Affinity chromatography robustness

Collins, M.O. and Yu, L. et al. (2005b) Robust enrichment of phosphorylated species in complex mixtures by sequential protein and peptide metal-affinity chromatography and analysis by tandem mass spectrometry. Sci. STKE 2005,16. 

We have developed an analogous, but more robust system which is not necessarily constraint by the aforementioned limitations. The obvious extension has been to couple an affinity-based separation with mass spectrometry. Hutchens et al. have shown that affinity probe surfaces can be ust to capture specific protein ligands allowing detection by laser desorption mass spectrometry (. The limitations to their technique have been that the surface area for ligand capture is quite small and salt (or detergent) contaminants are still problematic. Perfusive affinity resins, on the other hand, provide a tremendous surface area for binding. The nature and composition of the solvents required for affinity chromatography, however, are not directly compatible with mass spectrometric analysis. 

Immobilized metal affinity chromatography has been shown to be effective for isolating proteins from crude mixtures, as well as for selective separations of closely related proteins [2]. With respect to separation efficiency, IMAC compares well with biospecific affinity chromatography and the immobilized metalion complexes are much more robust than antibodies or enzymes. These factors make IMAC particularly well suited for scale-up to process scale chromatography. The main scale-up points to be aware of are the degree to which the column is metal saturated, the chelating agent content of the sample, and the potential of leached metal (or its interactions) within the product eluate. 

Example 1 Robustness of an Affinity Chromatography Step for Purification of a Coagulation Factor... 

The affinity chromatography process can be considered robust within the process parameter ranges studied using a qualified down-scale model. 

Affinity chromatography is based on selective non-covalent interaction between an analyte and specific molecules. It is very specific, but not very robust. It is often used in biochemistry in the purification of proteins bound to tags. These fusion proteins are labeled with compounds such as His-tags, biotin or antigens, which bind to the stationary phase specifically. After purification, some of these tags are usually removed and the pure protein is obtained. 

A wide range of chemical compounds have been imprinted successfully, ranging from small molecules,40 2 to large proteins and cells.43 MIPs have been developed for a variety of applications including chromatography,4445 solid-phase extraction (SPE),46 47 enzyme catalysis,48 sensor technology,44>49>50 biomimetic sensors,5153 and immunoassays.54-56 MIPs are robust, inexpensive and, in many cases, possess affinity and specificity that are suitable for industrial applications. The high specificity and... 

The mechanical and chemiceil stability of these methacrylate based adsorbents makes them attractive alternatives for process chromatography where a robust column packing is essential. They have been used for size separations and hydrophobic interaction chromatography and after derivatisation as affinity matrices and for ion exchange chromatography. 

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