The Challenges of Vaccine Transport and Storage
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The COVID-19 pandemic brought global attention to the process of developing a new vaccine – and with it, the crucial role of cold chain technologies for shipping and storage. Although different approaches to vaccine development were used, they all share a unifying challenge – the need for storage at low temperatures.
The need for refrigeration or ultra-low temperature storage for biological drugs and vaccines is not new, but demand increased considerably during the COVID-19 pandemic. This meant the cold chain industry needed to rapidly scale up production of equipment like ultra-low freezers while ensuring their reliability, a demand that has continued. It has also stimulated innovation in the industry, with consumers looking for more compact products with better energy efficiency and sophisticated monitoring systems to alert users of temperature fluctuations. For developing countries, where cold-chain infrastructure challenges predate the pandemic, researchers are pioneering new technologies such as solar-powered fridges to help prolong vaccine shelf-life.
Despite this progress, vaccines that are more stable and can be shipped and stored without refrigeration remains a key goal, and a wide range of approaches are being explored to achieve it.
Innovations in vaccine storage and shipping
Dr. Maria Croyle, professor of molecular pharmaceutics and drug delivery at the University of Texas, USA, is developing film matrices that could be used to transport live viruses without the cold chain. Her team recently set out to stabilize the adeno-associated virus (AAV) so it could be shipped without the need for dry ice or cold packs.1 There are several Food and Drug Administration (FDA) approved AAV products on the market; each must be stored and shipped frozen, and given to the patient within eight hours after thawing. If they thaw on the way to the clinic they must be discarded.
“We’re the first group to document shipment of an AAV vector across the US in a simple envelope without any cold packs or dry ice, and have the embedded virus deliver the genes in mice just as well as freshly prepared virus,” Croyle says. “We were also able to store live virus in the films for up to six months at room temperature without a reduction in their ability to deliver genes.”
One of the challenges Croyle’s lab need to overcome is the viscosity of the film formulation. “The formulation used to make the film matrix currently has the consistency of maple syrup, which cannot be easily pushed through a syringe,” says Croyle. “To address this issue, we had to modify the formulation to reduce the viscosity but still stabilize the virus at room temperature.” Unfortunately, the stability profile was better in the viscous formulation, but this taught the team something about the environment that AAV needs to remain stable. When they took virus stabilized in the viscous formulation and diluted it to the required dose, the dilution itself reduced the viscosity making it easier to push through a syringe.
They now plan to conduct additional testing in larger animals before the formulation is implemented in an AAV product currently in clinical testing. “The concept is not limited only to AAV vectors,” says Croyle. “We’re also working with collaborators to stabilize mRNA-based vaccines and a variety of livestock vaccines within our film, and plan to have a pilot production line built next year allowing us to mass produce the films in a rapid fashion.”
Storage challenges with RNA vaccines
The approval of the first messenger RNA (mRNA) vaccine for COVID-19 has opened the door to a new wave of RNA-based therapeutics. But the nature of RNA as a large, transient molecule that must be completely intact to function properly makes its stability a challenge for vaccine developers.
“mRNA is unstable because Nature designed it that way,” says Dr. Daan Crommelin, emeritus professor of pharmaceutical science at the University of Utrecht, the Netherlands. “These are large molecules and it takes just one break in a nucleotide chain to completely lose their activity.” Different solutions are being explored to address this issue, says Crommelin, who co-authored a recent review on the thermostability and storage of these products.2 “One approach is to look at whether you can stabilize the mRNA-lipid-nanoparticle (LNP) structure through freeze-drying and optimizing the choice of excipients such as lyoprotectants, or there’s a second option where you explore alternative ways of formulating the end product”, as discussed below.
The freeze-drying approach, lyophilization, is a technically challenging process, but it’s one being hotly pursued by companies in the field of RNA therapeutics. Kenneth Chien, professor in cell and molecular biology at the Karolinska Institute in Sweden, co-founder of Moderna and a member of eTheRNA’s board of directors, says that the capability to lyophilize complex mRNA molecules is already here, and he hopes to see this new technology available to everyone developing mRNA therapeutics, ushering in a new wave of vaccines and RNA therapeutics. “In a pandemic, where you need to get the vaccine to different locations that are challenging because of the current cold-chain requirements, the need for lyophilization is clear, but we also see there being a demand for rare diseases too, where manufacturers will need to make larger batches of product and store them because patients are distributed all over the world.”
How To Protect Frozen Patient Samples in the Event of a Freezer Malfunction
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Optimizing storage for LNPs
While the community waits for freeze-drying solutions, in the Irvine lab at the Massachusetts Institute of Technology, Dr. B.J. Kim had an idea for a summer project for her students, which ended up generating some key insights for optimizing LNP storage.3
“We were trying to find an effective way to store the RNA vaccines that we make in the lab, because we were continuously having to make them fresh for our research, and sometimes the timing for experiments didn’t work out,” she explains. “We also needed to ship vaccines out for experiments at other facilities, so we needed a validated way to store the particles. It just so happened that the pandemic hit us soon after starting this project, and we ended up yielding some very timely and important results.”
