( Purification of Murine IgG1 Using UNOs...)Purification of Murine IgG1 Using UNOsphere S and CHT ,,, Ceramic Hydroxyapatite Chromatography, Rev A

Purification of Murine IgG1 Using UNOsphere S and CHT Ceramic Hydroxyapatite Chromatography, Rev A

Henry Lai, PhD, and Samuel G Franklin, PhD, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547 USA

Introduction
Immunoglobulins (IgGs) from a variety of sources have been purified using the conventional process composed of a protein A chromatography step along with ion exchange chromatography and hydrophobic interaction chromatography steps (Jiskoot et al. 1989, Godfrey et al. 1993, Shadle et al. 1995, Ford et al. 2001). Protein A chromatography is normally applied as the first step in the process, yielding antibodies with very high purity in a single step. However, the disadvantages of protein A chromatography are: 1) antibody isomers are not well separated from different species; 2) protein A leakage requires additional chromatographic steps for protein A removal; and 3) protein A resins are substantially more expensive than ion exchange and ceramic hydroxyapatite supports.

Here we present an alternative process for antibody purification consisting of UNOsphere S chromatography and CHT ceramic hydroxyapatite chromatography. This process is simpler than the process utilizing protein A and avoids the leakage of a contaminating ligand (Jiskoot et al. 1989).

UNOsphere S support is a strong cation exchanger made from acrylamido and vinylic copolymers. It exhibits high protein binding capacity and low column backpressure at high linear velocity. Thus, it is a suitable capture resin in the downstream purification process. CHT ceramic hydroxyapatite support, with Ca²⁺ ions and PO₄
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