Journal of Chromatography B: Biomedical Sciences and Applications
Chromatographic behavior of mouse serum immunoglobulin G in protein G perfusion affinity chromatography
Introduction
Affinity chromatography (AC) has been widely used as an efficient method for the analysis, as well as for the large-scale separation, of biological macromolecules. The development of high-performance affinity chromatography (HPAC) makes broaden its applications in biotechnology. For the typical stationary phase, the first serious constraint to be considered is the problem of mass transfer, especially at high flow-rate. To improve mass transfer and speed up the separation process, in recent years perfusion chromatographic packings have been developed. The intraparticle convection in this technique enhances pore diffusivity and minimizes the mass transport effect in the stationary phase, so perfusion chromatography can achieve both the purification and rapid separation of proteins [1]. These packings have been successfully used in the affinity chromatography of proteins using immobilized ligands. For example, IgG was quickly determined by the protein A or protein G affinity packing [2]. The rapid characterization of the interaction between the human growth factor and its monoclonal antibody was conducted by protein G perfusion affinity chromatography [3]. Shigeo et al. [4] reported that a perfusion-type support in AC could be effectively used for the quick purification of bioactive materials secreted from cells. These investigations have mainly been concerned with studying the special features of these packings, such as short separation time, high accuracy, repeatability of analysis and operation at high flow-rate without reducing capacity.
Non-specific adsorption, irreversible adsorption and flow-rate effects are the main factors affecting the separation efficiency of AC. In this paper, the specificity of the protein G perfusion affinity column to mouse serum IgG in the loading solutions of various ionic strengths, the irreversible adsorption of IgG on the surface of protein G packings and the effects of elution solutions and flow-rate on affinity chromatographic behavior of IgG are discussed.
Section snippets
Apparatus and materials
The TSP liquid chromatograph (TSP, San Jose, CA, USA) consisted of a P4000 pump, an AS3000 autosampler and a Spectra FOCUS diode array detector. Chromatographic system control, data acquisition and chromatographic analysis were exerted with TSP PC 1000 Chromatography Manager software (3.0 version).
HPLC separations in high ionic strength solutions were carried out on an Alliance Waters 2690 Separation Module liquid chromatograph (Waters, Milford, MA, USA) with a Waters 996 PDA detector.
Non-specific binding and effect of loading solution
Some common contaminant proteins (BSA, transferrin and insulin) in serum as standard proteins were injected into the POROS G/M perfusion affinity column and eluted under the elution conditions studied. The results have indicated that these proteins have not been retained on the column at all and the column has exhibited no any non-specific bindings to these contaminant proteins. It is coincident with the results in the literature [2].
Protein G has a very high affinity for the Fc region of IgG,
Conclusions
Data presented in this paper indicate firstly that the protein G column showed no non-specific binding to the other proteins in serum but an irreversible adsorption to IgG under the investigation conditions. Secondly, variations of the elution solution composition and pH played an essential effect on the retention time and the peak shape of IgG to some extent. In different elution solution systems, ionic strength may have different effects on the chromatographic behavior of IgG. Thirdly, the
Acknowledgements
We are grateful to Waters China Ltd. in Beijing for providing us with the Alliance HPCL systems and to Anapure Bioscientific in Beijing for mouse sera as a free gift.
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