title: Protein binder for affinity purification of human immunoglobulin antibodies.
abstract: The 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.
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST) , Daejeon 305-701, Korea.
authors.forename: Woosung
authors.initials: W
authors.lastname: Heu
authors.forename: Jung-Min
authors.initials: JM
authors.lastname: Choi
authors.forename: Joong-Jae
authors.initials: JJ
authors.lastname: Lee
authors.forename: Sukyo
authors.initials: S
authors.lastname: Jeong
authors.forename: Hak-Sung
authors.initials: HS
authors.lastname: Kim
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification*; D016466:CHO Cells; D002151:Calorimetry; D002846:Chromatography, Affinity / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D000906:Antibodies; D007074:Immunoglobulin G