Ensilication and Thermal Stability of Proteins

Abstract

Stability of biological substances based on proteins, including vaccines, antibodies, and enzymes, is critically linked to its thermal environment. Temperature stress over time results in protein denaturation. Denaturation is a loss of structure and function in proteins. Their storage and distribution therefore relies on a “cold chain” of continuous refrigeration, which is costly and not always effective in medical and biological applications. Due to this issue, access to insulin for treating diabetes is still beyond the reach of millions of people around the world [1]. Diabetes is predicted to be the 7th leading cause of death in the next decade [2].This project therefore sought to develop an innovative protocol of “ensilication” ef- fectively enclosing protein in a deposited silica “cage” to protect the protein from the denaturing process. To evaluate this aim, a release protocol involving treatment with a dilute solution of acidified NaF was designed to release the ensilicated proteins into solution. As test subject, covalently bonded sol-gel silica network was first exam- ined to surround lysozyme, then applied to haemoglobin, a heterotetrameric protein with a complex tertiary and quaternary structure. Remarkably, insulin is profitably ensilicated (80%) here along with a mediator, chitosan.The mechanism for the formation of protein ensilication was investigated using Syn- chrotron SAXS and DLS. The structures of ensilicated protein were confirmed using multiple microscopy and spectrometry methods. In order to test the product’s stabil- ity, the ensilicated protein was subjected to heating at 100 ◦C for hours or long-term ambient temperature storage. Analysis of the proteins released from their ensilica- tion with a wide range of methods including SAXS, ELISA, CD and FT-IR revealed that even after exposure to such extreme temperatures, the protein structure remains consistent with that of the native protein. The results demonstrate that the process produces a storable solid protein-loaded material directly from solution, and may thus be suitable for use with proteins that do not tolerate lyophilisation. Ensilication offers the prospect of a solution to the “cold chain” problem for biological materials, such as insulin.

Date of Award	20 Dec 2017
Original language	English
Awarding Institution	University of Bath
Supervisor	Asel Sartbaeva (Supervisor) & Karen Edler (Supervisor)