Evaluation of Predictors of Protein Relative Stability Obtained by Solid-State Hydrogen/Deuterium Exchange Monitored by
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RESEARCH PAPER
Evaluation of Predictors of Protein Relative Stability Obtained by Solid-State Hydrogen/Deuterium Exchange Monitored by FTIR Rui Fang 1,2 & Wasfy Obeidat 1 & Michael J. Pikal 1 & Robin H. Bogner 1
Received: 17 December 2019 / Accepted: 28 July 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
ABSTRACT Purpose Hydrogen/deuterium (H/D) exchange over a range of temperatures suggests a protein structural/mobility transition in the solid state below the system glass transition temperature (Tg). The purpose of this study was to determine whether solid-state protein stability correlates with the difference between storage temperature and apparent Td where an abrupt change in mobility occurs, or alternatively, the extent of H/D exchange at a single temperature correlates directly to protein stability in lyophilized solids. Methods Solid-state H/D exchange was monitored by FTIR spectroscopy to study the extent of exchange and the apparent transition temperature in both pure recombinant human serum albumin (rHSA) and rHSA formulated with sucrose or trehalose. H/D exchange of freeze-dried formulations at 11% RH and temperatures from 30 to 80°C was monitored. Protein stability against aggregation at 40°C/11% RH for 6 months was assessed by size exclusion chromatography (SEC). Results Both sucrose and trehalose showed equivalent protection of protein secondary structure by FTIR. The rHSA:sucrose formulation showed superior long-term stability at 40°C by SEC over the trehalose formulation, but the apparent Td determined from H/D exchange was much higher in the trehalose formulation. Instead, the extent of H/D exchange (X∞) was lower in the sucrose formulation at the temperature of the stability studies (40°C) than found for the trehalose formulation, which was consistent with better stability in the sucrose formulation. In Loving Memory of Professor Michael J. Pikal * Robin H. Bogner [email protected] 1
University of Connecticut, Storrs, Connecticut, USA
2
Present address: Sterile and Specialty Products, Merck & Co., Inc, NJ Kenilworth, USA
Conclusions While apparent Td did not correlate with protein stability for rHSA, the extent of H/D exchange, X∞, did.
KEY WORDS Freeze-drying . FTIR . protein internal dynamics . protein stability . solid-state H/D exchange
INTRODUCTION Freeze-drying is the drying process of choice to improve the stability of parenterally administered proteins, which undergo significant physical and/or chemical degradation in solution during storage. In order to achieve optimal long-term stability, it is recommended that freeze-dried proteins be stored below the glass transition temperature (Tg) of the solid (i.e., an assembly of protein molecules or protein molecules dispersed in a matrix) (1,2). The Tg of such a molecularly miscible system marks the onset temperature of viscous flow on a long time scale around 10–10−4 s. However, there is an increasing body of work demonstrating that physical and/or chemical degradations still occur during storage at temp
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