Clinical and pre-clinical methods of heat-stabilization of human vaccines

Abstract

Vaccines have historically faced challenges regarding stability, especially in regions lacking a robust cold chain infrastructure. This review delves into established and emergent techniques to improve the thermostability of vaccines. We discuss the widely practiced lyophilization method, effectively transforming liquid vaccine formulations into a solid powdered state, enhancing storage and transportation ability. However, potential protein denaturation during lyophilization necessitates alternative stabilization methods. Cryoprotectants, namely starch and sugar molecules, have shown promise in protecting vaccine antigens and adjuvants from denaturation and augmenting the stability of biologics during freeze-drying. Biomineralization, a less studied yet innovative approach, utilizes inorganic or organic-inorganic hybrids to encapsulate biological components of vaccines with a particular emphasis on metal-organic coordination polymers. Encapsulation in organic matrices to form particles or microneedles have also been studied in the context of vaccine thermostability, showing some ability to store outside the cold-chain. Unfortunately, few of these techniques have advanced to clinical trials that evaluate differences in storage conditions. Nonetheless, early trials suggest that alternative storage techniques are viable and emphasize the need for more comprehensive studies. This review underscores the pressing need for heat-stable vaccines, especially in light of the increasing global distribution challenges. Combining traditional methods with novel approaches holds promise for the future adaptability of vaccine distribution and use.

Graphical Abstract

Introduction

$125–150 million per year is the predicted reduction in total systems costs for WHO and UNICEF partners if heat-stabilized vaccines are introduced for most of the Centers for Disease Control (CDC) recommended immunizations (Table 1). Just by creating a heat-stable pentavalent vaccine (Diphtheria, Pertussis, Tetanus, HBV, Hib), it is modeled that up to $10,945 per 100 patients can be saved.¹ These costs are primarily associated with vaccine purchase and the cold-chain storage cost required for transporting the vaccine to the site where it will be administered, with an approximately 8% cost associated with vaccine waste due to improper storage.²

Table 1.

List of recommended vaccines as outlined by the Centers for Disease Control (CDC) for all US children under 18.³

Required Human Vaccines
COVID-19
Diphtheria, pertussis, and tetanus (DPT)
Haemophilus influenzae type B (Hib)
Hepatitis A (HAV)
Hepatitis B (HBV)
Human Papillomavirus (HPV)
Influenza
Measles, Mumps, and Rubella (MMR)
Meningococcal
Meningococcal B
Pneumococcal
Polio
Respiratory syncytial virus (RSV)
Rotavirus
Varicella

The cold chain is a series of steps during the life-cycle of the vaccine, from making the vaccine to when it is administered to the patient. For each step in the cold-chain, the temperature must be maintained within the indicated range, and a documented record of the storage conditions of the vaccine must be upheld. This includes use of purpose-built units for refrigeration and freezing which offer more uniform temperature distribution and faster temperature recovery in addition to multiple built-in temperature sensors which significantly reduces the risk of vaccine freezing or thawing during transportation and long-term storage. Digital temperature sensors on equipment and vial monitors for cumulative heat exposure and freezing provide an indirect measure of vaccine potency and ensures consistent vaccine efficacy across the life-cycle of the vaccine. A majority of currently FDA approved vaccines require refrigerator storage (2–8°C), with a small fraction requiring freezer (−25°C and −15°C), and initially, the Pfizer-BioNTech COVID-19 vaccine requiring ultra cold storage (−80°C to −50°C) (Figure 1; Supplementary Table 1).

Figure 1.

Current FDA approved vaccines are sorted by vaccine type and adjuvant for a given pathogen/disease with storage temperature indicated text color. The vaccines are first sorted by type (mRNA, subunit, inactivated, live attenuated, combined) and then by adjuvant (e.g. alum, MF59, AS04) or non-protein subunit (i.e., glycoconjugate) when applicable. Light blue text indicates ultra-low freezer storage (−80°C to −60°C). Medium blue font indicated freezer storage (−50°C to −15°C). Black text indicates refrigerator storage (2°C to 8°C). Pathogen/disease with two colors indicates two available storage options. RSV = Respiratory syncytial virus, HBV = Hepatitis B, HPV = human papilloma virus, HiB = Haemophilus influenzae type b, JEV = Japanese encephalitis virus, MMR = Measles, mumps, and rubella, DPT = Diphtheria, pertussis, and tetanus, MPox = Monkey pox. More details are available in Supplementary Table 1.

