Purpose The goal of this study is to set an empirical baseline to map the structure-function relation of the antigens from the commercialized vaccine products. Methods To study the structural changes of protein antigens after adsorption several analytical tools including DLS, FTIR, Fluorescence, LD, and SEM have been used. Results All antigens have shown wide range of hydrodynamic diameter from 7 nm to 182 nm. Upon adjuvantation, the size distribution has become narrow, ranging from 10 to 12 μm, and has been driven by the derived diameter of aluminum phosphate (AlPO 4 ) adjuvant. Further to examine size and morphology of adsorbed antigens, SEM has been used. The SEM results have demonstrated that the AlPO 4 adjuvant suspension and adsorbed proteins consist of submicron particles that form a continuous porous surface. Diphtheria Toxoid (DT), Tetanus Toxoid (TT), and chemically-modified Filamentous Haemagglutinin (FHA) have shown surface adsorption to AlPO 4. Secondary structure alpha-helix and beta-sheet content of DT and TT has increased after adsorption to AlPO 4 adjuvant as shown by FTIR, whereas no significant changes were noted for other protein antigens. The results from Intrinsic Fluorescence have shown a structural rearrangement in DT and TT, consistent with the FTIR results. Multivalent vaccine product identity has been determined by FTIR as unique fingerprint spectrum. Conclusion The globular proteins such as DT and TT have shown changes in secondary structure upon adsorption to AlPO 4 , whereas fibrillar protein FHA has not been affected by adsorption. FTIR can be used as a lean technique to confirm product identity at different manufacturing sites.

Tuberculosis (TB) is one of the leading causes of death worldwide, making the development of effective TB vaccines a global priority. A TB vaccine consisting of a recombinant fusion protein, H4, combined with a novel synthetic cationic adjuvant, IC31, is currently being developed. The H4 fusion protein consists of two immunogenic mycobacterial antigens, Ag85 B and TB10.4, and the IC31 adjuvant is a mixture of KLK, a leucine-rich peptide (KLKL5KLK), and the oligodeoxynucleotide ODN1a, a TLR9 ligand. However, efficient and robust methods for assessing these formulated components are lacking. Here, we developed and optimized phase analysis light scattering (PALS), electrical sensing zone (ESZ), and Raman, FTIR, and CD spectroscopy methods to characterize the H4-IC31 vaccine formulation. PALS-measured conductivity and zeta potential values could differentiate between the similarly sized particles of IC31 adjuvant and the H4-IC31 vaccine candidate and could thereby serve as a control during vaccine formulation. In addition, zeta potential is indicative of the adjuvant to antigen ratio which is the key in the immunomodulatory response of the vaccine. ESZ was used as an orthogonal method to measure IC31 and H4-IC31 particle sizes. Raman, FTIR, and CD spectroscopy revealed structural changes in H4 protein and IC31 adjuvant, inducing an increase in both the β-sheet and random coil content as a result of adsorption. Furthermore, nanoDSF showed changes in the tertiary structure of H4 protein as a result of adjuvantation to IC31. Our findings demonstrate the applicability of biophysical methods to characterize vaccine components in the final H4-IC31 drug product without the requirement for desorption.

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1) Background: Traditionally, complex biological products such as vaccines presented unique challenges to implementation of even rudimentary characterization packages; thus, the product was defined almost exclusively by its manufacturing process. The advances in technology and analytical tools allowed the application of more comprehensive characterization packages for products such as adsorbed combination vaccines, which contain several antigens in a single formulation to protect against more than one disease, and may contain adjuvants and excipients. Aluminum phosphate (AlPO4) is a well-established adjuvant for enhancing the uptake of vaccines and to induce robust immunity against pathogens. During manufacturing, adjuvant is mixed with protein antigens which may in turn impact their higher order structure and stability. 2) Methods: To study the structural changes of protein antigens after adsorption several analytical tools including DLS, FTIR, Fluorescence, LD, and SEM were used. 3) Results: the AlPO4 adjuvant suspension consists of small submicron particles that form a continuous porous surface. Secondary structure alpha-helix and beta-sheet content of DT and TT increased after adsorption to AlPO4 adjuvant, whereas no significant changes were noted for other protein antigens. Interactions were noted between AlPO4 adjuvant and DT, TT, and FHA. 4) Conclusions: here we report for the first time the use of SEM for the visualization of adsorbed multivalent vaccine components. A unique signature profile detected for each multivalent vaccine by FTIR can be used as a lean in-process test to verify vaccine product composition and identity prior to filling.