Manufacturing development of recombinant S100A8/S100A9 for prevention of neonatal sepsis in preterm infants
SCHARRNBECK-DAVIS C. 1, VIEMANN D. 2, PIRR S. 3, VOGL T. 1
1 Institute of Immunology, University of Münster, Münster, Germany; 2 Department of Pediatrics, University Hospital of Würzburg, Würzburg, Germany; 3 Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, Hannover, Germany
The heterodimeric complex S100A8/S100A9 (Calprotectin) is an integral part of the innate immune response and belongs to the family of damage-associated molecular pattern molecules as it acts via binding to TLR4 expressing cells inducing pro-inflammatory immune responses. Contrary to the inflammatory effect in human adults, high S100A8/S100A9 serum levels induce innate immune programming in newborn infants whereas S100A8/S100A9 deficient states predispose neonates to hyper inflammation and late onset sepsis. Experimental data in mice indicate that feeding of pups with S100A8/S100A9 is a promising approach to overcome the risk of sepsis. A clinical study requires an appropriate expression system and purification methods to ensure a highly pure and endotoxin free protein. Escherichia coli BL21 cells transformed with a pET-vector containing the genomic sequences of human S100A8 or S100A9 is used as expression system to obtain a high protein yield. The protein was stored as insoluble inclusion bodies, thus, an extensive downstream process including cell lysis, solubilization, de- and renaturation, anionic exchange chromatography and gel-filtration was performed. Purified protein samples were analyzed by SDS-PAGE regarding purity and activity was tested by cytokine release of stimulated human monocytes. To optimize the downstream process, ammonium sulfate precipitation was performed resulting in a highly prepurified protein fraction. Another approach to optimize the purification process was to express both subunits in one vector. Co-expressing the subunits resulted in soluble S100A8/S100A9 complex in the supernatant simplifying the downstream process. In addition, the mammalian cell lines CHO-K1 and HEK293T were also used as alternative cell systems to produce high amounts of S100A8/S100A9.
By optimizing the current manufacturing process and strategy, the manufacturing of the protein complex S100A8/S100A9 in sufficient amounts and quality for clinical development seems feasible.