Evaluation of a chiral stationary phase based on mixed immobilized proteins

Development of a High Throughput Equilibrium Dialysis Method

“…The curvature of the plot indicates the existence of at least two independent binding sites with different intrinsic affinities:17 (1) high affinity, low capacity and (2) low affinity, high capacity. In the case of propranolol, the data correlate with a previously described two‐binding‐site model:18,20 high affinity binding to α 1 ‐acid glycoprotein (AGP) and a low affinity binding site to human serum albumin (HSA). The dissociation constant ( K d ) values are also in agreement between the present and the latter study, showing nanomolar binding affinity for high and micromolar affinity for low affinity binding sites.…”

HSA binding of HIV protease inhibitors: a high‐performance affinity chromatography study

“…The biochromatographic experiments were carried out with the packed epoxy silica-based column described above, in which HSA was covalently immobilized by the online procedure previously reported [7], with slight modifications. In brief, a solution of protein (10 mg/mL) in potassium PB buffer (50 mM, pH 7.1) containing 1 M ammonium sulphate was circulated through the epoxysilica column in closed circuit for 24 h. The column was then washed with 100 mL of potassium PB (50 mM, pH 7.1) followed by 100 mL of 1 M glycine dissolved in the same buffer.…”

Plasma Protein Binding and Volume of Distribution: Determination, Prediction and Use in Early Drug Discovery

“…[ [42][43][44][45] a Impacts on mass balance but should not affect concentration ratio if system is at equilibrium. By utilizing two centrifugations and using the proteinaceous retentate from a control plasma sample, it is possible to aid compound solubility and perturb non-specific binding, as demonstrated for a series of corticosteroids [27].…”

“…Chromatographic methodologies whereby HSA or AAG is chemically bonded to silica-based stationary phases were first reported by Wainer and colleagues about 15-20 years ago [42,43]. This approach measures a chromatographic retention factor (k 0 ) which is directly related to the proportion of molecules in the stationary phase and in the mobile phase, and so from this a percent bound value can be obtained describing the interaction between the immobilized protein and test compound of interest.…”