Robust design of adenovirus purification by two-column, simulated moving-bed, size-exclusion chromatography.
A simple, yet efficient, two-column simulated moving-bed (2CSMB) process for purifying adenovirus serotype 5 (Ad5) by size-exclusion chromatography (SEC) is presented and validated experimentally, and a general procedure for its robust design under parameter uncertainty is described. The pilot-scale run yielded a virus recovery of 86 percent and DNA and HCP clearances of 90 and 89 percent, respectively, without any fine tuning of the operating parameters. This performance compares very favorably against that of single-column batch chromatography for the same volume of size-exclusion resin. To improve the robustness of the 2CSMB-SEC process the best set of operating parameters is selected only among candidate solutions that are robust feasible, that is, remain feasible for all parameter perturbations within their uncertainty intervals. This robust approach to optimal design replaces the nominal problem by a worst case problem. Computational tractability is ensured by formulating the robust design problem with only the vertices of the uncertainty region that have the worst effect on the product purity and recovery. The robust design is exemplified on the case where the column volume and interparticle porosity are subject to uncertainty. As expected, to increase the robustness of the 2CSMB-SEC process it is necessary to reduce its productivity and increase its solvent consumption. Nevertheless, the design solution given by our robust approach is the least detrimental of all feasible operating conditions for the 2CSMB-SEC process.
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.
Piergiuseppe
P
Nestola
Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.
Ricardo J S
RJ
Silva
Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal.
Cristina
C
Peixoto
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.
Paula M
PM
Alves
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.
Manuel J T
MJ
Carrondo
Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; LAQV/REQUIMTE Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. Electronic address: pmota@fct.unl.pt.
José P B
JP
Mota
D000256:Adenoviridae / Q000302:isolation & purification*; D002850:Chromatography, Gel; D015203:Reproducibility of Results; D035501:Uncertainty
Robust design; Simulating moving bed; Size-exclusion chromatography; Two-column chromatography; Virus purification