Published:Journal of Chromatographic Science,
ISSN 0021-9665Volume
40, Number 6, July 2002, pp. 343-349
Epitope Affinity Chromatography and Biophysical Studies of Monoclonal
Antibodies and Recombinant Antibody Fragments
A. Murray[1], S. Missailidis[3],
and M.R. Price[2] [1]Neuroscience Research Group, School of Biomedical
Sciences, University of Nottingham Medical School, Queen’s Medical Centre,
Nottingham NG7 [2]UH, U.K., 2Cancer Research Laboratories, School
of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, U.K., [3]Chemistry Department, The Open University, Walton
Hall, Milton Keynes MK7 6AA, U.K.
Peptide epitope affinity chromatography is a powerful technique
for the purification of antibodies. This study aims to demonstrate the versatility
of the technique and to show how biophysical techniques such as circular dichroism
(CD) and fluorescence quenching (FQ) can aid the rational design of affinity
ligands and characterization of antibody-based reagents. The performance of
a number of peptide ligands for the purification of a range of different antibodies
and recombinant fragments is investigated by automated fast-protein liquid chromatography.
Purified products are analyzed for purity by sodium dodecyl sulfate–polyacrylamide
gel electrophoresis. They are then radiolabelled and the immunoreactivity is
determined. Ligands are analyzed for secondary structural characteristics by
CD and for binding affinity by FQ. Finally, a study is performed to investigate
the thermal stability of a recombinant antibody fragment by CD analysis. It
is found that simple ligand modifications such as the introduction of a C-terminal
cysteine residue can improve purification performance. The FQ studies show that
the modified peptide has a higher affinity for antibody. The CD analysis shows
that it has a tendency to dimerize because of the formation of disulfide bonds.
The versatility of epitope affinity is demonstrated through the purification
of a recombinant diabody (dbFv) and by the use of a separate peptide matrix
for the purification of an unrelated antibody. All studies result in antibody
preparations of high purity and immunoreactivity. The CD analysis of the dbFv
shows that it is denatured at 37°C and is therefore unsuitable as a targeting
reagent for use in humans in its present form. It is concluded that epitope
affinity chromatography coupled with biophysical analyses plays an important
role in the production and characterization of antibody-based reagents for targeted
diagnosis and therapy of human diseases.
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