Evaluation of an ion-exchange membrane for the purification of plasmid DNA.
Biotechnol Appl Biochem
; 37(Pt 3): 259-66, 2003 Jun.
Article
en En
| MEDLINE
| ID: mdl-12689337
ABSTRACT
Separation and purification of large quantities of plasmid DNA (pDNA) is a particularly difficult manufacturing issue because of the relatively low capacity, flow rate and purity observed using traditional bead-based chromatography. The objective of the present study was to evaluate the performance of anion-exchange membranes for the purification of pDNA from Escherichia coli lysate solution. The fate of host-cell protein and endotoxin relative to pDNA was measured and used to calculate recoveries, mass balances, dynamic capacities and purification factors as a function of the flow rate and loading volume of the lysate solution. Breakthrough curves were not sigmoidal and symmetric in shape. They rose sharply at first, and then slowly towards, but never reaching, saturation. Conversely, elution curves were independent of flow rate. pDNA bound tightly to the membranes, whereas protein and endotoxin did not. Dynamic binding capacity for pDNA was 20-25 times greater, and the flow rate was 55-550 times greater, than values observed for beads. However, some pDNA bound irreversibly to the membrane surface and was not removed completely during elution. The intrinsic rate of pDNA adsorption to the membrane was found to be rate-limiting, whereas effects of liquid-phase mass transfer and flow non-idealities were negligible. These results were interpreted using models of adsorption that included steric effects using the 'car-parking-problem' model, and surface residence time effects using the spreading model. This work demonstrated the advantages of ion-exchange membranes compared with beads for the purification of large biomolecules such as pDNA.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Plásmidos
/
Cromatografía por Intercambio Iónico
/
Análisis de Falla de Equipo
/
Membranas Artificiales
Tipo de estudio:
Evaluation_studies
/
Prognostic_studies
Idioma:
En
Revista:
Biotechnol Appl Biochem
Asunto de la revista:
BIOQUIMICA
/
BIOTECNOLOGIA
Año:
2003
Tipo del documento:
Article
País de afiliación:
Estados Unidos