Purification of bacterially expressed single chain Fv antibodies for clinical applications using metal chelate chromatography.
A new procedure is described for the purification of an anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), referred to as MFE-23, from bacterial supernatant. A simple insertion of a hexa-histidine tail fused at the C-terminus (MFE-23 His) provides an affinity tag which selectively binds to transition metal ions immobilised on an iminodiacetic acid (IDA) derivitised solid phase matrix. This method proved to be superior to standard CEA antigen affinity chromatography in the following ways. (1) A higher yield was produced (10 mg/l as opposed to 2.2 mg/l of bacterial supernatant). The latter figure was largely affected by the limited availability (size of the column) of immobilised CEA antigen. (2) Scale-up was relatively simple and less costly. (3) The risk of tumour derived antigen leaching from the column is eliminated. Results showed that immobilised Cu2+ ions were more effective than Ni2+ and Zn2+ ions in retaining the His tagged product giving a 90% pure product on elution. Clinical grade material was generated using size exclusion chromatography to remove aggregated material, and Detoxi gel to remove bacterial endotoxins. Validation assays to measure DNA, copper and endotoxins were performed to assess the levels of contaminants. MFE-23 His retained 84% antigen binding after 6 months storage at 4 degrees C and > 75% after radiolabelling. Further experiments confirmed that the His tail did not affect biodistribution and tumour localisation in nude mice bearing human colorectal tumour xenografts.
Casey
J L
JL
Department of Clinical Oncology, Royal Free Hospital Medical School, London, UK.
Keep
P A
PA
Chester
K A
KA
Robson
L
L
Hawkins
R E
RE
Begent
R H
RH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002614:Chelating Agents; D002846:Chromatography, Affinity / Q000379:methods*; D006639:Histidine / Q000235:genetics; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
D002614:Chelating Agents; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins; D006639:Histidine