Protein A affinity chromatography

Affinity chromatography and related techniques (e.g., thiol chromatography and IMAC) are widely used for preparative isolation because they enable a single protein or class of proteins to be selectively purified from very complex mixtures. They may be occasionally used as analytical tools. For example, protein A affinity chromatography has been used for quantitative analysis of immunoglobulins in ascites fluid.45 Information about surface-accessible histidine and phosphate groups may be obtained using IMAC. 

An example of virus clearance factors in chromatographic processes frequently used for purification of antibodies is given in Table 17. Lower clearance factors for protein A affinity chromatography have been found by Mariani and Tarditi128 when compared to results found with protein G by Walter and Allgaier.237 An explanation of this fact can be found in the fact that protein G requires harsher elution conditions than protein A. 

Zapata, G. (1999). Recovery of recombinant antibodies from unclarified Chinese hamster ovary cell culture fluid by expanded bed Protein A affinity chromatography. Proc. Waterside Monoclonal Conf. Norfolk, VA. 

The monoclonal antibodies (MAh) are purified by protein A-affinity chromatography from culture media or bioreactor fiuid (20). Purity of the monoclonal antibody is assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (XcellSurelock Mini-Cell with NuPage gel, 10% Bis-Tris, Invitrogen, Carlsbad, California). 

Fahrner, R., Whitney, D., Vanderlaan, M., Blank, G S., Performance comparison of protein A affinity-chromatography sorbents for purifying recombinant monoclonal antibodies. Biotechnol Appl Biochem 1999, 30, 121—128. 

Jungbauer A, Hahn R (2004). Enegineering protein A affinity chromatography. Curr. Opin. Drug Discov. Devel. 7 248-256. 

Carter-Franklin, J. N., Victa, C., McDonald, P. and Fahrner, R. Fragments of protein A eluted during protein A affinity chromatography. /. Chromatogr. 1163 105-111, 2007. 

Protein A affinity chromatography is currently the most often used procedure for purification of humanized or chimeric antibodies [52, 53], but also requires an acidic elution step that can sometimes cause unexpected aggregation or inactivation of antibodies [53-55]. Aptamer-bound IgGs are instead easily released from the aptamer resin under neutral pH conditions using simple elution buffers, such as an EDTA solution [49]. Combined with the aptamer s high specificity to hFcl, these additional potential purification advantages provides us with an alternative reagent for the mass purification of therapeutic antibodies, as previously described [49]. 

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