Validation of the Re-Use of Protein A Sepharose for the Purification of Monoclonal Antibodies

Abstract

The use of Protein A affinity chromatography at an early stage in the purification of a monoclonal antibody results in a high purity product with high yield. However, in order to operate the process in an economic manner it is often necessary to use a Protein A affinity column for a number of consecutive cycles to purify a single batch of antibody. An important aspect of process safety validation is the impact of repeated use of the column on the stability of the affinity ligand and hence on column performance and quality of the purified monoclonal antibody. A programme of work has been undertaken to study the effect of repeated use of a single column of Protein A Sepharose FF on the yield and quality of monoclonal antibody.

An automated chromatography system was used to carry out 25 sequential purification cycles. For each cycle the yield of antibody was measured by A₂₈₀ readings and the purity was determined by SDS-PAGE, isoelectric focussing and gel permeation HPLC analysis. The level of Protein A residues in the affinity purified monoclonal antibody was also determined for each cycle by ELISA.