Orthogonal Technologies for NISTmAb N-Glycan Structure Elucidation and Quantitation

Prien, Justin M.; Stöckmann, Henning; Albrecht, Simone; Millán-Martín, Silvia; Varatta, Matthew; Furtado, Marsha; Hosselet, Stephen; Wang, Meiyao; Formolo, Trina; Rudd, Pauline M.; Schiel, John E.

doi:10.1021/bk-2015-1201.ch004

Cited by 28 publications

(32 citation statements)

References 80 publications

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“…Overall, the structures identified and their relative amounts correlate quite closely to a previous multi-laboratory, multi-approach characterization study of the NIST mAb. 22 …”

Section: Resultsmentioning

confidence: 99%

Glycan characterization of the NIST RM monoclonal antibody using a total analytical solution: From sample preparation to data analysis

Hilliard

Alley

McManus

et al. 2017

mAbs

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Glycosylation is an important attribute of biopharmaceutical products to monitor from development through production. However, glycosylation analysis has traditionally been a time-consuming process with long sample preparation protocols and manual interpretation of the data. To address the challenges associated with glycan analysis, we developed a streamlined analytical solution that covers the entire process from sample preparation to data analysis. In this communication, we describe the complete analytical solution that begins with a simplified and fast N-linked glycan sample preparation protocol that can be completed in less than 1 hr. The sample preparation includes labelling with RapiFluor-MS tag to improve both fluorescence (FLR) and mass spectral (MS) sensitivities. Following HILIC-UPLC/FLR/MS analyses, the data are processed and a library search based on glucose units has been included to expedite the task of structural assignment. We then applied this total analytical solution to characterize the glycosylation of the NIST Reference Material mAb 8761. For this glycoprotein, we confidently identified 35 N-linked glycans and all three major classes, high mannose, complex, and hybrid, were present. The majority of the glycans were neutral and fucosylated; glycans featuring N-glycolylneuraminic acid and those with two galactoses connected via an α1,3-linkage were also identified.

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“…Overall, the structures identified and their relative amounts correlate quite closely to a previous multi-laboratory, multi-approach characterization study of the NIST mAb. 22 …”

Section: Resultsmentioning

confidence: 99%

Glycan characterization of the NIST RM monoclonal antibody using a total analytical solution: From sample preparation to data analysis

Hilliard

Alley

McManus

et al. 2017

mAbs

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“…Supplemental Table S3 lists all the quantified and derived values for NISTmAb and mod-NISTmAb for glycan compositions and glycan structures (24). Independent of this study, glycosylation of NIST-mAb was previously characterized by three laboratories using HILIC with fluorescence detection of 2-AB-labeled N-glycans and collectively found 24 glycan peaks (28,31). Another work using 1D-and 2D-LC-MS/MS for the analysis of glycopeptides found 60 glycan masses on NISTmAb (16).…”

Section: Overview Of Analytical Techniques For Mab Glycosylation Analmentioning

confidence: 95%

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods

Leoz

Duewer

Fung³

et al. 2020

Molecular & Cellular Proteomics

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Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

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“…The observed ratios of the glycoforms are in accordance with the literature findings. 36,37 The low-abundant proteoforms G0F-GIcNAc, G2F+Hex, and G2F +2Hex were only observed using FAIMS, and their detection and accurate relative quantitation are a good indicator of the heightened sensitivity afforded by FAIMS-MS.…”

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confidence: 99%

Direct measurement of light and heavy antibody chains using ion mobility and middle-down mass spectrometry

Melani

Srzentić

Gerbasi

et al. 2019

mAbs

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The analysis of monoclonal antibodies (mAbs) by a middle-down mass spectrometry (MS) approach is a growing field that attracts the attention of many researchers and biopharmaceutical companies. Usually, liquid fractionation techniques are used to separate mAbs polypeptides chains before MS analysis. Gas-phase fractionation techniques such as high-field asymmetric waveform ion mobility spectrometry (FAIMS) can replace liquid-based separations and reduce both analysis time and cost. Here, we present a rapid FAIMS tandem MS method capable of characterizing the polypeptide sequence of mAbs light and heavy chains in an unprecedented, easy, and fast fashion. This new method uses commercially available instruments and takes ~24 min, which is 40-60% faster than regular liquid chromatography-MS/MS analysis, to acquire fragmentation data using different dissociation methods.

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Orthogonal Technologies for NISTmAb N-Glycan Structure Elucidation and Quantitation

Cited by 28 publications

References 80 publications

Glycan characterization of the NIST RM monoclonal antibody using a total analytical solution: From sample preparation to data analysis

Glycan characterization of the NIST RM monoclonal antibody using a total analytical solution: From sample preparation to data analysis

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods

Direct measurement of light and heavy antibody chains using ion mobility and middle-down mass spectrometry

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