Using hydrogen/deuterium exchange mass spectrometry to study conformational changes in granulocyte colony stimulating factor upon PEGylation.
PEGylation is the covalent attachment of polyethylene glycol to proteins, and it can be used to alter immunogenicity, circulating half life and other properties of therapeutic proteins. To determine the impact of PEGylation on protein conformation, we applied hydrogen/deuterium exchange mass spectrometry (HDX MS) to analyze granulocyte colony stimulating factor (G-CSF) upon PEGylation as a model system. The combined use of HDX automation technology and data analysis software allowed reproducible and robust measurements of the deuterium incorporation levels for peptic peptides of both PEGylated and non-PEGylated G-CSF. The results indicated that significant differences in deuterium incorporation were induced by PEGylation of G-CSF, although the overall changes observed were quite small. PEGylation did not result in gross conformational rearrangement of G-CSF. The data complexity often encountered in HDX MS measurements was greatly reduced through a data processing and presentation format designed to facilitate the comparison process. This study demonstrates the practical utility of HDX MS for comparability studies, process monitoring, and protein therapeutic characterization in the biopharmaceutical industry.
Department of Bioanalytical and Discovery Analytical Sciences, Bristol-Myers Squibb, Princeton, NJ, USA. hui.wei@bms.com
Hui
H
Wei
Joomi
J
Ahn
Ying Qing
YQ
Yu
Adrienne
A
Tymiak
John R
JR
Engen
Guodong
G
Chen
D000595:Amino Acid Sequence; D041961:Deuterium Exchange Measurement / Q000379:methods*; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D010446:Peptide Fragments; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D011092:Polyethylene Glycols; C423652:pegylated granulocyte colony-stimulating factor