Stability studies of the vaccine adjuvant U-Omp19

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and lysosomal peptidases, increasing the amount of co-administered antigen that reaches the immune inductive 22 sites and its half-life inside cells, and it is able to stimulate antigen presenting cells in vivo. These activities enable 23 U-Omp19 to enhance the adaptive immune response to co-administrated antigens.

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To characterize the stability of U-Omp19, we have performed an extensive analysis of its physicochemical and 25 biological properties in a 3-year long-term stability study, and under potentially damaging freeze-thawing and 26 lyophilization stress processes. Results revealed that U-Omp19 retains its full protease inhibitor activity, its 27 monomeric state and its secondary structure even when stored in solution for 36 months or after multiple freeze-28 thawing cycles. Non-enzymatic hydrolysis resulted the major degradation pathway for storage in solution at 4°C 29 or room temperature which can be abrogated by lyophilization yet increasing protein tendency to form 30 aggregates.

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This information will play a key role in the development of a stable formulation of U-Omp19, allowing an

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Oral immunization offers several advantages over other routes of administration [1][2][3] . Mucosal vaccines are 36 highly effective providing both mucosal as well as systemic immunity [1][2][3][4][5] . This route of administration presents 37 logistical and regulatory advantages over parenterally given vaccines and may be the preferred choice for 38 emerging pathogens or the need of mass vaccination in case of a pandemic or epidemic. However, safety 39 regulatory issues remain to be attended; since the few current licensed oral vaccines are live attenuated or non-40 living whole-cell vaccines 1,2,4 . In this light, subunit vaccines lacking entire pathogens are considered to be safer 2 .

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Notwithstanding, some fundamental considerations must be upheld when developing subunit mucosal vaccines, 42 including poor immunogenicity, degradation of its constituents in the harsh mucosal environment, delivery of its 43 materials to mucosal immune inductive tissue, and modulation of the mucosal immune environment such that oral 44 tolerance does not develop [1][2][3][4] . These obstacles will be overcome by developing effective mucosal adjuvants 1,3,5 .

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Adjuvants are molecules or compounds that enhance the immune response to co-administered antigens (Ags) 46 after immunization. The major limiting factor for oral vaccine development is the restricted availability of mucosal  . U-Omp19 is able to stimulate cells of the immune system and also, it is capable of 64 partially inhibiting host gastrointestinal and endosomal proteases. These two main properties may explain its 65 adjuvant activity by protecting co-delivered antigens from degradation, increasing their half-life, promoting their 66 arrival to induction sites and enhancing the elicited antigen-specific immune response [9][10][11][12][13] .

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To be used as a drug, proteins must be stable during development, production and storage, preserving both 68 activity and structure. Since vaccines are prone to physical and chemical degradation during manufacturing,

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Omp19 solution were prepared. The mean of three absorbance spectra was recorded in the region between 220 108 and 320 nm (1.0 nm steps) and the sample protein content was determined based on the absorbance at λ = 280 109 nm and the dilution factor applied. The theoretical molar extinction coefficient of the purified protein (ε 280

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Size and aggregation state were monitored by DLS using a Zetasizer Nano-S (Malvern Instruments, UK). After

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To evaluate the stability of U-Omp19's protease inhibitor activity, its ability to inhibit α-chymotrypsin was  (Table 1). Though, U-Omp19 samples can be stored up to 3 weeks at RT or 1 month at 4 °C without 261 undergoing significant degradation ( Figure 4C).

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In order to investigate whether U-Omp19 fragmentation could be due to trace protease contamination, 263 additional work was conducted to screen excipients or sample treatments that may improve the stability of U-

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Neither the addition of a second purification step (SEC), nor sample treatment with the serine protease inhibitor 266 PMSF, the metal chelating agent EDTA or heat were able to reduce the observed proteolysis, suggesting that it   Figure 5B). Moreover, freeze-thaw cycles didn't induce significant protein aggregation ( Figure 5C).

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Together these results indicate that U-Omp19 can afford multiple cycles of freeze and thaw without undergoing 287 significant degradation, maintaining its physicochemical and biological properties unchanged.

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Lyophilization (freeze drying) of U-Omp19 overcomes fragmentation and allows room temperature 290 storage for at least 18 months but induces protein aggregation.

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Since protein fragmentation is the main degradation pathway that U-Omp19 undergoes when stored at room  modifications that could be introduced (e.g., site-directed mutagenesis) to improve its inherent stability.

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To achieve an overall stable formulation is the main target in any protein/drug formulation process. Protein . In this work, U-Omp19, was shown 389 to retain its full inhibitory activity, folding, integrity, while no significant aggregation was induced when subjected to 390 freezing ( Figure 5).

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Freeze-thaw is often explored as a forced degradation condition to determine the susceptibility of a protein to 392 temperature cycling, since low temperature, freeze-concentration, and ice formation damages may occur during 393 the process. Freeze-thawing is a common stress to which a protein drug can be exposed to deliberately or 394 accidentally, once or multiple times during manufacturing, shipping, and storage and may lead to protein

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