A Novel Purification Method for Monoclonal Antibodies

Abstract

Immobilized Protein A and Protein G are commonly used for immunoglobulin purification. Problems with these affinity methods include selective binding between different species and their subclasses ranging from strong binding to no binding at all, the number of steps involved in the purification protocol, and the harsh elution buffers containing primary amines at low pH. Here we report the use of a resin that overcomes the weaknesses of monoclonal antibody purification. The resin and protocol have been optimized to allow the extraction of non-relevant proteins from tissue culture supernatant and ascites fluid often present in high abundance such as albumin and transferrin, thereby enriching the final product with immunoglobulins without further diluting the sample. This flow-through purification approach can be completed in less than 15 minutes for volumes less than 30 ml and 2.1 hours for volumes up to 1L. These purification times are considerably shorter than the labor-intensive hours of affinity purification. The method uses a mild buffer at physiological pH, unlike immobilized Protein A and Protein G that require a harsh elution buffer that is known to denature antibodies due to low pH. Additionally, with our resin the purified antibody is in a buffer free of primary amines and is ready for use or further processing without subsequent dialysis. Monoclonal antibodies produced in tissue culture supernatant as well as mouse and rat ascites fluid have been used for purification studies with Melon Gel. The results in all cases of purity and recovery were better than or equal to those obtained with immobilized Protein A and Protein G. Pierce Biotechnology Inc.