A generalized purification step for viral particles using mannitol flocculation

Abstract

Vaccine manufacturing has conventionally been performed by the developed world using traditional unit operations like filtration and chromatography. There is currently a shift in the manufacturing of vaccines to the less developed world, requiring unit operations that reduce costs, increase recovery, and are amenable to continuous manufacturing. This work demonstrates that mannitol can be used as a flocculant for an enveloped and nonenveloped virus and can purify the virus from protein contaminants after microfiltration. The recovery of the virus ranges from 58 to 96% depending on virus, the filter pore size, and the starting concentration of the virus. Protein removal of 80% was achieved for the small nonenveloped virus using a 0.1 µm filter because proteins were not flocculated with the virus and flowed through the filter. It is hypothesized that mannitol dehydrates the viral surface by controlling the water structure surrounding the virus. Without the ability to become compact, as occurs with proteins, the virus aggregates in the presence of osmolytes and proteins do not. Osmolyte flocculation is a scalable process using high flux microfilters. It has been applied to both an enveloped and nonenveloped virus, making this process friendly to a variety of vaccine and gene therapy products. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1027-1035, 2018.

Osmolyte enhanced aqueous two‐phase system for virus purification

“…Changes in electrostatic and hydrophobic interactions can result in either intraparticle or interparticle interactions. The flexible smaller proteins are known to undergo conformational changes (intraparticle interactions) in the presence of osmoprotectants (Auton et al, 2011; Street et al, 2006) and comparatively rigid viruses have been shown to flocculate (interparticle interactions) (Gencoglu & Heldt, 2015; Gencoglu et al, 2014; Heldt et al, 2018) in the presence of similar osmolytes. In this study, the osmolytes induced interfacial interactions between virus particles and the PEG‐rich phase to improve virus recovery.…”

“…Osmoprotectants have been utilized to reduce intermolecular interactions, thereby inducing intramolecular interactions to stabilize protein biologics in final formulations (Lee 2000; Wlodarczyk et al, 2018). Conversely, osmolytes have been shown to induce intermolecular interactions to flocculate viruses (Gencoglu & Heldt, 2015; Gencoglu et al, 2014; Heldt et al, 2018), which aided in the development of filtration‐based methods for virus purification. The exact mechanism of osmolyte function in protein stabilization or viral flocculation, however, is not clearly understood.…”

Detection and thermal stabilization of virus based on surface properties

“…Osmolyte mannitol has been used as a flocculant for PPV and SINV and can purify the flocculated viruses from protein contaminants after microfiltration. A 58-96% virus recovery and an 80% of contaminated protein removal could be achieved [148].…”

“…Viruses tend to attract each other and form aggregations. The osmolyte effect on virus and protein stability is shown in Figure 2.14 [148]. Thus, protecting osmolytes can be used as virus flocculants [142,143].…”

“…Thus, protecting osmolytes can be used as virus flocculants [142,143]. The flocculation can also be used for virus purification [148]. Osmolyte mannitol has been used as a flocculant for PPV and SINV and can purify the flocculated viruses from protein contaminants after microfiltration.…”