(M1230-02-10) Probing Product Quality of Lyophilized Formulations Via Solid-State Nuclear Magnetic Resonance Spectroscopy

Purpose: The purpose of this novel technology is to use nuclear magnetic resonance (NMR) spectroscopy to non-destructively evaluate the product quality of lyophilized products. Freeze-drying biologics in solid matrices containing buffers, sugars, and surfactants is a common process to ensure their stability. Controlling the amount of water in the cake is critical to ensuring stability of the biologic. Some water is usually necessary in the sample to provide structural integrity for the biologic through hydrogen bonding interactions. These water molecules are bound and have little molecular mobility. However, too much water will allow the excipients to crystallize and can lead to degradation. This excess water is typically unbound and exhibits greater molecular mobility. This probe measures the static ¹H NMR signal of solid-state formulations after lyophilization. These protons are subject to signal broadening due to the dipole-dipole interactions present in the solid state. The resulting spectrum is therefore highly dependent on the molecular mobility of the formulation and residual water. Samples with excess free water will yield sharper signals due to increased molecular mobility. This information can be used to detect drying defects, spatial variations of drying within the lyophilization tray, and predict product stability. Since this system is adapted for 2R vials the measurement can be done directly after lyophilization or storage without disrupting the cake. This offers a fast, non-destructive method for product testing in contrast to traditional methods, which are usually destructive. 

Methods: This novel NMR probe measures the static ¹H signal of solid-state lyophilized formulations in a 300 MHz external magnetic field via a simple 90° pulse-acquire experiment. A one second pulse delay was used with a pulse width of 24 μs. An acquisition time of 25.6 ms was used with a dwell time of 25 μs. Typically 1000 total acquisitions are collected with a total experiment time of 17 minutes. A Tecmag Redstone console is used in these experiments. Samples containing varying ratios of Bovine Serum Albumin, sucrose, and trehalose were prepared using a SP Scientific Virtis Pro where the samples were frozen at -45 °C for 3 hours, followed by a primary drying at -35 °C for 48 hours, and a secondary drying at 10 °C for 3 hours. All samples were stored at -80 °C after lyophilization.

Results: Initial studies demonstrate the technique’s sensitivity to molecular mobility in model formulations containing ratios of bovine serum albumin (BSA), sucrose, trehalose, and mannitol. As expected, liquid D₂O with trace H₂O yields a sharp, intense peak due to high mobility. In contrast, solid systems exhibit reduced mobility and lower signal intensity. Rigid matrices, such as 4:1 trehalose: mannitol, produce signals near background levels. Notably, proof-of-concept experiments at time zero revealed a negative correlation between water content, but a positive correlation with molecular mobility across various BSA:sucrose ratios, highlighting its potential for formulation screening and stability prediction. Furthermore, sampling from various parts of the drying tray show measurable differences in mobility with the center vials exhibiting higher mobility. This suggests an ability for the probe to demonstrate the edge-center effect and can be used for freeze-drying optimization.

Conclusion: No current commercial technology offers predictive, non-destructive stability assessment of lyophilized products.¹ This SSNMR approach enables real-time, in-situ evaluation of dry powder formulations, with potential applications in formulation development, stability testing, and process optimization. This technology also can be easily adapted to a high throughput technique. As the pharmaceutical industry progresses toward advanced manufacturing and analytics, this technique could reduce waste, shorten time, and maximize data yield from individual samples.

References: 1. Wahl V, Khinast J, Paudel A 2016. Lyophilized Protein Powders: A review of analytical tools for root cause analysis of lot-to-lot variability. Trends in Analytical Chemisty 82:468-491.

spectrum of 1) Sealed empty vial; 2) 4:1 Mannitol – Trehalose; 3) 35% BSA – 65% Trehalose; 4)20% sucrose; 5) 10 uL water

Series of BSA to Sucrose ratios from 100% to 10% BSA.

(Left) Example heatmap of edge vs. center effect with sample vial locations. (Right) 1H NMR spectrum of sample vials containing 35% BSA and 65 trehalose with a total solids content of 5% (w/v%).