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09:47
Analysis of Histone Antibody Specificity with Peptide Microarrays
Published on: August 1, 2017
41.8K
Antibody characterization using novel ERLIC-MS/MS-based peptide mapping
Jing Zhen1, John Kim1, Ying Zhou1
1a Department of Analytical Sciences and Operations , Biologics CMC, Teva Pharmaceutical Industries , West Chester , PA , USA.
Mabs
|August 22, 2018
Summary
Electrostatic repulsion hydrophilic interaction chromatography (ERLIC) coupled with mass spectrometry (MS) offers a novel peptide mapping method for biologic therapeutics. This technique provides superior sequence coverage and characterization of challenging modifications like asparagine deamidation.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Proteomics
Background:
- Electrostatic repulsion hydrophilic interaction chromatography (ERLIC) coupled with mass spectrometry (MS) is gaining traction in proteomics.
- ERLIC is utilized for glycopeptide/phosphorylated peptide enrichment and sample fractionation.
Purpose of the Study:
- To introduce a novel ERLIC-MS/MS peptide mapping method for biologic therapeutics characterization.
- To evaluate ERLIC's performance against traditional reversed-phase liquid chromatography (RPLC)-MS/MS.
Main Methods:
- Development and application of an ERLIC-MS/MS based peptide mapping strategy.
- Characterization of denosumab, focusing on sequence coverage, terminal peptides, methionine oxidation, asparagine deamidation, and glycopeptides.
Main Results:
- Achieved 100% sequence coverage for denosumab light and heavy chains, outperforming RPLC-MS/MS for small peptides.
- Demonstrated superior separation and characterization of asparagine deamidated peptides, a known RPLC-MS/MS challenge.
- Successfully extended ERLIC applications from sample preparation to direct characterization of biologic therapeutics.
Conclusions:
- The developed ERLIC-MS/MS method is effective for denosumab characterization, offering complete sequence coverage.
- ERLIC-MS/MS serves as a valuable standalone or orthogonal method to RPLC-MS/MS for monoclonal antibody characterization.
- This approach enhances the reliability and confidence in bottom-up peptide mapping for biologic therapeutics analysis.
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