While considerable attention is focused on preventing vaccine exposure to temperatures above the optimal range, it is equally critical to guard against sub-optimal, particularly freezing, temperatures. This is especially pertinent in regions with severe winter conditions. The maintenance of appropriate temperature ranges is imperative not only during vaccine production and transportation but also at healthcare facilities where vaccinations are administered (Figure 2). Beyond the fundamental challenges of temperature control, geopolitical factors and climate change introduce additional layers of complexity. Geopolitical instability caused by war, conflict, or sanctions can disrupt vaccine supply chains, leading to increased vaccine wastage and cost. Similarly, extreme weather events, exacerbated by climate change, pose significant risks to transportation and storage infrastructure. These issues affect not only the immediate regions involved but also impact global research and development, manufacturing, and vaccine ability. This underscores the critical need for more thermally stable vaccines. Such advancements are vital to ensuring equitable vaccine access and the success of global immunization programs, particularly in the face of growing worldwide uncertainties.^{4, 5}

Figure 2.

Schematic of vaccine cold chain distribution process

At the heart of a vaccine is the antigen, which targets the immune response, and the adjuvant, which invokes an immune response. These antigens and adjuvants can be different aspects of the vaccine formulation or the same (e.g., virus). Adjuvants are coupled to the antigen in the case of live-attenuated and inactivated vaccines. Live-attenuated vaccines are a significant fraction of the FDA approved vaccines (Figure 1) and include formulations that protect against COVID-19, Mpox (monkeypox), tuberculosis, Ebola, Yellow Fever, and other diseases. Live-attenuated vaccines are among the most efficacious; indeed the Yellow Fever vaccine has been shown to protect for at least 47 years.⁶ However, live-attenuated vaccines are considered the least safe since they can reactivate within the host, particularly if the host is immunocompromised.⁷ Inactivated vaccines are considered more safe and contain a pathogen that has been heat or chemically killed. Influenza vaccines are most commonly inactivated viruses (Supplementary Table 1). The pathogen is no longer able to replicate, but often, inactivated vaccines require multiple boosting because they have decreased efficacy compared to a live-attenuated vaccine. A subunit-based vaccine is considered one of the safest and is comprised of parts of the pathogen (e.g., protein, carbohydrate), but these parts are often poorly immunogenic on their own and have to be co-delivered with an adjuvant to invoke a protective response. The last type of vaccine is a nucleic acid vaccine such as the COVID-19 mRNA lipid nanoparticle (LNP) vaccines. These vaccines are considered very safe because they cannot replicate in the host. Also they benefit from being able to be produced in a highly scalable manner, but have challenges associated with short-term protection, higher production cost compared to subunit vaccines, storage requirements at low temperatures, and carrier related immune responses (e.g. anti-polyethylene glycol [PEG], anti-lipid). ^8–18

There are only six FDA approved vaccine adjuvants: aluminum salts (often referred to as alum), AS04 (Monophosphoryl lipid A [MPLA] plus alum), MF59 (stabilized squalene emulsion), AS01 (MPLA with liposomal QS-21), CpG, and Matrix-M. In addition, another squalene emulsion (AS03) has been approved for pandemic use for the 2009 influenza pandemic. With the exception of two adjuvanted inactivated vaccines (Tick-Borne Encephalitis with alum and inactivated influenza with MF59), the adjuvants listed are used to boost protein or sugars isolated from pathogens. The storage conditions of vaccines are largely dependent on the antigen to prevent its denaturation, therefore ensuring consistent protective efficacy. In addition to antigen and adjuvant, there are residual amounts of the production vehicle used to grow the vaccine biologic as well as chemicals used in the process, chelating agents to limit microbial infection and product degradation, agents to adjust pH and antimicrobials (Supplementary Table 2).

