TY - JOUR
T1 - Rapid Conformational Analysis of Protein Drugs in Formulation by Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS)
AU - Esmail Nazari, Zeinab
AU - van de Weert, Marco
AU - Bou-Assaf, George
AU - Houde, Damian
AU - Weiskopf, Andrew
AU - Rand, Kasper Dyrberg
PY - 2016/11/1
Y1 - 2016/11/1
N2 - Hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) has become an established method for analysis of protein higher order structure. Here, we use HDX-MS methodology based on manual solid-phase extraction (SPE) to allow fast and simplified conformational analysis of proteins under pharmaceutically relevant formulation conditions. Of significant practical utility, the methodology allows global HDX-MS analyses to be performed without refrigeration or external cooling of the setup. In mode 1, we used dimethyl sulphoxide–containing solvents for SPE, allowing the HDX-MS analysis to be performed at acceptable back-exchange levels (<30%) without the need for cooling any components of the setup. In mode 2, SPE and chromatography were performed using fast isocratic elution at 0°C resulting in a back-exchange of 10%-30%. Real-world applicability was demonstrated by HDX-MS analyses of interferon-β-1a in formulation, using an internal HDX reference peptide (P7I) to control for any sample-to-sample variations in back-exchange. Advantages of the methodology include low sample use, optimized excipient removal using multiple solvents, and fast data acquisition. Our results indicate that HDX-MS can provide a reliable approach for fast conformation analysis of proteins in their intended formulations, which could facilitate an increased use of the technique in pharmaceutical development research.
AB - Hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) has become an established method for analysis of protein higher order structure. Here, we use HDX-MS methodology based on manual solid-phase extraction (SPE) to allow fast and simplified conformational analysis of proteins under pharmaceutically relevant formulation conditions. Of significant practical utility, the methodology allows global HDX-MS analyses to be performed without refrigeration or external cooling of the setup. In mode 1, we used dimethyl sulphoxide–containing solvents for SPE, allowing the HDX-MS analysis to be performed at acceptable back-exchange levels (<30%) without the need for cooling any components of the setup. In mode 2, SPE and chromatography were performed using fast isocratic elution at 0°C resulting in a back-exchange of 10%-30%. Real-world applicability was demonstrated by HDX-MS analyses of interferon-β-1a in formulation, using an internal HDX reference peptide (P7I) to control for any sample-to-sample variations in back-exchange. Advantages of the methodology include low sample use, optimized excipient removal using multiple solvents, and fast data acquisition. Our results indicate that HDX-MS can provide a reliable approach for fast conformation analysis of proteins in their intended formulations, which could facilitate an increased use of the technique in pharmaceutical development research.
U2 - 10.1016/j.xphs.2016.07.006
DO - 10.1016/j.xphs.2016.07.006
M3 - Journal article
SN - 0022-3549
VL - 105
SP - 3269
EP - 3277
JO - Journal of Pharmaceutical Sciences
JF - Journal of Pharmaceutical Sciences
IS - 11
ER -