Antibody characterization using novel ERLIC-MS/MS-based peptide mapping

ABSTRACT

Electrostatic repulsion hydrophilic interaction chromatography (ERLIC) coupled with mass spectrometry (MS) is a technique that is increasingly being used as a trapping/enrichment tool for glycopeptides/phosphorylated peptides or sample fractionation in proteomics research. Here, we describe a novel ERLIC-MS/MS-based peptide mapping method that was successfully used for the characterization of denosumab, in particular the analysis of sequence coverage, terminal peptides, methionine oxidation, asparagine deamidation and glycopeptides. Compared to reversed phase liquid chromatography (RPLC)-MS/MS methods, ERLIC demonstrated unique advantages in the retention of small peptides, resulting in 100% sequence coverage for both the light and heavy chains. It also demonstrated superior performance in the separation and characterization of asparagine deamidated peptides, which is known to be challenging by RPLC-MS/MS. The developed method can be used alone for peptide mapping-based characterization of monoclonal antibodies, or as an orthogonal method to complement the RPLC-MS/MS method. This study extends the applications of ERLIC from that of a trapping/fractioning column to biologic therapeutics characterization. The ERLIC-MS/MS method can enhance biologic therapeutics analysis with more reliability and confidence for bottom-up peptide mapping-based characterization.

Keywords:

• ERLIC
• antibody
• peptide mapping
• characterization
• deamidation
• methionine oxidation

Acknowledgments

We gratefully thank the advice from Amos Heckendorf (The Nest Group, Inc) and Andrew Alpert (PolyLC, Inc) on column selection and ERLIC background information. Jing Zhen also sincerely thanks Jacob Bongers for the internship training on mass spectrometry-based biologics characterization.

Supplemental material

Supplemental data for this article can be accessed here.

Abbreviations

CAN	=	Acetonitrile
CQA	=	Critical quality attributes
ERLIC-MS	=	Electrostatic repulsion hydrophilic interaction chromatography-mass spectrometry
LERLIC	=	Long-length electrostatic repulsion hydrophilic interaction chromatography
HILIC	=	Hydrophilic interaction chromatography
RPLC-MS	=	Reverse phase liquid chromatography – mass spectrometry