Measurement of drug-protein dissociation rates by high-performance affinity chromatography and peak profiling

John E. Schiel, Corey M. Ohnmacht, David S. Hage

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

The rate at which a drug or other small solute interacts with a protein is important in understanding the biological and pharmacokinetic behavior of these agents. One approach that has been developed for examining these rates involves the use of high-performance affinity chromatography (HPAC) and estimates of band-broadening through peak profiling. Previous work with this method has been based on a comparison of the statistical moments for a retained analyte versus nonretained species at a single, high flow rate to obtain information on stationary phase mass transfer. In this study an alternative approach was created that allows a broad range of flow rates to be used for examining solute-protein dissociation rates. Chromatographic theory was employed to derive equations that could be used with this approach on a single column, as well as with multiple columns to evaluate and correct for the impact of stagnant mobile phase mass transfer. The interaction of L-tryptophan with human serum albumin was used as a model system to test this method. A dissociation rate constant of 2.7 (±0.2) s-1 was obtained by this approach at pH 7.4 and 37°C, which was in good agreement with previous values determined by other methods. The techniques described in this report can be applied to other biomolecular systems and should be valuable for the determination of drug-protein dissociation rates.

Original languageEnglish (US)
Pages (from-to)4320-4333
Number of pages14
JournalAnalytical chemistry
Volume81
Issue number11
DOIs
StatePublished - Jun 1 2009

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Affinity chromatography
Mass transfer
Flow rate
Pharmaceutical Preparations
Proteins
Pharmacokinetics
Serum Albumin
Tryptophan
Rate constants

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Measurement of drug-protein dissociation rates by high-performance affinity chromatography and peak profiling. / Schiel, John E.; Ohnmacht, Corey M.; Hage, David S.

In: Analytical chemistry, Vol. 81, No. 11, 01.06.2009, p. 4320-4333.

Research output: Contribution to journalArticle

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