventional buffer systems, which necessitate labor-intensive offline fractionation, followed by MS analysis. To address these challenges, an innovative alternative strategy developed that directly couples native reversed-phase liquid (nRPLC) high-resolution Orbitrap online analysis (nRPLC-MS). In collaboration with Phenomenex, two types columns, a different hydrophobicity, were developed, allowing elution low concentration MS-friendly salt organic buffer. LC parameters optimized enhance detection intact under flow rate conditions. demonstrate feasibility platform characterizing ADCs, both interchain-linked (heterogeneous 0 8) site-specific ADCs evaluated. The method enables nondenatured separation simultaneous characterization species, strong correlation was observed between this approach HIC. integrated allows unbiased without postcolumn splitting or fractionation. Furthermore, comparisons commonly used methods (native SEC-MS RPLC-MS) have shown superior terms selectivity resolution achieved nRPLC method. Notably, unconjugated antibody (DAR0) successfully retained low-ionic-strength using Moreover, facilitated chromatographic positional isomers DAR4 conjugation linkages, not achievable traditional As result, holds great promise high-throughput screening across payload classes.

Language: Английский

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Current Analytical Strategies for mRNA-Based Therapeutics

Molecules, Journal Year: 2025, Volume and Issue: 30(7), P. 1629 - 1629

Published: April 6, 2025

Recent advancements in mRNA technology, utilized vaccines, immunotherapies, protein replacement therapies, and genome editing, have emerged as promising increasingly viable treatments. The rapid, potent, transient properties of mRNA-encoded proteins make them attractive tools for the effective treatment a variety conditions, ranging from infectious diseases to cancer single-gene disorders. capability rapid large-scale production therapeutics fueled global response COVID-19 pandemic. For clinical implementation, it is crucial deeply characterize control important attributes such purity/integrity, identity, structural quality features, functionality. This implies use powerful advanced analytical techniques characterization mRNA. Improvements electrophoresis, chromatography, mass spectrometry, sequencing, functionality assessments significantly enhanced detail information available product process characterization, well routine stability release testing. Here, we review latest mRNA-based therapeutics, typically employed by biopharmaceutical industry eventual market release.

Language: Английский

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Drug-to-Antibody Ratio Estimation via Proteoform Peak Integration in the Analysis of Antibody–Oligonucleotide Conjugates with Orbitrap Fourier Transform Mass Spectrometry

Analytical Chemistry, Journal Year: 2021, Volume and Issue: 93(38), P. 12930 - 12937

Published: Sept. 14, 2021

The therapeutic efficacy and pharmacokinetics of antibody–drug conjugates (ADCs) in general, antibody–oligonucleotide (AOCs) particular, depend on the drug-to-antibody ratio (DAR) distribution average value. DAR is considered a critical quality attribute, information pertaining to it needs be gathered during ADC/AOC development, production, storage. However, because high structural complexity samples, particularly initial drug-development stages, application current state-of-the-art mass spectrometric approaches can limited for analysis. Here, we demonstrate novel approach analysis complex following native size-exclusion chromatography Orbitrap Fourier transform spectrometry (FTMS). based integration proteoform-level spectral peaks order provide an estimate its value with less than 10% error. peak performed via truncation Orbitrap's unreduced time-domain ion signals (transients) before spectra generation FT processing. Transient recording processing are undertaken using external data acquisition system, FTMS Booster X2, coupled Q Exactive HF instrument. This has been applied whole subunit-level trastuzumab oligonucleotides. obtained results indicate that sample purification or simplification procedures, example, deglycosylation, could omitted minimized prior analysis, streamlining process.

Language: Английский

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Recent advances in structural mass spectrometry methods in the context of biosimilarity assessment: from sequence heterogeneities to higher order structures

Journal of Pharmaceutical and Biomedical Analysis, Journal Year: 2023, Volume and Issue: 236, P. 115696 - 115696

Published: Sept. 9, 2023

Language: Английский

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Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry

JACS Au, Journal Year: 2021, Volume and Issue: 1(12), P. 2385 - 2393

Published: Nov. 29, 2021

In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting ability native mass spectrometry inform about structure and dynamics. How behavior intact proteins in gas phase depends on presence distribution ionizable surface residues has been difficult answer because multiple chargeable sites are present virtually all proteins. Turning engineering, we show that side chains completely dispensable for charging under conditions, if present, they preferential protonation sites. The absence results identical state distributions native-like denaturing while coexisting conformers can be distinguished using ion mobility separation. An excess chains, other hand, effectively modulates stability. fact, moving single group dramatically alter gas-phase conformation ion. We conclude although sum charges governed solely by Coulombic terms, their locations affect stability phase.

