Understanding the process-induced formation of minor conformational variants of Erwinia chrysanthemil-asparaginase.

During Erwinia chrysanthemil-asparaginase (ErA) manufacture, minor conformational variants are formed that elute in the acidic region of the analytical ion-exchange HPLC chromatogram. These variants retain enzymatic activity and form part of the biopharmaceutical product, but must be kept within acceptable limits through controlled operation of the manufacturing process. The high isoelectric point of the ErA native tetramer (8.6) leads to certain process steps being operated in the alkaline pH region. Previously, the formation of these species during processing was not fully understood. In this work, in-process samples were analysed, and alkaline pH (8-9) and hold time were found to be the governing parameters. Formation of ErA acidic species was found to be accelerated at higher pH values and longer hold times, suggesting potential control strategies for the manufacturing process. However, the kinetics of ErA conformational variant formation were found to be slow (0.15-0.25 percent per day at pH 8.5). Changes in the ErA melt temperature (Tm) with pH as determined by both differential scanning calorimetry and differential scanning fluorimetry were found to be predictive of the tendency to form the IEX-HPLC acidic species during processing. Biopharmaceutical process developers should be aware of such changes to proteins and build relevant control strategies into process validation plans.

Human dendritic cells conditioned with Staphylococcus aureus enterotoxin B promote TH2 cell polarization.

BACKGROUND: Immune surveillance against microbes at sites of interface with environment involves immediate recognition of pathogen-associated molecular patterns by dendritic cells (DCs). According to their first-line position, DCs are key parameters for the establishment of an appropriate innate and adaptive response against pathogens to avoid disease development. Even though their role in pathogenesis is well known, bacterial toxins have been less examined for their ability to drive DC activation and T-cell polarization. OBJECTIVE: We made the assumption that early conditioning of DCs with Staphylococcus aureus enterotoxins could take part in T-cell polarization. METHODS: Human monocyte-derived DCs were stimulated with S aureus enterotoxin B (SEB) and characterized with respect to secretion of inflammatory cytokines and their ability to drive polarization of naive allogenic T cells. RESULTS: We demonstrated that SEB induced maturation of DCs and that SEB-activated DCs secreted high levels of IL-2 but no IL-12p70, contrary to LPS-activated ones. Accordingly, we further showed that SEB-activated DCs were able to drive polarization of naive T cells into the T(H)2 subset. By using highly purified SEB and Toll-like receptor (TLR)-2 stably transfected Human Embryonic Kidney (HEK) 293 cells, we demonstrated for the first time the ability of SEB to induce TLR2 signaling. Furthermore, the involvement of SEB-TLR2 interaction in activation of dendritic cells was supported by neutralizing activity of anti-TLR2 antibodies. CONCLUSION: Altogether, our findings reinforce the notion that bacterial toxins may appear as new pathogen-associated molecular patterns, which could play a major role in inflammation and bacterial pathologies.