Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn.
Susceptibility of methionine residues to oxidation is a significant issue of protein therapeutics. Methionine oxidation may limit the product's clinical efficacy or stability. We have studied kinetics of methionine oxidation in the Fc portion of the human IgG2 and its impact on the interaction with FcRn and Protein A. Our results confirm previously published observations for IgG1 that two analogous solvent-exposed methionine residues in IgG2, Met 252 and Met 428, oxidize more readily than the other methionine residue, Met 358, which is buried inside the Fc. Met 397, which is not present in IgG1 but in IgG2, oxidizes at similar rate as Met 358. Oxidation of two labile methionines, Met 252 and Met 428, weakens the binding of the intact antibody with Protein A and FcRn, two natural protein binding partners. Both of these binding partners share the same binding site on the Fc. Additionally, our results shows that Protein A may serve as a convenient and inexpensive surrogate for FcRn binding measurements.
Pan
Hai
H
Analytical and Formulation Sciences, Amgen Inc., Thousand Oaks, California 91320-1799, USA. hpan@amgen.com
Chen
Kenneth
K
Chu
Liping
L
Kinderman
Francis
F
Apostol
Izydor
I
Huang
Gang
G
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D055767:Immobilized Proteins / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008715:Methionine / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011485:Protein Binding; D011961:Receptors, Fc / Q000378:metabolism*; D016415:Sequence Alignment; D013205:Staphylococcal Protein A / Q000378:metabolism*; D020122:tert-Butylhydroperoxide / Q000737:chemistry
D015395:Histocompatibility Antigens Class I; D055767:Immobilized Proteins; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011961:Receptors, Fc; D013205:Staphylococcal Protein A; D020122:tert-Butylhydroperoxide; D008715:Methionine; C100688:Fc receptor, neonatal