Sterilizing filtration of plasmid DNA: effects of plasmid concentration, molecular weight, and conformation
| Autor: | Watson Matthew P, Sangeetha L. Sagar, John O. Konz, Winters Michael A |
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| Rok vydání: | 2006 |
| Předmět: |
Chromatography
Molecular mass Concentration effect Reproducibility of Results DNA Biology Buffers Molecular biology Models Biological law.invention Molecular Weight chemistry.chemical_compound Plasmid chemistry Plasmid dna law Yield (chemistry) Pressure Supercoiled plasmid Nucleic Acid Conformation Filtration Biotechnology Plasmids |
| Zdroj: | Biotechnology progress. 22(2) |
| ISSN: | 8756-7938 |
| Popis: | Plasmid DNA purification development has been driven by the increased need for large quantities of highly purified, sterile plasmid DNA for clinical studies. Detailed characterization and development of the terminal sterile filtration process step is often limited due to time constraints and the scarcity of sufficient quantities of purified plasmid. However, the large size of the plasmid molecule and variations in conformation can lead to significant yield losses if this process step is not optimized. In this work, the gradual pore-plugging model of flow decay was found to be valid for plasmid DNA by using an ultra scaledown apparatus (1-4 cm(2) filter area). Filtration capacity was found to be insensitive to pressure. Multiple filter types were screened and both source and composition of materials were found to affect filter capacity dramatically. The filter capacity for plasmid was improved by increasing plasmid concentrations as well as by modifying buffer conditions to reduce the apparent size of the plasmid. Filtration capacities varied over a greater than 2 log range when plasmids with sizes ranging from 5.5 to 11 kb and supercoiled plasmid content of 55-95% were explored. Larger plasmids and feeds with lower supercoiled contents led to reduced capacities. These results can be used to define conditions for scale-up of plasmid sterile filtration, as evidenced by processing a 30 g lot using a filtration area of 1,000 cm(2), with a 96% yield, based on filtration capacity data from 4 cm(2) test filters. |
| Databáze: | OpenAIRE |
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