Internal Fragments Enhance Middle-Down Mass Spectrometry Structural Characterization of Monoclonal Antibodies and Antibody-Drug Conjugates

Analytical Chemistry, Journal Year: 2024, Volume and Issue: 96(6), P. 2491 - 2499

Published: Jan. 31, 2024

Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) are important large biotherapeutics (∼150 kDa) high structural complexity that require extensive sequence structure characterization. Middle-down mass spectrometry (MD-MS) is an emerging technique sequences maps subunits larger than those released by trypsinolysis. It avoids potentially introducing artifactual modifications may occur in bottom-up MS while achieving higher coverage compared to top-down MS. However, returning complete information MD-MS still challenging. Here, we show assigning internal fragments direct infusion of a mAb ADC substantially improves their For the reduced NIST mAb, including recovers nearly 100% accessing middle region inaccessible terminal fragments. The identification glycosylations can also be improved after inclusion lysine-linked IgG1-DM1 ADC, considering increases DM1 conjugation sites 80%, comparable reported 83% achieved peptide mapping on same (Luo et al. Anal. Chem. 2016, 88, 695–702). This study expands our work application fragment assignments mAbs ADCs extended other heterogeneous therapeutic molecules such as multispecifics fusion proteins for more widespread applications.

Language: Английский

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“Lab of the Future”─Today: Fully Automated System for High-Throughput Mass Spectrometry Analysis of Biotherapeutics

Journal of the American Society for Mass Spectrometry, Journal Year: 2023, Volume and Issue: 34(6), P. 1073 - 1085

Published: May 15, 2023

Here we describe a state-of-the-art, integrated, multi-instrument automated system designed to execute methods involved in mass spectrometry characterization of biotherapeutics. The includes liquid and microplate handling robotics utilities, integrated LC-MS, along with data analysis software, perform sample purification, preparation, as seamless unit. process begins tip-based purification target proteins from expression cell-line supernatants, which is initiated once the samples are loaded onto metadata retrieved our corporate aggregation system. Subsequently, purified protein prepared for MS, including deglycosylation reduction steps intact reduced analysis, proteolytic digestions, desalting, buffer exchange via centrifugation peptide map analysis. then into LC-MS instrumentation acquisition. acquired raw initially stored on local area network storage that monitored by watcher scripts upload MS cloud-based servers. processed appropriately configured workflows such database search mapping or charge deconvolution undigested proteins. results verified formatted expert curation directly cloud. Finally, curated appended accompany biotherapeutic cell lines subsequent processes.

Language: Английский

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Facilitating high throughput bispecific antibody production and potential applications within biopharmaceutical discovery workflows

mAbs, Journal Year: 2024, Volume and Issue: 16(1)

Published: Feb. 21, 2024

A major driver for the recent investment surge in bispecific antibody (bsAb) platforms and products is multitude of distinct mechanisms action that bsAbs offer compared to a combination two monoclonal antibodies. Four bsAb were granted first regulatory approvals US or EU during 2023 biopharmaceutical industry pipeline brimming with candidates across broad range therapeutic applications. In previously reported discovery campaigns, following hypothesis-based choice specific target proteins, selections screening activities have often been performed mono-specific formats. The conversion modalities has usually positioned toward end process involved small numbers lead molecules, largely due challenges expressing, purifying, analyzing large bsAbs. this review, we discuss emerging strategies facilitate production expanded panels, focusing particularly upon combinatorial methods generate matrices. Such technologies will enable in. formats at earlier stages not only widening accessible protein space maximize chances success, but also advancing empirical bi-target validation assess initial selection hypotheses.

Language: Английский

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A Robust and Sensitive New Peak Detection and Identification Method for Mass Spectrometry-Based Differential Analysis in Biologics Characterization

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

Published: Jan. 8, 2025

New peak detection (NPD) is a significant component of the multiattribute method (MAM) for MS use to facilitate quality attributes exhibiting abnormal ratio changes, vanishing attributes, or newly emerging attributes. However, challenges remain get balanced sensitivity and minimize false positives in NPD. In this study, we have developed robust NPD identification enhance 10-fold (0.5% spike-in) compared previously reported work while maintaining controlled via statistics-driven experimental design utilizing three control samples product-specific peptide library. This not only enables MAM replace conventional analytical methods attribute control, but also provides new objective way performing differential analysis LC-MS-based experiments at different stages biopharmaceutics process development.

