Purification and characterization of a single-chain chimeric anti-p185 antibody expressed by CHO-GS system.
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.
Wang
Jing
J
School of Life Science, University of Science and Technology of China, Hefei, Anhui 230027, China.
Shi
Yu
Y
Liu
Yanqi
Y
Hu
Siyi
S
Ma
Jun
J
Liu
Jing
J
Cheng
Liansheng
L
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000915:Antibody Affinity; D000920:Antibody-Dependent Cell Cytotoxicity; D001667:Binding, Competitive; D016466:CHO Cells; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000628:therapy; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
D000911:Antibodies, Monoclonal; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins