title: Purification of recombinant adenovirus type 3 dodecahedric virus-like particles for biomedical applications using short monolithic columns.
abstract: Adenovirus type 3 dodecahedric virus-like particles (Ad3 VLP) are an interesting delivery vector. They penetrate animal cells in culture very efficiently and up to 300,000 Ad3 VLP can be observed in one cell. The purification of such particles usually consists of several steps. In these work we describe the method development and optimization for the purification of Ad3 VLP using the Convective Interaction Media analytical columns (CIMac). Results obtained with the CIMac were compared to the already established two-step purification protocol for Ad3 VLP based on sucrose density gradient ultracentifugation and the Q-Sepharose ion-exchange column. Pure, concentrated and bioactive VLP were obtained and characterized by several analytical methods. The recovery of the Ad3 VLP was more than 50% and the purified fraction was almost completely depleted of DNA; less than 1% of DNA was present. The purification protocol was shortened from five days to one day and remarkably high penetration efficacy of the CIMac-purified vector was retained. Additionally, CIMac QA analytical column has proven to be applicable for the final and in-process control of various Ad3 VLP samples.
authors.affiliation: BIA Separations, doo, Ljubljana, Slovenia.
authors.forename: Lidija
authors.initials: L
authors.lastname: Urbas
authors.forename: Barbara Lah
authors.initials: BL
authors.lastname: Jarc
authors.forename: Miloš
authors.initials: M
authors.lastname: Barut
authors.forename: Monika
authors.initials: M
authors.lastname: Zochowska
authors.forename: Jadwiga
authors.initials: J
authors.lastname: Chroboczek
authors.forename: Boris
authors.initials: B
authors.lastname: Pihlar
authors.forename: Ewa
authors.initials: E
authors.lastname: Szolajska
mesh_terms: D000260:Adenoviruses, Human / Q000737:chemistry / Q000302:isolation & purification*; D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange / Q000295:instrumentation / Q000379:methods*; D004274:DNA, Recombinant / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D018411:Spodoptera; D014771:Virion / Q000737:chemistry / Q000302:isolation & purification*; D014776:Virus Cultivation / Q000379:methods*
chemical_list: D004274:DNA, Recombinant