Affinity chromatography specific desorption

In the downstream processing of bioprocesses, fixed-bed adsorbers are used extensively both for the recovery of a target and for the removal of contaminants. Moreover, their performance can be estimated from the breakthrough curve, as stated in Chapter 11. The break time tg is given by Equation 11.13, and the extent of the adsorption capacity of the fixed bed utilized at the break point and loss of adsorbate can be calculated from the break time and the adsorption equilibrium. Affinity chromatography, as weii as some ion-exchange chromatography, are operated as specific adsorption and desorption steps, and the overall performance is affected by the column capacity available at the break point and the total operation time. 

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. 

As shown in Figure 11.10, the operations in affinity chromatography are regarded as highly specific adsorption and desorption steps. Thus, the overall performance is much affected by the break point, an estimation of which can be made as described in Section 11.5.2. 

In the present paper, we report high-performance affinity chromatography of thrombin in presence of AT III and Hep, using two types of resins as stationary phases either heparin-like PSSO or AT Ill-like PAOM. In order to differentiate their mechanisms of interaction with thrombin, we examined the chromatographic behavior of thrombin in the presence, or in the absence of AT III and/or heparin. Finally, thrombin was injected on the columns at low ionic strength. The desorption of bound thrombin from the two solid surfaces was then carried out using AT III, heparin and the AT Ill-Hep complex, to elucidate the specificity of the interactions involved. 

Surface enhanced laser desorption/ionization (SE-LDI) is a variant of MALDI in which the MALDI probe is derivatized with various substances that have affinity for the analyte. The probes are then used to extract the analyte directly from mixtures thus avoiding sample loss through more complicated procedures such as column chromatography. Contaminants can be washed from the probe with appropriate buffers or solvents leaving the purified analyte ready for analysis. Many adsorbents have been used typical examples are hydrophobic or ionic compounds, enzymes, various receptors, antibodies, and nucleic acids. Although most applications have been reported with proteins, the technique is potentially applicable to any type of compound for which a specific adsorbent can be attached to the probe. 

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