Although there are other formulation efforts that can reduce vaccine administration costs (e.g., needle-free vaccination), heat-stabilization of vaccines has been identified as the most significant transformative formulation advancement needed in the administration of these life-saving technologies in resource-limited settings worldwide. The most commonly employed physical-chemical methods to heat-stabilize vaccines apply one or more of the following techniques: lyophilization, cryoprotectants, biomineralization, or encapsulation in organic matrices. In addition to these, protein modifications (e.g., directed evolution) have been used to create more stable antigens; ^19–22 however, this method will not be discussed in this review. This review will address physical-chemical techniques with respect to pre-clinical studies and also review clinical trials which evaluate human vaccines that are heat stabilized. There is a comprehensive review on heat-stabilized veterinary vaccines by Fanelli et al.²³

Lyophilization

Lyophilization or freeze-drying is a process used in vaccine manufacturing to remove water and form a powder that can be resuspended with a sterile diluent at the time of administration (Figure 3). By removing water it can reduce the likelihood of microbial growth and buffer the effects of temperature fluctuations. With the process of lyophilization, the advantages of having a dry formulation can be accomplished without the use of elevated temperatures (i.e. heating) that would significantly denature the biologics in the formulation.²⁴ The first step in lyophilization is to freeze the liquid mixture that contains the vaccine. Then, pressure is significantly reduced, as well as temperature, to sublimate the frozen water. Temperature and pressure are then stabilized to form a dry powder. The powder is then sealed and often stored with desiccants to ensure it remains dry. This process is often used alone in the manufacturing of vaccines, but in the development of heat-stable vaccines, it is often used in tandem with another method (e.g., cryoprotectants, biomineralization, encapsulation).²⁵

Figure 3.

Schematic of generalized lyophilization (freeze-drying) process used for drying of vaccine formulations.

Lyophilization, while a critical process in vaccine manufacturing, presents several challenges. A primary concern is the potential denaturation of antigens during freeze-drying. This can lead to a reduced protective response or correlates of protection (e.g., antibody titers, neutralizing antibodies, cellular responses). Furthermore, the equipment and energy demands for lyophilization contribute to higher production costs due to the increased time and resources required. Consequently, various modifications in the lyophilization process have been proposed. These include altering the geometry, sequence, and timing/duration in which lyophilization is performed. The most basic changes regard whether vaccine components are dried in the vial or a planar sheet (shelf drying). Rather than having a cylindrical volume to cool, the thermodynamics of having a thin plane often in intimate contact with a cooling metal sheet (over a glass vial) can result in rapid and uniform freeze-drying. Changes in the speed and intensity of pressure and temperature in the process can also result in changes in vaccine efficacy, as reported with herpes simplex virus 2 (HSV-2)²⁶ and other vaccines.²⁴ The balance between product quality and cost effective production must be maintained during the scale up process. Another innovative approach is the expression of vaccine elements in yeast, followed by whole-cell lyophilization, to enhance the protection of biological components during the process.²⁷ Furthermore, lyophilization can be used to create foams through sacrificial excipients that sublimate with the water,^{28, 29} and can also form thin films, typically with highly viscous solutions, that can be hydrated prior to injection.^{30, 31} These diverse methods underscore the complexity and adaptability of lyophilization in vaccine production, yet also highlight the need for careful consideration of each vaccine’s unique requirements.

Lyophilization is the most commonly used method for stabilization of vaccines, and it has been shown to increase the heat stabilization for the rotavirus,³² influenza,³³ Polio,³⁴ and other vaccines.³³ Terrinoni et al.³⁵ evaluated inactivated Hikojima whole cell/cholera toxin B subunit oral cholera vaccine freeze dried through two different methods. In the first method, 1.5mL aliquots were placed at 80 °C for 2.5 h and then freeze-dried for 18 h under vacuum at 0.010 mbar in a planar lyophilizer with a shelf temperature of −5°C. The second group was handled in the same manner as group 1, but an additional final drying step for 30 min at 0.001 mbar was added, and the shelf-temperature was raised to 37 °C before the vials were sealed. Although there were some differences in antibody (IgA, IgG) titer between the two groups, with the second group typically performing at or below the first group, the vaccine lyophilized by either method resulted in greater heat-stability than the liquid vaccine that is recommended to be stored between 2–8°C. The lyophilized vaccine illustrated stability after 26 months of storage at 4 °C or 25 °C and after 8 months at 40 °C.³⁵ This study demonstrated the heat-stability that can be afforded with vaccine lyophilization.

