Using Analytical Quality by Design to improve analytical method development in vaccines quality control: Application to an optimized quantitative high-performance anion-exchange chromatographic method.

Analytical quality by design (AQbD) is an enhanced approach for the development of analytical methods. AQbD has received much industrial interest, being the subject of several recently published draft guidelines. This article demonstrates the application of AQbD to determine the quantity of non-adsorbed polysaccharide polyribosyl ribitol phosphate (PRP) and percentage of depolymerized PRP in a commercial hexavalent liquid vaccine, and establishment of an analytical control strategy (ACS). The quantification method developed is high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection, preceded by ultracentrifugation (sample preparation) for separation of the depolymerized polysaccharide from the native adsorbed polysaccharide. The first step was to develop the analytical target profile (ATP) which defines the purpose of the analytical measurement as well as the development scope. As a second step, risk assessment tools were used for identification and ranking of the critical method variables (CMVs) which have a potential impact on method performance if not controlled. Based on a multivariate Design of Experiments (DoE) approach, a proposed method operational design region (MODR) was determined for seven CMVs. Finally, the ACS was established from the understanding of the analytical method and the robustness study. This article focuses on robust and operational ranges of critical parameters linked to the ultracentrifugation and chromatographic steps for depolymerized polysaccharide content control. The design space proposed for CMVs corresponds to the ranges that ensure a product that complies with the previously established precision criteria (±2% equivalent to ± 10 % around the product criterion, which is 20 % for depolymerized polysaccharide control limit). The following design space was established from the DoE statistical modeling for ultracentrifugation critical parameters: [483,000-520,000] g for speed, [11-19]°C for temperature, [29-34] minutes for duration, and from extemporaneous to 8 min for holding time before supernatant recuperation after the ultracentrifugation. For chromatographic critical parameters, the MODR is [2-6] psi for mobile phase helium pressure, [0-7] days for mobile phase storage time, and [0-3] days for samples storage time in the autosampler at 5 °C. Methods optimized using the AQbD approach provide strong justifications during regulatory filing for the selection of analytical CMVs, and for the ACS to be applied during the lifecycle management of the method.

mRNA extraction from lipid nanoparticles.

Messenger RiboNucleic Acid (mRNA) vaccines have recently shown considerable promises for both prophylactic and therapeutic vaccines. These vaccines do not carry an antigen but the information for producing it using the cell machinery, turning the human body into an antigen factory. However, mRNA is an unstable molecule, susceptible to physical, chemical and enzymatic degradation by exo- and endonucleases. If the mRNA is degraded, it can no longer be translated correctly into the antigen of interest and the vaccine lose its efficacy. To protect from nucleases degradation and allow it to get into the cells, mRNA can be encapsulated in lipid nanoparticles (LNPs). As part of the manufacturing process, the quality of the mRNAs should be controlled before the encapsulation (at the drug substance stage) as well as after formulation on the final vaccine product (at the drug product stage). Therefore, it is necessary to be able to extract the mRNA from the LNPs, that is to deformulate the final vaccine product. In this work, different deformulation methods have been compared: spin column extraction, magnetic particle extraction, organic extraction, and direct disruption. Advantages and disadvantages of each of these methods are highlighted.