The instability of mRNA vaccines presents significant challenges for their storage, transportation, and large-scale distribution, particularly in resource-limited countries. Recently, freeze-drying (lyophilization) has been considered as a promising approach for preserving mRNA vaccine efficacy. This formulation technique enhances the long-term stability of mRNA vaccines by converting them into a stable dry powder. The purpose of this review is to provide an overview of the current knowledge on the progress of freeze-drying techniques for mRNA vaccines, with emphasis on the associated challenges. This review highlights the factors influencing the stability of freeze-dried mRNA vaccines and provides a comprehensive overview of the formulation components, including excipients, buffers, and surfactants, as well as the process parameters and storage conditions that aim to improve stability and shelf-life. By providing these insights, this review supports the advancement of more robust, scalable, and efficient lyophilization protocols, ultimately addressing the stability limitations of mRNA vaccines and enhancing their global accessibility.

The thermostability of vaccines, particularly enveloped viral vectored vaccines, remains a challenge to their delivery wherever needed. The freeze-drying of viral vectored vaccines is a promising approach but remains challenging due to the water removal process from the outer and inner parts of the virus. In the case of enveloped viruses, freeze-drying induces increased stress on the envelope, which often leads to the inactivation of the virus. In this study, we designed a method to freeze-dry a recombinant vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. Since the envelope of VSV is composed of 50% lipids and 50% protein, the formulation study focused on both the protein and lipid portions of the vector. Formulations were prepared primarily using sucrose, trehalose, and sorbitol as cryoprotectants; mannitol as a lyoprotectant; and histidine as a buffer. Initially, the infectivity of rVSV-SARS-CoV-2 and the cake stability were investigated at different final moisture content levels. High recovery of the infectious viral titer (~0.5 to 1 log loss) was found at 3–6% moisture content, with no deterioration in the freeze-dried cakes. To further minimize infectious viral titer loss, the composition and concentration of the excipients were studied. An increase from 5 to 10% in both the cryoprotectants and lyoprotectant, together with the addition of 0.5% gelatin, resulted in the improved recovery of the infectious virus titer and stable cake formation. Moreover, the secondary drying temperature of the freeze-drying process showed a significant impact on the infectivity of rVSV-SARS-CoV-2. The infectivity of the vector declined drastically when the temperature was raised above 20 °C. Throughout a long-term stability study, formulations containing 10% sugar (sucrose/trehalose), 10% mannitol, 0.5% gelatin, and 10 mM histidine showed satisfactory stability for six months at 2–8 °C. The development of this freeze-drying process and the optimized formulation minimize the need for a costly cold chain distribution system.

The instability of mRNA-LNP vaccines presents significant challenges in storage, transportation, and large-scale distribution, particularly in resource-constrained countries. Recently, freeze-drying (lyophilization) has been considered as a promising approach for preserving efficacy and enhancing the long-term stability of mRNA vaccines by converting mRNA-LNP formulations into a stable dry powder. The purpose of this review is to provide an overview of the current knowledge on the optimization and progress of freeze-drying techniques for mRNA-LNP vaccines, with a particular emphasis on the associated challenges. This review highlights the factors influencing the stability of freeze-dried mRNA-LNP vaccines and provides a comprehensive overview of the formulation components like excipients (cryoprotectants and lyoprotectants), buffers, surfactants and also the process parameters, and storage conditions. By providing these insights, this review supports the advancement of more robust, scalable, and efficient lyophilization protocols, ultimately addressing the stability limitations of mRNA-LNP vaccines and enhancing their global accessibility.

