Fast quantitation of recombinant plasminogen activator inhibitor type 1 inbacterial lysates by micropellicular reversed-phase liquid chromatography

A rapid reversed-phase HPLC assay is described for quantitating recombinant plasminogen activator inhibitor type 1 (PAI-1) in cultures of Escherichia coli. The assay utilized a short nonporous micropellicular C-18 column with an acetonitrile gradient in 0.1% trifluoroacetic acid. The selective resol ution of recombinant PAI-1 from major host proteins occurred within 1.3 min . Regeneration of initial chromatography conditions was fast and allowed su ccessive injections every 3 min. The assay's limit of detection for recombi nant PAI-1 was 0.5 mu g in 5-mu l injections of bacterial lysates and the a ssay was linear to 20 mu g. The assay's apparent recovery was between 94 an d 108% throughout the quantitative range. In a direct comparison to gel ele ctrophoresis the reversed-phase assay proved superior in monitoring recombi nant PAI-1 titers in cultures of E. coli. (C) 2000 Elsevier Science B.V. Al l rights reserved.