Design and Optimization of a Purification Process for MY32/Ls Protein Solubilizing Inclusion Bodies for a New Vaccine Against Sea Lice

Abstract

Sea lice are the most problematic marine pathogens the salmon industry has to deal with, significantly affecting Europe and America. The worst offenders are genera: Pseudocaligus, Caligus, and Lepeophtheirus. Over €305 million in losses are estimated. Recent results have suggested that subolesin/akirin/myosin32 are good candidate antigens for the control of arthropod infestations such as sea lice. The aim of this study was to design and optimize the purification step of MY32/Ls protein to obtain the active pharmaceutical ingredient against sea lice. Non-chromatographic purification strategies were employed, based on published works, to establish rupture, washing, solubilization, and refolding conditions. A disruption process using a bead mill was established. Cell culture volume was flushed through the mill eight times. Three 60-minute process steps for washing inclusion bodies were performed, and the fourth washing step was a 20-minute process. The first two washes with a Tris-HCl (50 mM) buffer were performed using a non-ionic surfactant (Triton X-100). For the third wash, the surfactant was omitted. The fourth washing step was carried out at a pH of 10.5. The inclusion body solubilization process, in an alkaline condition of pH 12.5, lasted 90 minutes. For the refolding step, Tween 80 was used as a stabilizing additive. The API obtained had a better immune response (IgG) than the positive control, with higher purity.