Liquid formulations have been successfully used in many viral vector vaccines including influenza (Flu), hepatitis B, polio (IPV), Ebola, and COVID-19 vaccines. The main advantage of liquid formulations over lyophilized formulations is that they are cost-effective, as well as easier to manufacture and distribute. However, studies have shown that the liquid formulations of enveloped viral vector vaccines are not stable over extended periods of time. In this study, we explored the development of the liquid formulations of an enveloped recombinant Vesicular Stomatitis Virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. To do so, we used a design of experiments (DOE) method, which allowed us to assess the stability dynamics of the viral vector in an effective manner. An initial stability study showed that trehalose, gelatin, and histidine were effective at maintaining functional viral titers during freeze–thaw stress and at different temperatures (−20, 4, 20, and 37 °C). These preliminary data helped to identify critical factors for the subsequent implementation of the DOE method that incorporated a stress condition at 37 °C. We used the DOE results to identify the optimal liquid formulations under the selected accelerated stress conditions, which then guided the identification of long-term storage conditions for further evaluation. In the long-term stability study, we identified several liquid formulations made of sugar (sucrose, trehalose, and sorbitol), gelatin, and a histidine buffer that resulted in the improved stability of rVSV-SARS-CoV-2 at 4 °C for six months. This study highlights an effective approach for the development of liquid formulations for viral vector vaccines, contributing significantly to the existing knowledge on enveloped viral vector thermostability.

Background: Thermostability remains a key bottleneck for equitable access to mRNA-LNPs vaccines, mainly due to cold-chain requirements. Objectives and methods: Here, we optimized freeze-drying formulations by screening excipients (sugars, sugar alcohols, and proteins) and buffers to preserve mRNA-LNPs as solid formulations under ambient and refrigerated conditions. Physicochemical properties (size, polydispersity index [PDI], and encapsulation efficiency [EE]) and functional integrity, assessed by fluorescence-based in vitro transfection assays, were evaluated during long-term storage of up to six months. Results: Preliminary screening identified 20% sucrose and trehalose with Tris or histidine buffers as optimal for preserving physicochemical properties during freeze-drying, including high encapsulation efficiency (>90%), particle size (~200 nm), and low polydispersity (PDI < 0.2). Mannitol, gelatin, and PBS-based buffers showed adverse effects. At 4 °C, formulations F1–F3 maintained physicochemical stability and functional transfection activity for up to four months. In contrast, 20 °C storage caused progressive destabilization, with increased size, PDI, and encapsulation loss (>60% by six months). Among all formulations, 20% sucrose with 5 mM Tris (F1) showed the most robust preservation of physicochemical integrity and in vitro transfection efficiency under refrigerated and ambient conditions. Conclusions: Sugars outperformed sugar alcohols and gelatin as cryoprotectants. All formulations were stable, including functionally active at 4 °C for up to four months, while a sucrose/Tris formulation retained acceptable stability at 20 °C. Overall, the results demonstrate the feasibility of storing mRNA drug products as solid formulations at non-freezing temperatures.

Thermostability of vaccines, particularly enveloped viral vectored vaccines, remains a challenge for their delivery wherever needed. Freeze-drying of viral vectored vaccines is a prom-ising approach but remains challenging due to the water removal process from the outer and in-ner parts of the virus. In the case of enveloped viruses, freeze-drying induces increased stresses on the envelope, which often leads to inactivation of the virus. In this study, we design a method to freeze-dry a recombinant Vesicular Stomatitis Virus (VSV) expressing the SARS-CoV-2 spike glycoprotein. Since the envelope of VSV is composed of 50% lipid and 50% protein, the formula-tion study focused on both the protein and lipid portions of the vector. Formulations were pre-pared primarily using sucrose, trehalose, and sorbitol as cryoprotectants; mannitol as a lyopro-tectant; and histidine as a buffer. Initially, the infectivity of rVSV-SARS-CoV-2 and cake stability were investigated at different final moisture contents. High recovery of the infectious viral titer ( 0.5 to 1 log loss) was found at 3-6% moisture content with no deterioration of the freeze-dried cakes. To further minimize infectious viral titer loss, the composition and concentration of the ex-cipients were studied. An increase from 5 to 10% of both cryoprotectants and lyoprotectant, to-gether with the addition of 0.5% gelatin, resulted in the improved recovery of infectious virus titer and stable cake formation. Moreover, the secondary drying temperature of the freeze-drying pro-cess showed a significant impact on the infectivity of rVSV-SARS-CoV-2. The infectivity of the vector declined drastically when the temperature was raised above 20°C. Throughout a long-term stability study, formulations containing 10% sugar (sucrose/trehalose), 10% mannitol, 0.5% gelatin, and 10 mM of histidine showed satisfactory stability for six months at 2-8°C. The development of this freeze-drying process and the optimized formulation minimizes the need for a costly cold chain distribution system.