title: The LC/MS analysis of glycation of IgG molecules in sucrose containing formulations.
abstract: Glycation of a recombinant monoclonal IgG2 molecule, in sucrose containing liquid formulations, was studied using reversed-phase LC/MS analysis of the intact IgG, the F(ab')2 fragments and after complete tryptic digestion. The extent of glycation in sucrose containing formulations was monitored at different temperatures over a period of 21 months using the Hexose index (Hex(I)). Hex(I) represents the average number of hexose molecules per molecule of IgG and was calculated by using the intensity values of peaks corresponding to hexose isoforms in the deconvoluted mass spectra. The rate of glycation in mildly acidic sucrose containing formulations was proportional to the incubation temperature. No glycation was observed in sucrose containing formulations incubated at 4 degrees C even after 18 months. However, when the same formulations were incubated at 37 degrees C glycation was observed after just 1 month. The glycation sites were mapped to 10 lysine residues distributed throughout the molecule. The amino terminal end of the light chain was also shown to contain glycation. The surface accessibility of the lysine side chain could influence its susceptibility to glycation.
authors.affiliation: Department of Pharmaceutics, Amgen Inc., 1201 Amgen Court West, Seattle, Washington 99119-3105, USA. hgadgil@amgen.com
authors.forename: Himanshu S
authors.initials: HS
authors.lastname: Gadgil
authors.forename: Pavel V
authors.initials: PV
authors.lastname: Bondarenko
authors.forename: Gary
authors.initials: G
authors.lastname: Pipes
authors.forename: Doug
authors.initials: D
authors.lastname: Rehder
authors.forename: Arnold
authors.initials: A
authors.lastname: McAuley
authors.forename: Natalie
authors.initials: N
authors.lastname: Perico
authors.forename: Tom
authors.initials: T
authors.lastname: Dillon
authors.forename: Margaret
authors.initials: M
authors.lastname: Ricci
authors.forename: Michael
authors.initials: M
authors.lastname: Treuheit
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002626:Chemistry, Pharmaceutical; D002853:Chromatography, Liquid; D005079:Excipients / Q000737:chemistry*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D010449:Peptide Mapping; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013395:Sucrose / Q000737:chemistry*; D013678:Technology, Pharmaceutical / Q000379:methods*; D013696:Temperature; D013997:Time Factors; D014357:Trypsin
chemical_list: D000911:Antibodies, Monoclonal; D005079:Excipients; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D013395:Sucrose; D014357:Trypsin; D008239:Lysine