Process and Formulation Effects on Protein Structure in Lyophilized Solids Using Mass Spectrometric Methods

• Published in: Journal of pharmaceutical sciences Vol. 105; no. 5; pp. 1684 - 1692
• Main Authors: Iyer, Lavanya K., Sacha, Gregory A., Moorthy, Balakrishnan S., Nail, Steven L., Topp, Elizabeth M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2016
• Subjects: Animals; controlled nucleation; Drug Compounding; Freeze Drying - methods; FTIR; Horses; hydrogen-deuterium exchange; lyophilization; mass spectrometry; Mass Spectrometry - methods; microscopy; Myoglobin - analysis; Myoglobin - chemistry; photolytic labeling; protein formulation; protein structure; Protein Structure, Secondary; solid-state; Tandem Mass Spectrometry - methods; X-Ray Diffraction - methods; protein structure; protein formulation; hydrogen-deuterium exchange; lyophilization; controlled nucleation; photolytic labeling; FTIR; mass spectrometry; microscopy; solid-state
• ISSN: 0022-3549; 1520-6017
• DOI: 10.1016/j.xphs.2016.02.033

Myoglobin (Mb) was lyophilized in the absence (Mb-A) and presence (Mb-B) of sucrose in a pilot-scale lyophilizer with or without controlled ice nucleation. Cake morphology was characterized using scanning electron microscopy, and changes in protein structure were monitored using solid-state Fourier-transform infrared spectroscopy, solid-state hydrogen-deuterium exchange–mass spectrometry, and solid-state photolytic labeling–mass spectrometry (ssPL-MS). The results showed greater variability in nucleation temperature and irregular cake structure for formulations lyophilized without controlled nucleation. Controlled nucleation resulted in nucleation at ∼(−5°C) and uniform cake structure. Formulations containing sucrose showed better retention of protein structure by all measures than formulations without sucrose. Samples lyophilized with and without controlled nucleation were similar by most measures of protein structure. However, ssPL-MS showed the greatest photoleucine incorporation and more labeled regions for Mb-B lyophilized with controlled nucleation. The data support the use of solid-state hydrogen-deuterium exchange–mass spectrometry and ssPL-MS to study formulation and process-induced conformational changes in lyophilized proteins.