title: Successful application of monolithic innovative technology using a carbonyldiimidazole disk to purify supercoiled plasmid DNA suitable for pharmaceutical applications.
abstract: The growing demand on plasmid DNA (pDNA) manufacture for therapeutic applications requires a final product with higher quality and quantity, spending the least time. Most of the current processes for pDNA production use at least one chromatographic step, which often constitutes a key-step in the purification sequence. Monolithic stationary phases are new alternatives to the conventional matrices, which offer fast separation of pDNA due to their excellent mass transfer properties and their high binding capacity for large molecules, as pDNA. However, the efficient recovery of pure pDNA focuses on a suitable balance of the feedstock, adsorbent and mobile phase properties. To satisfy the increasing demand for pharmaceutical grade plasmids, we developed a novel downstream process which overcomes the bottlenecks of common lab-scale techniques while complying with all regulatory requirements. This work reports an integrative approach using the carbonyldiimidazole monolith to efficiently purify the supercoiled (sc) pDNA active conformation from other plasmid topologies and Escherichia coli impurities present in a clarified lysate. The monolith specificity and selectivity was also assessed by performing experiments with plasmids of several sizes of 2.7, 6.05 and 7.4 kilo base pairs (kbp), verifying the applicability to purify different plasmids. Hence, the process yield of the pDNA purification step using the CDI monolith was 89%, with an extremely reduced level of impurities (endotoxins and gDNA), which was reflected in good transfection experiments of the sc plasmid DNA sample. Overall, the analytical results and transfection studies performed with the pDNA sample purified with this monolithic enabling technology, confirmed the suitability of this pDNA to be used in pharmaceutical applications.
authors: CICS-UBI-Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Covilhã, Portugal.
authors: A
authors: A
authors: Sousa
authors: C T
authors: CT
authors: Tomaz
authors: F
authors: F
authors: Sousa
authors: J A
authors: JA
authors: Queiroz
mesh_terms: D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods; D004278:DNA, Superhelical / Q000302:isolation & purification* / Q000592:standards; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli; D015316:Genetic Therapy; D007093:Imidazoles / Q000737:chemistry*; D010957:Plasmids / Q000302:isolation & purification* / Q000592:standards; D012680:Sensitivity and Specificity; D014162:Transfection; D019444:Vaccines, DNA / Q000737:chemistry / Q000235:genetics
chemical_list: D004278:DNA, Superhelical; D007093:Imidazoles; D019444:Vaccines, DNA; C006900:N,N-carbonyldiimidazole