Cryoprotectants

To help mitigate some of the biologic denaturation that can occur during the lyophilization process, cryoprotectants are often used (Table 2). Sugars and starches are the most commonly used cryoprotectants, with trehalose, dextran, and pullulan being the three most commonly used pre-clinically (Figure 4). Trehalose is a disaccharide comprised of two molecules of glucose that is abundantly found in plants and non-mammalian animals and is used in several antibody formulations as an excipient.^{42, 43} Dextran is a poly-glucose molecule available in a wide range of molecular weights and is FDA approved as a plasma expander and an excipient in antibody formulations.^{43, 44} Pullulan is a polymer comprised of repeat maltotriose units derived from yeast.⁴⁵ By adding cryoprotects, the glass transition temperature of the formulation is increased, which can offer better protein stability as the biologics are frozen and dried.⁴⁰ The mixture’s viscosity is also changed, which can help protect during the lyophilization process.³⁰ The increased viscosity better supports formation of a glassy matrix to protect the biologics during the freezing process. Once dried, this glassy matrix forms a protective barrier that provides heat stability.⁴⁰ Table 2 presents several infectious disease vaccines evaluated pre-clinically for heat-stabilization. Several viral vectors (e.g., adenovirus, adeno-associated virus) that can be used for infectious disease vaccines, gene therapy, and other applications have also been evaluated for heat stability.^{30, 46–49}

Table 2.

Preclinical vaccines that use cryoprotect to create a heat-stabilized formulation.

Pathogen	Type	Formulation	Route	Cryoprotectant	Temperature (duration)	Outcome	Ref
Bacillus anthracis	Subunit	Recombinant protective antigen with alum and GLA	SC	Mannitol (113 mg), trehalose (9.5 w/v%)	40 °C (16 weeks)	Neutralizing antibody was improved compared to liquid vaccine	36
Hepatitis B vaccine	Subunit	HepB surface antigen with alum (Shanvac-B)	IP	Trehalose with dextran or hetastarch	37 °C (15 months)	Increased seroconversion (antibodies) of heat-stable formulation	37
Ricin Toxin A	Subunit	Protein with alum (RiVax)	SC	Trehalose (4, 8 or 12 w/v%)	40 °C (4 weeks)	Protein stability was retained after storage.	38
Meningitis	Subunit protein–polysaccharide conjugate	Meningitis A protein-polysaccharide conjugate (MenA)	IP	Trehalose with dextran or hetastarch	40 °C (20 weeks) or 60 °C (2 weeks)	The polysaccharide was degraded at elevated conditions	37
Mycobacterium tuberculosis	Subunit with Adjuvant	ID93 recombinant protein with GLA-SE squalene emulsion with TLR-4 agonist glucopyranosyl lipid A	IM	Trehalose (8.1–10%)	4, 37, 50 °C (3 months)	The thermostable single-vial vaccine generated greater antibody and cellular responses than the non-thermostable two-vial vaccine in humans.	39
Influenza	Inactivated virus	Inactivated influenza A	PV	Pullulan (10 wt%), trehalose (0.5M), or mixture of both	40 °C (2 months)	No observable change in antibody titers	40
Herpes Simplex Virus type 2	Live-attenuated	Virus	PV	Pullulan (10 wt%), trehalose (0.5M), or mixture of both	40 °C (2 months)	A loss in efficacy, as measured by antibody titers, was observed	40
Salmonella enterica serovar Typhi	Live-attenuated	Attenuated bacteria	IN or IP	Trehalose, methionine, and gelatin	25 °C (12 weeks)	No significant loss in antibody titer	28
Mycobacterium tuberculosis	Live-attenuated	Vesicular stomatitis virus (VSV)-vectors for Ag85A	IM	Trehalose and trehalose/dextran 3:1	37 °C (15 days)	Vaccine immunogenicity was maintained in heat stable form but not liquid formulation	41

SC = subcutaneous. IM = Intramuscular. IN = Intranasal. PV = Vaginal route. IP = intraperitoneal.

