Manufacturing of large biomolecules such as viral vectors used in emerging gene therapies suffers from low product yields due to limitations of traditional resin-based chromatography in downstream processing. Ultrafiltration based purification techniques are being considered for purification of such viral vectors by separating these viral vectors from impurities such as host cell proteins. Improvements in yields of viral vectors have so far concentrated on the design of new chromatography stationary phases such as monoliths, membrane adsorbers, nanofibers and gigaporous resins. Improvement in the architecture of ultrafiltration membranes has not been studied for application in viral vector purifications. Porous anodic alumina (PAA) membranes, exhibit narrow pore size distribution and straight through pore channels compared to traditional polymeric membranes which have broad pore size distributions and tortuous channels. The present work evaluated porous anodic alumina membranes for potential applications in virus ultrafiltration using model protein solutes. Protein nanoparticles of 80-90nm diameter and thyroglobulin of 20 nm diameter were used as the physical mimics for two of the most commonly used viral vectors, Adenovirus and Adeno-associated viruses respectively and a small protein of 8 nm, bovine serum albumin was used as the model impurity. A reproducible and high yielding protocol was developed for the synthesis of protein nanoparticles from bovine serum albumin using a de-solvation process. Based on comparable hydraulic permeability, dextran sieving curve and mean pore size 20 nm rated PAA membrane and 300 kDa rated polymeric ultrafiltration membranes were compared for filterability of the model solutes. PAA membranes were found to have superior fouling resistance (1.5-2.5 times higher values of recoverable membrane permeability) and up to 4 times higher transmission than the polymeric membranes for large model solutes. These findings were attributed to the differences in the membrane architecture resulting in different sieving behaviour. PAA membranes were found to be susceptible to leaky transmissions of large solutes due to the presence of surface defects. Separation performance of binary mixtures of model solutes was studied using a diafiltration process. Electrostatic interactions and transmembrane pressure were identified as crucial process parameters to improve separation performance of the alumina membranes. Lot-to-lot variations in the alumina membranes were also characterised using electron microscopy and were found to influence the separation performance. PAA membranes were found to be compatible for virus processing as similar infectivity recovery of 60-70% was observed for both PAA and the polymeric membranes along with 60 % removal of the impurities.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:763319 |
| Date | January 2018 |
| Creators | Sharma, A. |
| Contributors | Bracewell, D. G. |
| 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/10061755/ |
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