MAB Purification General Protocol
This example demonstrates the effectiveness of using a high selectivity affinity chromatography technique as a capture step, since only a second gel filtration polishing step was needed to achieve the required level of purity. The objective of this work was to produce an efficient, routine procedure for monoclonal antibody purification. A more detailed description of this work can be found in Application Note 18-1128-93.
Target Molecule
Mouse monoclonal IgG 1 antibodies.
Source Material
Cell culture supernatant.
Sample Extraction and Clarification
Salt concentration and pH were adjusted to those of the binding buffer in the capture step. Samples were filtered through a 0.45 µm filter before chromatography.
Capture
Affinity or ion exchange chromatography are particularly suitable for samples such as cell culture supernatants as they are binding techniques which concentrate the target protein and significantly reduce sample volume. For monoclonal anti-body purification capture of the target protein can be achieved by using a highly selective affinity chromatography medium. In this example a HiTrap rProtein A column was used. Although general standard protocols were supplied with this pre-packed columns, it was decided to further optimise the binding and elution conditions for the specific target molecule. Most mouse monoclonal antibodies of the IgG 1 sub-class require high salt concentrations to bind to immobilised Protein A, therefore a salt concentration was selected which gave the largest elution peak area and absence of antibodies in the flow-through. Results from the scouting for optimal binding conditions are shown in Figure 1. Scouting for the optimum elution pH also helped to improve antibody recovery. Optimisation of binding and elution conditions gave a well resolved peak containing IgG 1 , as shown in Figure 2.
Fig. 1. Automatic scouting for optimal binding conditions.
Fig. 2. Optimised capture step on HiTrap rProtein A.
Intermediate Purification
No intermediate purification was required as the high selectivity of the capture step also removed contaminating proteins and low-molecular substances giving a highly efficient purification.
Figure 3: The final purification step.
Polishing
In most antibody preparations there is a possibility that IgG aggregates and/or dimers are present. It was therefore essential to include a gel filtration polishing step, despite the high degree of purity achieved during capture. The polishing step removes low or trace levels of contaminants. Superdex 200 prep grade gel filtration media was selected as it has the most suitable molecular weight separation range for IgG antibodies. | 