Figure 4.

Chemical structure of three commonly used cryoprotectants: Trehalose, dextran, and pullulan.

Biomineralization

Biomineralization is the process of adding inorganic or organic-inorganic hybrid materials to biologics. In nature, biomineralization can be seen in shells, bones, and teeth. In the pre-clinical evaluation of heat-stable vaccines, this process has been shown in metal-organic coordination polymers, ensilication, and the use of inorganic gels (Table 3). As a field, biomineralization is less well studied than cryoprotectants, but these approaches offer significant heat-stabilization and are evaluated at higher temperatures than cryoprotectants. With biomineralization, it is thought that the inorganic containing element surrounds the biologics of the vaccine, protecting them from the damage of high heat, such as boiling temperatures, by dissipating the heat more readily through its inorganic (often metal) molecules.⁵¹ In some cases, crystallization sites have been added to the vaccine element (e.g. virus) to facilitate the encapsulation of the biologic.⁵⁰ A popular approach is the use of metal and organic coordination polymers.^50–52 These platforms form a polymer comprised of an inorganic metal linked with an organic (usually dipeptide) linker. They can form one-dimensional, or 2D and 3D structures (Figure 5) with 2D and 3D structures referred to as metal-organic frameworks (MOFs).^{55, 56} Interestingly, Hendy et al. show with a zinc-carnosine complexation polymer that incorporates the protein antigen at the time of formation results in reduced thermostability compared to protein added after the polymer is formed, with the protein complexing to the zinc via its HIS-tag.⁵² This would imply that encapsulation is not required for heat-stability for this biomineralization method. Biomineralization is a nascent approach to heat-stable vaccines and certainly will continue to grow in exciting ways as the field progresses.

Table 3.

Preclinical vaccines that use biomineralization to create a heat-stabilized formulation.

Pathogen	Type	Formulation	Route	Biomineralization Technique	Temperature (duration)	Outcome	Ref
Human enterovirus type 71	Live-attenuated	Human enterovirus	SC	Calcium phosphate	26 °C (9 days) 37 °C (1 week)	A significant decrease in neutralizing antibodies and IFN-γ secreting cells after antigen recall was observed in nonmineralized formulation compared to mineralized one	50
Tobacco mosaic virus	Live-attenuated	TMV	SC	ZIF-8 Metal organic framework	100 °C (20 min)	Encapsulation in the ZIF-8 increased residence time and offered comparable antibody titers compared to unencapsulated	51
Influenza	Subunit	Designer hemagglutinin	IM	Zinc-Carnosine complexation polymer	40°C (3 months) 24°C (6 months)	Cytotoxicity and TNF-a production in dendritic cells was not significantly different from -20°C control. No significant differences in binding during a competitive ELISA were observed with storage at 40°C	52
Cryptococcus neoformans	Subunit	Protective antigen from C. neoformans	SC	C. neoformans coated silica particles	20 °C (1 hour) 95 °C (1 hour)	IgG titer was similar in ensilicated albumin vaccine and ensilicated C. neoformans vaccine protected similarly as unensilicated vaccine	53
Horse-radish peroxidase (HRP)	Subunit	HRP	NA	ZIF-8 Metal organic framework	80 °C (1 hour) 153 °C (1 hour)	Enzyme activity was retained compared to untreated control	50
Foot and mouth disease virus (FMDV)	Subunit	FMDV Virus-like particle (VLP)	IM	Calcium phosphate	25 °C (8 days) 37 °C (4 days)	Biomineralized VLPs produced high antibody and neutralizing antibody titer and protection against challenge	54

SC = subcutaneous. IM = Intramuscular. NA = not applicable

Figure 5.

