Protein Protocols Protein Protocols

Affinity Purification of Monoclonal Antibodies 1121
163
Affinity Purification Techniques for Monoclonal Antibodies
Alexander Schwarz
1. Introduction
Monoclonal antibodies have many applications in biotechnology such as immunoaffinity
chromatography, immunodiagnostics, immunotherapy, drug targeting, biosensors,
and many others. For all these purposes homogeneous antibody preparation
are needed. Affinity chromatography, which relies on the specific interaction between
an immobilized ligand and a particular molecule sought to be purified, is a wellknown
technique to purify proteins from solution. Three different affinity techniques
for the one-step purification of antibodies—protein A, thiophilic adsorption, and
immobilized metal affinity chromatography—are described in this chapter.
1.1. Protein A Chromatography
Protein A is a cell wall component of Staphylococcus aureas. Protein A consists
of a single polypeptide chain in the form of a cylinder, which contains five highly
homologous antibody binding domains. The binding site for protein A is located on the
Fc portion of antibodies of the immunoglobulin G (IgG) class (1). Binding occurs
through an induced hydrophobic fit and is promoted by addition of salts such as sodium
citrate or sodium sulfate. At the center of the Fc binding site as well as on protein A
reside histidine residues. At alkaline pH, these residues are uncharged and hydrophobic,
strengthening the interaction between protein A and the antibody. As the pH is
shifted to acidic values, these residues become charged and repel each other. Differences
in the pH-dependent elution properties (Table 1) are seen between antibodies
from different classes as well as different species due to minor differences in the binding
sites. These differences can be successfully exploited in the separation of contaminating
bovine IgG from mouse IgG, as shown in Subheading 1.3.
The major attraction in using protein A is its simplicity. The supernatant is adsorbed
onto a protein A gel, the gel is washed, and the antibody is eluted at an acidic pH. The
antibody recovered has a purity of >90%, often with full recovery of biological activity.
The method described in Subheading 3.1. provides a more detailed description of
the purification for high-affinity antibodies.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
1121