Language: Английский

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Ion–Ion Charge Reduction Addresses Multiple Challenges Common to Denaturing Intact Mass Analysis

Analytical Chemistry, Journal Year: 2022, Volume and Issue: 94(9), P. 3930 - 3938

Published: Feb. 21, 2022

Complete LC–MS-based protein primary sequence characterization requires measurement of intact profiles under denaturing and/or reducing conditions. To address issues overcharging unstructured proteins acidic, conditions and sample heterogeneity (macro- micro-scales) which often confound mass analysis a wide variety samples, we propose the use broadband isolation entire charge state distributions followed by ion–ion proton transfer reduction, have termed "full scan PTCR" (fsPTCR). Using rapid size exclusion chromatography coupled to fsPTCR-Orbitrap MS time-resolved deconvolution data analysis, demonstrate strategy for method optimization, leading significant analytical advantages over conventional MS1. Denaturing flexible bacterial translation initiation factor 2 (91 kDa) using fsPTCR reduced showed an 11-fold gain in S/N compared Analysis fsPTCR-MS microheterogeneous glycoprotein fetuin revealed twice as many proteoforms MS1 (112 vs 56). In macroheterogeneous mixture ranging from 14 148 kDa, provided more than 10-fold increased sensitivity quantitative accuracy diluted bovine serum albumin (66 kDa). Finally, our shows that collisional gas pressure is key parameter can be utilized during retain or remove larger acquired spectra.

Language: Английский

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High-Throughput Mass Spectrometry for Biopharma: A Universal Modality and Target Independent Analytical Method for Accurate Biomolecule Characterization

Journal of the American Society for Mass Spectrometry, Journal Year: 2022, Volume and Issue: 33(11), P. 2191 - 2198

Published: Oct. 7, 2022

Reversed-phase liquid chromatographic mass spectrometry (rpLC-MS) is a universal, platformed, and essential analytical technique within pharmaceutical biopharmaceutical research. Typical rpLC method gradient times can range from 5 to 20 min. As monoclonal antibody (mAb) therapies continue evolve bispecific antibodies (BsAbs) become more established, research stage engineering panels will clearly in size. Therefore, high-throughput (HT) MS automated deconvolution methods are key for success. Additionally, newer therapeutics such as T-cell engagers nucleic acid-based modalities also require characterization. Herein, we present modality target agnostic HT solid-phase extraction (SPE) that affords the analysis of 96-well plate 41.4 min, compared traditional rpLC-MS would typically take 14.4 h. The described accurately determine molecular weights monodispersed highly polydispersed biotherapeutic species membrane proteins; levels glycosylation, glycation, formylation; detect chain mispairing; accurate drug-to-antibody ratio values.

Language: Английский

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UniDec Processing Pipeline for Rapid Analysis of Biotherapeutic Mass Spectrometry Data

Analytical Chemistry, Journal Year: 2023, Volume and Issue: 95(30), P. 11491 - 11498

Published: July 21, 2023

Recent advances in native mass spectrometry (MS) and denatured intact protein MS have made these techniques essential for biotherapeutic characterization. As analysis has increased throughput scale, new data workflows are needed to provide rapid quantitation from large datasets. Here, we describe the UniDec processing pipeline (UPP) of batched data. UPP is built into software package, which provides fast processing, deconvolution, peak detection. The user programming interfaces read a spreadsheet that contains file names, deconvolution parameters, settings. After iterating through analyzing each file, it returns results HTML reports. We demonstrate use measure correct pairing percentage on set bispecific antibody drug-to-antibody ratios antibody–drug conjugates. Moreover, because free open-source, users can easily build this platform create customized calculations. Thus, flexible workflow be deployed diverse settings wide range applications.

Language: Английский

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Surface Modified Nano-Electrospray Needles Improve Sensitivity for Native Mass Spectrometry

Journal of the American Society for Mass Spectrometry, Journal Year: 2022, Volume and Issue: 33(6), P. 1031 - 1037

Published: May 19, 2022

Native mass spectrometry (MS) and charge detection-mass (CD-MS) have become versatile tools for characterizing a wide range of proteins macromolecular complexes. Both commonly use nanoelectrospray ionization (nESI) from pulled borosilicate needles, but some analytes are known to nonspecifically adsorb the glass, which may lower sensitivity limit quality data. To improve native MS CD-MS, we modified surface nESI needles with inert modifiers, including polyethylene-glycol. We found that modification improved signal intensity CD-MS adeno-associated viral capsids. Based on mechanistic comparisons, hypothesize improvement is more likely due an increased flow rate coated ESI rather than less nonspecific adsorption. In any case, these surface-modified provide simple inexpensive method improving challenging analytes.

Language: Английский

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