Novel purification method of virus-like particles containing the Hepatitis B virus Surface antigen expressed in the methylotrophic yeast Pichia pastoris

LIMERES MJ; GOMEZ ER; NUSBLAT AD; CUESTAS ML

Congreso; Reunión conjunta de Sociedades de Biociencias; 2017

Virus-like particles (VLPs) are self-assembled systems composed of antigenic proteins, lacking genetic information, that are frequently used for large-scale vaccine production. For instance, currently used hepatitis B vaccines are based on VLPs derived from different eukaryotic systems, such as Pichia pastoris. These spherical empty particles are composed of many molecules of the viral S protein (HBsAg). Attempts to purify such immunogen have shown that its antigenicity is sensitive to ammonium precipitation, acidic pH, high ionic strength and PEG; and requires several chromatographic steps. Here, we describe a novel purification method that reduces costs, time-consuming steps and makes the process more efficient, simpler and quicker than others.VLPs containing the wild-type HBsAg were intracellularly expressed in P. pastoris. Transformed cells were grown in shaker flasks and were then lysed by disruption using glass beads. Purification was performed by a first step of adsorption-desorption on colloidal silica followed by ultracentrifugation on sucrose cushion, dialysis against PBS buffer and concentration with AMICON 10K centrifuge tubes. Each purification step was analyzed by Coomassie and silver-stained SDS-PAGE gels. The presence of HBsAg was determined by mass spectrometry (MS) and its antigenicity by chemiluminescent microparticle immunoassay (CMIA). Finally, the assembly of the subviral particles was evidenced by transmission electron microscopy (TEM).Results showed high levels of expression and purity of HBsAg. The antigenicity of the VLPs was maintained according to CMIA results and its identity was corroborated by MS. Finally, the typical morphological characteristics of the HBsAg-VLPs were observed by TEM.In conclusion, the whole purification process described in the present work avoided possible alterations of the morphology and antigenic properties of the HBsAg-VLPs and it was simpler and cheaper than the conventional ones used in industry.