Comprehensive Multidimensional Liquid Chromatography–Mass Spectrometry for the Characterization of Charge Variants of a Bispecific Antibody

Grunert, Ingrid; Heinrich, Katrin; Hingar, Michael; Ernst, Juliane; Winter, Martin; Bomans, Katrin; Wagner, Katharina; Fevre, Arnaud; Reusch, Dietmar; Wuhrer, Manfred; Bulau, Patrick

doi:10.1021/jasms.2c00296

Cited by 11 publications

(10 citation statements)

References 28 publications

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“…However, to date, there has been a lack of an analytical workflow dedicated to in-depth characterization of mAb charge variants. Often, analytical efforts are focused on the characterization throughput or new charge variant identification, , but not on comprehensive characterization of mAb charge variants that is needed to sufficiently explain the observed charge heterogeneity. Consequently, the existing workflows relied either on untargeted or targeted peptide mapping analysis.…”

Section: Resultsmentioning

confidence: 99%

In-Depth Characterization of Acidic Variants Induced by Metal-Catalyzed Oxidation in a Recombinant Monoclonal Antibody

Yang¹,

Zhang²,

Gan³

et al. 2023

Anal. Chem.

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Characterization of antibody charge heterogeneity is an important task for antibody drug development. Recently, a correlation between acidic charge heterogeneity and metalcatalyzed oxidation has been observed for antibody drugs. However, to date, the acidic variants induced by metal-catalyzed oxidation have not been elucidated. In addition, it is challenging to satisfactorily explain the induced acidic charge heterogeneity, as the existing analytical workflows, which relied on either untargeted or targeted peptide mapping analysis, could lead to incomplete identification of the acidic variants. In this work, we present a new characterization workflow by combining untargeted and targeted analyses to thoroughly identify and characterize the induced acidic variants in a highly oxidized IgG1 antibody. As a part of this workflow, a tryptic peptide mapping method was also developed for accurate determination of the relative extent of site-specific carbonylation, where a new hydrazone reduction procedure was established to minimize under-quantitation artifacts caused by incomplete reduction of hydrazones during sample preparation. In summary, we identified 28 site-specific oxidation products, which are located on 26 residues and of 11 different modification types, as the sources of the induced acidic charge heterogeneity. Many of the oxidation products were reported for the first time in antibody drugs. More importantly, this study provides new insights to understanding acidic charge heterogeneity of antibody drugs in the biotechnology industry. Additionally, the characterization workflow presented in this study can be applied as a platform approach in the biotechnology industry to better address the need for in-depth characterization of antibody charge variants.

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Section: Resultsmentioning

confidence: 99%

In-Depth Characterization of Acidic Variants Induced by Metal-Catalyzed Oxidation in a Recombinant Monoclonal Antibody

Yang¹,

Zhang²,

Gan³

et al. 2023

Anal. Chem.

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show abstract

“…Importantly, as therapeutic modalities have become more complex, the mass distributions also confirm the correct assembly of the products. For example, native/intact MS is useful for elucidating the correct pairing of bispecific antibodies and the correct assembly of more complex structures. − Over the past few decades, the demand for bispecific antibody analysis has increased to thousands of samples per year. New modalities and antibody formats are often developed, leading to a variety of projects for intact mass analysis, which may include light chain ratio optimization, antibody pairing combinations, CDR swapping panels, and purification strategies.…”

Section: Introductionmentioning

confidence: 99%

UniDec Processing Pipeline for Rapid Analysis of Biotherapeutic Mass Spectrometry Data

Phung¹,

Bakalarski²,

Hinkle³

et al. 2023

Anal. Chem.

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

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“…For example, native/intact MS is useful for elucidating the correct pairing of bispecific antibodies and correct assembly of more complex structures. [4][5][6][7] In developing and optimizing bispecific antibody pairing strategies, minimizing undesired species, including mis-paired antibodies and homodimers, is crucial in any bispecific therapeutic platform.…”

Section: Introductionmentioning

confidence: 99%

The UniDec Processing Pipeline for Rapid Analysis of Biotherapeutic Mass Spectrometry Data

Phung¹,

Bakalarski²,

Hinkle³

et al. 2023

Preprint

View full text Add to dashboard Cite

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

show abstract

Comprehensive Multidimensional Liquid Chromatography–Mass Spectrometry for the Characterization of Charge Variants of a Bispecific Antibody

Cited by 11 publications

References 28 publications

In-Depth Characterization of Acidic Variants Induced by Metal-Catalyzed Oxidation in a Recombinant Monoclonal Antibody

In-Depth Characterization of Acidic Variants Induced by Metal-Catalyzed Oxidation in a Recombinant Monoclonal Antibody

UniDec Processing Pipeline for Rapid Analysis of Biotherapeutic Mass Spectrometry Data

The UniDec Processing Pipeline for Rapid Analysis of Biotherapeutic Mass Spectrometry Data

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