The record-breaking rollout of the COVID-19 vaccines just nine months after the start of the pandemic was nothing short of miraculous. Soon enough, though, the cracks in the world’s distribution infrastructure began to show. The mRNA-based vaccines required incredibly low temperatures to remain viable, but as the vaccines rolled out to the public, reports of wasted doses that had thawed in transport peppered the news. 

Most vaccines and biologic-based drugs in use today require some form of cold storage and transport. Often, this cold chain infrastructure is not available in resource limited or remote locations, making effective vaccination campaigns and drug distribution difficult.

“Access to medicines is a big issue all around the globe, and to be honest, most of the cost of these medicines is wrapped up in how we store them,” said Maria Croyle, a pharmacist and drug formulation expert at the University of Texas at Austin.

With her experience using freeze drying to stabilize viruses for gene therapy, Croyle knew that there had to be a solution. One night, as she and her husband were watching a documentary about the preservative and stabilizing qualities of amber, it hit her.

“We were like, ‘Is that it? We need to create our own amber,’” she said. 

After years of research and thousands of different combinations of sugars, polymers, and surfactants, Croyle and her team developed a thin film technology that can encapsulate live virus vaccines, bacteria, gene therapies, and even antibody-based drugs. These thin films, which Croyle now also develops at her company Jurata Thin Film, remain stable at room temperature for months and can be administered orally, nasally, or resuspended in liquid for injection, revolutionizing the way vaccines and biologics are made and broadening their accessibility.

An unflavored gummy bear

When Croyle started her laboratory in 2000, she had her heart set on creating stable viral vectors for gene therapies, but two months into setting up her new office, a patient died in a high-profile gene therapy clinical trial, halting much of the funding to gene therapy research.

“I had to sit in my office and look at the wall and go, ‘What am I going to do now?’” she said. “Viruses are used for a lot of things, and vaccines is one of them, so we just turned the gears a little bit.”

Soon after, Croyle presented some of her formulation research at a conference where it caught the eye of Ebola researcher Heinz Feldmann who led the Special Pathogens Program of the National Microbiology Laboratory at the Public Health Agency of Canada at the time and now leads the Laboratory of Virology at the Rocky Mountain Laboratories for the National Institutes of Health.

“I didn’t really know anything about Ebola, except that it was scary,” said Croyle. As she and Feldmann spoke over the phone, he described the need for a vaccine that could be easily distributed to rural and remote areas without spoiling in the daytime humidity and heat or in the frigid evening temperatures characteristic of Africa and sub-Saharan Africa.

Most importantly, he said, they needed a vaccine that didn’t require a needle. Injectable vaccines require trained medical personnel to administer them, increasing the resources needed and cost associated with vaccination.

Croyle and her team got to work. They tried thousands of different combinations of materials until they finally found a mix that worked. She and her team then spread the mix over molds that looked like little contact lenses and allowed them to dry. 

Each mold represented a single dose, but “it could potentially contain enough vaccines for ten doses. If we needed something for 1000 doses, it would essentially be the size of the sheet of paper,” Croyle added. “It is definitely a resource saving approach.”

Croyle described the final film as “not fully solid, and it's not a jelly. It's somewhere in between.” Because one of the components is a sugar compound, the oral formulation of the films tastes a little sweet.

“I tasted it. To my knowledge, I'm the only person who admitted [to tasting] it,” Croyle laughed. “It's very pleasant. It's almost like an unflavored gummy bear.”

They tested their adenovirus-based Ebola thin film vaccine in mice and guinea pigs and saw that it protected the animals from an Ebola infection (1). They then moved into nonhuman primates and found that when they delivered their vaccine nasally, all of the vaccinated macaques survived a lethal dose of Ebola (2).

“I remember seeing the data, and I was like, ‘Wow, this is so cool!’” said Gary Kobinger, a virologist then at the Public Health Agency of Canada who was one of Croyle’s early collaborators and coauthors. He now leads the Galveston National Laboratory at the University of Texas. “She was really a trailblazer,” he added. “At the time, she was the only one that I knew who was looking at really detailed formulation of vaccines to improve their potency.”

