Taylor Dispersion Analysis to support lipid-nanoparticle formulations for mRNA vaccines.

Lipid nanoparticles (LNPs) are currently the most advanced non-viral clinically approved messenger ribonucleic acid (mRNA) delivery systems. The ability of a mRNA vaccine to have a therapeutic effect is related to the capacity of LNPs to deliver the nucleic acid intact into cells. The role of LNPs is to protect mRNA, especially from degradation by ribonucleases (RNases) and to allow it to access the cytoplasm of cells where it can be translated into the protein of interest. LNPs enter cells by endocytosis and their size is a critical parameter impacting their cellular internalization. In this work, we studied different formulation process parameters impacting LNPs size. Taylor dispersion analysis (TDA) was used to determine the LNPs size and size distribution and the results were compared with those obtained by Dynamic Light Scattering (DLS). TDA was also used to study both the degradation of mRNA in the presence of RNases and the percentage of mRNA encapsulation within LNPs.

Size and Charge Characterization of Lipid Nanoparticles for mRNA Vaccines.

Messenger RNA vaccines have come into the spotlight as a promising and adaptive alternative to conventional vaccine approaches. The efficacy of mRNA vaccines relies on the ability of mRNA to reach the cytoplasm of cells, where it can be translated into proteins of interest, allowing it to trigger the immune response. However, unprotected mRNA is unstable and susceptible to degradation by exo- and endonucleases, and its negative charges are electrostatically repulsed by the anionic cell membranes. Therefore, mRNA needs a delivery system that protects the nucleic acid from degradation and allows it to enter into the cells. Lipid nanoparticles (LNPs) represent a nonviral leading vector for mRNA delivery. Physicochemical parameters of LNPs, including their size and their charge, directly impact their in vivo behavior and, therefore, their cellular internalization. In this work, Taylor dispersion analysis (TDA) was used as a new methodology for the characterization of the size and polydispersity of LNPs, and capillary electrophoresis (CE) was used for the determination of LNP global charge. The results obtained were compared with those obtained by dynamic light scattering (DLS) and laser Doppler electrophoresis (LDE).

Antigen-Adjuvant Interactions in Vaccines by Taylor Dispersion Analysis: Size Characterization and Binding Parameters.

Vaccine adjuvants are immunostimulatory substances used to improve and modulate the immune response induced by antigens. A better understanding of the antigen-adjuvant interactions is necessary to develop future effective vaccine. In this study, Taylor dispersion analysis (TDA) was successfully implemented to characterize the interactions between a polymeric adjuvant (poly(acrylic acid), SPA09) and a vaccine antigen in development for the treatment of Staphylococcus aureus. TDA allowed one to rapidly determine both (i) the size of the antigen-adjuvant complexes under physiological conditions and (ii) the percentage of free antigen in the adjuvant/antigen mixture at equilibrium and finally get the interaction parameters (stoichiometry and binding constant). The complex sizes obtained by TDA were compared to the results obtained by transmission electron microscopy, and the binding parameters were compared to results previously obtained by frontal analysis continuous capillary electrophoresis.

Study of Interactions between Antigens and Polymeric Adjuvants in Vaccines by Frontal Analysis Continuous Capillary Electrophoresis.

Vaccine adjuvants are used to enhance the immune response induced by antigens that have insufficient immunostimulatory capabilities. The present work aims at developing a frontal analysis continuous capillary electrophoresis (FACCE) methodology for the study of antigen-adjuvant interactions in vaccine products. After method optimization using three cationic model proteins, namely lysozyme, cytochrome c, and ribonuclease A, FACCE was successfully implemented to quantify the free antigen and thus to determine the interaction parameters (stoichiometry and binding constant) between an anionic polymeric adjuvant (polyacrylic acid, SPA09) and a cationic vaccine antigen in development for the treatment for Staphylococcus aureus. The influence of the ionic strength of the medium on the interactions was investigated. A strong dependence of the binding parameters with the ionic strength was observed. The concentration of the polymeric adjuvant was also found to significantly modify the ionic strength of the formulation, the extent of which could be estimated and corrected.

Determination of molecular size parameters and quantification of polyacrylic acid by high performance size-exclusion chromatography with triple detection.