SEM images of ZnCar based complexes. (A) ZnCar MOF prepared in (dimethyl fumarate solvate) synthesized with reaction mixture stirring according to Katsoulidis et al. (B-D) ZnCar coordination polymer (1D) prepared in HEPES buffer pH = 7.4. (B) Blank ZnCar coordination polymer. Reprinted with permission from Eckshtain-Levi, M.; Batty, C. J.; Lifshits, L. M.; McCammitt, B.; Moore, K. M.; Amouzougan, E. A.; Stiepel, R. T.; Duggan, E.; Ross, T. M.; Bachelder, E. M.; Ainslie, K. M. Metal-Organic Coordination Polymer for Delivery of a Subunit Broadly Acting Influenza Vaccine. ACS Appl Mater Interfaces 2022, 14 (25), 28548–28558. DOI: 10.1021/acsami.2c04671. Copyright 2022 American Chemical Society. Vaccine antigen or adjuvant-loaded complexes (C) CpG, and (D) COBRA Hemagglutinin Y2 (scale bars represent 2 μm). Synthesized by mixing a ZnCar suspension with an aliquot of HA, CpG, or COBRA Y2 to promote adsorption. Reprinted with permission from Hendy, D. A.; Lifshits, L. M.; Batty, C. J.; Carlock, M. A.; Ross, T. M.; Mousa, J. J.; Bachelder, E. M.; Ainslie, K. M. Zinc Carnosine Metal-Organic Coordination Polymer as a Potent Broadly Active Influenza Vaccine Platform with in Vitro Shelf-Stability. Mol Pharm 2023. DOI: 10.1021/acs.molpharmaceut.3c00424. Copyright 2023 American Chemical Society. ⁵⁷;^{56, 58}

Encapsulation in organic matrices

Vaccine elements have been effectively encapsulated into organic matrices including polymers and lipids since their initial report in 1976 (Table 4).^{7, 64–67} Often, these platforms are used to protect the vaccine cargo from degradation, control the release of the encapsulated agents for an extended period, and/or enhance delivery to immune cells of interest. Additionally, some of these materials can also stabilize the vaccine elements outside the cold chain. Several types of formulations exist that encapsulate vaccine elements into a polymeric matrix or lipid particle (Figure 6); ⁷ they can also be covalently bound to the surface.^{68, 69} Vaccine elements are most commonly encapsulated into spherical particles that are (polymeric nano/microparticles [NPs]), nucleic acid complexing LNPs, or virosomes). Virosomes are liposomes containing virus elements (e.g., proteins, inactivated viruses) that have lipid-anchored antigens on the surface of the particles.⁶⁰ Beyond these spherical forms, vaccine elements have also been incorporated into microneedles.

Table 4.

Preclinical vaccines that use encapsulation in organic matrices to create a heat-stabilized formulation.

Pathogen	Type	Formulation	Route	Encapsulation Technique	Temperature (duration)	Outcome	Ref
Influenza	Live-attenuated	Several Influenza virus strains	IN	Spray dried formulation in gelatin	37 °C (4.5 months)	Antibody and neutralization titers were comparable with sucrose and gelatin containing formulations.	29
Influenza	Inactivated	Inactivated PR8 influenza	ID	Microneedles of PVA and water with sucrose	60 °C (4 months)	Microneedles protected better than in solution and had comparable neutralization to IM vaccination.	59
HIV	Subunit	Gp41	IN, PO, SL	Virosome with trehalose	40 °C (3 months)	Antibody titers were maintained when stored at elevated temperatures.	60
Influenza	Subunit	Designer hemagglutinin	IM	Acetalated dextran microparticles via homogenization	40°C (3 months) 24°C (6 months)	IFN-γ production in dendritic cells was similar to ideally stored particles. Neutralization and cytokine production after antigen recall also did not differ from ideally stored particles.	61
Coronavirus	mRNA	RBD expressing mRNA in LNPs	IM	LNPs with ionizable lipid 2-hexyldecyl 6-(ethyl(3-((2-hexyldecyl)oxy)-2-hydroxypropyl)amino)hexanoate	37 °C (30 days)	Higher humoral and cellular immunity was observed with the new lipid compared to ALC-0315	62
Coronavirus	mRNA	RBD expressing mRNA in LNPs	ID	LNPs in PVP: PVA microneedles	26 °C (6 months)	Antibody titers were on the order of IM vaccination after prime-boost for only the ideally stored microneedles	63

ID = intradermal. IM = Intramuscular. PO = Oral. SL = sublingual. PVA = polyvinyl alcohol. PVP = polyvinylpyrrolidone. LNPs = lipid nanoparticles. RBD = receptor binding domain.

