Lyophilized mRNA Vaccines Advance to Clinical Trials

Lyophilized mRNA Vaccines Advance to Clinical Trials
Posted in: Therapeutics

The remarkable success of the COVID-19 vaccines developed by Pfizer/BioNTech and Moderna is due to the combination of two technology platforms: mRNA vaccinology and lipid nanoparticle (LNP) delivery (Labouta et al., 2022). LNPs protect the encapsulated mRNA active ingredient (Figure 1), enable cellular uptake, and permit the release of the mRNA for translation into protein immunogens (Melamed et al., 2022). 

FIGURE 1. Depiction of mRNA-LNP components, degradation factors, and storage. Taken from Schoenmaker et al. 2021 and free to use under CC BY 4.0 license.

The urgency of the COVID-19 pandemic precluded conventional real-time stability/potency studies for different storage conditions. The requirement for ultra-low dry-ice (-80 oC) temperature for storage and shipping limited the number and geographical locations of adequately equipped end-use facilities. 

The pandemic urgency also precluded conventional studies of excipient (i.e., additives) usage, which would be required for lyophilization. Yet, these studies have been ongoing, and now lyophilized mRNA-LNP vaccines are in clinical studies, which, as Science said, “could bring the pandemic’s star vaccine technology to more of the world.”

Why Lyophilize?

Molecules in solution are subject to various degradation reactions. For mRNA, this is principally the pH-dependent hydrolysis of phosphorus-oxygen bonds in linkages between nucleotides. While the rate of chemical degradation in solution decreases with lower temperatures, such reactions in the solid-state are exceedingly slow. Consequently, solid-state storage of mRNA and other macromolecular biopharmaceuticals is preferred, providing that dissolution for administration is not problematic. 

Lyophilization, or freeze-drying, is the process of freezing a substance, putting it under a vacuum, then removing almost all water so that the ice changes from solid directly to vapor. As reviewed elsewhere (Allison et al. 2000), removing water from a solution of lipid/nucleic acid complexes usually leads to a glassy mass or viscous “goo” that does not readily dissolve in aqueous vehicles used for vaccine administration. This problem is generally solved by finding a suitable excipient, typically a sugar (e.g., sucrose or trehalose), which, when added to the mRNA-LNP solution, leads to a fluffy “cake” of lyophilized vaccine that can be reconstituted as needed (Figure 2). This permits long-term storage, shipping, and handling under convenient conditions. However, until recently, no studies demonstrated the long-term stability and efficacy of a lyophilized mRNA-LNP for vaccine usage.

FIGURE 2. Typical freeze-dried “cakes” of formulations with excipients in vials. Taken from Ogienko et al. 2022 and free to use under CC BY 4.0 license.

Lyophilization Provides Long-Term Stability for mRNA-LNP Vaccines

The first proof-of-concept for the long-term stability of a lyophilized mRNA-LNP vaccine was published in February 2022 by a research team (Muramatsu et al.) that included mRNA vaccinology experts Katalin Karikó and Norbert Pardi. Lyophilization of mRNA-LNP formulations was less straightforward than solutions of small molecules because mRNA-LNPs are complex multi-component structures (Figure 1). These nanoparticle structures are carefully assembled from specific types of lipids at certain ratios using well-defined processes. Numerous physicochemical parameters such as particle size and polydispersity are critical to biological performance and must be retained during the lyophilization process and subsequent storage.

Briefly, mRNAs for firefly luciferase or the influenza virus hemagglutinin antigen were in vitro co-transcriptionally synthesized using TriLink CleanCap® reagent and N1-methylpseudouridine (m1Ψ)-5′-triphosphate (Henderson et al. 2021). Each mRNA was encapsulated in an LNP and lyophilized with the help of sucrose and maltose as excipients. Lyophilized mRNA-LNP was stored under various conditions.

The physicochemical properties of these lyophilized materials did not significantly change after storage for 12 weeks at room temperature or 24 weeks at 4 oC. Compared to the original storage recommendations provided by the U.S. CDC for the Pfizer/BioNTech COVID-19 vaccine, which specified no more than 31 days at 2-8 oC before mixing with normal saline, lyophilization has significantly extended such storage. 

Additional real-time physicochemical stability data will need to be obtained to warrant even longer times for refrigerated storage before reconstitution for administration. As for retention of vaccine activity, comparative studies by Muramatsu et al. in mice demonstrated that these reconstituted and injected mRNA-LNPs maintain their encoded-antigen expression and immunogenicity. The following sections outline several clinical studies aimed at establishing the immunogenicity of lyophilized mRNA-LNPs in humans, which will hopefully provide results leading to much higher vaccination rates in non-developed areas.  

