Endotoxin reduction from biotech silk material inhibits the production of anti-silk antibodies in mice.

Eliminating endotoxins is a common problem in the development of biotechnologically produced pharmaceuticals or biomaterials. Residual endotoxins in the final sample may hamper the properties of the product or induce severe adverse effects. Developing an effective downstream purification protocol that ensures a lack of minimal endotoxin content in the final product can be a challenging task. In our previous studies, we developed nanospheres produced from bioengineered silks. Despite their good overall biocompatibility, in vivo characterization of spheres showed mild activation of the immune system (mainly in terms of anti-silk antibody production). Herein, we examined, if the endotoxins delivered with the silk spheres might have contributed to activating the adaptive immune response. We investigated various commercially available methods for endotoxin removal that can be applied as an extra step in downstream endotoxin removal from MS1-type silk proteins. We selected a method that allowed for a 10-fold reduction of endotoxin content in soluble silk and 2-fold in the final product (silk spheres). The reduced level of endotoxins improved the biocompatibility of the silk spheres as these particles induced negligible titers of anti-silk antibodies in an in vivo immune study. Since endotoxins can enhance life-threatening immune responses, it is crucial to optimize the method of their removal before clinical use not only of silk-based products but also of other biomolecules produced biotechnologically.