The presence of even trace amounts of aggregate in immunoglobulin biopharmaceuticals is of significant concem during all phases of drug development, from process design to quality control. The formation of aggregates can adversely impact process economics by causing decreased product yield, peak broadening (requiring the addition of polishing steps), and loss of activity (Gagnon 1996). Aggregate formation can also adversely affect product safety because it can cause complement activation or even anaphylaxis upon injection (Ritchie and Navolotskaia 1996, WHO Report 1995).
Most chromatographic techniques would be expected to be able to remove aggregated or multimeric species of immunoglobulin from the monomer. However, techniques such as ion exchange and hydrophobic interaction chromatography may also induce the formation of additional aggregate or multimer due to increased protein concentration or changes in salt concentration, pH, or both required for elution. Size exclusion chromatography does not suffer from these disadvantages, but does result in significant dilution of the product. If large quantities of product are required, large size exclusion columns, concentration steps, and support equipment will add considerably to production costs. In some cases, additional product loss due to denaturation may occur during dilution.
CHT Ceramic Hydroxyapatite exhibits electrostatic, repulsive, and coordinate covalent bond formation in interacting with protein species. We have explored its potential in removing aggregate from a purified IgG4 biopharmaceutical known to contain aggregate, and report here on a simple and effective process to do so.