title: Downstream processing of triple layered rotavirus like particles.
abstract: Rotavirus like particles (RLPs) constitute a potential vaccine for the prevention of rotavirus disease, responsible for the death of more than half a million children each year. Increasing demands for pre-clinical trials material require the development of reproducible, scaleable and cost-effective purification strategies as alternatives to the traditional laboratory scale CsCl density gradient ultracentrifugation methods commonly used for the purification of these complex particles. Self-assembled virus like particles (VLPs) composed by VP2, VP6 and VP7 rotavirus proteins (VLPs 2/6/7) were produced in 5l scale using the insect cells/baculovirus expression system. A purification process using depth filtration, ultrafiltration and size exclusion chromatography as stepwise unit operations was developed. Removal of non-assembled rotavirus proteins, concurrently formed particles (RLP 2/6), particle aggregates and products of particle degradation due to shear was achieved. Particle stability during storage was studied and assessed using size exclusion chromatography as an analytical tool. Formulations containing either glycerol (10% v/v) or trehalose (0.5 M) were able to maintain 75% of intact triple layered VLPs, at 4 degrees C, up to 4 months. The overall recovery yield was 37% with removal of 95% of host cell proteins and 99% of the host cell DNA, constituting a promising strategy for the downstream processing of other VLPs.
authors.affiliation: IBET/ITQB, Apartado 12, P-2780-901 Oeiras, Portugal.
authors.forename: C
authors.initials: C
authors.lastname: Peixoto
authors.forename: M F Q
authors.initials: MF
authors.lastname: Sousa
authors.forename: A C
authors.initials: AC
authors.lastname: Silva
authors.forename: M J T
authors.initials: MJ
authors.lastname: Carrondo
authors.forename: P M
authors.initials: PM
authors.lastname: Alves
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D036022:Capsid Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012401:Rotavirus / Q000235:genetics / Q000648:ultrastructure; D012400:Rotavirus Infections / Q000235:genetics / Q000517:prevention & control; D022243:Rotavirus Vaccines / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use; D018411:Spodoptera / Q000166:cytology / Q000235:genetics; D019065:Virus Assembly / Q000235:genetics
chemical_list: D036022:Capsid Proteins; D011994:Recombinant Proteins; D022243:Rotavirus Vaccines