Pulse Proteolysis: An Orthogonal Tool for Protein Formulation Screening.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Pharmaceutical Sciences

Background:

• Predicting protein formulation stability is challenging, often requiring lengthy studies.
• Developing rapid analytical methods for stability prediction is crucial for biopharmaceutical development.

Purpose of the Study:

• To evaluate pulse proteolysis as a predictive analytical tool for protein solution-state stability.
• To compare pulse proteolysis with standard biophysical techniques for assessing formulation stability.

Main Methods:

• Four proteins were formulated with varying buffers and excipients.
• Urea-induced unfolding followed by "pulse" proteolysis was performed.
• Proteolytic resistance was quantified using capillary electrophoresis-sodium dodecyl sulfate (CE-SDS).
• Orthogonal biophysical methods (DSF, DSC, DLS, CD, fluorescence) were employed.
• Stability was monitored via size exclusion chromatography (SEC) under storage conditions.

Main Results:

• Formulations with higher proteolytic resistance showed increased monomer content and thermal stability.
• Pulse proteolysis demonstrated a strong correlation with SEC stability data.
• Standard biophysical techniques showed poor correlation with SEC stability data.

Conclusions:

• Pulse proteolysis is a quantitative and predictive tool for assessing protein conformational stability.
• This method can reliably rank-order different protein formulations.
• Pulse proteolysis provides an orthogonal approach to traditional stability assessment methods.