Figure 6.

Polymeric and lipid platforms as well as commonly used methods to make some of these platforms.

Polymeric NPs are often produced through emulsion processes involving homogenization or sonication. While these methods effectively encapsulate vaccine elements, they impose shear stress, which can denature protein antigens, reducing neutralizing titers and lowering the overall vaccine efficacy.^70–72 Emulsion based methods are further limited by being batch processes, which increase manufacturing costs compared to continuous processing methods such as microfluidics,⁷³ electrospray,⁷¹ and spray drying.⁷⁴ However, these methods are not without drawbacks of their own. For example, similar to emulsion methods, microfluidic devices produce particles in a continuous phase, which is often aqueous and can result in diffusion (and loss) of hydrophobic vaccine elements, reducing the overall encapsulation efficiency of the cargo.⁷⁵ This is in contrast to electrospray and spray drying techniques where the continuous phase is air and, therefore, loss of encapsulates is limited. Electrospray and spray drying are two distinct methods used for particle generation, with the former operating at room temperature and the latter at elevated temperatures. Despite the thermal differences, spray drying is a more common manufacturing technique for polymeric particles. Spray drying has been used to generate pre-clinical formulations for subunit HBV,³⁷ adjuvanted subunit anthrax,⁷⁶ glycol-conjugate meningitis,³⁷ live-attenuated measles,⁷⁷ and live-attenuated influenza vaccine,²⁹.

LNPs are the most recent vaccine platform to be FDA approved, with the Moderna and Pfizer COVID-19 formulations. The ionic interaction of mRNA and charged lipids results in a self-assembled nanoparticle that can be formed continuously through microfluidics.⁷⁸ These lipid complexes have existed for a long time,⁷⁹ but the addition of novel ionizable lipids in the LNPs, as well as more stable mRNA constructs, has resulted in enhanced protective efficacy and translation of the platforms for clinical use. However, LNPs face storage challenges, illustrated by the initial ultra-cold storage requirements for Pfizer’s vaccine (−90°C and −60°C), later revised to freezer storage (−50°C and −15°C).⁸⁰ Modifications in LNPs have been explored to improve their storage long term at regular refrigeration (4 °C) by changing out the ionizable or helper lipids in the LNPs.^{18, 62} To further stabilize them, LNPs have also been encapsulated in polymeric microneedles.⁶³

Microneedles, often produced via micro molding, ^{81, 82} present a promising needle-free alternative for vaccine delivery, potentially suitable for self-administration. They are particularly beneficial for resource limited settings. Microneedle patches are typically small, covering an area on the order of only a few centimeters. ⁸² While this size is ideal for easy application, it limits the amount of antigen and adjuvant that can be loaded; especially when a secondary carrier is used (e.g., LNPs), the antigen load is even more significantly reduced. Nonetheless, microneedles are promising for heat-stable formulations and have shown protection against coronavirus⁶³ and influenza⁵⁹ when stored at elevated temperatures.

