Disulfide bond assignment of an IgG1 monoclonal antibody by LC–MS with post-column partial reduction

Li, Xiaojuan; Wang, Fengqiang; Xu, Wei; May, Kimberly; Richardson, Daisy; Liu, Hongcheng

doi:10.1016/j.ab.2013.01.020

Cited by 21 publications

(35 citation statements)

References 30 publications

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“…Post-column chemical reduction of disulfide-bonded proteolytic digests typically involves separation of the non-reduced protein digests by reversed phase liquid chromatography, and the eluates from the column are mixed (via a mixing-tee) with an optimized amount of a reducing agent (e.g TCEP) to partially reduce the disulfide bonds, as demonstrated by Li et al [98, 99]. Since only partial disulfide bond reduction occurs after LC separation, disulfide-linked peptides with intact disulfide bonds and their corresponding Cys-containing peptides that resulted from the disulfide bond reduction would have the same retention time and would both be detected in the same full scan mass spectrum.…”

Section: Liquid Chromatography-mass Spectrometric Disulfide Bond Amentioning

confidence: 99%

“…Since only partial disulfide bond reduction occurs after LC separation, disulfide-linked peptides with intact disulfide bonds and their corresponding Cys-containing peptides that resulted from the disulfide bond reduction would have the same retention time and would both be detected in the same full scan mass spectrum. To identify disulfide-bonded peptides, the extracted ion chromatograms of all Cys-containing peptides are compared, and the Cys-containing peptides that have the same retention time are assigned as disulfide bonded [98, 99]. The assignments are confirmed by the presence of a disulfide-linked peptide at the same retention time with a molecular mass corresponding to the sum of the molecular masses of the two Cys-containing peptides minus 2 Da.…”

Section: Liquid Chromatography-mass Spectrometric Disulfide Bond Amentioning

confidence: 99%

“…The assignments are confirmed by the presence of a disulfide-linked peptide at the same retention time with a molecular mass corresponding to the sum of the molecular masses of the two Cys-containing peptides minus 2 Da. Scrambled disulfide bonds are readily assigned in the same manner [99]. Liu and coworkers [100] used this approach to map the disulfide bonds of an IgG2 antibody, but instead of using only LC-MS data, LC-MS/MS (CID) data were used for unambiguous characterization of the LC peaks of the disulfide bonded peptides and those of their corresponding Cys-containing peptides.…”

Section: Liquid Chromatography-mass Spectrometric Disulfide Bond Amentioning

confidence: 99%

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Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins

2017

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Disulfide bonds are important structural moieties of proteins: they ensure proper folding, provide stability, and ensure proper function. With the increasing use of proteins for biotherapeutics, particularly monoclonal antibodies, which are highly disulfide bonded, it is now important to confirm the correct disulfide bond connectivity and to verify the presence, or absence, of disulfide bond variants in the protein therapeutics. These studies help to ensure safety and efficacy. Hence, disulfide bonds are among the critical quality attributes of proteins that have to be monitored closely during the development of biotherapeutics. However, disulfide bond analysis is challenging because of the complexity of the biomolecules. Mass spectrometry (MS) has been the go-to analytical tool for the characterization of such complex biomolecules, and several methods have been reported to meet the challenging task of mapping disulfide bonds in proteins. In this review, we describe the relevant, recent MS-based techniques and provide important considerations needed for efficient disulfide bond analysis in proteins. The review focuses on methods for proper sample preparation, fragmentation techniques for disulfide bond analysis, recent disulfide bond mapping methods based on the fragmentation techniques, and automated algorithms designed for rapid analysis of disulfide bonds from liquid chromatography-MS/MS data. Researchers involved in method development for protein characterization can use the information herein to facilitate development of new MS-based methods for protein disulfide bond analysis. In addition, individuals characterizing biotherapeutics, especially by disulfide bond mapping in antibodies, can use this review to choose the best strategies for disulfide bond assignment of their biologic products. Graphical Abstract This review, describing characterization methods for disulfide bonds in proteins, focuses on three critical components: sample preparation, mass spectrometry data, and software tools.

