Sterile filtration is considered as a final step in processing pharmaceutical grade plasmid DNA. During the development of filtration process, fundamental understanding on the mechanism of fouling and the DNA degradation is critical to improve filtration performance. This study focuses on the fouling and degradation of large plasmid DNA (> 20 kb) in sterilising grade filters. Scanning electron microscopy (SEM) was applied to investigate the mechanism of fouling of pGEc47 plasmid DNA (56 kb) in sterile filter. The investigation contributes to the fundamental understanding of the behaviour of large plasmid molecules during filtration through 0.22 µm membrane. The SEM images suggested that the fouling was due to entrapment of plasmids on the surface of membrane that created meshes of plasmids DNA. The severe form of the superposition of DNA meshes blocked the entrance of pores and restricted the passage of other incoming plasmid molecules. The observations of cross sections of the membrane showed that after filtration with 200 µg of plasmid, the blockage of internal pores was detected. Quantitative analysis of the progression of fouling using digital image processing technique suggested that the transition of fouling occurred. During filtration of 50 µg plasmid, the blockage was due to superposition of DNA molecules. However, after filtration of 200 µg plasmid, complete blockage of the pores was observed. In order to understand the blockage of membrane by plasmid DNA, the mechanism of fouling of pQR150 (20 kb) and pGEc47 (56 kb) plasmids during constant pressure filtration inside 0.22 µm PVDF membrane is experimentally investigated. The decline of filtrate flux as function of time is analysed using the framework of classical and combined blocking models (Bolton et al., 2006). The results for both plasmids indicate a transition between fouling mechanisms. Initially, during early part of the filtration, the intermediate blocking model provided the best fit of the experimental results suggesting that fouling of the membrane was mainly caused by deposition of particles onto its surface. Afterwards, the result trends were best captured by the standard blocking model indicating that internal fouling of membrane was the dominant fouling mechanism. A study of the transmission (Cf /C0) of both plasmids shows a significant reduction of plasmid transmission which coincides with the transition of the fouling mechanism from intermediate to standard blocking. The study elucidates the applicability of filtration blocking model to explain the fouling behaviour of large plasmid DNA during sterile filtration. The loss of plasmid is also related to the degradation of supercoiled (SC) plasmid to other topologies such as open circular and linear DNA. The degradation is correlated to fluid stresses in bioprocessing equipments that may contribute to the breakage of the phosphodiester bond between bases in the double-helical structure of DNA. The computational fluid dynamics (CFD) simulation was carried out to estimate the magnitude of elongational strain rate inside 0.22 µm polyvinylidene fluoride (PVDF) membrane which is a critical for plasmid DNA degradation. The results were compared with the critical elongational strain rate for DNA breakage and the experimental data that correlates with the loss of 20 kb plasmid DNA. Two approaches have been developed to determine the strain rates inside the membrane, which are the macro- and micro-scale models. During the filtration of plasmid at 5 and 8 psi transmembrane pressures, the macro-scale model estimated the average elongational strain rates up to 1x103 s-1. Furthermore, the micro-scale model detected wide range of local elongational strain rates up to 1x105 s-1. However, local elongational strain rates below ~4x103 s-1 were detected in most of regions of the membrane, which is below the critical elongational strain rate for DNA breakage.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:587721 |
| Date | January 2013 |
| Creators | Affandy, A. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1383802/ |
Page generated in 0.0133 seconds