Protein A Resins
Protein A is a bacterial protein from Staphylococcus aureus, with the capacity to bind mammalian antibodies of class immunoglobulin G (IgG) with high affinity. The gene for Protein A has been cloned and expressed in Escherichia coli (6, 7) allowing for the production of large quantities of recombinant Protein A.
Although recombinant Protein A is widely used as an affinity ligand for the capture and purification of antibodies, its sensitivity to alkaline conditions prevents the use of rigorous and cost-effective CIP and sanitization protocols based on NaOH.
Compared to conventional Protein A resins, one of the affinity chromatography resins investigated, Resin 2 (MabSelect SuRe, Cytiva) is based on a modified alkali-tolerant Protein A ligand. Through protein engineering, the amino acids in one of the IgG-binding domains particularly sensitive to alkali were identified and substituted with more stable ones.
A novel prototype Resin 3 offers an increased dynamic binding capacity (DBC) at a slightly longer residence time.
Evaluating capacity and reusability
Table I shows the Protein A resins that were compared for performance and cost-efficiency. According to the vendor, Resin 3 exhibits higher DBC than Resin 2 at longer residence times (8). A residence time of 6 min is expected to give a DBC (at 10% breakthrough) of approximately 60 g antibody per liter resin. In this study, the authors were able to confirm this behavior, and for further studies a residence time of 6 min and a loading of 50 g antibody per liter resin, corresponding to approximately 80% of the DBC (at 10% breakthrough), were selected.
Table 1
Table II presents the outline of the lifetime study. An amount of cell-culture supernatant corresponding to 50-g antibody per liter resin was applied to the column at 6 min. residence time. This was followed by a two-step washing procedure to remove unbound particles. Bound antibody was eluted with 50 mM acetic acid and the column resin was cleaned in place with five column volumes (CV) of 0.1 M NaOH.
Table two
