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.

A Generic Procedure for the Isolation of pH- and Magnesium-Responsive Chicken scFvs for Downstream Purification of Human Antibodies.

Affinity chromatography provides an excellent platform for protein purification, which is a key step in the large scale downstream processing of therapeutic monoclonal antibodies (Mabs). Protein A chromatography constitutes the gold standard for Mab purification. However, the required acidic conditions (2.8-3.5) for elution from the affinity matrix limit their applicability, particularly for next generation antibodies and antibody fusion proteins, since denaturation and irreversible aggregation can occur due to the acidic buffer conditions. Here we describe a generic procedure for the generation of antigen-specific chromatography ligands with tailor-made elution conditions. To this end, we generated a scFv-library based on mRNA from a chicken immunized with human Fc. The antibody repertoire was displayed on yeast Saccharomyces cerevisiae screened via FACS toward pH- and magnesium-responsive scFvs which specifically recognize human IgG antibodies. Isolated scFvs were reformatted, produced in Escherichia coli and immobilized on NHS-agarose columns. Several scFvs were identified that mediated antibody binding at neutral pH and antibody recovery at pH values of 4.5 and higher or even at neutral pH upon MgCl2 exposure. The iterative screening methodology established here is generally amenable to the straightforward isolation of stimulus-responsive antibodies that may become valuable tools for a variety of applications.

Simultaneous separation and analysis of water- and fat-soluble vitamins on multi-modal reversed-phase weak anion exchange material by HPLC-UV.

Several methods for the separation of vitamins on HPLC columns were already validated in the last 20 years. However, most of the techniques focus on separating either fat- or water-soluble vitamins and only few methods are intended to separate lipophilic and hydrophilic vitamins simultaneously. A mixed-mode reversed-phase weak anion exchange (RP-WAX) stationary phase was developed in our laboratory in order to address such mixture of analytes with different chemical characteristics, which are difficult to separate on standard columns. The high versatility in usage of the RP-WAX chromatographic material allowed a baseline separation of ten vitamins within a single run, seven water-soluble and three fat-soluble, using three different chromatographic modes: some positively charged vitamins are eluted in ion exclusion and ion repulsion modes whereas the negatively charged molecules are eluted in the ion exchange mechanism. The non-charged molecules are eluted in a classical reversed-phase mode, regarding their polarities. The method was validated for the vitamin analysis in tablets, evaluating selectivity, robustness, linearity, accuracy, and precision. The validated method was finally employed for the analysis of the vitamin content of some commercially available supplement tablets.

Statistical optimization of the silylation reaction of a mercaptosilane with silanol groups on the surface of silica gel.

Thiol-modified silica is often used as an intermediate product for further synthesis of modified stationary phases for chromatography or purification processes. Different conditions were used to synthesize such thiol-modified particles, but systematic optimizations remained scarce. In this study the reaction conditions for the synthesis of mercaptopropyl-modified silica were optimized. The general synthetic method consists in slurrying the silica gel in toluene before adding 3-mercaptopropyldimethoxymethylsilane together with a tertiary amine as catalyst (here dimethylaminopyridine). Reaction time and temperature were optimized using a full factorial design of experiment (DoE) from 3 to 25h with temperature varying between 45 and 105 degrees C. The surface coverage of the silica with mercaptopropyl-groups was analyzed by two different ways (elemental analysis and chemical surface reaction with 2,2'-dipyridyl disulfide followed by HPLC-UV analysis of stoichiometrically liberated pyridyl-2-thione). We obtained a three-dimensional (3D) plot of the surface coverage as a function of reaction time and temperature. The arch-shaped hyperplane allowed us to determine an optimum with regard to time and temperature, which yields to the highest surface coverage possible. We also verified that the increase of the surface coverage does not lead to a decrease of the stability of the surface modification by subjecting the gels to treatment with high temperature and acidic conditions. The stability was monitored by different chromatographic methods. Moreover, (29)Si cross-polarization-magic angle spinning (CP-MAS) NMR spectra of materials prepared by different conditions allowed to confirm that the Si species on the surface were essentially the same, while there was only a minute difference in signal intensities for the individual Si species for materials obtained by distinct temperatures.