The interaction between protein A and immunoglobulin G (IgG) was studied at a variety of pH values using a surface plasmon resonance (SPR) device, which provides real time kinetic data without labelling or molecular alteration. This study was carried out due to the large scale use of Protein A affinity chromatography for the purification of IgG for pharmaceutical purposes, and is one of the most costly steps in the purification process. The results produced were largely in line with those produced in previous literature with binding remaining strong between pH 7.4 and 5.0, although the association rate decreased as pH decreased. Below pH 5.0, the rate of IgG elution markedly increased, with pH 3.5 showing near full elution seconds after the association phase of the SPR interaction finished. Problems were encountered with non-specific binding between the SPR sensor chip and IgG occurring under a variety of conditions, requiring various remedies. However, no complete interactions were successfully carried out under pH 5.0, so the results obtained below this value were obtained by binding at pH 7.4 and then elution at the desired pH.
The data showed binding behaviour that was most successfully explained by a three-site model, each with a binding ratio of 1:1. The binding ratio is questionable given that Protein A and IgG typically bind at a ratio of 1:2 but may be explained by the sites being independent of one another and thus no secondary attachment is observed. A variety of models were fitted to the data but only two- and three-site models fitted the experimental data, with the three-site model being a more accurate and robust fit across pH changes. A multiple site model seems intuitively correct given the six different binding sites that Protein A has for interaction with IgG. The models produced have potential applications in a larger model of Protein A affinity chromatography, although a number of additional factors would need to be taken into account, such as mass transfer effects and the IgG concentration gradient.
| Identifer | oai:union.ndltd.org:canterbury.ac.nz/oai:ir.canterbury.ac.nz:10092/7952 |
| Date | January 2013 |
| Creators | Plummer, Ben Thomas |
| Publisher | University of Canterbury. Chemical and Process Engineering |
| Source Sets | University of Canterbury |
| Language | English |
| Detected Language | English |
| Type | Electronic thesis or dissertation, Text |
| Rights | Copyright Ben Thomas Plummer, http://library.canterbury.ac.nz/thesis/etheses_copyright.shtml |
| Relation | NZCU |
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