Researchers at the University of Bath, working with collaborators at the University of Newcastle, have developed a method for stabilizing proteins that could remove the need to refrigerate life-saving vaccines during transportation and storage, and potentially prevent half of the world’s vaccine doses from having to be discarded. The team’s technique for tailor-fitting a vaccine with a silica coat—a process known as ensilication—could effectively increase access to vaccines for millions of children around the world. Having demonstrated the technology in lab settings two years ago, the researchers have now reported on experiments in mice, which have shown how the ensilication technology can stabilize vaccines in real-world settings.
“This is really exciting data because it shows us that ensilication preserves not just the structure of the vaccine proteins but also the function—the immunogenicity,” said project leader Asel Sartbaeva, PhD, at University of Bath’s department of chemistry. Sartbaeva and colleagues reported on in vivo tests with the ensilication technology, in a paper published in Scientific Reports, titled, “Ensilicated tetanus antigen retains immunogenicity: in vivo study and time-resolved SAXS characterization.”
Many biopharmaceuticals are proteins that are not stable ex vivo, the authors wrote. As a result, they must be stabilized to increase shelf-life, and this is commonly achieved by regulating the temperature at which they are stored and transported. For example, vaccine formulations that are stable at 2–8°C can be ineffective at higher temperatures or if they are frozen and then thawed, because the proteins become unstable and start to denature. This can mean that millions of children around the world miss out on life-saving inoculations because of the challenges of end-to-end cold chain distribution, which requires a vaccine to be refrigerated from the moment of manufacturing to the endpoint destination.
“Within cold chain transportation, from manufacturing to endpoint destination, temperature control has proven challenging,” the team noted. “Fluctuations in temperature, heating/freezing, can affect biopharmaceuticals resulting in aggregation and protein unfolding, leading to loss of potency. This is a serious issue in global public health, as it is a major hindrance to achieving universal childhood vaccination worldwide.”
The ensilication technology developed by the U.K. scientists effectively wraps the proteins in a silica shell, which prevents the molecules from degrading if they are heated, even up to 100°C, or stored at room temperature for up to three years.
For their newly reported studies with the technology, the team sent both ensilicated and regular samples of the tetanus vaccine from Bath to Newcastle through the postal system. It’s a journey of over 300 miles, which by post takes a day or two. The tetanus toxin C fragment (TTCF) is a component of the tetanus toxoid used in the diphtheria, tetanus, and pertussis (DTP) vaccine.
Doses of either the ensilicated, or unprotected vaccine were then injected into mice, to see if they were still effective. To confirm the protective capacity of ensilication in vivo, we immunized mice with native or denatured TTCF, or with TTCF after ensilication, and obtained serum samples. The ensilicated material used during this study was stored in powdered form for one month at room temperature and then transported using commercially available means without any specialized equipment,” the investigators wrote.
The results showed that while the ensilicated vaccine triggered an immune response in the recipient animals, demonstrating that it was still active, no immune response was detected in mice injected with unprotected doses of the vaccine, indicating that it had been damaged in transit. “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,” the researchers commented. “Using TTCF as a model, we demonstrate that ensilication could be a solution to overcome the challenges of biopharmaceutical ‘cold chain’ transportation.”
They separately used a technology called time-resolved Small Angle X-ray Scattering (SAXS) to investigate the formation of TTCF-silica nanoparticles, with a view to providing some insight into the ensilication process and its protective effect on proteins. “Our results reveal ensilication to be a staged diffusion-limited cluster aggregation (DLCA) type reaction,” they explained. “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.”
The researchers also pointed out that while ensilication can be used to preserve proteins, the current technology requires laboratory conditions for the silica removal and subsequent dialysis, which wouldn’t be suitable for real-world clinical situations, particularly in low-income countries. However, they noted, “for injectable bioharmaceuitcals, such as vaccines, we are working toward alternative release methods included in an all-in-one device which we hope to prototype in the near future.”
“This project has focused on tetanus, which is part of the DTP (diphtheria, tetanus, and pertussis) vaccine given to young children in three doses,” added Sartbaeva. “Next, we will be working on developing a thermally stable vaccine for diphtheria, and then pertussis. Eventually, we want to create a silica cage for the whole DTP trivalent vaccine, so that every child in the world can be given DTP without having to rely on cold chain distribution.”
Silica is an inorganic, nontoxic material, and Sartbaeva estimates that ensilicated vaccines could be used for humans within 5–15 years. She hopes that applying the technology to silica-wrap proteins will eventually be adopted to store and transport all childhood vaccines, as well as other protein-based products, such as antibodies and enzymes. “Ultimately, we want to make important medicines stable so they can be more widely available,” she said. “The aim is to eradicate vaccine-preventable diseases in low-income countries by using thermally stable vaccines and cutting out dependence on cold chain.”