title: Adenovirus purification by two-column, size-exclusion, simulated countercurrent chromatography.
abstract: Adenovirus serotype 5 (Ad5) was successfully separated by size-exclusion chromatography (SEC) using a simple, yet efficient, two-column, quasi-continuous, simulated moving-bed process operated in an open-loop configuration. The operating cycle is divided into two identical half-cycles, each of them consisting of the following sequence of sub-steps: (i) elution of the upstream column and direction of the effluent of the downstream column to waste; (ii) elution of the upstream column and redirection of its effluent to waste while the downstream column is fed with the clarified bioreaction bulk and its effluent collected as purified product; (iii) operation of the system as in step (i) but collecting the effluent of the downstream column as product; (iv) elution of the upstream column and direction of its effluent to waste while the flow through the downstream column is temporarily halted. Clearance of impurities, namely DNA and host cell protein (HCP), were experimentally assessed. The pilot-scale run yielded a virus recovery of 86%, and a clearance of 90% and 89% for DNA and HCP, respectively, without any fine tunning of the predetermined operating parameters. These figures compare very favorably against single-column batch chromatography for the same volume of size-exclusion resin. However, and most importantly, the virus yield was increased from 57% for the batch system to 86% for the two-column SEC process because of internal recycling of the mixed fractions of contaminated Ad5, even though the two-column process was operated strictly in an open-loop configuration. And last, but not least, the productivity was increased by 6-fold with the two-column process. In conclusion, the main drawbacks of size-exclusion chromatography, namely low productivity and low product titer, were overcome to a considerable extent by an innovative two-column configuration that keeps the mixed fractions inside the system at all times.
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal.
authors.forename: Piergiuseppe
authors.initials: P
authors.lastname: Nestola
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.
authors.forename: Ricardo J S
authors.initials: RJ
authors.lastname: Silva
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.
authors.forename: Cristina
authors.initials: C
authors.lastname: Peixoto
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal.
authors.forename: Paula M
authors.initials: PM
authors.lastname: Alves
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal; Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal.
authors.forename: Manuel J T
authors.initials: MJ
authors.lastname: Carrondo
authors.affiliation: Requimte/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal; Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.
authors.forename: José P B
authors.initials: JP
authors.lastname: Mota
mesh_terms: D000256:Adenoviridae / Q000302:isolation & purification*; D002850:Chromatography, Gel; D003377:Countercurrent Distribution; D004279:DNA, Viral / Q000302:isolation & purification; D057809:HEK293 Cells; D006801:Humans; D014771:Virion / Q000302:isolation & purification
chemical_list: D004279:DNA, Viral
keywords: Adenovirus; Downstream processing; Simulating moving bed; Size exclusion chromatography; Two-column chromatography; Virus purification