Top-Down Characterization and Intact Mass Quantitation of a Monoclonal Antibody Drug from Serum by Use of a Quadrupole TOF MS System Equipped with Electron-Activated Dissociation

Kellie, John F.; Schneck, Nicole A.; Causon, Jason; Baba, Takashi; Mehl, John T; Pohl, Kerstin I

doi:10.1021/jasms.2c00206

Cited by 9 publications

(10 citation statements)

References 28 publications

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“…For comparison, a very recent in vivo study combining immunoaffinity and middle-down MS/MS [70], indicates a sequence coverage of around 30% for the fragmentation of the Lc of a therapeutic mAb by ECD for 1 mg injected on column. In our case, this sequence coverage is obtained for only 42 ng on column, showing that our workflow is much more sensitive.…”

Section: Middle-down Ms/ms Coupled To Immunocapturementioning

confidence: 99%

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

Dhenin

Lafont

Dupré

et al. 2023

Preprint

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

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Section: Middle-down Ms/ms Coupled To Immunocapturementioning

confidence: 99%

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

Dhenin

Lafont

Dupré

et al. 2023

Preprint

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“…In addition, the sensitivity of the mass spectrometer is enhanced using the Zeno trap pulsing . EIEIO has played an essential role in detailed structure annotation for various biomolecules, such as peptides , and lipids, including phosphatidylcholines, , sphingomyelin, , triacylglycerols. , EIEIO has demonstrated significant value in the complete structural elucidation of singly charged peptides with post-translational modifications, a challenge often encountered with the widely employed fragmentation technique of collision-induced dissociation (CID) mode . Besides, combined with differential mobility spectrometry (DMS), EIEIO can differentiate constitutional isomers, cis/trans isomers, and diastereomers of eicosanoids .…”

Section: Introductionmentioning

confidence: 99%

Enhancing Metabolome Annotation by Electron Impact Excitation of Ions from Organics-Molecular Networking

Wang,

Sun,

Wang

et al. 2024

Anal. Chem.

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Liquid chromatography−high-resolution mass spectrometry (LC−HRMS) is widely used in untargeted metabolomics, but large-scale and high-accuracy metabolite annotation remains a challenge due to the complex nature of biological samples. Recently introduced electron impact excitation of ions from organics (EIEIO) fragmentation can generate information-rich fragment ions. However, effective utilization of EIEIO tandem mass spectrometry (MS/MS) is hindered by the lack of reference spectral databases. Molecular networking (MN) shows great promise in large-scale metabolome annotation, but enhancing the correlation between spectral and structural similarity is essential to fully exploring the benefits of MN annotation. In this study, a novel approach was proposed to enhance metabolite annotation in untargeted metabolomics using EIEIO and MN. MS/MS spectra were acquired in EIEIO and collision-induced dissociation (CID) modes for over 400 reference metabolites. The study revealed a stronger correlation between the EIEIO spectra and metabolite structure. Moreover, the EIEIO spectral network outperformed the CID spectral network in capturing structural analogues. The annotation performance of the structural similarity network for untargeted LC−MS/MS was evaluated. For the spiked NIST SRM 1950 human plasma, the annotation coverage and accuracy were 72.94 and 74.19%, respectively. A total of 2337 metabolite features were successfully annotated in NIST SRM 1950 human plasma, which was twice that of LC−CID MS/MS. Finally, the developed method was applied to investigate prostate cancer. A total of 87 significantly differential metabolites were annotated. This study combining EIEIO and MN makes a valuable contribution to improving metabolome annotation.

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“…We have previously examined the use of top-down MS for antibodies immunocapture from serum. 20 Building on this as a case study, we demonstrate top-down MS on a clipped biotherapeutic and assay background proteins. The first example entails a complex fusion protein biotherapeutic that was post-spiked into assayprocessed clinical serum.…”

Section: Introductionmentioning

confidence: 99%

“…Top-down identification and characterization approaches are well-established in the proteomics community. , The same approaches may be utilized for biotherapeutics. , For in-life studies, catabolites are identified based on intact mass only; while likely correct, there may be instances where statistically confident identification based on top-down fragmentation provides definitive evidence of the specific amino acid site for molecule catabolism or amino acid site hydrolysis (termed “clipping” throughout this article). We have previously examined the use of top-down MS for antibodies immunocapture from serum . Building on this as a case study, we demonstrate top-down MS on a clipped biotherapeutic and assay background proteins.…”

Section: Introductionmentioning

confidence: 99%

Protein LC-MS Tools for the Next Generation of Biotherapeutic Analyses from Preclinical and Clinical Serum

Schneck¹,

Mehl²,

Kellie³

2023

J. Am. Soc. Mass Spectrom.

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LC-MS analysis of therapeutic antibodies and other biotherapeutics from in-life studies (e.g., serum/plasma) has evolved from simple peptide digestion to peptide mapping and intact mass monitoring. From more advanced analytical approaches, a deeper understanding as to the fate of the biotherapeutic in vivo is gained. Here, we examine the next generation of approaches to facilitate the most comprehensive understanding of large molecule drug fate in circulation. Three case studies are presented: (1) use of relative and absolute calibration curves for biotherapeutic quantitation from the same sample set; (2) top-down mass spectrometry applied to bioanalytical assays; (3) biotherapeutic protein complexes from serum analyzed by native protein MS. We anticipate that these approaches will be further adapted and applied by other research groups.

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Top-Down Characterization and Intact Mass Quantitation of a Monoclonal Antibody Drug from Serum by Use of a Quadrupole TOF MS System Equipped with Electron-Activated Dissociation

Cited by 9 publications

References 28 publications

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

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

Enhancing Metabolome Annotation by Electron Impact Excitation of Ions from Organics-Molecular Networking

Protein LC-MS Tools for the Next Generation of Biotherapeutic Analyses from Preclinical and Clinical Serum

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