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Lysozyme-Sucrose Interactions in the Solid State: Glass Transition, Denaturation, and the Effect of Residual Water
Malmö University, Sweden.
Malmö University, Sweden; Lund University, Sweden.
Malmö University, Sweden; Lund University, Sweden: NovoNordiskA/S, Denmark.
Malmö University, Sweden; Lund University, Sweden.
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2023 (English)In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392Article in journal (Refereed) Epub ahead of print
Abstract [en]

The freeze-drying of proteins, along with excipients, offers a solution for increasing the shelf-life of protein pharmaceuticals. Using differential scanning calorimetry, thermogravimetric analysis, sorption calorimetry, and synchrotron small-angle X-ray scattering (SAXS), we have characterized the properties at low (re)hydration levels of the protein lysozyme, which was freeze-dried together with the excipient sucrose. We observe that the residual moisture content in these samples increases with the addition of lysozyme. This results from an increase in equilibrium water content with lysozyme concentration at constant water activity. Furthermore, we also observed an increase in the glass transition temperature (Tg) of the mixtures with increasing lysozyme concentration. Analysis of the heat capacity step of the mixtures indicates that lysozyme does not participate in the glass transition of the sucrose matrix; as a result, the observed increase in the Tg of the mixtures is the consequence of the confinement of the amorphous sucrose domains in the interstitial space between the lysozyme molecules. Sorption calorimetry experiments demonstrate that the hydration behavior of this formulation is similar to that of the pure amorphous sucrose, while the presence of lysozyme only shifts the sucrose transitions. SAXS analysis of amorphous lysozyme-sucrose mixtures and unfolding of lysozyme in this environment show that prior to unfolding, the size and shape of lysozyme in a solid sucrose matrix are consistent with its native state in an aqueous solution. The results obtained from our study will provide a better understanding of the low hydration behavior of protein-excipient mixtures and support the improved formulation of biologics. © 2023 The Authors. 

Place, publisher, year, edition, pages
American Chemical Society , 2023.
Keywords [en]
differential scanning calorimetry, glass transition, hydration, proteins, small-angle X-ray scattering, solid-state formulations
National Category
Physical Chemistry
Identifiers
URN: urn:nbn:se:ri:diva-66144DOI: 10.1021/acs.molpharmaceut.3c00403Scopus ID: 2-s2.0-85168498460OAI: oai:DiVA.org:ri-66144DiVA, id: diva2:1795465
Note

This research was funded by the Swedish Governmental Agency for InnovationSystems (VINNOVA) and was carried out within the competence center NextBioForm.Mr. Shuai Bai (Lund University) is thanked for freeze-dryinglysozyme−sucrose samples. The SAXS measurements were performed on the CoSAXS beam lineat MAX IV (Lund,Sweden) under the proposal 20200788. The research conducted at MAXIV, a Swedish national user facility, is supported by the Swedish Research Counci lunder contract 2018-07152, the Swedish Governmental Agency for Innovation Systems under contract No. 2018-04969, and Formas under contract 2019-02496.

Available from: 2023-09-08 Created: 2023-09-08 Last updated: 2023-09-08Bibliographically approved

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Millqvist-Fureby, Anna

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