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Protein Binder for Affinity Purification of Human Immunoglobulin Antibodies
journal contribution
posted on 2014-06-17, 00:00 authored by Woosung Heu, Jung-Min Choi, Joong-Jae Lee, Sukyo Jeong, Hak-Sung KimThe importance of a downstream process
for the purification of
immunoglobulin antibodies is increasing with the growing application
of monoclonal antibodies in many different areas. Although protein
A is most commonly used for the affinity purification of antibodies,
certain properties could be further improved: higher stability in
alkaline solution and milder elution condition. Herein, we present
the development of Fc-specific repebody by modular engineering approach
and its potential as an affinity ligand for purification of human
immunoglobulin antibodies. We previously developed the repebody scaffold
composed of Leucine-rich repeat (LRR) modules. The scaffold was shown
to be highly stable over a wide range of pH and temperature, exhibiting
a modular architecture. We first selected a repebody that binds the
Fc fragment of human immunoglobulin G (IgG) through a phage display
and increased its binding affinity up to 1.9 × 10–7 M in a module-by-module approach. The utility of the Fc-specific
repebody was demonstrated by the performance of an immobilized repebody
in affinity purification of antibodies from a mammalian cell-cultured
medium. Bound-antibodies on an immobilized repebody were shown to
be eluted at pH 4.0 with high purity (>94.6%) and recovery yield
(>95.7%).
The immobilized repebody allowed a repetitive purification process
more than ten times without any loss of binding capability. The repebody
remained almost intact even after incubation with 0.5 M NaOH for 15
days. The present approach could be effectively used for developing
a repeat module-based binder for other target molecules for affinity
purification.