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Ryan M Kramer, Michelle C Archer, Mark T Orr, Natasha Dubois Cauwelaert, Elyse A Beebe, Po-wei D Huang, Quinton M Dowling, Alicia M Schwartz, Dawn M Fedor, Thomas S Vedvick, Christopher B Fox Infectious Disease Research Institute, Seattle, WA, USA Background: Adjuvants have the potential to increase the efficacy of protein-based vaccines but need to be maintained within specific temperature and storage conditions. Lyophilization can be used to increase the thermostability of protein pharmaceuticals; however, no marketed vaccine that contains an adjuvant is currently lyophilized, and lyophilization of oil-in-water nanoemulsion adjuvants presents a specific challenge. We have previously demonstrated the feasibility of lyophilizing a candidate adjuvanted protein vaccine against Mycobacterium tuberculosis (Mtb), ID93 + GLA-SE, and the subsequent improvement of thermostability; however, further development is required to prevent physicochemical changes and degradation of the TLR4 agonist glucopyranosyl lipid adjuvant formulated in an oil-in-water nanoemulsion (SE). Materials and methods: In this study, we took a systematic approach to the development of a thermostable product by first identifying compatible solution conditions and stabilizing excipients for both antigen and adjuvant. Next, we applied a design-of-experiments approach to identify stable lyophilized drug product formulations. Results: We identified specific formulations that contain disaccharide or a combination of disaccharide and mannitol that can achieve substantially improved thermostability and maintain immunogenicity in a mouse model when tested in accelerated and real-time stability studies. Conclusion: These efforts will aid in the development of a platform formulation for use with other similar vaccines. Keywords: adjuvant, lyophilization, tuberculosis, formulation development, design of experiments, controlled temperature chain, GRAS

Cyclic GMP-AMP synthase (cGAS) is a pattern recognition receptor critical for the innate immune response to intracellular pathogens, DNA damage, tumorigenesis and senescence. Binding to double-stranded DNA (dsDNA) induces conformational changes in cGAS that activate the enzyme to produce 2'-3' cyclic GMP-AMP (cGAMP), a second messenger that initiates a potent interferon (IFN) response through its receptor, STING. Here, we combined two-state computational design with informatics-guided design to create constitutively active, dsDNA ligand-independent cGAS (CA-cGAS). We identified CA-cGAS mutants with IFN-stimulating activity approaching that of dsDNA-stimulated wild-type cGAS. DNA-independent adoption of the active conformation was directly confirmed by X-ray crystallography. In vivo expression of CA-cGAS in tumor cells resulted in STING-dependent tumor regression, demonstrating that the designed proteins have therapeutically relevant biological activity. Our work provides a general framework for stabilizing active conformations of enzymes and provides CA-cGAS variants that could be useful as genetically encoded adjuvants and tools for understanding inflammatory diseases., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Although substantial effort has been made in the development of next-generation recombinant vaccine systems, maintenance of a cold chain is still typically required and remains a critical challenge in effective vaccine distribution. The ability to engineer alternative containment systems that improve distribution and administration represents potentially significant enhancements to vaccination strategies. In this work, we evaluate the ability to successfully lyophilize a previously demonstrated thermostable tuberculosis vaccine formulation (ID93 + GLA-SE) in a cartridge format compared to a traditional vial container format. Due to differences in the shape of the container formats, a novel apparatus was developed to facilitate lyophilization in a cartridge. Following lyophilization, the lyophilizate was assessed visually, by determining residual moisture content, and by collecting melting profiles. Reconstituted formulations were assayed for particle size, protein presence, and GLA content. Based on assessment of the lyophilizate, the multicomponent vaccine was successfully lyophilized in both formats. Also, the physicochemical properties of the major components in the formulation, including antigen and adjuvant, were retained after lyophilization in either format. Ultimately, this study demonstrates that complex formulations can be lyophilized in alternative container formats to the standard pharmaceutical glass vial, potentially helping to increase the distribution of vaccines.

Dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) are two orthogonal and complementary methods of measuring size of particles in a sample. These technologies use the theory of Brownian motion by analyzing the random changes of light intensity scattered by particles in solution. Both techniques can be used to characterize particle size distribution of proteins and formulations in the nanometer to low micron range.Each method has benefits over the other. DLS is a quick and simple measurement that is ideal for monodisperse particles and can also analyze a distribution of particles over a wide range of sizes. NTA provides a size distribution that is less susceptible to the influence of a few large particles, and has the added benefit of being able to measure particle concentration. Here we describe methods for measuring the particle size and concentration of an oil-in-water nanoemulsion.

Fourier transform infrared (FTIR) spectroscopy is widely used in the pharmaceutical industry for process monitoring, compositional quantification, and characterization of critical quality attributes in complex mixtures. Advantages over other spectroscopic measurements include ease of sample preparation, quantification of multiple components from a single measurement, and the ability to quantify optically opaque samples. This method describes the use of a multivariate model for quantifying a TLR4 agonist (GLA) adsorbed onto aluminum oxyhydroxide (Alhydrogel ® ) using FTIR spectroscopy that may be adapted to quantify other complex aluminum based adjuvant mixtures.

Aluminum salts have a long history as safe and effective vaccine adjuvants. In addition, aluminum salts have high adsorptive capacities for vaccine antigens and adjuvant molecules, for example, Toll-like receptor 4 (TLR4) agonists. However, the physicochemical properties of aluminum salts make direct quantitation of adsorbed molecules challenging. Typical methods for quantifying adsorbed molecules require advanced instrumentation, extreme sample processing, often destroy the sample, or rely on an indirect measurement. A simple, direct, and quantitative method for analysis of adsorbed adjuvant molecules is needed. This report presents a method utilizing Fourier transform infrared spectroscopy with a ZnSe-attenuated total reflectance attachment to directly measure low levels (<30 μg/mL) of TLR4 agonists adsorbed on aluminum salts with minimal sample preparation., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)