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<p>Lyophilization is widely used to stabilize and enhance the shelf-life of protein drug products, including monoclonal antibodies (mAbs), bi- and tri-specific antibodies, antibody fragments, enzymes, etc. Disaccharides such as trehalose and sucrose are commonly used as stabilizers in lyophilized protein formulations. For these disaccharide excipients to be good stabilizers, it requires formation of rigid, immobile matrix where proteins are homogenously distributed. In this study, we investigated the relationship between molecular mobility, homogeneity, and long-term stability in lyophilized formulations of a mAb with trehalose or sucrose as stabilizers. We used solid-state nuclear magnetic resonance spectroscopy (ssNMR) to measure <sup>1</sup>H T₁ and <sup>1</sup>H T₁<sub>ρ</sub> relaxation times, probing the mobility and homogeneity of the components at 20–50&#xa0;nm and 2–5&#xa0;nm scales, respectively. Formulations with higher disaccharide content showed longer <sup>1</sup>H T₁ relaxation times and greater monomer retention, consistent with a more rigid glassy matrix and enhanced stability. Relaxation times of mAb and disaccharide were closely matched in most formulations, indicating good homogeneity, which was essential for stability. However, exceptions were observed in samples where disaccharide crystallization occurred, leading to phase separation and inhomogeneity in the lyophilized matrix. Despite having long <sup>1</sup>H T₁ times, these samples showed reduced stability, underscoring the importance of maintaining both low mobility and homogeneous amorphous structure. Overall, this study demonstrates that ssNMR relaxation measurements are effective predictors of lyophilized protein stability as they provides information on both matrix rigidity and component homogeneity in the solid state.</p>

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Predicting the stability of lyophilized formulations containing a monoclonal antibody and sucrose/trehalose using solid-state NMR spectroscopy

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月6日

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  • Rachana Sapkota,
  • Isabella Perez Meija,
  • Cole W. Tower,
  • Tianyun Zhang,
  • Harshil K. Renawala,
  • Eric J. Munson

摘要

Lyophilization is widely used to stabilize and enhance the shelf-life of protein drug products, including monoclonal antibodies (mAbs), bi- and tri-specific antibodies, antibody fragments, enzymes, etc. Disaccharides such as trehalose and sucrose are commonly used as stabilizers in lyophilized protein formulations. For these disaccharide excipients to be good stabilizers, it requires formation of rigid, immobile matrix where proteins are homogenously distributed. In this study, we investigated the relationship between molecular mobility, homogeneity, and long-term stability in lyophilized formulations of a mAb with trehalose or sucrose as stabilizers. We used solid-state nuclear magnetic resonance spectroscopy (ssNMR) to measure 1H T₁ and 1H T₁ρ relaxation times, probing the mobility and homogeneity of the components at 20–50 nm and 2–5 nm scales, respectively. Formulations with higher disaccharide content showed longer 1H T₁ relaxation times and greater monomer retention, consistent with a more rigid glassy matrix and enhanced stability. Relaxation times of mAb and disaccharide were closely matched in most formulations, indicating good homogeneity, which was essential for stability. However, exceptions were observed in samples where disaccharide crystallization occurred, leading to phase separation and inhomogeneity in the lyophilized matrix. Despite having long 1H T₁ times, these samples showed reduced stability, underscoring the importance of maintaining both low mobility and homogeneous amorphous structure. Overall, this study demonstrates that ssNMR relaxation measurements are effective predictors of lyophilized protein stability as they provides information on both matrix rigidity and component homogeneity in the solid state.