Affinity chromatography methodology

Several affinity screening methodologies that include MS-based readout and work under protein-excess conditions have been developed in the past decade [1]. Some examples include affinity selection/mass spectrometry (ASMS Abbott Labs [10]), size exclusion chromatography with LC-ESI-MS (see Chapter 2 and 3 [11-19]), the use of coupled or non-coupled pulsed ultra-filtration/mass spectrometry (summarized in this chapter [11, 20-23]), restricted access phase chromatography (see Chapter 5 [24, 25]), capillary electrophoresis [26, 27], target shift mass spectrometry [28], and multitarget affinity/specificity screening (MASS, see Chapter 10 [29, 30]). 

Neoglycoproteins are suitable affinants for affinity chromatography of carbohydrate-binding proteins. As they can be obtained easily and inexpensively in large amounts, and as the methodology for attaching... 

Affinity chromatography combines the analytical and chemical capacities of chemically bonded stationary phases and immobilized enzymes. Technology and methodology of both techniques are joined in the development of affinity stationary phases. Since steric requirements are even more determining than in simple immobilized enzyme systems, spacer molecules have great importance in these modifications. Commonly used spacer arms are summarized in figure 8.3. 

In procedures involving affinity chromatography, which may use biological components such as mAbs, appropriate measures should be taken to ensure that no contamination arises from its use, such as adventitious viruses, that could threaten the safety of the final product. The more commonly used methodologies to determine the levels of contamination are presented in Section 13.4.8. 

M18. Mahley, R. W., and Weisgraber, K. H., Subfractionation of high density lipoproteins into two metabolically distinct subclasses by heparin affinity chromatography and Geon-Pevikon electrophoresis. In Report of the High Density Lipoprotein Methodology Workshop (K. Lippel, ed.), pp. 356-366. U.S. Department of Health, Education and Welfare. NIH publication No. 79-1661, Bethesda, Md., 1979. 

All individual methodologies described in the following, such as ion exchange, hydrophobic interaction, and affinity chromatography, are potentially usable in packed-bed mode as well as in fluid-bed mode if the solid phase sorbent meets the specific requirements of density and particle size. The only exception today is gel filtration, which requires a large number of plates for an acceptable resolution that the fluid bed cannot guarantee. 

Methodology Prior to experimentation the stationary phase constmct is prepared and poured into the column. Notably, the columns used for affinity chromatography tend to be shorter than those used for other column chromatography methods. Once the stationary phase construct has settled (packed) in the column, a loading buffer is passed through to equilibrate the system before addition of loading buffer... 

With respect to their application, aptamers were selected in the past mainly for their use as therapeutic agents. In addition to the therapeutic field, aptamers have been then used in several analytical methodologies, such as affinity chromatography, capillary electrophoresis (14), mass spectrometry (15), or biosensors (1). These aptamers-based methods have been mainly employed in the clinical area for the development of diagnostic assays. 

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