title: Towards purification of adenoviral vectors based on membrane technology.
abstract: Given increasing applications of recombinant adenoviruses for gene therapy and vaccination, there is a need for highly robust and fast purification platforms for their large scale manufacture. Traditional chromatographic methods using resins as matrices have several limitations such as high-pressure drops and slow processing rates due to pore diffusion and channelling of the feed through the bed. In contrast, membrane adsorbers offer the advantage of fast, gentle, and effective isolation. Furthermore, membranes are easy to use, no column packing is needed and, when used as disposables, no cleaning validation is necessary, representing a substantial advantage to meet cGMP requirements. In this work, a strategy for purification of adenovirus vectors from cell-culture bulks fully based on membrane devices is presented. Ultrafiltration membranes with molecular weight cutoffs of 300, 500, and 750 kDa were tested for the concentration of cell-culture supernatant after an initial clarification step. The results show that the use of ultrafiltration/diafiltration membranes not only concentrates the virus but also leads to the removal of 90% of host cell DNA and proteins in the retentate. Two membrane adsorbers (Sartobind Q and Sartobind anion direct) were evaluated for adenovirus vectors capture and purification. To define the best operating conditions, the effect of pH, conductivity, and recirculation of load bulk on the recovery yield of infectious adenoviruses were evaluated. Sartobind anion direct allows for higher recovery yields (up to 62%) of infectious adenoviruses than Sartobind Q; identical ratios between total and infectious adenoviruses (TP/IP) were achieved for both membrane adsorbers. The overall recovery yield of the process is approximately 52%; this work credits membrane technology as an alternative for the concentration and purification of adenoviruses and as a promising solution for downstream processing of other viral vectors.
authors.affiliation: ITQB-UNL/IBET, Oeiras, Portugal.
authors.forename: Cristina
authors.initials: C
authors.lastname: Peixoto
authors.forename: Tiago B
authors.initials: TB
authors.lastname: Ferreira
authors.forename: Marcos F Q
authors.initials: MF
authors.lastname: Sousa
authors.forename: Manuel J T
authors.initials: MJ
authors.lastname: Carrondo
authors.forename: Paula M
authors.initials: PM
authors.lastname: Alves
mesh_terms: D000256:Adenoviridae / Q000235:genetics* / Q000302:isolation & purification*; D000327:Adsorption; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D013499:Surface Properties; D013997:Time Factors; D014462:Ultrafiltration / Q000295:instrumentation / Q000379:methods*