Language: Английский

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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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Development of an Automated, High-Throughput Methodology for Native Mass Spectrometry and Collision-Induced Unfolding

Analytical Chemistry, Journal Year: 2023, Volume and Issue: 95(45), P. 16717 - 16724

Published: Nov. 4, 2023

Native ion mobility mass spectrometry (nIM-MS) has emerged as a useful technology for the rapid evaluation of biomolecular structures. When combined with collisional activation in collision-induced unfolding (CIU) experiment, nIM-MS experimentation can be leveraged to gain greater insight into conformation and stability. However, CIU remain throughput limited due nonautomated sample preparation introduction. Here, we explore use RapidFire robotic handling system develop an automated, high-throughput methodology nMS CIU. We describe native RapidFire-MS (nRapidFire-MS) capable performing online desalting introduction little 10 s per sample. CIU, our nRapidFire-MS approach used collect fingerprints 30 following by using size exclusion chromatography cartridges. compared data collected standard approaches, signals recorded exhibit identical collision cross sections, indicating that same conformational populations are tracked two approaches. Our further suggest extended study variety classes, including proteins protein complexes ranging from 5 300 kDa oligonucleotides. Furthermore, acquired biotherapeutics potential enable analyses biopharmaceutical samples. conclude discussing enabling development future assays catalyzing breakthroughs engineering, inhibitor discovery, formulation biotherapeutics.

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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Cardiolipin Regulates the Activity of the Mitochondrial ABC Transporter ABCB10

Biochemistry, Journal Year: 2023, Volume and Issue: 62(21), P. 3159 - 3165

Published: Oct. 9, 2023

The ATP-binding cassette (ABC) transporter ABCB10 resides in the inner membrane of mitochondria and is implicated erythropoiesis. Mitochondria from different cell types share some specific characteristics, one which high abundance cardiolipin. Although previous studies have provided insight into ABCB10, affinity selectivity this toward lipids, particularly those found mitochondria, remain poorly understood. Here, native mass spectrometry used to directly monitor binding events lipids human ABCB10. results reveal that binds avidly cardiolipin with an significantly higher than other phospholipids. first three display positive cooperativity, suggestive cardiolipin-binding sites on Phosphatidic acid second-best binder investigated. bulk phosphatidylcholine phosphatidylethanolamine, weakest for Other bind a similar affinity. Functional assays show regulates ATPase activity dose-dependent fashion. was also impacted presence but lesser extent Taken together, has cardiolipin, lipid transporter.

Language: Английский

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Monitoring mAb proteoforms in mouse plasma using an automated immunocapture combined with top‐down and middle‐down mass spectrometry

PROTEOMICS, Journal Year: 2023, Volume and Issue: 24(3-4)

Published: July 21, 2023

Abstract Monoclonal antibodies (mAbs) have established themselves as the leading biopharmaceutical therapeutic modality. Once developability of a mAb drug candidate has been assessed, an important step is to check its in vivo stability through pharmacokinetics (PK) studies. The gold standard ligand‐binding assay (LBA) and liquid chromatography‐mass spectrometry (LC‐MS) performed at peptide level (bottom‐up approach). However, these analytical techniques do not allow address different proteoforms that can arise from biotransformation. In recent years, top‐down middle‐down mass approaches gained popularity characterize proteins proteoform but are yet widely used for PK We propose here workflow based on automated immunocapture followed by chromatography‐tandem (LC‐MS/MS) spiked mouse plasma. demonstrate applicability our large concentration range using pembrolizumab model. also compare performance two state‐of‐the‐art Orbitrap platforms (Tribrid Eclipse Exploris 480) added value accurate sensitive characterization plasma highlighted.

Language: Английский

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A Research Journey: Over a Decade of Denaturing and Native-MS Analyses of Hydrophobic and Membrane Proteins in Amgen Therapeutic Discovery

Journal of the American Society for Mass Spectrometry, Journal Year: 2023, Volume and Issue: 34(10), P. 2413 - 2431

Published: Aug. 29, 2023

Membrane proteins and associated complexes currently comprise the majority of therapeutic targets remain among most challenging classes for analytical characterization. Through long-term strategic collaborations forged between industrial academic research groups, there has been tremendous progress in advancing membrane protein mass spectrometry (MS) methods their concomitant application to Amgen project progression. Herein, I will describe a detailed personal account how electrospray ionization (ESI) native (nMS), ion mobility-MS (IM-MS), reversed phase liquid chromatographic (RPLC-MS), high-throughput solid extraction spectrometry, matrix-assisted laser desorption were developed, optimized, validated within Research, importantly, these applied hydrophobic analyses ultimately support Additionally, discuss all highly important productive collaborative efforts, both internal external academic, which key generating samples, methods, data described herein. also some early previously unpublished nano-ESI (nESI) native-MS from Research University California Los Angeles (UCLA) collaboration. present examples real-life biotherapeutic projects that supported by MS (and IM) techniques start describing initial nESI nMS experiments performed at 2011 on empty nanodisc molecules, using quadrupole time-of-flight MS, progressed 15 Tesla Fourier transform cyclotron resonance UCLA. Then are monomeric multimeric acquired tandem-MS modes, multiple activation, resulting dramatic spectral simplification. Also is we investigated far less established published subject, denaturing RPLC-MS analysis proteins, developed robust reproducible method capable effective separation differing only presence or absence an N-terminal post translational modification. evolution aforementioned into method. Finally, give my opinion developments area needs evolve state where it can be biopharmaceutical environment routine support.

Language: Английский

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