Complete mapping of disulfide linkages for etanercept products by multi-enzyme digestion coupled with LC-MS/MS using multi-fragmentations including CID and ETD

Huang, Lijuan; Chiang, Chun-Yi; Chen, Shunli; Wei, Shih-Yao; Chen, Shu Hui

doi:10.1016/j.jfda.2018.11.007

Cited by 12 publications

(8 citation statements)

References 29 publications

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“…In addition, Huang et al. reported how multiple fragmentations including CID and ETD can be implemented to characterize disulfide linkages, including the TNFR portion (22 disulfide bonds) of two etanercept products (Enbrel ® and TuNEX ® ) [65].…”

Section: Analytical Methods For Fc‐fusion Proteins Characterizationmentioning

confidence: 99%

“…Based on these studies, peptide mapping combined with multiple collisional dissociation techniques is a suitable tool for the comparison of biosimilar and innovator product glycosylation heterogeneities. In addition, Huang et al reported how multiple fragmentations including CID and ETD can be implemented to characterize disulfide linkages, including the TNFR portion (22 disulfide bonds) of two etanercept products (Enbrel R and TuNEX R ) [65].…”

Section: Confirmation Of Primary Structure By Ms Analysismentioning

confidence: 99%

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Therapeutic Fc‐fusion proteins: Current analytical strategies

Duivelshof

Murisier

Camperi

et al. 2020

J of Separation Science

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Fc-Fusion proteins represent a successful class of biopharmaceutical products, with already 13 drugs approved in the European Union and United States as well as three biosimilar versions of etanercept. Fc-Fusion products combine tailored pharmacological properties of biological ligands, together with multiple functions of the fragment crystallizable domain of immunoglobulins. There is a great diversity in terms of possible biological ligands, including the extracellular domains of natural receptors, functionally active peptides, recombinant enzymes, and genetically engineered binding constructs acting as cytokine traps. Due to their highly diverse structures, the analytical characterization of Fc-Fusion proteins is far more complex than that of monoclonal antibodies and requires the use and development of additional product-specific methods over conventional generic/platform methods. This can be explained, for example, by the presence of numerous sialic acids, leading to high diversity in terms of iso-

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Section: Analytical Methods For Fc‐fusion Proteins Characterizationmentioning

confidence: 99%

Section: Confirmation Of Primary Structure By Ms Analysismentioning

confidence: 99%

Therapeutic Fc‐fusion proteins: Current analytical strategies

Duivelshof

Murisier

Camperi

et al. 2020

J of Separation Science

View full text Add to dashboard Cite

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“…Although etanercept has only two-thirds of the molecular weight of IgG1 monoclonal antibody (100 kDa), it has 26 disulfide bonds, three sites of N-glycosylation and 13 sites of Oglycosylation. 23 HPLC coupled to an Orbitrap mass analyzer was used for peptide mapping analysis. This study resulted in excellent sequence coverage and successful identification of the PTMs of three etanercept analogues.…”

Section: Introductionmentioning

confidence: 99%

“…The complex structure of the etanercept used in this study brings great challenges to quality evaluation. Although etanercept has only two‐thirds of the molecular weight of IgG1 monoclonal antibody (100 kDa), it has 26 disulfide bonds, three sites of N‐glycosylation and 13 sites of O‐glycosylation 23 . HPLC coupled to an Orbitrap mass analyzer was used for peptide mapping analysis.…”

Section: Introductionmentioning

confidence: 99%

Integrating ultra‐high‐performance liquid chromatography tandem mass spectrometry and imaged capillary isoelectric focusing for in‐depth characterization of complex fusion proteins

