Application of Dual Protease Column for HDX-MS Analysis of Monoclonal Antibodies

A co-immobilized, dual protease column was developed and implemented to more efficiently digest IgG molecules for hydrogen/deuterium exchange mass spectrometry (HDX-MS). The low-pH proteolytic enzymes pepsin and type XIII protease from Aspergillus were packed into a single column to most effectively...

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Bibliographic Details
Published in:Journal of pharmaceutical sciences 2017-02, Vol.106 (2), p.530-536
Main Authors: Nirudodhi, Sasidhar N., Sperry, Justin B., Rouse, Jason C., Carroll, James A.
Format: Article
Language:English
Subjects:
analytical chemistry
Animals
Antibodies, Monoclonal - chemistry
Antibodies, Monoclonal - metabolism
Aspergillus - enzymology
biotechnology
CHO Cells
Chromatography, High Pressure Liquid - methods
Cricetulus
Deuterium Exchange Measurement
Enzymes, Immobilized - metabolism
HPLC
Immunoglobulin G - chemistry
Immunoglobulin G - metabolism
mass spectrometry
Mass Spectrometry - methods
Peptide Hydrolases - metabolism
protein structure
Proteolysis
Sequence Analysis, Protein - methods
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Summary:A co-immobilized, dual protease column was developed and implemented to more efficiently digest IgG molecules for hydrogen/deuterium exchange mass spectrometry (HDX-MS). The low-pH proteolytic enzymes pepsin and type XIII protease from Aspergillus were packed into a single column to most effectively combine the complementary specificities. The method was optimized using an IgG2 monoclonal antibody as a substrate because they are known to be more difficult to efficiently digest. The general applicability of the method was then demonstrated using IgG1 and IgG4 mAbs. The dual protease column and optimized method yielded improved digestion efficiency, as measured by the increased number of smaller, overlapping peptides in comparison with pepsin or type XIII alone, making HDX-MS more suitable for measuring deuterium uptake with higher resolution. The enhanced digestion efficiency and increased sequence coverage enables the routine application of HDX-MS to all therapeutic IgG molecules for investigations of higher order structure, especially when posttranslational and storage-induced modifications are detected, providing further product understanding for structure–function relationships and ultimately ensuring clinical safety and efficacy.
ISSN:0022-3549
1520-6017
DOI:10.1016/j.xphs.2016.10.023