title: HIC resolution of an IgG1 with an oxidized Trp in a complementarity determining region.
abstract: Susceptibility of tryptophan (Trp) in a complementarity-determining region (CDR) to oxidation is a significant issue for recombinant monoclonal antibody (mAb) therapeutics due to the clinical efficacy and stability concerns. Here we present a case study using hydrophobic interaction chromatography (HIC) to separate an oxidized Trp containing population of an IgG1. The best separation was achieved using dual Dionex ProPac HIC-10 columns, and the oxidized Trp population was isolated as a separated pre-peak. Peptide map analysis revealed that the oxidized Trp is located in a heavy chain CDR. In addition, the HIC method was capable of monitoring the oxidation status of the CDR Trp, as the oxidation rate of the CDR Trp measured by HIC directly correlated with the results of the peptide maps. The same method conditions were also capable of separating oxidized methionine (Met) and isomerization/deamidation products, which co-elute as another pre-peak at a different retention time from the oxidized Trp species. These observations indicate that the HIC procedure can be utilized to monitor the oxidative status of the CDR Trp in the IgG1.
authors: Department of Pharma Technical Development, Genentech, Oceanside, CA 92056, USA.
authors: Daniel
authors: D
authors: Boyd
authors: Timothy
authors: T
authors: Kaschak
authors: Boxu
authors: B
authors: Yan
mesh_terms: D000818:Animals; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D022801:Complementarity Determining Regions / Q000737:chemistry* / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D014357:Trypsin / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
chemical_list: D022801:Complementarity Determining Regions; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D014364:Tryptophan; D008715:Methionine; D014357:Trypsin