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Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders
Published on: April 14, 2015
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Optimizing the Formulation and Lyophilization Process for a Fragment Antigen Binding (Fab) Protein Using Solid-State
Lokesh Kumar1, Karthik Balakrishna Chandrababu2, Shenbaga Moorthy Balakrishnan2
1Pharmaceutical Development, Genentech Inc. , San Francisco , California 94080 , United States.
Molecular Pharmaceutics
|October 1, 2019
Summary
Solid-state hydrogen-deuterium exchange with mass spectrometry (ssHDX-MS) effectively predicts lyophilized protein stability. This method rapidly screens fragment antigen binding protein (Fab) formulations and lyophilization cycles, reducing the need for extensive stress testing.
Area of Science:
- Analytical Chemistry
- Biopharmaceutical Science
- Protein Formulation
Background:
- Lyophilization is critical for stabilizing protein therapeutics like fragment antigen binding proteins (Fabs).
- Optimizing lyophilization formulations and cycles is essential for ensuring long-term drug product stability.
- Traditional methods for assessing stability often involve time-consuming stress degradation studies.
Purpose of the Study:
- To evaluate solid-state hydrogen-deuterium exchange with mass spectrometry (ssHDX-MS) as a rapid analytical tool.
- To screen and optimize lyophilized Fab formulations and lyophilization cycles.
- To correlate ssHDX-MS deuterium incorporation with solid-state stress degradation.
Main Methods:
- ssHDX-MS was applied to lyophilized Fab formulations with varying stabilizers and ratios under controlled conditions.
- Deuterium incorporation was measured by mass spectrometry.
- Solid-state stress degradation was assessed using size exclusion chromatography (SEC) and ion-exchange chromatography (IEC) at 50 °C.
- The impact of controlled nucleation (CN), uncontrolled nucleation (UCN), and annealing (AN) lyophilization processes was evaluated.
Main Results:
- ssHDX-MS deuterium incorporation correlated with solid-state stress degradation for different Fab formulations.
- Formulations with lower deuterium incorporation exhibited less aggregation and charge modification.
- For low concentration Fabs (2.5 mg/mL), ssHDX-MS results aligned with solid-state stability differences observed across lyophilization processes (AN < CN < UCN).
- No significant effect of ice nucleation was observed for high concentration Fabs (25 mg/mL).
Conclusions:
- ssHDX-MS is a valuable and rapid method for screening and optimizing lyophilized Fab formulations.
- The technique accurately predicts solid-state stability and can guide the selection of optimal lyophilization cycles.
- ssHDX-MS reduces the reliance on lengthy stress degradation studies, accelerating drug development timelines.
