Sean M McClure1, Patrick L Ahl2, Jeffrey T Blue2. 1. Center for Materials Science and Engineering, Merck Sharp & Dohme Corp, WP75B-210, 770 Sumneytown Pike, West Point, PA, 19486, USA. sean.mcclure@merck.com. 2. Vaccine Drug Product Development, Merck Sharp & Dohme Corp, West Point, PA, 19486, USA.
Abstract
PURPOSE: The purpose was to evaluate DSF for high throughput screening of protein thermal stability (unfolding/ aggregation) across a wide range of formulations. Particular focus was exploring PROTEOSTAT® - a commercially available fluorescent rotor dye - for detection of aggregation in surfactant containing formulations. Commonly used hydrophobic dyes (e.g. SYPRO™ Orange) interact with surfactants, complicating DSF measurements. METHODS: CRM197 formulations were prepared and analyzed in standard 96-well plate rT-PCR system, using SYPRO™ Orange and PROTEOSTAT® dyes. Orthogonal techniques (DLS and IPF) are employed to confirm unfolding/aggregation in selected formulations. Selected formulations are subjected to non-thermal stresses (stirring and shaking) in plate based format to characterize aggregation with PROTEOSTAT®. RESULTS: Agreement is observed between SYPRO™ Orange (unfolding) and PROTEOSTAT® (aggregation) DSF melt temperatures across wide range of non-surfactant formulations. PROTEOSTAT® can clearly detect temperature induced aggregation in low concentration (0.2 mg/mL) CRM197 formulations containing surfactant. PROTEOSTAT® can be used to explore aggregation due to non-thermal stresses in plate based format amenable to high throughput screening. CONCLUSIONS: DSF measurements with complementary extrinsic dyes (PROTEOSTAT®, SYPRO™ Orange) are suitable for high throughput screening of antigen thermal stability, across a wide range of relevant formulation conditions - including surfactants -with standard, plate based rT-PCR instrumentation.
PURPOSE: The purpose was to evaluate DSF for high throughput screening of protein thermal stability (unfolding/ aggregation) across a wide range of formulations. Particular focus was exploring PROTEOSTAT® - a commercially available fluorescent rotor dye - for detection of aggregation in surfactant containing formulations. Commonly used hydrophobic dyes (e.g. SYPRO™ Orange) interact with surfactants, complicating DSF measurements. METHODS: CRM197 formulations were prepared and analyzed in standard 96-well plate rT-PCR system, using SYPRO™ Orange and PROTEOSTAT® dyes. Orthogonal techniques (DLS and IPF) are employed to confirm unfolding/aggregation in selected formulations. Selected formulations are subjected to non-thermal stresses (stirring and shaking) in plate based format to characterize aggregation with PROTEOSTAT®. RESULTS: Agreement is observed between SYPRO™ Orange (unfolding) and PROTEOSTAT® (aggregation) DSF melt temperatures across wide range of non-surfactant formulations. PROTEOSTAT® can clearly detect temperature induced aggregation in low concentration (0.2 mg/mL) CRM197 formulations containing surfactant. PROTEOSTAT® can be used to explore aggregation due to non-thermal stresses in plate based format amenable to high throughput screening. CONCLUSIONS: DSF measurements with complementary extrinsic dyes (PROTEOSTAT®, SYPRO™ Orange) are suitable for high throughput screening of antigen thermal stability, across a wide range of relevant formulation conditions - including surfactants -with standard, plate based rT-PCR instrumentation.
Authors: Alexander A Maskevich; Vitali I Stsiapura; Valeriy A Kuzmitsky; Irina M Kuznetsova; Olga I Povarova; Vladimir N Uversky; Konstantin K Turoverov Journal: J Proteome Res Date: 2007-02-17 Impact factor: 4.466
Authors: Vitali I Stsiapura; Alexander A Maskevich; Valery A Kuzmitsky; Vladimir N Uversky; Irina M Kuznetsova; Konstantin K Turoverov Journal: J Phys Chem B Date: 2008-12-11 Impact factor: 2.991
Authors: Kari Kopra; Salla Valtonen; Randa Mahran; Jonas N Kapp; Nazia Hassan; William Gillette; Bryce Dennis; Lianbo Li; Kenneth D Westover; Andreas Plückthun; Harri Härmä Journal: Int J Mol Sci Date: 2022-06-26 Impact factor: 6.208