Affinity chromatography with immobilized protein A from Staphylococcus aureus or from recombinant sources has been used for the purification of immunoglobulins since the 1970s (Gagnon 1996). Much of its popularity is due to the simplicity of the method itself, the high product purity and recovery, and protein A's selectivity to IgG class and species. Good clearance of DNA, virus, and endotoxin is often obtained, plus the immobilized protein A is relatively resistant to chemical regeneration and sanitization procedures.
Despite these advantages, conditioned media and cellular lysates frequently contain varying amounts of proteases, which can release fragments of protein A from immobilizing supports (Bloom et al. 1989). This is of great concern to the diagnostics and biopharmaceutical industries due to the wide range of immunomodulatory effects associated with protein A and its fragments (Gagnon 1996).
A number of chromatographic methods have been used to separate protein A from IgG. Cation exchange chromatography is attractive because conditions can be devised in which protein A does not bind to the support while IgG is retained. However, process development is often required and chaotropic agents may be needed to disrupt the protein A-IgG complex. Anion exchange chromatography has been used, but suffers from the disadvantage that the strength of the protein A-IgG association is enhanced by elevated pH. Since both IgG and protein A are retained, coelution of protein A fragments with IgG can occur (Bloom et al. 1989). Use of hydrophobic interaction chromatography for protein A-IgG separation has also been reported. Process development is again required since high salt concentrations increase the affinity of protein A for IgG. Coelution of protein A and IgG is common, reducing contaminant clearance and product yield.
Mariani et al. (1989) developed a simple method for the purification of IgG1 in the presence of protein A on hydroxyapatite (Bio-Rad HPHT). They started with an ascites fluid ammonium sulfate fraction. In recent years, HPHT has been replaced by the more robust CHT ceramic hydroxyapatite, which was designed for process-scale separations. In this report, we show that CHT can resolve IgG1 from an IgG1-protein A complex in unfractionated conditioned media. We have also optimized conditions for the separation of protein A and an IgG4 by cation exchange chromatography on UNOsphere™ S support.