Rapid evaluation for heterogeneities in monoclonal antibodies by liquid chromatography/mass spectrometry with a column-switching system

Kuribayashi, Ryosuke; Hashii, Noritaka; Harazono, Akira; Kawasaki, Nana

doi:10.1016/j.jpba.2012.04.005

Cited by 19 publications

(12 citation statements)

References 30 publications

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“…Investigations at the intact protein level have the advantage of requiring less sample handling, but glycan analyses of intact proteins by chromatography and MS are still in their infancy compared with measurements at the peptide and glycan level. Most commonly, these mass spectrometric analyses are performed under denaturing conditions, with the protein eluted in an acidified organic water mixture using LC-MS, 20 - 22 prior to ionization by electrospray. Current TOF mass analyzers provide sufficient resolving power to resolve (less complex) glycan profiles on the intact proteins in a qualitative and relative quantitative manner.…”

Section: Introductionmentioning

confidence: 99%

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Rosati

Bremer

Schuurman

et al. 2013

mAbs

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Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies

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Section: Introductionmentioning

confidence: 99%

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Rosati

Bremer

Schuurman

et al. 2013

mAbs

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show abstract

“…Pretreatment processing is also needed for LC/MS analysis. The pretreatment methods include ultrafiltration, extraction, solid phase extraction [36,37], and column switch [38].…”

Section: Characteristics Of Gas Chromatography-mass Spectrometrymentioning

confidence: 99%

Novel methodologies in analysis of small molecule biomarkers and living cells

Chen

Zhu

2014

Tumor Biol.

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Enzyme-linked immuno-sorbent assay (ELISA) is widely used for biomarker detection. A good biomarker can distinguish patients from healthy or benign diseases. However, the ELISA method is not suitable for small molecule or trace substance detection. Along with the development of new technologies, an increasing level of biomaterials, especially small molecules, will be identified as novel biomarkers. Quantitative immuno-PCR, chromatography-mass spectrometry, and nucleic acid aptamer are emerging methodologies for detection of small molecule biomarkers, even in living cells. In this review, we focus on these novel technologies and their potential for small molecule biomarkers and living cell analysis.

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“…primary structure and posttranslational modifications (PTMs)) and conformation . Although relatively rapid analytical methods using MS as a PAT tool are available for determining primary structure and PTMs, no published study has described the development of a PAT tool for evaluating the disulfide bond proteoform of biopharmaceuticals during manufacturing. We chose mecasermin as a model, because the properties of its oxidized (folded) and reduced (unfolded) forms are known in detail .…”

Section: Introductionmentioning

confidence: 99%

A novel rapid analysis using mass spectrometry to evaluate downstream refolding of recombinant human insulin-like growth factor-1 (mecasermin)

Furuki

Toyo’oka

Yamaguchi

2017

Rapid Commun Mass Spectrom

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Rapid evaluation for heterogeneities in monoclonal antibodies by liquid chromatography/mass spectrometry with a column-switching system

Cited by 19 publications

References 30 publications

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Novel methodologies in analysis of small molecule biomarkers and living cells

A novel rapid analysis using mass spectrometry to evaluate downstream refolding of recombinant human insulin-like growth factor-1 (mecasermin)

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