Grasmeijer N, Stankovic M, de Waard H, Frijlink H W, Hinrichs W L J
Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, AV Groningen, The Netherlands.
Biochim Biophys Acta. 2013 Apr;1834(4):763-9. doi: 10.1016/j.bbapap.2013.01.020. Epub 2013 Jan 27.
The aim of this study was to elucidate the role of the two main mechanisms used to explain the stabilization of proteins by sugar glasses during drying and subsequent storage: the vitrification and the water replacement theory. Although in literature protein stability is often attributed to either vitrification or water replacement, both mechanisms could play a role and they should be considered simultaneously. A model protein, alkaline phosphatase, was incorporated in either inulin or trehalose by spray drying. To study the storage stability at different glass transition temperatures, a buffer which acts as a plasticizer, ammediol, was incorporated in the sugar glasses. At low glass transition temperatures (<50°C), the enzymatic activity of the protein strongly decreased during storage at 60°C. Protein stability increased when the glass transition temperature was raised considerably above the storage temperature. This increased stability could be attributed to vitrification. A further increase of the glass transition temperature did not further improve stability. In conclusion, vitrification plays a dominant role in stabilization at glass transition temperatures up to 10 to 20°C above storage temperature, depending on whether trehalose or inulin is used. On the other hand, the water replacement mechanism predominantly determines stability at higher glass transition temperatures.
玻璃化转变和水置换理论。尽管在文献中蛋白质稳定性常被归因于玻璃化转变或水置换，但这两种机制可能都发挥作用，应同时予以考虑。通过喷雾干燥将模型蛋白碱性磷酸酶掺入菊粉或海藻糖中。为研究在不同玻璃化转变温度下的储存稳定性，在糖类玻璃态中加入一种作为增塑剂的缓冲剂——2-氨基-2-甲基-1,3-丙二醇。在低玻璃化转变温度（<50°C）下，蛋白质的酶活性在60°C储存期间大幅下降。当玻璃化转变温度大幅高于储存温度时，蛋白质稳定性增加。这种稳定性增加可归因于玻璃化转变。玻璃化转变温度的进一步升高并未进一步提高稳定性。总之，根据使用的是海藻糖还是菊粉，在高于储存温度10至20°C的玻璃化转变温度下，玻璃化转变在稳定化过程中起主导作用。另一方面，在较高的玻璃化转变温度下，水置换机制主要决定稳定性。
