title: Double-peak elution profile of a monoclonal antibody in cation exchange chromatography is caused by histidine-protonation-based charge variants.
abstract: We have systemically investigated unusual elution behaviors of an IgG4 (mAb A) in cation exchange chromatography (CEX). This mAb A exhibited two elution peaks under certain conditions when being purified by several strong CEX columns. When either of the two peaks was isolated and re-injected on the same column, the similar pattern was observed again during elution. The protein distribution between the two peaks could be altered by NaCl concentration in the feed, or NaCl concentration in wash buffer, or elution pH, suggesting two pH-associated strong-and-weak binding configurations. The protein distributions under different pH values showed good correlation with protonated/un-protonated fractions of a histidine residue. These results suggest that the double-peak elution profile associates with histidine-protonation-based charge variants. By conducting pepsin digestion, amino-acid specific chemical modifications, peptide mapping, and measuring the effects of elution residence time, a histidine in the variable fragment (Fab) was identified to be the root cause. Besides double-peak pattern, mAb A can also exhibit peak-shouldering or single elution peak on different CEX resins, reflecting different resins' resolving capability on protonated/un-protonated forms. This work characterizes a novel cause for unusual elution behaviors in CEX and also provides alternative avenues of purification development for mAbs with similar behaviors.
authors.affiliation: Purification Process Sciences, Medimmune LLC, One Medimmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Haibin
authors.initials: H
authors.lastname: Luo
authors.affiliation: Analytical Biochemistry Development, Medimmune LLC, One Medimmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Mingyan
authors.initials: M
authors.lastname: Cao
authors.affiliation: Purification Process Sciences, Medimmune LLC, One Medimmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Kelcy
authors.initials: K
authors.lastname: Newell
authors.affiliation: Purification Process Sciences, Medimmune LLC, One Medimmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Afdahl
authors.affiliation: Analytical Biochemistry Development, Medimmune LLC, One Medimmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Jihong
authors.initials: J
authors.lastname: Wang
authors.affiliation: Purification Process Sciences, Medimmune LLC, One Medimmune Way, Gaithersburg, MD 20878, USA.
authors.forename: William K
authors.initials: WK
authors.lastname: Wang
authors.affiliation: Purification Process Sciences, Medimmune LLC, One Medimmune Way, Gaithersburg, MD 20878, USA. Electronic address: Liyu@Medimmune.com.
authors.forename: Yuling
authors.initials: Y
authors.lastname: Li
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis*; D002021:Buffers; D002411:Cation Exchange Resins; D002852:Chromatography, Ion Exchange / Q000379:methods; D006639:Histidine / Q000737:chemistry*; D007074:Immunoglobulin G / Q000032:analysis*; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D010449:Peptide Mapping
chemical_list: D000911:Antibodies, Monoclonal; D002021:Buffers; D002411:Cation Exchange Resins; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D006639:Histidine
keywords: Cation exchange chromatography (CEX); DEPC; Double-peak; F(ab′)(2); Histidine protonation; Hydroxylamine; Monoclonal antibody (mAb); Peptide mapping