title: Effect of the conformational stability of the CH2 domain on the aggregation and peptide cleavage of a humanized IgG.
abstract: To examine the effect of the conformational stability of the CH2 domain on aggregation and peptide cleavage of a humanized IgG1, we carried out size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses of incubated sample solutions. By comparing the residual percentage of monomer after incubation at 60 and 80°C at various pH levels, we found that aggregation and peptide cleavage of the humanized IgG1 occurred during long incubation at 60°C under acidic conditions. Next, we confirmed cleavage of the Asp272-Pro273 peptide bond in the CH2 domain. Comparison of the cleavage rates of the IgG1 monomer and a peptide containing the same Asp-Pro sequence revealed that the conformational stability of the CH2 domain retards cleavage of the Asp272-Pro273 peptide bond at 60°C and pH 4.0. The finding of aggregation and peptide cleavage of the humanized IgG1 after long incubation at 60°C under acidic conditions was supported by another finding: there were lower unfolding temperatures of the CH2 domain at pH 4.0 and 5.0. We conclude that the conformational stability of the CH2 domain is closely related to aggregation and peptide cleavage of the humanized IgG1 under acidic conditions. We also found that the 2-[N-morpholino] ethane sulfonate buffer inhibits aggregation of the IgG1 at pH 4.0-5.0 and 7.0-8.0.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Kyushu University, 3-3-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kameoka
authors.forename: Tadashi
authors.initials: T
authors.lastname: Ueda
authors.forename: Taiji
authors.initials: T
authors.lastname: Imoto
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002021:Buffers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D055503:Protein Multimerization; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D012996:Solutions; D013696:Temperature
chemical_list: D002021:Buffers; D007074:Immunoglobulin G; D010446:Peptide Fragments; D012996:Solutions