title: Separation of post-translational modifications in monoclonal antibodies by exploiting subtle conformational changes under mildly acidic conditions.
abstract: Chromatographic separation plays a key role in the identification, quantification, and characterization of protein variants. Here we describe separation of species containing two post-translational modifications (glycosylation and methionine oxidation) in the Fc fragment of a monoclonal antibody. The method is based on cation-exchange chromatography under mildly acidic conditions that destabilize mainly the CH2 domain. Our data suggest that the separation is not mediated by the chemical modification itself, but rather by subtle structural changes induced by the chemical modification in the domain-decoupled conformation that monoclonal antibodies adopt around pH 4. Compared to other procedures already described in the literature, this method demonstrates an improved separation and allows purification of species in the native fold for additional functional characterization. This approach of separation under conditions where the protein assumes an alternative conformation could find a more general utility for the separation of chemical modifications in proteins.
authors.affiliation: Merck Research Laboratories, West Point, PA 19486, USA.
authors.forename: Shiyi
authors.initials: S
authors.lastname: Wang
authors.forename: Roxana
authors.initials: R
authors.lastname: Ionescu
authors.forename: Norbert
authors.initials: N
authors.lastname: Peekhaus
authors.forename: Jin-Yu
authors.initials: JY
authors.lastname: Leung
authors.forename: Sha
authors.initials: S
authors.lastname: Ha
authors.forename: Josef
authors.initials: J
authors.lastname: Vlasak
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D002942:Circular Dichroism; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011487:Protein Conformation; D011489:Protein Denaturation; D011499:Protein Processing, Post-Translational; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
chemical_list: D000911:Antibodies, Monoclonal; D008715:Methionine