Purification of a Chimeric Simian – Human Immunodeficiency Virus-Like Nanoparticle from HEK293 Cell Culture

Abstract

Virus-like nanoparticles (VNPs) have a promising future in vaccination, gene therapy and drug delivery. These artificial vectors consist only of the recombinantly produced virus shell without any viral genetic information packaged inside. Similarly to viruses, the structural proteins that comprise a VNP can spontaneously self-assemble to form the particle or can assemble through several intermediate steps. This work focus on the purification of complex self-assembled nanoparticles from cell cultures. A chimeric Simian – Human Immunodeficiency virus-like nanoparticle was constructed by fusion of Simian matrix protein (p17) and HIV-1 p6 protein. This fusion protein assembles as spherical nanoparticles of ∼80 nm diameter which are released to the culture medium when expressed in HEK 293T cells. A simple two-step purification process was developed to purify these enveloped chimeric particles. Diafiltration using a 300 kDa cut-off PPV (polyvinylpyrrolidone) membrane operating in tangential flow was initially performed to recover and concentrate enveloped nanoparticles released to the cell culture medium while simultaneously promoting buffer exchange and conditioning. Concentrated nanoparticles were further purified by ion exchange chromatography using strong anion exchangers being eluted from the column at ∼34 mS/cm conductivity, pH of 8.