Biological substances based on proteins, including vaccines, antibodies, and enzymes, typically degrade at room temperature over time due to denaturation, as proteins unfold with loss of secondary and tertiary structure. Their storage and distribution therefore relies on a “cold chain” of continuous refrigeration; this is costly and not always effective, as any break in the chain leads to rapid loss of effectiveness and potency. Efforts have been made to make vaccines thermally stable using treatments including freeze-drying (lyophilisation), biomineralisation, and encapsulation in sugar glass and organic polymers. Here for the first time we show that proteins can be enclosed in a deposited silica “cage”, rendering them stable against denaturing thermal treatment and long-term ambient-temperature storage, and subsequently released into solution with their structure and function intact. This “ensilication” method produces a storable solid protein-loaded material without the need for desiccation or freeze-drying. Ensilication offers the prospect of a solution to the “cold chain” problem for biological materials, in particular for vaccines.

There is an urgent need for the development of vaccine thermostabilisation methodologies as the maintenance of a continuous and reliable cold chain remains a major hurdle to the global distribution of safe and effective vaccines. Ensilication, a method that encases proteins in a resistant silica cage has been shown to physically prevent the thermal denaturation of a number of model proteins. In this study we investigate the utility of this promising approach in improving the thermal stability of antigens and vaccine conjugates highly relevant to the development of candidate tuberculosis vaccines, including antigen 85b conjugated with the Staphylococcus aureus -protein based adjuvant Sbi. Here we analyse the sensitivity of these constructs to thermal denaturation and demonstrate for the first time the benefits of ensilication in conferring these vaccine-relevant proteins with protection against temperature-induced loss of structure and function without the need for refrigeration. Our results reveal the potential of ensilication in facilitating the storage and transport of vaccines at ambient temperatures in the future and therefore in delivering life-saving vaccines globally, and in particular to remote areas of developing countries where disease rates are often highest.

our recently developed ensilication approach can physically stabilize proteins in silica without use of a pre-formed particle matrix. Stabilisation is done by tailor fitting individual proteins with a silica coat using a modified sol-gel process. Biopharmaceuticals, e.g. liquid-formulated vaccines with adjuvants, frequently have poor thermal stability; heating and/or freezing impairs their potency. As a result, there is an increase in the prevalence of vaccine-preventable diseases in low-income countries even when there are means to combat them. one of the root causes lies in the problematic vaccine 'cold chain' distribution. We believe that ensilication can improve vaccine availability by enabling transportation without refrigeration. Here, we show that ensilication stabilizes tetanus toxin c fragment (ttcf), a component of the tetanus toxoid present in the diphtheria, tetanus and pertussis (Dtp) vaccine. experimental in vivo immunization data show that the ensilicated material can be stored, transported at ambient temperatures, and even heat-treated without compromising the immunogenic properties of TTCF. To further our understanding of the ensilication process and its protective effect on proteins, we have also studied the formation of ttcf-silica nanoparticles via time-resolved Small Angle X-ray Scattering (SAXS). Our results reveal ensilication to be a staged diffusion-limited cluster aggregation (DLcA) type reaction. An early stage (tens of seconds) in which individual proteins are coated with silica is followed by a subsequent stage (several minutes) in which the protein-containing silica nanoparticles aggregate into larger clusters. our results suggest that we could utilize this technology for vaccines, therapeutics or other biopharmaceuticals that are not compatible with lyophilization. Biopharmaceuticals are biologically derived active compounds intended for therapeutic or diagnostic usage. Examples include monoclonal antibodies, vaccines, cells and blood components. Many of these are proteins and not stable ex vivo. They must be stabilised to increase their shelf-life. The main approach in stabilisation of biopharmaceuticals is regulating temperature. Maintaining low (2-8 °C) temperatures retains native states of these compounds. Many protein-based biopharmaceuticals are vulnerable not only to heating but also to freezing 1-3 , which can disrupt protein structure and lead to denaturation on thawing. Within cold chain transportation 1,2,4-7 , from manufacturing to endpoint destination, temperature control has proven challenging. Fluctuations in temperature, heating/freezing 1 , can affect biopharmaceuticals resulting in aggregation and protein unfolding, leading to loss of potency. This is a serious issue in global public health 8-10

Ensilication is a novel method of protein thermal stabilisation using silica. It uses a modified sol–gel process which tailor fits a protective silica shell around the protein to enable room temperature storage of biopharmaceuticals.

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