As Croyle and her team prepared their nonhuman primate vaccine results for publication, the 2014 Ebola outbreak in West Africa had just reached the United States.

“We saw that happening, and it increased our sense of urgency and also validated that we were doing the right thing. This is something that can be useful,” said Croyle. While their vaccine performed well in macaques, it was not ready to treat people in that Ebola outbreak, but Croyle and her team knew that they had made something important.

“At that point, we were like, ‘Well, it's great. We made an Ebola vaccine, but what else could this be used for?”

Three-year-old vaccines and stable gene therapy

To find more applications for their thin films, Croyle and her team simply looked in their laboratory freezers.

“What do you keep in the -80 freezer or a -20 freezer even, and what can we do to move that to room temperature stability?” she asked.

They mixed antibodies, bacteria, and viruses into their film solutions, and they found that they could stabilize them at room temperature for months (3). Even when they made adjustments to their film formula to accommodate viruses other than adenoviruses, such as influenza, they found that mice vaccinated with an influenza-optimized thin film vaccine developed a strong neutralizing antibody response.

To be honest, at the three-year mark, we sort of forgot about it. My lab manager husband was walking through the lab, and he's like, ‘Hey, there's still some films in here!’ 
– Maria Croyle, University of Texas at Austin and Jurata Thin Film

Most exciting, though, was the moment when they realized that they still had some films of their original Ebola vaccine formulation that had been sitting at room temperature for the past three years. During their original experiments, they had made big batches of films and tested a few at a time at regular timepoints to assess their activity.

“To be honest, at the three-year mark, we sort of forgot about it. My lab manager husband was walking through the lab, and he’s like, ‘Hey, there’s still some films in here!’” said Croyle. When they checked these forgotten films, the films still released the active adenovirus vaccine (3). “The three-year time point really led me to believe that, okay, I think we have something.”

Partnering with the microbiologist and immunologist Jude Samulski and biotech entrepreneur Sheila Mikhail, Croyle founded the company Jurata Thin Film to develop the technology further. The name “Jurata,” she noted, is the name of a village off the coast of Poland in the Baltic Sea that is the world’s largest source of natural amber.

With the team’s expertise in thin film formulations, they want to help stabilize technology like vaccines and gene therapies that other groups had developed. They recently partnered with scientists at the gene therapy company AskBio to use the thin film technology to stabilize one of their adeno-associated virus (AAV) vectors.

Because scientists typically deliver gene therapy via injection, the Jurata Thin Film team needed to figure out how to convert a film that was originally designed for an oral administration to an injectable one. When they rehydrated the original formulation, it had the consistency of maple syrup, which was much too viscous to inject.

“We had to find a sweet spot where we could reduce that viscosity and make it easy to inject, but also maintain the stability that we needed at room temperature. And, we did that,” said Croyle. The team then mixed their reconfigured films with AskBio’s AAV, made the films, put them in a Ziploc bag, and mailed them in a standard envelope from Croyle’s laboratory in Texas to the AskBio scientists in North Carolina.

Scientists typically store gene therapy vectors at -80°C, and once these vectors thaw to room temperature, they must be used immediately. Re-freezing and thawing them destroys their activity. Instead, when stored in the thin films, the AAVs endured 16 successive freeze-thaw cycles and still retained 100% activity (4).

“When we started testing this thin film formulation, we saw that there was a broad range of temperatures where we could preserve the potency of the vector. We were super excited about it,” said Anna Tretiakova, a scientist at AskBio and a coauthor of the paper.

Tretiakova and her team then injected mice with the thin film formulation that had been stored at either 4°C for 150 days or at 25°C for 100 days. When the scientists compared the performance of the films with the standard AAV formulation stored at -80°C, they found that the biodistribution and transgene expression of the AAVs from the thin films were just as good as the standard AAVs kept at -80°C.

The temperature stability of the AAVs in the thin films will make shipping gene therapies to scientific collaborators and clinical trial sites much less expensive by eliminating the need for freezing during shipping.

