Mechanisms and Applications of Solid-State Hydrogen Deuterium Exchange
Abstract
To prolong their long-term stability, protein molecules are commonly dispensed as lyophilized powders to be reconstituted before use. Evaluating the stability of these biomolecules in the solid state is routinely done by using various analytical techniques such as glass transition temperature, residual moisture content and other spectroscopic techniques. However, these techniques often show poor correlation with long term storage stability studies. As a result, time intensive long term storage stability studies are still the golden standard for evaluating protein formulations in the solid state. Over the past few years, our lab has developed solid-state hydrogen deuterium exchangemass spectrometry (ssHDX-MS) as an analytical tool that probes the backbone of a protein molecule in the solid state. ssHDX-MS gives a snapshot of protein-matrix interactions in the solid state and has a quick turnaround of a few weeks as opposed to a few months for accelerated stability testing. Additionally, various studies in the past have demonstrated that ssHDX-MS can be used for a wide range of biomolecules and shows strong correlation to long term stability studies routinely employed. The main aim of this dissertation is to provide an initial understanding of the mechanism behind ssHDX-MS in structured protein formulations. Specifically, this dissertation is an attempt at studying the effects of various experimental variables on the ssHDX-MS of myoglobin formulations as well as demonstrating the utility of this analytical technique. Firstly, the effects of varying temperature and relative humidity on ssHDX-MS of myoglobin formulations is studied with the help of statistical modeling. Secondly, the effects of pressure on ssHDX-MS of myoglobin formulations are evaluated at an intact and peptide digest levels. Finally, ssHDX-MS is used as a characterization tool to evaluate the effects of two different lyophilization methods on the structure and stability of myoglobin formulations. The results of studies described in this dissertation show ssHDX-MS to be sensitive to changes in experimental parameters, namely temperature, relative humidity, pressure, and excipients. Additionally, ssHDX-MS results were in good agreement with other routinely employed analytical and stability testing techniques when used to compare the effects of two lyophilization methods on myoglobin formulations.
Degree
Ph.D.
Advisors
Topp, Purdue University.
Subject Area
Analytical chemistry|Atmospheric sciences|Biophysics|Chemistry|Medical imaging
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