Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches

Camperi, Julien; Goyon, Alexandre; Guillarme, Davy; Zhang, Kelly; Stella, Cinzia

doi:10.1039/d0an01963a

Cited by 54 publications

(39 citation statements)

References 129 publications

(170 reference statements)

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“…To overcome these issues, on-line comprehensive two-dimensional liquid chromatography (LC × LC or 2D) emerged as an attractive technique. [72][73][74] 2D LC combines two different chromatography columns, and thereby enables a rapid and efficient analysis to provide greater resolution for the resulting chromatogram and obtain more information within a single injection. Furthermore, this combination system can be coupled with MS (2D LC/MS) to provide a wide variety of options for ADC characterization.…”

Section: Summary and Future Landscapementioning

confidence: 99%

Recent Advances in Drug–Antibody Ratio Determination of Antibody–Drug Conjugates

Matsuda

Mendelsohn

2021

Chem. Pharm. Bull.

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Antibody-drug conjugates (ADCs) are biopharmaceuticals produced by chemically linking small molecules (payloads) to antibodies that possess specific affinity for the target cell. The ADCs currently on the commercially market are the result of a stochastic conjugation of highly-potent payloads to multiple sites on the monoclonal antibody, resulting in a heterogeneous drug-antibody ratio (DAR) and drug distribution. The heterogeneity inherent to ADCs not produced site-specifically may not only be detrimental to the quality of the drug but also is less-desirable from the perspective of regulatory science. An ideal method or unified approach used to measure the DAR for ADCs, a critical aspect of their analysis and characterization, has not yet been established in the ADC field and remains an often-challenging issue for bioanalytical chemists. In this review we describe, compare, and evaluate the characteristics of various DAR determination methods for ADCs featuring recently reported technologies. The future landscape of bioconjugate DAR analysis is also discussed.

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Section: Summary and Future Landscapementioning

confidence: 99%

Recent Advances in Drug–Antibody Ratio Determination of Antibody–Drug Conjugates

Matsuda

Mendelsohn

2021

Chem. Pharm. Bull.

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“…In addition to single MC, the report by Zhou et al disclosed tandem membrane chromatography, which reduced the purification time with a minimal number of mobile phases. This combination of two different chromatographies has become a popular topic in the analytical field [59,60]. This attractive technique is termed two-dimensional liquid chromatography (LC × LC or 2D).…”

Section: Membrane Chromatographymentioning

confidence: 99%

Current approaches for the purification of antibody–drug conjugates

Matsuda

2021

J of Separation Science

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In the past two decades, antibody-drug conjugates have gained increasing attention because they expand the therapeutic index when compared with that of traditional chemotherapies. Antibody-drug conjugates are highly complex structures consisting of antibodies covalently conjugated with small-molecule cytotoxic drugs. The complex structure of antibody-drug conjugates makes chemistry, manufacturing, and control difficult. In contrast to antibody production, distinct purification methods following conjugation of antibodies with druglinkers are required for the manufacturing. For process development of antibody drug conjugates, the drug-to-antibody ratio, free drug-linkers, and aggregates are critical quality attributes that must be strictly controlled and removed by appropriate purification techniques. In this review, features of various purification methods used to purify antibody drug conjugates are described and evaluated. The future landscape of the antibody-conjugates field is also discussed briefly.

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“… 2 − 4 To provide sufficient quality of biopharmaceutical products, the U.S. Food and Drug Administration (FDA) recommends the characterization and monitoring of critical quality attributes (CQAs) directly at the peptide level. 5 , 6 To comply with the requirements, peptide mapping analysis has become a standard method for characterizing the primary structure of biopharmaceuticals and thus the accurate identification of post-translational modifications (PTMs). 5 , 7 , 8 Nevertheless, this analysis requires a labor-intense and time-consuming manual sample preparation.…”

Section: Introductionmentioning

confidence: 99%

“… 5 , 6 To comply with the requirements, peptide mapping analysis has become a standard method for characterizing the primary structure of biopharmaceuticals and thus the accurate identification of post-translational modifications (PTMs). 5 , 7 , 8 Nevertheless, this analysis requires a labor-intense and time-consuming manual sample preparation. 9 To increase efficiency, two approaches toward method automation were established.…”

Section: Introductionmentioning

confidence: 99%

mD-UPLC-MS/MS: Next Generation of mAb Characterization by Multidimensional Ultraperformance Liquid Chromatography-Mass Spectrometry and Parallel On-Column LysC and Trypsin Digestion

et al. 2022

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For the past few years, multidimensional liquid chromatography-mass spectrometry (LC-MS) systems have been commonly used to characterize post-translational modifications (PTMs) of therapeutic antibodies (mAbs). In most cases, this is performed by fractionation of charge variants by ion-exchange chromatography and subsequent online LC-MS peptide mapping analysis. In this study, we developed a multidimensional ultra-performance-liquid-chromatography-mass spectrometry system (mD-UPLC-MS/MS) for PTM characterization and quantification, allowing both rapid analysis and decreased risk of artificial modifications during sample preparation. We implemented UPLC columns for peptide mapping analysis, facilitating the linkage between mD-LC and routine LC-MS workflows. Furthermore, the introduced system incorporates a novel in-parallel trypsin and LysC on-column digestion setup, followed by a combined peptide mapping analysis. This parallel digestion with different enzymes enhances characterization by generating two distinct peptides. Using this approach, a low retentive ethylene oxide adduct of a bispecific antibody was successfully characterized within this study. In summary, our approach allows versatile and rapid analysis of PTMs, enabling efficient characterization of therapeutic molecules.

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Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches

Abstract: This review presents an overview of current analytical trends in antibody characterization by multidimensional LC-MS approaches.

Cited by 54 publications

References 129 publications

Recent Advances in Drug–Antibody Ratio Determination of Antibody–Drug Conjugates

Recent Advances in Drug–Antibody Ratio Determination of Antibody–Drug Conjugates

Current approaches for the purification of antibody–drug conjugates

mD-UPLC-MS/MS: Next Generation of mAb Characterization by Multidimensional Ultraperformance Liquid Chromatography-Mass Spectrometry and Parallel On-Column LysC and Trypsin Digestion

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