Synthetic polyelectrolytes are a broad class of vaccine adjuvants. Among them, polyacrylic acid (PAA), a polyanionic polymer, is currently evaluated by Sanofi Pasteur. As chain length is considered to be a critical quality attribute for adjuvant properties of PAA, measurement of precise and accurate molecular size parameters is important for these polymers. In the field of synthetic polymer chemistry, methods for determination of molecular size parameters are well defined. Specifically, high performance size-exclusion chromatography (HPSEC) with multi-detection system is a method of choice. This paper describes the development of HPSEC method to well characterize and precisely quantify PAA in different adjuvant formulations. A first set of characterizations were made, with determination of dn/dc coefficient, which enabled the determination of weight- and number-average molecular weight, viscosimetric radius, and intrinsic viscosity. In-depth characterization was also made with branching study through the use of Mark-Houwink parameter determination. The quantification method was also evaluated according to validation method-like criteria: limit of detection and limit of quantification, repeatability, accuracy, and specificity with recombinant surface glycoprotein gB from human cytomegalovirus (CMV-gB) as model antigen.

Separation of three strains of polio virus by capillary zone electrophoresis and study of their interaction with aluminum oxyhydroxide.

The development of combination vaccines is essential to reduce the number of injections, shorten vaccination schedules and increase vaccination coverage. Vaccine adjuvants are used to modulate and enhance the immune response induced by the antigens. To support the development of combination vaccines, the study of antigen-adjuvant interactions in the final vaccine formulations is required as interaction competitions may take place between the different antigens. In the present work, a capillary zone electrophoresis (CZE) methodology was firstly optimized on six model proteins, namely bovine serum albumin, ß-lactoglobulin, myoglobin, ribonuclease A, cytochrome C and lysozyme. A cationic dynamic coating (polybrene) and a zwitterionic amino acid additive (ß-alanine) in the background electrolyte were used to reduce the phenomena of protein adsorption on the inner wall of the capillary and thus optimize the separation efficiency of the proteins. The developed methodology was then used to separate three strains from inactivated polio virus, each strain being a whole virus composed of copies of 4 viral proteins and study their interaction with aluminum oxyhydroxide. The antigen-adjuvant interactions could be modulated by addition of phosphate ions playing the role of competitors for the poliovirus.

Design and preclinical characterization of a novel vaccine adjuvant formulation consisting of a synthetic TLR4 agonist in a thermoreversible squalene emulsion.

We describe the development, analytical characterization, stability and preclinical efficacy of AF04, a combination adjuvant comprising the synthetic toll-like receptor 4 (TLR4) agonist, E6020, formulated in AF03, a thermoreversible squalene emulsion. By using AF04 with the recombinant major outer membrane protein of Chlamydia trachomatis (Ct-MOMP) and with the recombinant surface glycoprotein gB from human cytomegalovirus (CMV-gB) as model antigens, we show that AF03 and E6020 can synergize to augment specific antibody and Th-1 cellular immune responses in mice. In terms of formulation, we observe that the method used to incorporate E6020 into AF03 affects its partition between the oil and water phases of the emulsion which in turn has a significant impact on the tolerability (IV pyrogenicity test in rabbits) of this novel adjuvant combination.

Characterization of surfactants in an oil-in-water emulsion-based vaccine adjuvant using MS and HPLC-MS: structural analysis and quantification.

Mass spectrometry (MS) and high performance liquid chromatography coupled to mass spectrometry (HPLC-MS) techniques were developed to characterize two surfactants, cetheareth-12 and sorbitan oleate, used to manufacture AF03, an emulsified oil-in-water (O/W) adjuvant. MS was first used to characterize the chemical structure and determine the composition of the two surfactants. The two surfactants appeared as complex products, in particular with respect to the nature of the fatty alcohols and the distribution of the number of ethylene oxides in cetheareth-12, and with respect to the different sorbitan-bound fatty acids (oleic, linoleic and palmitic acids) in sorbitan oleate. Subsequently, once the ions of interest were determined and selected, HPLC-MS was developed and optimized to quantify and to "quality control" the two surfactants as raw materials and as ingredients in the final O/W emulsion bulk and filled products.