Clinical Trials with Heat-stabilized Vaccines

Only a limited number of the aforementioned technologies have been translated into registered clinical trials where storage conditions are evaluated. A majority of the trials listed to evaluate thermostability involve the conventional formulation stored at an alternative temperature (NCT00169455, NCT01762930, NCT01559597, and NCT03433482) (Table 5). Most of these studies indicated that an alternative storage temperature resulted in similar outcomes compared to the ideally stored vaccine, particularly regarding serum antibodies. This displayed that alternative storage schedules are viable for live-attenuated, inactivated, and subunit-conjugate vaccines and indicates that changes in storage should be more readily evaluated clinically. National clinical trial number 02728869 (NCT02728869) ^{32, 84} developed a heat stable live-attenuated oral rotavirus (HSRV) vaccine based on the FDA approved RotaTeq vaccine. To identify a heat-stable formulation, they first iterated pre-clinically using different buffering agents, bulking agents, cryoprotectants, amino acids, and cations to identify a stabilized vaccine containing HEPES buffer, sucrose, polyvinylpyrrolidone, L- arginine and calcium chloride dihydrate.³² The addition of these excipients allowed for storage at 45 °C for up to 9 months and at 37 °C for up to 12 months. The HSRV vaccine was developed in the Hilleman Laboratories and has now been licensed.⁸⁸

Table 5.

Clinical trials registered at ClinicalTrials.org discuss storage, heat-stability, or cold-chain with respect to vaccines and have experimental arms which compare vaccines stored at different storage conditions.

Pathogen	Trial Number and References	Temperature (duration)	Formulation	Status	Phase	State & Completion Date	Outcome
Rotavirus	NCT00169455 83	37°C (7 days)	Lyophilized Oral Live Attenuated Human Rotavirus (RIX4414) Vaccine	Completed	Phase 3	Mar/2005 – Dec/2005	Lyophilized vaccine stored at 37°C for 7 days before reconstitution had similar immunogenicity to the vaccine stored at 2–8 °C.
Rotavirus	NCT02728869 32, 84	45 °C (9 months) 37 °C (12 months)	RotaTeq live attenuate oral virus with lyophilized cake comprised of HEPES, Sucrose, PVP, L- Arginine, and CaCl2•2H20	Completed	Phase I/II	Jun/2016 – Apr/2017	Similar adverse events to placebo and in infants. Anti-rotavirus IgA titers were similar to the heat-stable versus non-heat stable vaccine.
Cholera	NCT01762930 85	2–8 °C, 25 °C, 37 °C, or 42 °C (14 days)	Shanchol inactivated whole-cell oral vaccine	Completed	Phase 2	Jun/2012 – Dec/2015	Similar titers at days 7 and 21 for serum IgG and IgA responses were similar for all temperatures up to 14 days.
Tetanus	NCT01559597 86	40 °C (<30 days)	Tetanus toxoid Conjugate	Completed	NA	Nov/2012 – Mar/2013	Similar antibody titers were observed between the standard and elevated temperature groups.
Meningitis	NCT03433482 87	2–8 °C (30 months)	MenACWY (Menveo) conjugate	Completed	Phase 2	Aug/2018 – Dec/2019	Comparable outcomes between conventional and 30 month stored vaccines.

PVP = polyvinylpyrrolidone.

Conclusions

There are several methods that have been shown pre-clinically to successfully store vaccines outside typical storage temperatures. The established method of lyophilization has been instrumental in the transformation of vaccines into a powder form, enhancing both storage longevity and transport efficiency. Yet, its propensity for protein denaturation poses potential threats to vaccine efficacy. Cryoprotectants have emerged as a countermeasure, augmenting the glass transition temperature, and enhancing formulation viscosity, thus safeguarding stability during freeze-drying. Concurrently, biomineralization, though less explored, represents an innovative approach, employing inorganic or organic-inorganic hybrids to potentially enhance heat stability without necessitating complete biologic encapsulation. Encapsulation in lipid or polymer matrices have also been used to protect vaccines at elevated temperatures. Vaccine elements can be encapsulated via several methods for polymeric particles (e.g. spray drying, electrospraying), lipid nanoparticles (e.g. microfluidics) and microneedles (e.g. micromolding) with new methods being generated continuously such as 3D printing. Despite the burgeoning promise of these techniques, a limited number have transitioned to the clinical trial stage. However, preliminary trials underscore the viability of alternative storage conditions, emphasizing the need for further, more rigorous studies. As global demand intensifies for effective vaccine distribution, especially in regions with limited cold chain infrastructure, the fusion of traditional methods with newer ones like cryoprotectant use and biomineralization is encouraging. As more techniques undergo clinical testing, the future of vaccine distribution appears promisingly adaptable.