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

Abstract

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 (SEM) and changes in protein structure were monitored using solid-state Fourier-transform infrared spectroscopy (ssFTIR), solid-state hydrogen-deuterium exchange-mass spectrometry (ssHDX-MS) 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 pLeu incorporation and more labeled regions for Mb-B lyophilized with controlled nucleation. The data support the use of ssHDX-MS and ssPL-MS to study formulation and process-induced conformational changes in lyophilized proteins.

Digital Twin for Lyophilization by Process Modeling in Manufacturing of Biologics

“…For these expensive and sensitive products it is important to design a safe and reliable process. During lyophilization the protein is exposed to a variety of stresses like pH changes, freeze-concentration, denaturation and aggregation caused by dehydration [5,10,11]. Other phenomena that can lead to protein denaturation during freeze drying are cold denaturation [12] and denaturation induced by interactions with ice-water interface [13,14].…”

“…Therefore an adequate formulation has to be used to protect the protein and ensure product integrity and drug activity [15]. On the one hand different components can be used to optimize the formulation but on the other hand the choice of excipients has an impact on the drying time [10,11]. Various reviews deal with the topic of excipients in lyophilization processes [11,[16][17][18].…”

“…Here an annealing step can be incorporated to alter the ice crystal morphology [21]. This step is critical for 2 of 29 protein solutions since it comprises several stresses like cold temperatures, freeze-concentration and ice formation that are critical to protein stability [10,11,15,22]. A review for the freezing step has been published [22].…”

Optimizing the Formulation and Lyophilization Process for a Fragment Antigen Binding (Fab) Protein Using Solid-State Hydrogen–Deuterium Exchange Mass Spectrometry (ssHDX-MS)

“…Similarly, Iyer et al evaluated ssHDX-MS of myoglobin/sucrose formulation (1:1 w/w; 70 μM myoglobin) lyophilized using UCN vs CN. No significant differences in deuterium incorporation on ssHDX-MS were observed . The effects of lyophilization processing differences on stress degradation were not evaluated in either of these studies, however.…”

“…No significant differences in deuterium incorporation on ssHDX-MS were observed. 14 The effects of lyophilization processing differences on stress degradation were not evaluated in either of these studies, however. The studies presented here further evaluated the capability of ssHDX-MS to detect differences in lyophilization processing for a model Fab and related these differences to protein degradation on storage.…”