Fan

Zhang

et al. 2023

Rapid Comm Mass Spectrometry

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Rationale Fc‐fusion proteins represent a successful class of biopharmaceutical products, which combine the tailored pharmacological properties of biological ligands with the multiple functions of the fragment crystallizable domain of immunoglobulins. There is great diversity in terms of possible biological ligands creating highly diverse structures, therefore the analytical characterization of fusion proteins is far more complex than that of monoclonal antibodies and requires the use and development of additional product‐specific methods over conventional generic/platform methods. Methods Employing etanercept analogues as studied fusion proteins, the Orbitrap mass analyzer with ultra‐high performance liquid chromatography (UHPLC–MS) and imaged capillary isoelectric focusing (icIEF) were utilized for the in‐depth fusion protein characterization. Results The amino acid sequence coverage, peptide mapping, and post‐translational modifications of etanercept analogues were analyzed by UHPLC–MS. The post‐translational modification results were complemented by imaged capillary isoelectric focusing to produce quality research on etanercept analogues. Conclusions The developed workflow integrating UHPLC–MS and icIEF provided an innovative strategy for characterizing complex fusion proteins in the process of quality control and manufacturing.

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“…These methods have been used as a standalone MS/MS technique or combined with CID to improve sequence coverage. Multifragmentation approaches including CID and ETD have been applied to achieve complete mapping of rather complicated disulfide linkages of etanercept products . These mapping processes often require intensive labor for manual confirmation.…”

Section: Introductionmentioning

confidence: 99%

Comprehensive Workflow for Mapping Disulfide Linkages Including Free Thiols and Error Checking by On-Line UV-Induced Precolumn Reduction and Spiked Control

Kuo

Wei

et al. 2020

Anal. Chem.

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Mapping highly complicated disulfide linkages and free thiols via liquid chromatography–tandem mass spectrometry (LC–MS2) is challenging because of the difficulties in optimizing sample preparation to acquire critical MS data and detecting mispairings. Herein, we report a highly efficient and comprehensive workflow using an on-line UV-induced precolumn reduction tandem mass spectrometry (UV-LC–MS2) coupled with two-stage data analysis and spiked control. UV-LC–MS2 features a gradient run of acetonitrile containing a tunable percentage of photoinitiators (acetone/alcohol) that drives the sample to the MS through a UV-flow cell and reverse phase column to separate UV-induced products for subsequent fragmentation via low energy collision-induced dissociation. This allowed the alkylated thiol-containing and UV-reduced cysteine-containing peptides to be identified by a nontargeted database search. Expected or unexpected disulfide/thiol mapping was then carried out based on the search results, and data were derived from partially reduced species by photochemical reaction. Complete assignments of native and scrambled disulfide linkages of insulin, α-lactalbumin, and bovine serum albumin (BSA) as well as the free C34-BSA were demonstrated using none or single enzyme digestion. This workflow was applied to characterize unknown disulfide/thiol patterns of the recombinant cyclophilin 1 monomer (rTvCyP1 mono) from the human pathogen Trichomonas vaginalis. α-Lactalbumin was judiciously chosen as a spiked control to minimize mispairings due to sample preparation. rTvCyP1 was determined to contain a high percentage of thiol (>80%). The rest of rTvCyP1 mono were identified to contain two disulfide/thiol patterns, of which C41–C169 linkage was confirmed to exist as C53–C181 in rTvCyP2, a homologue of rTvCyP1. This platform identifies heterogeneous protein disulfide/thiol patterns in a de-novo fashion with artifact control, opening up an opportunity to characterize crude proteins for many applications.

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Complete mapping of disulfide linkages for etanercept products by multi-enzyme digestion coupled with LC-MS/MS using multi-fragmentations including CID and ETD

Cited by 12 publications

References 29 publications

Therapeutic Fc‐fusion proteins: Current analytical strategies

Therapeutic Fc‐fusion proteins: Current analytical strategies

Integrating ultra‐high‐performance liquid chromatography tandem mass spectrometry and imaged capillary isoelectric focusing for in‐depth characterization of complex fusion proteins

Comprehensive Workflow for Mapping Disulfide Linkages Including Free Thiols and Error Checking by On-Line UV-Induced Precolumn Reduction and Spiked Control

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