title: Characterization of asparagine 330 deamidation in an Fc-fragment of IgG1 using cation exchange chromatography and peptide mapping.
abstract: Deamidation is one of the most common degradation pathways for proteins and frequently occurs at "hot spots" with Asn-Gly, Asn-Ser or Asn-Thr sequences. Occasionally, deamidation may occur at other motifs if the local protein structure can participate or assist in the formation of the succinimide intermediate. Here we report the use of a chymotryptic peptide mapping method to identify and characterize a deamidated form of an IgG1 which was observed as an acidic peak in the cation exchange chromatography (CEX). The antibody was formulated in sodium acetate buffer, pH 5.3 and this deamidated form was observed mainly under thermal stress conditions. It was found that the IgG1 molecule with deamidation in the Fc region at asparagine residue 330 (in a Val-Ser-Asn-Lys motif) is the predominant form in this CEX peak, and was missed by tryptic mapping because the peptides are hydrophilic and elute near the void volume. In addition, a domain-based CEX method using papain digestion was developed to monitor the Asn 330 deamidation. These methods revealed that the Fc deamidation occurs mainly at Asn 330 in the VSNK motif at pH 5.3, whereas at pH 7.5, deamidation occurs predominantly at Asn 389 and Asn 394 in the NGQPENNYK motif.
authors.affiliation: Department of Protein Analytical Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA. Electronic address: yonghuaz@gene.com.
authors.forename: Yonghua Taylor
authors.initials: YT
authors.lastname: Zhang
authors.affiliation: Department of Protein Analytical Chemistry, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hu
authors.affiliation: Late Stage Pharmaceutical Development, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Pace
authors.affiliation: Late Stage Pharmaceutical Development, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Rita
authors.initials: R
authors.lastname: Wong
authors.affiliation: Late Stage Pharmaceutical Development, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Y John
authors.initials: YJ
authors.lastname: Wang
authors.affiliation: Late Stage Pharmaceutical Development, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Yung-Hsiang
authors.initials: YH
authors.lastname: Kao
mesh_terms: D000577:Amides / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D001216:Asparagine / Q000737:chemistry* / Q000378:metabolism; D016466:CHO Cells; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D006863:Hydrogen-Ion Concentration; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D055550:Protein Stability
chemical_list: D000577:Amides; D000911:Antibodies, Monoclonal; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D001216:Asparagine
keywords: Cation exchange chromatography; Deamidation; Digestion strategies; IgG; Peptide mapping