Characterisation of factors contributing to the performance of nonwoven fibrous matrices as substrates for adenovirus vectored vaccine stabilisation.

Adenovirus vectors offer a platform technology for vaccine development. The value of the platform has been proven during the COVID-19 pandemic. Although good stability at 2-8 °C is an advantage of the platform, non-cold-chain distribution would have substantial advantages, in particular in low-income countries. We have previously reported a novel, potentially less expensive thermostabilisation approach using a combination of simple sugars and glass micro-fibrous matrix, achieving excellent recovery of adenovirus-vectored vaccines after storage at temperatures as high as 45 °C. This matrix is, however, prone to fragmentation and so not suitable for clinical translation. Here, we report an investigation of alternative fibrous matrices which might be suitable for clinical use. A number of commercially-available matrices permitted good protein recovery, quality of sugar glass and moisture content of the dried product but did not achieve the thermostabilisation performance of the original glass fibre matrix. We therefore further investigated physical and chemical characteristics of the glass fibre matrix and its components, finding that the polyvinyl alcohol present in the glass fibre matrix assists vaccine stability. This finding enabled us to identify a potentially biocompatible matrix with encouraging performance. We discuss remaining challenges for transfer of the technology into clinical use, including reliability of process performance.

Excipient selection for thermally stable enveloped and non-enveloped viral vaccine platforms in dry powders.

Two enveloped viral vectors, vesicular stomatitis virus and influenza virus, and a non-enveloped viral vector, human adenovirus type 5, were encapsulated by spray drying to enhance thermal stability.Results with these candidates led to the hypothesis that stability performance of chosen excipients may be less virus-specific, as previously postulated in the literature, and more differentiated based on whether the virus has a lipid envelope. Spray dried samples were characterized for their thermal properties, RNA viability and in vitro viral activity after storage at 37 °C for up to 30 days or at 45 °C for up to 3 days. The enveloped viral vectors, as a group, were more thermally stable in trehalose while the non-enveloped viral vector showed higher activity with mannitol as the primary excipient in blends. Trehalose shows strong hydrogen bonds with the envelope's lipid membrane than the other carbohydrates, more effectively replacing water molecules while maintaining the fluidity of the membrane. Conversely, the small size of mannitol molecules was attributed to the more effective hydrogen bonding between water and the protein capsid of non-enveloped viral vectors. In all cases, a matrix with high glass transition temperature contributed to thermal stabilization through vitrification. This work suggests that carbohydrate stabilizer selection may be more dependent on the envelope rather than the specific viral vector, which, if universally true, will provide a guideline for future formulation development.

Fast, selective and quantitative protein profiling of adenovirus-vector based vaccines by ultra-performance liquid chromatography.

A method for the quantitative determination of the protein composition of adenovirus-vector based vaccines was developed. The final method used RP-UPLC with UV absorbance detection, a C4 column (300 Å, 1.7 µm, 2.1 × 150 mm), and a water- acetonitrile (ACN) gradient containing trifluoroacetic acid (TFA) as ion-pairing agent. The chromatographic resolution between the various adenovirus proteins was optimized by studying the effect of the TFA concentration and the column temperature, applying a full factorial design of experiments. A reproducible baseline separation of all relevant adenovirus proteins could be achieved within 17 min run time. Samples containing adenovirus particles could be directly injected into the UPLC system without sample pretreatment. The viruses reproducibly dissociate into proteins in the UPLC system upon contact with the mobile phase containing ACN. The new RP-UPLC method was successfully validated for protein profiling and relative quantification of proteins in vaccine products based on adenovirus vector types 26 and 35. The intermediate precision of the relative peak areas of all proteins was between 1% and 14% RSD, except for the peak assigned to protein V (26% RSD). The method proved to be stability indicating with respect to thermal and oxidation stress of the adenovirus-vector based vaccine and was successfully implemented for the characterization of adenovirus-based products.

Additives for vaccine storage to improve thermal stability of adenoviruses from hours to months.

Up to 80% of the cost of vaccination programmes is due to the cold chain problem (that is, keeping vaccines cold). Inexpensive, biocompatible additives to slow down the degradation of virus particles would address the problem. Here we propose and characterize additives that, already at very low concentrations, improve the storage time of adenovirus type 5. Anionic gold nanoparticles (10-8-10-6 M) or polyethylene glycol (PEG, molecular weight ∼8,000 Da, 10-7-10-4 M) increase the half-life of a green fluorescent protein expressing adenovirus from ∼48 h to 21 days at 37 °C (from 7 to >30 days at room temperature). They replicate the known stabilizing effect of sucrose, but at several orders of magnitude lower concentrations. PEG and sucrose maintained immunogenicity in vivo for viruses stored for 10 days at 37 °C. To achieve rational design of viral-vaccine stabilizers, our approach is aided by simplified quantitative models based on a single rate-limiting step.