Purification and characterization of a single-chain chimeric anti-p185 antibody expressed by CHO-GS system

Protein Expr Purif. 2005 May;41(1):68-76. doi: 10.1016/j.pep.2004.11.007.

Abstract

Monoclonal antibody A21 reacts specifically with the extracellular domain of p185c-erbB-2 oncoprotein, a member of the epidermal growth factor receptor family. In a previous study, we constructed a single-chain chimeric antibody, assembled using an A21 single-chain Fv antibody and a human IgG1 Fc fragment. In this study, we expressed this chimeric antibody using a CHO-GS system, and developed a simple and efficient method for its purification. After only one step using affinity purification, the recovery rate and purity of the antibody attained was 60 and 91%, respectively. After a second step, using reverse phase HPLC purification, the purity was above 99%. The high purity of the recombinant antibody allowed us to identify a number of its intrinsic molecular properties, including antigen binding activity, measurement of affinity constant, N-terminal sequencing, and mass spectrometer analysis. These results further augment the potential of this recombinant antibody to be a drug candidate for cancer therapy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal / chemistry
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / isolation & purification*
  • Antibodies, Monoclonal / metabolism
  • Antibody Affinity
  • Antibody-Dependent Cell Cytotoxicity
  • Binding, Competitive
  • CHO Cells
  • Chromatography, Affinity
  • Chromatography, High Pressure Liquid
  • Cricetinae
  • Gene Expression
  • Humans
  • In Vitro Techniques
  • Mice
  • Molecular Sequence Data
  • Neoplasms / therapy
  • Receptor, ErbB-2 / immunology*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Transfection

Substances

  • Antibodies, Monoclonal
  • Recombinant Fusion Proteins
  • Receptor, ErbB-2