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Section: Liquid Chromatography-mass Spectrometric Disulfide Bond Amentioning

confidence: 99%

Section: Liquid Chromatography-mass Spectrometric Disulfide Bond Amentioning

confidence: 99%

Section: Liquid Chromatography-mass Spectrometric Disulfide Bond Amentioning

confidence: 99%

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Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins

2017

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“…Structurally, immunoglobulin G1 (IgG1) antibodies contain four interchain disulfides [two hinge and two LC (light chain)–heavy chain (HC) connections], whereas IgG2 antibodies have been reported to exist in three different disulfide isoforms that differ in the configuration of the six interchain disulfides . Solvent accessibility has been reported to affect the reduction potential of IgG interchain disulfide bonds .…”

Section: Introductionmentioning

confidence: 99%

“…Structurally, immunoglobulin G1 (IgG1) antibodies contain four interchain disulfides [two hinge and two LC (light chain)heavy chain (HC) connections], whereas IgG2 antibodies have been reported to exist in three different disulfide isoforms that differ in the configuration of the six interchain disulfides. [6][7][8][9] Solvent accessibility has been reported to affect the reduction potential of IgG interchain disulfide bonds. [10][11][12][13] In agreement with these reports, the preferred conjugation sites for an IgG1 occurs in the LC-HC cysteines involved in interchain connections, as well as those involved in the hinge disulfide bonding.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Cysteine-Linked Conjugation Profiles of Immunoglobulin G1 and Immunoglobulin G2 Antibody–Drug Conjugates

Wiggins¹,

Liu-Shin²,

Yamaguchi

et al. 2015

Journal of Pharmaceutical Sciences

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A passive membrane system for on‐line mass spectrometry reagent addition

Zarkovic

Borden

Krogh

et al. 2023

Rapid Comm Mass Spectrometry

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Rationale: Post-separation addition of chemical modifiers in liquid chromatographymass spectrometry is widely used for improving ionization sensitivity and selectivity. This is typically accomplished using a post-column T-junction, which can result in sample dilution and imperfect mixing. We present a passive semi-permeable hollow fiber membrane approach for the addition of chemical modifiers that avoids these issues.Methods: Model compounds were directly infused by flow injection to an electrospray ionization triple quadrupole mass spectrometer after passing through a polydimethylsiloxane hollow fiber membrane. Ionization enhancement reagents were introduced into the flowing stream by membrane permeation from aqueous solutions. Ionization enhancement from volatile acids and bases in positive and negative electrospray ionization was evaluated to assess the feasibility of this approach.Results: The membrane-based apparatus resulted in relative ionization enhancement factors of up to 14Â, depending upon the analyte, reagent, and ionization mode used. Ionization enhancement signal stability is reasonable (relative standard deviation of 5-7%) for extended periods from the same reagent solution, and minimal analyte dilution is observed. A proof-of-concept demonstration of the chromatographic "trifluoroacetic acid fix" strategy is presented.Conclusions: An on-line mass spectrometry ionization reagent addition method with potential post-chromatography reagent addition applications was developed using a hollow fiber polydimethylsiloxane membrane. This approach offers a promising alternative to traditional methods requiring additional hardware such as pumps and T-junctions that can result in sample dilution and imperfect reagent mixing. | INTRODUCTIONThe post-separation infusion of derivatizing reagents and chemical modifiers in liquid chromatography-mass spectrometry (LC/MS) workflows has been widely used to enhance ionization without altering chromatographic performance. This is done because optimal separation solvent systems can be quite different from those needed for efficient ionization. 1,2 Reagents are frequently introduced to the LC eluent via a T-junction, infusing reagent via a second pumping system. Other post-column addition techniques (still utilizing secondary liquid handling pumps) include the use of reaction coils 3 and triaxial electrospray probes. 4 Although analyte ionization is

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Disulfide bond assignment of an IgG1 monoclonal antibody by LC–MS with post-column partial reduction

Cited by 21 publications

References 30 publications

Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins

Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins

Characterization of Cysteine-Linked Conjugation Profiles of Immunoglobulin G1 and Immunoglobulin G2 Antibody–Drug Conjugates

A passive membrane system for on‐line mass spectrometry reagent addition

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