“Sometimes you have collaborators in Europe, and you have to ship on dry ice. If the box gets held up at customs, your dry ice could evaporate, and you could lose the entire prep,” said Tretiakova. “A lot of these logistical challenges could simply be eliminated with this technology.”

Less breath strip, more contact lens

The next step for Croyle and her thin films is manufacturing. At the moment, her lab is well-equipped to create the films and send them to collaborators for testing specific applications.

“We can come in at say eight o'clock in the morning, mix the film matrix with the vaccine, and spread it out in molds,” said Croyle. “It's ready to peel by two o'clock in the afternoon, package, and we can hit the last FedEx pickup of the day. It really is rapidly deployable.” But, she added, that pipeline is not going to be sufficient for long. “It's cute that everything happens in my lab, but can we really make 50,000 doses a day or more?”

It's cute that everything happens in my lab, but can we really make 50,000 doses a day or more? 
– Maria Croyle, University of Texas at Austin and Jurata Thin Film

She and her colleagues at Jurata Thin Film reached out to multiple manufacturers to find a way to produce the thin films at scale. Most manufacturing steps involve freeze drying, but because the thin films don’t need to be frozen, those facilities wouldn’t work. On the other end of the spectrum, Croyle and her team talked with people who make oral films like breath strips that dissolve on the tongue, but those manufacturing steps required very high temperatures that would kill their virus-based vaccine or gene therapy.

In the end, Croyle found a surprising partner.

“We actually were paired with a contact lens maker who understands what we do,” she said. “By the end of [January 2023], we will have a benchtop machine where we can now make films that are packaged properly.” Like disposable contact lenses, the thin films will be encased in a peelable foil container. Croyle has set the goal of having a pilot manufacturing setup in place by the end of the summer of 2023.

While the injectable drugs and vaccines are the standard mode of administration today, Croyle has not given up on oral formulations yet. In her academic laboratory, she and her team are working on a grant that she named, “Cinnamon and Spice as Adjuvants are Nice?” based on an idea sparked by one of her pharmacy students. 

The student was originally from Nigeria, and she mentioned that the common flavorings like bubblegum and strawberry that American companies use to flavor their medications are extremely off-putting to the African palette. Instead, flavors like cinnamon were much easier to tolerate. Another one of Croyle’s students then looked through the scientific literature and found that different types of flavorings can trigger a small, but non-harmful, inflammatory response in the mouth.

“We have actually found some things like cinnamon, menthol, and things like that, that can increase the potency of vaccines that are given by the oral route, so that's also very exciting,” said Croyle.

For Croyle, the most rewarding part of her work on thin film technology has been to see it become a reality. She described seeing the recently constructed Tesla Gigafactory in Austin, and “when we drove by it, I started to tear up, and my husband's like, ‘What's wrong with you?’” Croyle laughed. “I said, ‘That was somebody's dream. And it's there. It's real.’ So, I think as we go through different stages of Jurata, it's just very gratifying.”

As the scientists in her lab and at Jurata Thin Film gear up to manufacture more of their thin films and eventually test them in clinical trials, they expect to see even more happy tears from their leader.

“Everybody's like, ‘If that was your thing with the Gigafactory, what are you going to do when we do our first clinical trial?’” Croyle said. “I'm sure there'll be some emotion there as well.”

References

1. Choi, J.H. et al. A Single Sublingual Dose of an Adenovirus-Based Vaccine Protects against Lethal Ebola Challenge in Mice and Guinea Pigs. Mol Pharmaceutics  9, 156-167 (2012).
2. Choi, J.H. et al. A Single Dose Respiratory Recombinant Adenovirus-Based Vaccine Provides Long-Term Protection for Non-Human Primates from Lethal Ebola Infection. Mol Pharmaceutics  12, 2712-2731 (2015).
3. Bajrovic, I. et al. Novel technology for storage and distribution of live vaccines and other biological medicines at ambient temperature. Sci Adv  6, eaau4819 (2020).
4. Doan, T.N.K. et al. Thermostability and in vivo performance of AAV9 in a film matrix. Commun Med  2, 148 (2022).