Size and molecular flexibility of sugars determine the storage stability of freeze-dried proteins

Tonnis, W. F., Mensink, M. A., de Jager, A., Maarschalk, K. V. D. V., Frijlink, H. W. & Hinrichs, W. L. J., Mar-2015, In : Molecular pharmaceutics. 12, 3, p. 684-694 11 p.

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  • Size and Molecular Flexibility of Sugars Determine the Storage Stability of Freeze-Dried Proteins

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Protein-based biopharmaceuticals are generally produced as aqueous solutions and stored refrigerated to obtain sufficient shelf life. Alternatively, proteins may be freeze-dried in the presence of sugars to allow storage stability at ambient conditions for prolonged periods. However, to act as a stabilizer, these sugars should remain in the glassy state during storage. This requires a sufficiently high glass transition temperature (Tg). Furthermore, the sugars should be able to replace the hydrogen bonds between the protein and water during drying. Frequently used disaccharides are characterized by a relatively low Tg, rendering them sensitive to plasticizing effects of residual water, which strongly reduces the Tgvalues of the formulation. Larger sugars generally have higher Tgs, but it is assumed that these sugars are limited in their ability to interact with the protein due to steric hindrance. In this paper, the size and molecular flexibility of sugars was related to their ability to stabilize proteins. Four diverse proteins varying in size from 6 kDa to 540 kDa were freeze-dried in the presence of different sugars varying in size and molecular flexibility. Subsequently, the different samples were subjected to an accelerated stability test. Using protein specific assays and intrinsic fluorescence, stability of the proteins was monitored. It was found that the smallest sugar (disaccharide trehalose) best preserved the proteins, but also that the Tgof the formulations was only just high enough to maintain sufficient vitrification. When trehalose-based formulations are exposed to high relative humidities, water uptake by the product reduces the Tgs too much. In that respect, sugars with higher Tgs are desired. Addition of polysaccharide dextran 70 kDa to trehalose greatly increased the Tgof the formulation. Moreover, this combination also improved the stability of the proteins compared to dextran only formulations. The molecularly flexible oligosaccharide inulin 4 kDa provided better stabilization than the similarly sized but molecularly rigid oligosaccharide dextran 6 kDa. In conclusion, the results of this study indicate that size and molecular flexibility of sugars affect their ability to stabilize proteins. As long as they maintain vitrified, smaller and molecularly more flexible sugars are less affected by steric hindrance and thus better capable at stabilizing proteins.
Original languageEnglish
Pages (from-to)684-694
Number of pages11
JournalMolecular pharmaceutics
Issue number3
Publication statusPublished - Mar-2015


  • lyophilization, oligosaccharides, mobility, dextran, trehalose, inulin, stabilization, vitrification, water replacement, steric hindrance, GLASS-TRANSITION, SOLID-STATE, STABILIZATION, TREHALOSE, DYNAMICS, DEXTRAN, PHARMACEUTICALS, SPECTROSCOPY, MECHANISMS, PARTICLES

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