BioNTech/Pfizer Clinical Trials of a Lyophilized COVID-19 mRNA-LNP Vaccine

BioNTech and Pfizer recently started a Phase 3 clinical trial (NCT04816669) to evaluate the safety, tolerability, and immunogenicity of a two-dose schedule of multiple formulations of their approved COVID-19 vaccine (BNT162b2, Comirnaty®). 

Briefly, this study, performed in healthy adults aged 18 to 55 years in the U.S., will compare the immune response of lyophilized BNT162b2 presented in single-dose vials to that of bulk frozen-liquid BNT162b2 in multidose vials, the currently approved way of storage for administration. The study will also compare an additional dose of frozen liquid BNT162b2 in participants who already received the two-dose schedule of lyophilized BNT162b2.

EyeGene Clinical Trial of a Lyophilized COVID-19 mRNA-LNP Vaccine

In March 2021, the Korean pharmaceutical company EyeGene (Hong et al.) posted a preprint reporting the development of its lyophilized mRNA vaccine against SARS-CoV-2. Their vaccine’s mRNA was custom synthesized by TriLink with CleanCap reagent and the modified NTP, 5-methoxyuridine.

To formulate these mRNAs for delivery, PEG-free cationic liposomes were prepared with DOTAP, DOPE, and cholesterol using a thin-film method (Wui et al., 2019). The resultant lipid film was rehydrated, homogenized, and combined with each mRNA to encapsulate it; the mRNA-LNP was then lyophilized for storage. Although no storage data were reported, EyeGene stated that their COVID vaccine is stored or shipped refrigerated (2-8 oC) and does not require ultra-low temperature storage. Of note, these LNPs do not contain PEG, a composition that differs from the Pfizer/BioNTech and Moderna vaccines that employ LNPs with a PEG-lipid component. 

According to Korea Biomedical Review, in September 2021, EyeGene received Korean regulatory approval for a Phase 1/2a trial of this candidate lyophilized mRNA-LNP COVID-19 vaccine. In their Phase 1 study, 45 healthy adults are divided into three dose groups to evaluate the safety, tolerability, and dose-finding. Phase 2a will be informed by the Phase 1 results but will have two dose groups to further assess immunogenicity, safety, and tolerability in 125 healthy adults. 

In October 2021, TriLink announced that it expects to supply its proprietary CleanCap mRNA capping technology and the modified uridine triphosphate to EyeGene to produce its COVID-19 vaccine. 

Moderna Clinical Trials of a Lyophilized Cytomegalovirus mRNA Vaccine

Cytomegalovirus (CMV) is a genus of viruses in the family Herpesviridae that use humans and monkeys as natural hosts. CMV is enveloped and has a highly symmetric capsid surrounding its double-stranded DNA genome. CMV can be transmitted from mother to child during pregnancy, birth, or breastfeeding. When a baby is infected with CMV prior to delivery, it is known as a congenital CMV infection. Most babies with congenital CMV infection have no symptoms, yet, about 10% have birth defects that range from mild to extremely severe.

In November 2021, Moderna presented an update on the clinical development of its CMV vaccine (mRNA-1647). This vaccine is comprised of six different antigen-encoding mRNAs (John et al., 2018). The initial LNP formulation of mRNA-1647 used in Phase 1 was replaced by a lyophilized dose that allows 5 oC storage and ≥18 months of shelf-life.

ClinicalTrials.Gov lists two studies for lyophilized mRNA-1647 reconstituted in normal saline. NCT05105048 is a Phase 1 study to evaluate the safety, reactogenicity, and immunogenicity of mRNA-1647 in healthy CMV-seronegative or CMV-seropositive adult patients in the U.S. This is followed by a similar Phase 3 trial (NCT05085366) of 6,900 patients that includes an evaluation of the efficacy of mRNA-1647.

Concluding Comments

Clearly, lyophilization provides vast improvements in the storage and distribution of mRNA-LNP vaccines. This technology will allow RNA vaccines to expand their availability to all corners globally. 

Importantly, TriLink recently launched its proprietary CleanCap technology as GMP-grade reagents. CleanCap is the fastest and easiest way to produce a 5’ Cap 1 mRNA with 95% efficiency, offering a modern alternative to enzymatic capping. With GMP grade now available, CleanCap reagents are an immediate solution for swiftly scaling from clinical to commercial therapeutics production.

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