Recombinant protein drugs are an important part of the biopharmaceutical industry, providing treatments for a variety of diseases and generating billions in annual sales. Most protein drugs are given by injection. Because many proteins are not sufficiently stable in solution to provide an acceptable shelf-life, they are often marketed in solid forms, typically as lyophilized (freeze-dried) amorphous powders that are reconstituted prior to administration. Over the past decade, ~40% of newly FDA-approved biologics have been marketed in solid forms. Degradation processes of various kinds can still occur in the solid state, however. Few analytical methods can provide high resolution information on the solid-state environment of the protein, forcing the industry to rely on time-consuming stability studies (6-24 months) to optimize formulation and process, and to ensure the stability of the final product. 

We have applied hydrogen deuterium exchange with mass spectrometric analysis (HDX-MS) to lyophilized proteins (ssHDXX-MS) in an attempt to address this gap. In ssHDX-MS, a lyophilized  solid powder is exposed to D2O in the vapor phase at controlled temperature and relative humidity. Over time, the samples are reconstituted and assayed for total deuterium incorporation using mass spectrometry. With proteolytic digestion, sites of deuterium incorporation can be resolved to peptide level. Our results have shown that, In many cases, total deuterium incorporation is highly correlated to protein stability on storage (e.g., aggregation), supporting the use of ssHDX-MS as a screening tool for formulation and process development for lyophilized protein drug products. This correlation also suggests that the hydrogen bond network in the solid state is a critical determinant of storage stability, including both the intramolecular hydrogen bonds that contribute to protein structure and the intermolecular hydrogen bonds between the protein and components of the matrix. The protein thus serves as a reporter of the hydrogen bond network in the solid powder in these ssHDX-MS studies. This presentation will provide background on protein drugs, present key ssHDX-MS results to date, and propose mechanistic interpretations.

 

Zoom link: https://bristol-ac-uk.zoom.us/j/94983629930