Protein Thermal Stabilization and Delivery via Adsorption onto Porous Silicas

Abstract

The cold chain is a problem that affects the whole pharmaceutical industry: in 2019, 45% of new FDA approved drugs required refrigeration or freezing. It is estimated that biopharma loses around 35 billion dollars every year due to failures in temperature control logistics. Proteins, the main components of vaccines, enzymes and many pharmaceuticals, are sensitive to denaturing and losing their conformational structure when exposed to thermal stress. This is why most pharmaceutical products require a temperature-controlled supply chain to ensure that heat exposure does not disrupt the intermolecular interaction needed to maintain protein structure and activity. The aim of this study is to develop a more sustainable technology via protein adsorption onto mesoporous silica, in order to improve the protein’s thermal stability. Two proteins were investigated: Invasion Plasmid Antigen D (IpaD) and Lactoferrin (Lf). Circular dichroism (CD) confirmed that the adsorbed IpaD and Lf after the heat treatment maintained a native secondary structure rich in α-helical content. In contrast, the unprotected proteins after heat treatment lost their secondary structure. Isotherms modeled using Langmuir, Freundlich and Temkin models demonstrated that the adsorption of IpaD and Lf onto silicas is best fit by the Langmuir model. Silica pore size was determined to be a key factor in protein adsorption. If pores are less than 15 nm, adsorption is negligible. If the pores are between 15-25 nm, then monolayer coverage is achieved and IpaD is protected from thermal denaturing. If pores are larger than 25 nm the adsorption is multilayer coverage and it is easier to remove the protein from the silica due to a less developed hydrogen bond network. In addition, this work investigates the protein release from silica using non-ionic detergents (Triton X-100, Tween 20, 40 and 80). Lactoferrin desorption tests with non-ionic detergents showed a trend for increased Lf recovery with increased detergent hydrophobicity. Triton X-100 and Tween 80 were the best detergents for Lf desorption, recovering over 80% of the initial protein. This research provides strong evidence that proteins can be thermally stabilized on mesoporous silica and efficiently released from the silica using non-ionic detergents.