title: Rates and impact of human antibody glycation in vivo.
abstract: Glycation of immunoglobulin G (IgG) can result from incubation with a reducing sugar in vitro or during circulation in vivo. Upon injection of a recombinantly produced human therapeutic IgG into humans, changes in the glycation levels could be observed as a function of circulation time. Mass changes on the individual IgG polypeptide chains as the results of glycation were determined using reversed-phase liquid chromatography/mass spectrometry. Changes to the light and heavy chains were low but easily detectable at 0.00092 and 0.0021 glucose (Glc) additions per chain per day, respectively. Levels of glycation found on the Fc portion of IgG isolated from healthy subjects, using a similar analytical approach, were on average 0.045 Glc molecules per fragment. In vivo glycation rates could be approximated in vitro by modeling the physiological glycation reaction with a simplified incubation containing physiological Glc concentrations, pH and temperature but with a high concentration of a single purified IgG. To test the impact of glycation on IgG function, highly glycated IgG1 and IgG2 were prepared containing on average 42-49 Glc molecules per IgG. Binding to FcγIIIa receptors, neonatal Fc receptor or protein A was similar or identical to the non-glycated IgG controls. Although the modifications were well distributed throughout the protein sequence, and at high enough levels to affect the elution position by size-exclusion chromatography, no changes in the tested Fc functions were observed.
authors.affiliation: Department of Process and Product Development, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Goetze
authors.forename: Y Diana
authors.initials: YD
authors.lastname: Liu
authors.forename: Thomas
authors.initials: T
authors.lastname: Arroll
authors.forename: Lily
authors.initials: L
authors.lastname: Chu
authors.forename: Gregory C
authors.initials: GC
authors.lastname: Flynn
mesh_terms: D056148:Chromatography, Reverse-Phase; D005947:Glucose / Q000737:chemistry*; D006031:Glycosylation; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D013058:Mass Spectrometry; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D011961:Receptors, Fc / Q000737:chemistry / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000378:metabolism; D013696:Temperature
chemical_list: D015395:Histocompatibility Antigens Class I; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011961:Receptors, Fc; D013205:Staphylococcal Protein A; D005947:Glucose; C100688:Fc receptor, neonatal