Two-step purification of monoclonal IgG

The increasing demand for monoclonal antibodies (MAb) as biopharmaceuticals has promoted the development of cell cultures with increased expression levels. As a consequence, the demand for more efficient purification processes has increased. This application note describes a two-step process for MAb purification based on MabSelect SuReTM and CaptoTM adhere, a strong multimodal anion exchanger. The two-step process is applied to the purification of monoclonal IgG 1 antibodies from CHO cell culture supernatant and the results are compared with data from a process based on a three-step process. If necessary, Capto adhere can be used as a second or third polishing step in any MAb purification platform. Introduction Over the last 20 years, the use of antibody titers in mammalian cell culture has increased dramatically. Recent industry information reports antibody titers from 1 to 5 g/L. The associated increase in contaminant levels, such as host cell proteins (HCP), viruses, aggregates, and protein A, leads to new challenges for MAb purification, and upgraded processes with efficient protein purification steps are required. Large-scale purification of MAbs usually consists of three chromatographic steps. Initially the MAb is captured using protein A affinity chromatography, giving a product with high purity, typically 99%. The product is then further polished by cationic and anionic exchange chromatography. The GE Healthcare chromatography media toolbox simplifies this process. MabSelect SuRe, characterized by alkaline stability, enhanced protease resistance and a generic elution profile, is used for the initial protein A capture step. MabSelect SuRe LX has been further developed from MabSelect SuRe to give even higher binding capacity at longer residence time. The medium of choice for polishing is Capto adhere. Capto adhere is a strong multimodal anion exchanger that offers a different selectivity compared to traditional ion exchangers, and is designed for intermediate purification and polishing of MAbs. Removal of protein A leakage, aggregates, host cell proteins, nucleic acids, and viruses is performed in flowthrough mode. With Capto adhere it is possible to replace the two postprotein A steps with a single polishing step, and end up with a two-step process (Fig 1). If necessary, Capto adhere can be used in combination with other media in a three step process. An alternative intermediate step is represented by cation exchange chromatography using Capto S. MabSelect SuRe MabSelect SuRe LX Capto SP ImpRes Capto S Capto Q Capto adhere Capto Q, Capto adhere, Capto Q ImpRes Virus inactivation & filtration Virus filtration and final formulation Capto adhere Cell removal Cell culture Fig 1. GE Healthcare chromatography media toolbox. Application note 28-9078-92 AD Process-scale antibody purifcation GE Healthcare Life Sciences