Jl. Casey et al., PURIFICATION OF BACTERIALLY EXPRESSED SINGLE-CHAIN FV ANTIBODIES FOR CLINICAL-APPLICATIONS USING METAL CHELATE CHROMATOGRAPHY, Journal of immunological methods, 179(1), 1995, pp. 105-116
A new procedure is described for the purification of an anti-carcinoem
bryonic antigen (CEA) single chain Fv (scFv), referred to as MFE-23, f
rom bacterial supernatant. A simple insertion of a hexa-histidine tail
fused at the C-terminus (MFE-23 His) provides an affinity tag which s
electively binds to transition metal ions immobilised on an iminodiace
tic acid (IDA) derivitised solid phase matrix. This method proved to b
e superior to standard CEA antigen affinity chromatography in the foll
owing 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 affecte
d by the limited availability (size of the column) of immobilised CEA
antigen. (2) Scale-up was relatively simple and less costly. (3) The r
isk 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 e
xclusion chromatography to remove aggregated material, and Detoxi gel
to remove bacterial endotoxins. Validation assays to measure DNA, copp
er and endotoxins were performed to assess the levels of contaminants.
MFE-23 His retained 84% antigen binding after 6 months storage at 4 d
egrees C and >75% after radiolabelling. Further experiments confirmed
that the His tail did not affect biodistribution and tumour localisati
on in nude mice bearing human colorectal tumour xenografts.