Improved characterization of trastuzumab deruxtecan with PTCR and internal fragments implemented in middle-down MS workflows

Beaumal, Corentin; Deslignière, Evolène; Diemer, Hélène; Carapito, Christine; Cianférani, Sarah; Hernandez-Alba, Oscar

doi:10.1007/s00216-023-05059-x

Cited by 5 publications

(2 citation statements)

References 48 publications

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“…Specifically, the inclusion of internal fragment assignments has shown to be valuable in the TD-MS analysis of mAbs and ADCs, which motivates us to explore the employment of internal fragments in MD-MS of mAbs and ADCs to obtain more comprehensive characterization results . A recent study applied MD-MS aided with internal fragment analysis using online denaturing LC-MS/MS to characterize a cysteine-linked therapeutic site-specific ADC, achieving sequence coverages ranging from 70 to 90% …”

Section: Introductionmentioning

confidence: 99%

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

Wei,

Lantz,

Ogorzalek Loo

et al. 2024

Anal. Chem.

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

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Section: Introductionmentioning

confidence: 99%

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

Wei,

Lantz,

Ogorzalek Loo

et al. 2024

Anal. Chem.

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“…The EThcD tandem mass spectrometry method combines the application of ETD to the polyprotonated peptides as precursors, followed by HCD applied for the whole array of the primary fragment ions and the survived molecular precursors. The EThcD approach has been proposed in Albert Heck laboratory and showed itself very useful for localization of phosphosites − and ribosylation sites, − protein lipidation, characterization of antibody–drug conjugates, and monoclonal antibodies analysis. − The approach also works with glycopeptides, − including even automated identification . It can be applied for proteoform analysis − and for differentiation of isomeric Leu/Ile residues. , …”

Section: Introductionmentioning

confidence: 99%

EThcD as a Unique Tool for the Top-Down De Novo Sequencing of Intact Natural Ranid Amphibian Peptides

Samgina,

Vasileva,

Zubarev

et al. 2024

Anal. Chem.

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De novo sequencing of any novel peptide/protein is a difficult task. Full sequence coverage, isomeric amino acid residues, inter-and intramolecular S−S bonds, and numerous other post-translational modifications make the investigators employ various chemical modifications, providing a variety of specific fragmentation MS n patterns. The chemical processes are timeconsuming, and their yields never reach 100%, while the subsequent purification often leads to the loss of minor components of the initial peptide mixture. Here, we present the advantages of the EThcD method that enables establishing the full sequence of natural intact peptides of ranid frogs in de novo topdown mode without any chemical modifications. The method provides complete sequence coverage, including the cyclic disulfide section, and reliable identification of isomeric leucine/isoleucine residues. The proposed approach demonstrated its efficiency in the analysis of peptidomes of ranid frogs from several populations of Rana arvalis, Rana temporaria, and Pelophylax esculentus complexes.

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Towards a universal method for middle-down analysis of antibodies via proton transfer charge reduction—Orbitrap mass spectrometry

Oates,

Lieu,

Kline

et al. 2024

Anal Bioanal Chem

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Improved characterization of trastuzumab deruxtecan with PTCR and internal fragments implemented in middle-down MS workflows

Cited by 5 publications

References 48 publications

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

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

EThcD as a Unique Tool for the Top-Down De Novo Sequencing of Intact Natural Ranid Amphibian Peptides

Towards a universal method for middle-down analysis of antibodies via proton transfer charge reduction—Orbitrap mass spectrometry

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