title: Rapid Conformational Analysis of Protein Drugs in Formulation by Hydrogen/Deuterium Exchange Mass Spectrometry.
abstract: 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.
authors: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 2100, Denmark.
authors: Zeinab
authors: Z
authors: E Nazari
authors: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 2100, Denmark.
authors: Marco
authors: M
authors: van de Weert
authors: Department of Protein Pharmaceutical Development, Biogen, Cambridge, Massachusetts 02142.
authors: George
authors: G
authors: Bou-Assaf
authors: Department of Protein Pharmaceutical Development, Biogen, Cambridge, Massachusetts 02142.
authors: Damian
authors: D
authors: Houde
authors: Department of Protein Pharmaceutical Development, Biogen, Cambridge, Massachusetts 02142.
authors: Andrew
authors: A
authors: Weiskopf
authors: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 2100, Denmark. Electronic address: Kasper.Rand@sund.ku.dk.
authors: Kasper
authors: K
authors: D Rand
mesh_terms: D000804:Angiotensin II / Q000032:analysis* / Q000737:chemistry; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D041961:Deuterium Exchange Measurement / Q000379:methods*; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D006801:Humans; D007328:Insulin / Q000032:analysis* / Q000737:chemistry; D000068556:Interferon beta-1a / Q000032:analysis* / Q000737:chemistry; D011487:Protein Conformation; D052616:Solid Phase Extraction / Q000379:methods; D013997:Time Factors
chemical_list: D007328:Insulin; D000804:Angiotensin II; D000068556:Interferon beta-1a; D004121:Dimethyl Sulfoxide
keywords: LC-MS; analytical biochemistry; analytical chemistry; biopharmaceuticals characterization; formulation; mass spectrometry; physical stability; protein folding/refolding; protein formulation; protein structure