Development of versatile affinity-based system for one step purification process: Case of Group A Streptococcus vaccine.

Affinity capture is one of the most attractive strategies for simplifying downstream processing. Although it is a key mainstream approach for antibody purification, the same is not true for other biologics such as vaccines, mainly due to the lack of suitable affinity material. In this study, a novel custom affinity system is introduced permitting widespread adoption of affinity capture for the purification of biologics beyond antibodies. This is illustrated here by the development of a one-step purification process of a mutant form of streptolysin O (SLO), a vaccine candidate against Streptococcus pyogenes infection. The system consists of the association of custom ligands based on the Nanofitin protein scaffold, with Eshmuno® industry-grade chromatography medium. The Nanofitins were selected for their specificity to the target product. The newly developed affinity medium was used at different column sizes to monitor scalability from process development (1 ml) and robustness verification (5 ml) to pilot (133 ml) and technical (469 ml) runs. The single-step affinity purification consistently delivered high purity product (above > 90%) and improved performances compared with the current three-step process: reduced process time and footprint (3 to 1 step) and increased product yields (0.31 g vs. 0.04 g of SLO per kg of harvest broth). The custom affinity system herein described can potentially be applied to any biologic for which a specific Nanofitin is identified, thus establishing a platform with a strong impact on the manufacturing of vaccines and other biological targets.

Effects of Organic and Inorganic Nitrogen on the Growth and Production of Domoic Acid by Pseudo-nitzschia multiseries and P. australis (Bacillariophyceae) in Culture.

Over the last century, human activities have altered the global nitrogen cycle, and anthropogenic inputs of both inorganic and organic nitrogen species have increased around the world, causing significant changes to the functioning of aquatic ecosystems. The increasing frequency of Pseudo-nitzschia spp. in estuarine and coastal waters reinforces the need to understand better the environmental control of its growth and domoic acid (DA) production. Here, we document Pseudo-nitzschia spp. growth and toxicity on a large set of inorganic and organic nitrogen (nitrate, ammonium, urea, glutamate, glutamine, arginine and taurine). Our study focused on two species isolated from European coastal waters: P. multiseries CCL70 and P. australis PNC1. The nitrogen sources induced broad differences between the two species with respect to growth rate, biomass and cellular DA, but no specific variation could be attributed to any of the inorganic or organic nitrogen substrates. Enrichment with ammonium resulted in an enhanced growth rate and cell yield, whereas glutamate did not support the growth of P. multiseries. Arginine, glutamine and taurine enabled good growth of P. australis, but without toxin production. The highest DA content was produced when P. multiseries grew with urea and P. australis grew with glutamate. For both species, growth rate was not correlated with DA content but more toxin was produced when the nitrogen source could not sustain a high biomass. A significant negative correlation was found between cell biomass and DA content in P. australis. This study shows that Pseudo-nitzschia can readily utilize organic nitrogen in the form of amino acids, and confirms that both inorganic and organic nitrogen affect growth and DA production. Our results contribute to our understanding of the ecophysiology of Pseudo-nitzschia spp. and may help to predict toxic events in the natural environment.