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.

Recombination between polioviruses and co-circulating Coxsackie A viruses: role in the emergence of pathogenic vaccine-derived polioviruses.

Ten outbreaks of poliomyelitis caused by pathogenic circulating vaccine-derived polioviruses (cVDPVs) have recently been reported in different regions of the world. Two of these outbreaks occurred in Madagascar. Most cVDPVs were recombinants of mutated poliovaccine strains and other unidentified enteroviruses of species C. We previously reported that a type 2 cVDPV isolated during an outbreak in Madagascar was co-circulating with coxsackieviruses A17 (CA17) and that sequences in the 3' half of the cVDPV and CA17 genomes were related. The goal of this study was to investigate whether these CA17 isolates can act as recombination partners of poliovirus and subsequently to evaluate the major effects of recombination events on the phenotype of the recombinants. We first cloned the infectious cDNA of a Madagascar CA17 isolate. We then generated recombinant constructs combining the genetic material of this CA17 isolate with that of the type 2 vaccine strain and that of the type 2 cVDPV. Our results showed that poliovirus/CA17 recombinants are viable. The recombinant in which the 3' half of the vaccine strain genome had been replaced by that of the CA17 genome yielded larger plaques and was less temperature sensitive than its parental strains. The virus in which the 3' portion of the cVDPV genome was replaced by the 3' half of the CA17 genome was almost as neurovirulent as the cVDPV in transgenic mice expressing the poliovirus cellular receptor gene. The co-circulation in children and genetic recombination of viruses, differing in their pathogenicity for humans and in certain other biological properties such as receptor usage, can lead to the generation of pathogenic recombinants, thus constituting an interesting model of viral evolution and emergence.