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.

A potent novel vaccine adjuvant based on straight polyacrylate.

A structure-activity study was conducted to identify the structural characteristics underlying the adjuvant activity of straight (i.e. non-crosslinked) polyacrylate polymers (PAAs) in order to select a new PAA adjuvant candidate for future clinical development. The study revealed that the adjuvant effect of PAA was mainly influenced by polymer size (Mw) and dose. Maximal effects were obtained with large PAAs above 350 kDa and doses above 100 µg in mice. Small PAAs below 10 kDa had virtually no adjuvant effect. HPSEC analysis revealed that PAA polydispersity index and ramification had less impact on adjuvanticity. Heat stability studies indicated that residual persulfate could be detrimental to PAA stability. Hence, this impurity was systematically eliminated by diafiltration along with small Mw PAAs and residual acrylic acid that could potentially affect product safety, potency and stability. The selected PAA, termed SPA09, displayed an adjuvant effect that was superior to that of a standard emulsion adjuvant when tested with CMV-gB in mice, even in the absence of binding to the antigen. The induced immune response was dominated by strong IFNγ, IgG2c and virus neutralizing titers. The activity of SPA09 was then confirmed on human cells via the innate immune module of the human MIMIC® system.

Individualized Fortification Influences the Osmolality of Human Milk.

Background: Fortification of human milk (HM) increases its osmolality, which is associated with an increased risk of necrotizing enterocolitis. The impact of new fortifiers on osmolality is not well-known, nor are the kinetics regarding the increase in osmolality. Aim: To determine the optimum fortifier composition for HM fortification by measuring the osmolality of fortified HM made with three powder multicomponent fortifiers (MCFs) and a protein fortifier (PF). Methods: The osmolality of HM was assessed at 2 (H2) and 24 (H24) h after fortification to compare the effects of MCF (MCF1-3) and PF used in quantities that ensured that infants' nutrient needs would be met (MCF: 4 g/100 ml HM; PF: 0.5 g or 1 g/100 ml HM). To evaluate the early kinetics associated with the osmolality increase, the osmolality of HM fortified with MCF1 or MCF2 was also measured at 0, 1, 5, 10, 15, 20, 30, 40, 50, 60, 90, and 120 min after fortification. Results: The osmolality increased significantly immediately after fortification, depending on the type of fortification used and the quantity of MCF and PF used, rather than the time elapsed after fortification. The maximum value at H24 was 484 mOsm/kg. The mean increase in osmolality between H2 and H24 was 3.1% (p < 0.01) (range: 0.2-10.8%). Most of the increase (>70%) occurred immediately after fortification. Conclusion: When choosing a fortifier, its effect on HM osmolality should be considered. As most of the increase in osmolality occurred immediately, bedside fortification is not useful to prevent the increase in osmolality, and further research should focus on improving fortifier composition.

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.

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.