Significant Improvements in the Performance of an Established Affinity Chromatography Procedure Employed to Purify a Monoclonal Antibody in 100 Purification Cycles
Abstract Protein A-Sepharose affinity chromatography is a very successful method for the purification of immunoglobulins for pharmaceutical use. However, the chromatography efficiency and lifetime of this method have to be always adjusted to specific chromatography conditions (biological source, buffers, flow rates, antibody properties, temperature, protein concentration, cleaning protocol, etc). This study sought to demonstrate improvements in the performance of an established affinity chromatography procedure employed to purify the CB.Hep-1 monoclonal antibody (mAb) used in the purification of the active pharmaceutical ingredient of a Hepatitis B vaccine. In conclusion, the relative poor mAb recovery observed in 150 mM PBS; pH 8.0/100 mM citric acid; pH 3.0 buffer system conditions was attributed to the inefficacy of the elution buffer to disrupt completely interactions between the matrix and mAb. In this regard, retention of the CB.Hep-1 mAb into the matrix was helped by the ligand coupled to the matrix and not by unspecific interactions. The 1.5M glycine-NaOH/3M NaCl; pH 9.0/200 mM glycine-HCl; pH 2.5 buffer system significantly improved the affinity chromatography recovery without affecting mAb purity, molecular homogeneity, ligand leakage and mouse DNA content in 100 purification cycles. Thus, application of 1.5M glycine-NaOH/3M NaCl; pH 9.0/200 mM glycine-HCl; pH 2.5 as buffer system allowed the reduction of the CB.Hep-1 mAb and Hepatitis B vaccine costs, respectively.