They systematically varied parameters known to be important: storage temperature, concentration of cryoprotectant and the type of buffer and then compared the immune response elicited by the vaccine under the different storage conditions with freshly prepared vaccine LNPs. “We found that for the particular LNP-RNA vaccines that we make in the lab, storing the particles in 10% w/v sucrose, with phosphate-buffered saline and at -20°C was the optimal method, with storage temperature being the most dominant factor,” explains Kim. This validated what has been seen with the COVID-19 mRNA vaccines, but it also revealed other important insights. “We realized that just looking at the size of particles before and after storage is not a good indicator of the vaccine functionality, because you may get RNA leakage or degradation even though the structural integrity of the particle remains.”
In addition to mRNA, Kim and colleagues also carried out the work using self-replicating or self-amplifying RNA, which makes copies of itself once inside the body, amplifying production of the encoded protein. These RNA molecules are about 10 times longer than a typical mRNA sequence. “We found there are some differences in storage properties between mRNA and self-replicating RNAs that we saw most clearly at -80°C,” says Kim. “While the mRNA-loaded LNPs were stored just fine at -80°C, the self-replicating RNA-loaded LNPs aggregated catastrophically.” The team believes it’s probably related to differences in how the lipids pack together and organize with the RNA, and while there’s not yet a good technology for investigating the internal structure of an LPN, they are keen to study this phenomenon further.
Other approaches to improve thermostability
Another approach to tackling the thermostability issues of RNA is to have on-the-spot mixing of the two main components – the mRNA in one vial, and a carrier system in another. “If you take positively charged colloidal systems that are eager to pick up the mRNA, then this can be mixed at the bedside,” says Crommelin. “This is a particularly good alternative for cancer vaccines that are patient-specific, because otherwise you have to make LNPs for each patient every time they need them, which makes it more laborious, costly and wasteful. It’s easier to mix them as you need.”
The Access to Advanced Health Institute (AAHI), a non-profit biotech research institute focused on global health, has taken this carrier concept a step further and created a self-amplifying RNA vaccine against COVID-19 that can be freeze-dried and is stable for at least 6 months at room temperature, and at least 10 months if refrigerated.4 The vaccine uses a nanostructured lipid carrier (NLC) that was first developed to deliver a Zika virus vaccine.5 The carrier is mixed with the self-amplifying RNA and then used immediately or freeze-dried. After freeze-drying and storage at either 4 °C or 25 °C the vaccine was still able to elicit an immune response – generating specific mouse IgG antibodies against SARS-CoV-2 – to a level comparable with the freshly mixed vaccine.
“We focus on the practical and perhaps less glamorous side of vaccine science,” says Dr. Emily Voigt, principal scientist at AAHI and lead author of the study: “We realized before the pandemic that while RNA vaccines had significant benefits for pandemic response, significant drawbacks – particularly their complexity of manufacturing and stability – would cause them to be difficult to manufacture and distribute worldwide. So, we focused on developing a simpler, potent, and stable RNA vaccine technology that we applied to Covid.” The vaccine is now in clinical trials in South Africa, and is straightforward to manufacture at existing vaccine manufacturing facilities worldwide without specialty equipment. “Our goal for this platform is to get our vaccine products to the people who need them for this and for the next pandemic, keeping cost and stability in mind,” Voigt adds.
Looking to the future
To truly unlock the potential of mRNA and other RNA vaccines, the technology to manufacture them will need to be more widely accessible across the globe to reduce the burden on the cold chain. “It’s more likely that vaccines will be made on site in areas where the pandemic begins,” says Chien. “My prediction is we’ll see manufacturing facilities springing up in China, Southeast Asia and in Africa because the capabilities of making GMP grade mRNA will be faster, cheaper and more widely available.”
Although these facilities will still require aspects of the cold chain for shipping and storing vaccine components, the whole manufacturing process is also likely to be more efficient through automation. For example, the World Health Organization has been working on an automated tabletop set-up for a GMP facility, where users can plug and play the vaccine components. “The approach is not going to work for all mRNA drugs and vaccines, but I do think the process of manufacturing mRNA vaccines will soon become routine like production of antibodies and other recombinant proteins, as the cost of the enzymes, RNA templates and modified nucleotides drop in a few years. It’s a rapidly growing field – one of the most rapidly moving areas in biology,” concludes Chien.
1. Doan TNK, Le MD, Bajrovic I, et al. Thermostability and in vivo performance of AAV9 in a film matrix. Commun Med.2022;2(1):148. doi: 10.1038/s43856-022-00212-6
2. Blenke EO, Örnskov E, Schöneich C, et al. The storage and in-use stability of mRNA vaccines and therapeutics: Not a cold case. JPharmSci. 2022;0(0). doi: 10.1016/j.xphs.2022.11.001
3. Kim B, Hosn RR, Remba T, et al. Optimization of storage conditions for lipid nanoparticle-formulated self-replicating RNA vaccines. J Control Release. 2023;353:241-253. doi: 10.1016/j.jconrel.2022.11.022
4. Voigt EA, Gerhardt A, Hanson D, et al. A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability. npj Vaccines. 2022;7(1):1-13. doi: 10.1038/s41541-022-00549-y
5. Erasmus JH, Khandhar AP, Guderian J, et al. A nanostructured lipid carrier for delivery of a replicating viral RNA provides single, low-dose protection against Zika. Mol Ther. 2018;26(10):2507-2522. doi: 10.1016/j.ymthe.2018.07.010