Development of Robust Freeze-Drying Process for Long-Term Stability of rVSV-SARS-CoV-2 Vaccine.

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.

Enabling mRNA Therapeutics: Current Landscape and Challenges in Manufacturing.

Recent advances and discoveries in the structure and role of mRNA as well as novel lipid-based delivery modalities have enabled the advancement of mRNA therapeutics into the clinical trial space. The manufacturing of these products is relatively simple and eliminates many of the challenges associated with cell culture production of viral delivery systems for gene and cell therapy applications, allowing rapid production of mRNA for personalized treatments, cancer therapies, protein replacement and gene editing. The success of mRNA vaccines during the COVID-19 pandemic highlighted the immense potential of this technology as a vaccination platform, but there are still particular challenges to establish mRNA as a widespread therapeutic tool. Immunostimulatory byproducts can pose a barrier for chronic treatments and different production scales may need to be considered for these applications. Moreover, long-term storage of mRNA products is notoriously difficult. This review provides a detailed overview of the manufacturing steps for mRNA therapeutics, including sequence design, DNA template preparation, mRNA production and formulation, while identifying the challenges remaining in the dose requirements, long-term storage and immunotolerance of the product.