Extended characterization of unpaired cysteines in an IgG1 monoclonal antibody by LC-MS analysis

Li, Xiaojuan; Xiao, Li; Kochert, Brent A.; Donnelly, Daniel P.; Gao, Xinliu; Richardson, Douglas D.

doi:10.1016/j.ab.2021.114172

Cited by 6 publications

(12 citation statements)

References 27 publications

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“…Digestion procedure (2) with PNGase F: 100 μg of CD24Fc fusion protein was digested with SialEXO and OpeRATOR enzymes at an enzyme-to-protein ratio of 1 U/μg for both enzymes in 20 mM Tris-HCl digestion buffer at pH 7.5 with PNGase F enzyme at an enzyme-to-protein ratio of 0.1 U/μg and incubated at 37 °C overnight with shaking. Digestion procedure (3) with PNGase F and limited Lys-C digestion: After completion of all steps of digestion procedure (2), 0.25 μg of Lys-C enzyme was added at 1:400 enzyme-to-protein ratio and incubated at 37 °C for an additional 20 min . The glycopeptides generated from the above three digestion procedures were enriched by using graphite columns and dried using a vacuum evaporator.…”

Section: Methodsmentioning

confidence: 99%

“…Digestion procedure (3) with PNGase F and limited Lys-C digestion: After completion of all steps of digestion procedure (2), 0.25 μg of Lys-C enzyme was added at 1:400 enzyme-to-protein ratio and incubated at 37 °C for an additional 20 min. 31 The glycopeptides generated from the above three digestion procedures were enriched by using graphite columns and dried using a vacuum evaporator. The glycopeptides were separated on a Waters Acquity UPLC Glycoprotein Amide (1.7 μm, 2.1 X150 mm) column using a Vanquish UPLC system coupled with a Thermo Eclipse mass spectrometer for an LC-MS/MS EThcD analysis.…”

Section: ■ Introductionmentioning

confidence: 99%

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Precise O-Glycosylation Site Localization of CD24Fc by LC-MS Workflows

Wilmanowski

Gao

et al. 2022

Anal. Chem.

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CD24Fc is a homodimeric recombinant Fc-fusion protein comprised of human CD24 connected to immunoglobulin G1 (IgG1) Fc fragment. CD24 is heavily glycosylated, and its biological function is considered mainly mediated by its glycosylation. Identification of the O-glycosylation sites would facilitate an in-depth understanding of the functional role of O-glycans in CD24. However, the presence of clustered mucin-type O-glycans together with N-glycans makes the determination of O-glycosylation sites and abundance very challenging. In this study, two sets of liquid chromatography-mass spectrometry (LC-MS) workflows were developed for the comprehensive characterization of O-glycosylation in CD24: (1) Fractionation and collision-induced dissociation (CID) workflow involving multienzyme digestion, fractionation, OpeRATOR/SialEXO digestion, and CID analysis; (2) Direct OpeRATOR/SialEXO digestion followed by electron-transfer/higher-energy collision dissociation (EThcD) analysis. The precise O-glycosylation sites were identified in CD24 for the first time, and the site occupancy was assessed. A total of 12 O-glycosylation sites were identified. Seven glycosylation sites were identified by both workflows, and five additional sites were identified only by the EThcD workflow. The predominant O-glycosylation site in CD24 was Thr25 followed by Thr15. The CID workflow provided an overall relative quantitation of O-glycoforms at the CD24 level and site localization for singly O-glycosylated peptides. The EThcD workflow directly identified glycosylation sites by tandem mass spectrometry (MS/MS) for singly, doubly, and triply O-glycosylated peptides. Together, both workflows validated each other’s results and can be applied to a complex mucin-type O-glycosylation site analysis of other glycoproteins and Fc-fusion therapeutics.

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Section: Methodsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Precise O-Glycosylation Site Localization of CD24Fc by LC-MS Workflows

Wilmanowski

Gao

et al. 2022

Anal. Chem.

