Identification of multiple sources of charge heterogeneity in a recombinant antibody

J Chromatogr B Biomed Sci Appl. 2001 Mar 10;752(2):233-45. doi: 10.1016/s0378-4347(00)00548-x.

Abstract

Seven forms of a therapeutic recombinant antibody that binds to the her2/neu gene product were resolved by cation-exchange chromatography. Structural differences were assigned by peptide mapping and HIC after papain digestion. Deamidation of light chain asparagine 30 to aspartate in one or both light chains is responsible for two acidic forms. A low potency form is due to isomerization of heavy chain aspartate 102; the Asp102 succinimide is also present in a basic peak fraction. Forms with both Asn30 deamidation and Asp102 isomerization modifications were isolated. Deamidation of heavy chain Asn55 to isoaspartate was also detected. Isoelectric focusing in a polyacrylamide gel was used to verify the assignments. All modifications were found in complementarity determining regions.

MeSH terms

  • Amino Acid Sequence
  • Antibodies / chemistry*
  • Antibodies / immunology
  • Chromatography, High Pressure Liquid
  • Chromatography, Ion Exchange
  • Isoelectric Focusing
  • Molecular Sequence Data
  • Peptide Mapping
  • Receptor, ErbB-2 / immunology*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / immunology
  • Trypsin / chemistry

Substances

  • Antibodies
  • Recombinant Proteins
  • Receptor, ErbB-2
  • Trypsin