(M1230-09-60) Predicting the Stability of Lyophilized Formulations Containing a Monoclonal Antibody and Sucrose/Trehalose Using Solid-State NMR Spectroscopy

Purpose: The long-term stability of lyophilized protein formulations depends on both the physical state of the matrix and the interactions between the protein and excipients. Methods like SEC can track protein degradation over time, but do not provide molecular-level insights into the formulation environment. Solid-state NMR (ssNMR) offers the ability to probe molecular mobility and miscibility in amorphous systems through relaxation measurements, potentially revealing mechanisms that govern protein stability. The purpose of this study is to assess matrix mobility and miscibility in lyophilized protein formulations using ssNMR, and to determine how these properties correlate with long-term protein stability.

Methods: We studied two sets of formulations: monoclonal antibody (mAb)-trehalose and mAb-Sucrose formulations. Both sets of formulations contained 10, 20, 30, 50, 70, 90 %w/w of trehalose/sucrose. Formulations were lyophilized and stored at 50°C post lyophilization. Residual moisture content was analyzed by Karl fisher titration. The monomer (%) was analyzed using Size exclusion chromatography (SEC) on an Agilent infinity 1200ii system. ssNMR data were collected using a Bruker Avance 400 MHz and PhenixNMR HX MAS probe.

Results: ¹H T₁ and ¹H T₁ρ relaxation times, indicative of matrix mobility and component miscibility in lyophilized systems, were measured for samples containing mAb and trehalose/sucrose using solid-state NMR. In the formulations with trehalose, the relaxation times, ¹H T₁ of both mAb and trehalose increased as a function of trehalose content. For instance, the ¹H T₁ values for trehalose and mAb were 1.47 sand 1.48 s in the 90mAb_10trehalose sample, increasing to 4.75 s and 4.6 s in the 10mAb_90trehalose formulation. This increase indicates greater matrix rigidity with higher trehalose levels. The similar T₁ values for mAb and trehalose suggest that the formulations are homogeneous on a 20–50 nm length scale. Similarly, comparable ¹H T₁ρ relaxation times between mAb and trehalose across all trehalose formulations indicate homogeneity on a smaller 2–5 nm scale. A similar trend was observed in sucrose-containing formulations, where both ¹H T₁ and ¹H T₁ρ relaxation times increased with sucrose content, suggesting that a higher weight fraction of sucrose also reduces matrix mobility. The similar relaxation times for mAb and sucrose in most formulations indicate homogeneity on both the 20–50 nm and 2–5 nm length scales. However, the 10mAb_90sucrose sample exhibited inhomogeneity on the 2–5 nm scale, as indicated by divergent ¹H T₁ρ relaxation times between mAb and sucrose. This may be attributed to a plasticizing effect of residual moisture, potentially leading to sucrose crystallization over time. In trehalose-containing samples, size exclusion chromatography (SEC) over six months revealed a correlation between protein content and monomer loss, as shown in Fig. 1. The 10mAb_90trehalose formulation retained the highest monomer content, while the 90mAb_10trehalose formulation showed the greatest loss. A similar trend was observed for sucrose-containing samples, with one exception: the 10mAb_90sucrose sample showed the highest monomer loss among all formulations, likely due to its high moisture content (1.36%), which may have promoted sucrose crystallization and subsequent mAb aggregation. Overall, ¹H T₁ and ¹H T₁ρ relaxation times offer mechanistic insights into the stability of lyophilized formulations. Longer and similar relaxation times for both mAb and trehalose/sucrose indicate a more rigid and homogeneous matrix, which supports protein stability. In contrast, formulations with lower trehalose/sucrose content (10%, 20%, and 30% w/w) remain homogeneous but exhibit monomer loss due to increased matrix mobility, as reflected by shorter relaxation times.

Conclusion: Solid-state NMR relaxation measurements (¹H T₁ and ¹H T₁ρ) effectively characterize matrix mobility and miscibility in lyophilized protein formulations. Formulations with higher trehalose/sucrose content exhibit reduced molecular mobility and more rigid, homogeneous matrices, which correlate with greater long-term protein stability. In contrast, formulations with lower sugar content, despite being miscible, show higher mobility and reduced stability. Additionally, moisture-induced crystallization, as seen in the 10mAb/90sucrose sample, can override the stabilizing effects of high sugar content. These findings demonstrate that ssNMR provides mechanistic insight and serve as a predictive tool for protein stability.

Acknowledgements: Munson Lab Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University The mAb was supplied by Merck & Co., Inc., Rahway, NJ, USA, and is gratefully acknowledged