Rangan Mallik

Analysis of drug-protein binding by ultrafast affinity chromatography using immobilized human serum albumin

Human serum albumin (HSA) was explored for use as a stationary phase and ligand in affinity microcolumns for the ultrafast extraction of free drug fractions and the use of this information for the analysis of drug-protein binding. Warfarin, imipramine, and ibuprofen were used as model analytes in this study. It was found that greater than 95% extraction of all these drugs could be achieved in as little as 250 ms on HSA microcolumns. The retained drug fraction was then eluted from the same column under isocratic conditions, giving elution in less than 40 s when working at 4.5 mL/min. The chromatographic behavior of this system gave a good fit with that predicted by computer simulations based on a reversible, saturable model for the binding of an injected drug with immobilized HSA. The free fractions measured by this method were found to be comparable to those determined by ultrafiltration, and equilibrium constants estimated by this approach gave good agreement with literature values. Advantages of this method include its speed and the relatively low cost of microcolumns that contain HSA. The ability of HSA to bind many types of drugs also creates the possibility of using the same affinity microcolumn to study and measure the free fractions for a variety of pharmaceutical agents. These properties make this technique appealing for use in drug-binding studies and in the high-throughput screening of new drug candidates.

Immobilization of α1-acid glycoprotein for chromatographic studies of drug–protein binding II. correction for errors in association constant measurements

A new method for the immobilization of alpha(1)-acid glycoprotein (AGP) in high-performance liquid chromatography (HPLC) columns was recently described for applications such as drug binding studies. Part of this earlier work used self-competition zonal elution studies to measure association equilibrium constants between immobilized AGP and R- or S-propranolol. It was later found that analysis of these data by a common equation derived for linear elution conditions gave erroneous values for experiments actually conducted under nonlinear conditions. This report discusses the nature of this error and uses frontal analysis to estimate the true binding strength between R- and S-propranolol and HPLC columns containing immobilized AGP.

Development of immunoaffinity restricted access media for rapid extractions of low-mass analytes.

Restricted access media using antibodies as immobilized ligands were developed for the rapid and selective capture of small analytes by immunoextraction, giving rise to materials referred to as immunoaffinity restricted access media (IA-RAM). To make such a material, intact antibodies for the desired target were first immobilized onto porous silica, with antibodies at or near the outer surface of the support then being treated with papain (or a related agent) to release and remove their binding domains. The result was a support in which only antibodies deep within the pores remained intact and able to bind to the target. Items evaluated in the development of such media included the immobilization method used for the antibodies, the pore size of the support, and the amount of papain and time that were used for support treatment. A theoretical model was also developed to describe the extent of binding domain removal based on the measured polypeptide content of the IA-RAM support before and after treatment with papain. The final optimized conditions for making the IA-RAM supports were used to prepare columns that contained antifluorescein antibodies. Injections of fluorescein and fluorescein-labeled bovine serum albumin onto these IA-RAM columns gave selective and quantitative extraction of fluorescein in 1-2 s. This approach can be used with other antibodies and low-mass targets and should be valuable for such applications as the rapid separation of drugs from drug-protein complexes or the isolation of labeled/modified peptides from intact proteins that contain the same modification or label.