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“…This has since been successfully applied to determine free-Cys levels in therapeutic recombinant coagulation factor VIII products ( Arsiccio et al, 2022 ). A recent development of this method forgoes the use of a fully NEM-alkylated mAb standard and directly quantified NEM-labeled free-Cys with IAA-labeled Cys derived from disulfide bonds ( Li et al, 2021 ). However, it should be noted that the ionization properties of NEM- and IAA-labeled peptides will not be comparable and may potentially lead to inaccurate quantitation which should be addressed in detailed method validation.…”

Section: Wholly Mass Spectrometry Methodsmentioning

confidence: 99%

A Review of Methodologies for the Detection, Quantitation, and Localization of Free Cysteine in Recombinant Proteins: A Focus on Therapeutic Monoclonal Antibodies

Metcalfe

2022

Front. Mol. Biosci.

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Free-cysteine residues in recombinant biotherapeutics such as monoclonal antibodies can arise from incorrect cellular processing of disulfide bonds during synthesis or by reduction of disulfide bonds during the harvest and purification stage of manufacture. Free cysteines can affect potency, induce aggregation, and decrease the stability of therapeutic proteins, and the levels and positions of free cysteines in proteins are closely monitored by both manufacturers and regulators to ensure safety and efficacy. This review summarizes the latest methodologies for the detection and quantification of free cysteines.

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“…S-S bonds have low dissociation energies (~60 kcal/mol) and are hence more prone to cleavage when exposed to reducing agents or when subjected to stress conditions [9]. In addition to S-S bond cleavage, cysteine residues are also known to be highly susceptible to several other modifications including disulfide scrambling, racemization, cysteinylation, bridging to additional LC, and trisulfide formation [9,10], all of which could be CQAs that affect the binding, potency, pharmacokinetic profiles, and immunogenicity of the antibody. The presence of improper disulfide bond profiles during manufacturing [11,12] might increase molecular heterogeneity, impact the potency of the molecule [2], and affect manufacturing process yield [13].…”

Section: Introductionmentioning

confidence: 99%

“…For more details, we refer the reader to an excellent review of this topic [18]. For IgG1 antibodies, reports based on spectroscopic and mass spectrometric methods have reported the abundance of free sulfhydryl states on HC:C22 [10,19], HC:C96 [10,19], Unpaired cysteine residues or free sulfhydryl groups are primarily a result of (a) incomplete disulfide bond formation during antibody synthesis/assembly and (b) the reductive cleavage of existing disulfide bonds. State-of-the-art methods for free cysteine characterization primarily use liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS/MS) methods to detect, locate, and quantify the levels of free thiols.…”

Section: Introductionmentioning

confidence: 99%

Heterogeneity in Disulfide Bond Reduction in IgG1 Antibodies Is Governed by Solvent Accessibility of the Cysteines

Natesan,

Dykstra,

Banerjee

et al. 2023

Antibodies

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We studied unpaired cysteine levels and disulfide bond susceptibility in four different γ-immunoglobulin antibodies using liquid chromatography–mass spectrometry. Our choice of differential alkylating agents ensures that the differential peaks are non-overlapping, thus allowing us to accurately quantify free cysteine levels. For each cysteine residue, we observed no more than 5% to be unpaired, and the free cysteine levels across antibodies were slightly higher in those containing lambda light chains. Interchain and hinge residues were highly susceptible to reducing stresses and showed a 100–1000-fold higher rate of reduction compared to intrachain cysteines. Estimations of the solvent-accessible surface for individual cysteines in IgG1, using an implicit all-atom molecular dynamics simulation, show that interchain and hinge cysteines have >1000-fold higher solvent accessibility compared to intrachain cysteines. Further analyses show that solvent accessibility and the rate of reduction are linearly correlated. Our work clearly establishes the fact that a cysteine’s accessibility to the surrounding solvent is one of the primary determinants of its disulfide bond stability.

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Extended characterization of unpaired cysteines in an IgG1 monoclonal antibody by LC-MS analysis

Cited by 6 publications

References 27 publications

Precise O-Glycosylation Site Localization of CD24Fc by LC-MS Workflows

Precise O-Glycosylation Site Localization of CD24Fc by LC-MS Workflows

A Review of Methodologies for the Detection, Quantitation, and Localization of Free Cysteine in Recombinant Proteins: A Focus on Therapeutic Monoclonal Antibodies

Heterogeneity in Disulfide Bond Reduction in IgG1 Antibodies Is Governed by Solvent Accessibility of the Cysteines

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