Purification of Mouse IgG1 on Protein A and the Measurement of Contaminating Protein A

Abstract

A new affinity matrix (PROSEP-A High Capacity) has been developed for the purification of immunoglobulin G (IgG). The matrix consists of protein A, covalently bound to controlled pore glass. The binding capacity of PROSEP-A High Capacity for mouse IgGl in various binding buffers of different pH and ionic strength has been determined. The binding capacity was in the region of 10 to 21mg/ml depending on the pH and ionic strength of the binding buffer. The association constants of PROSEP-A and protein A bound to sepharose for mouse IgGl were determined and values of 1.15 x 10⁵M⁻¹) and 0.97 x 10⁵(M⁻¹) were obtained respectively. The equilibration time for binding mouse IgG1 to PROSEP-A High Capacity was determined to be about 20 minutes and a sharp breakthrough curve was observed for the adsorption of mouse IgG1 to PROSEP-A High Capacity. A sensitive enzyme immunoassay was developed to measure the amount of protein A contaminating the purified antibody. The assay has a sensitivity of lng protein A per milligram IgG1 and the leakage of protein A from PROSEP-A High Capacity in the presence of mouse IgG1 was between 2–5 ng protein A per milligram antibody.