The manufacture of high quality virus at an industrial scale remains a challenge. In the present study, the ability of nanofiber adsorbent functionalised with quaternary (Q) amine ligands to purify adenovirus 5 (Ad5) from crude feeds was investigated. The hypothesis for this study is that nanofibres modified with Q amine ligands will enhance purification of Ad5, enabling recovery of the virus at high, infectious yields from crude cell lysate feeds. The nanofiber adsorbent technology are non-woven regenerated cellulose nanofibers that present an open pore structure 0.2-2.0µm with large inter-fiber space and shallow bed height. These are features previously reported to be advantageous for separation of viral vectors. The upstream process for propagation of Ad5 stocks involves use of a HEK293 cell line that can allow proliferation of the virus as it contains the E1 gene deleted from Ad5. Optimal culture conditions for reproducible production of infective Ad5 from HEK293 are poorly characterised. Therefore, we performed investigations of the influence of cell passage (P: P2, P5, P10), metabolic activity (mitochondrial activity: MTT assay), and rate of proliferation on generation of infective Ad5. HEK293 passage influenced the yield of Ad5 produced. Cells of a low passage (P2) generated lower amounts of virus than those of higher passage (P5, P10). This was due to lower growth rates of P2 cells than P5 and P10 cells. The intermediate passage cells (P10) presented with the highest growth rates that were significantly greater (P ≤ 0.05) than that of P2 cells. The mitochondrial activity of P2 cells were lower than that of P10 cells but P5 cells presented with greater mitochondrial activity than P10 cells. HEK293 cells were used at passages 10-15 to propagate Ad5 for the nanofibre optimisation and screening phases of the study. The Q amine modified nanofibres are novel and therefore preliminary investigations of their function for separation of proteins and viruses are necessary. Conditions for the use of nanofibre were optimised initially by assessing the ability of those matrices to purify bovine serum albumin (BSA) and thyroglobulin. High recovery separations of BSA and thyroglobulin were achieved with nanofibres compared to Q-Sepharose and POROS chromatography materials. A number of gradient elution methods were applied to purify Ad5 from a clarified bulk feed lysate. Ad5 was purified from a clarified bulk feed and protein VIII, a protein marker for infective mature capsids was identified (via mass spectrometry) in a high salt elution fraction. Our study is the first to describe evidence for identification of a cleaved mature capsid protein in a preparative chromatographic purification step. Evaluations of the effects of process variables that may induce stress on Ad5 during separation procedures showed that high salt concentrations in elution buffers and increased flow rates did not affect Ad5 recovery and infective yield. Lowering the Ad5 feed volume from 50mL to 20 mL lead to improvements in resolution of eluted virus peaks and increases in infective Ad5 yields from 69% to 78%. Further optimisation of nanofiber-mediated purification of Ad5 and screening of nanofibers functionalised with Q amine ligands at low (440 µg/mol), medium (750 µg/mol) and high (1029 µg/mol) densities was performed. Clarified crude and filtered (500 kDa hollow fiber tangential flow filtration system) Ad5 feeds were purified using nanofibers at low, medium and high Q amine ligand densities. Results showed that by maintaining short process times, infective Ad5 recoveries of over 90% were achieved and those are the highest infective recoveries of Ad5 achieved to date. Prolonged adsorption durations on Q amine nanofibers showed significant losses in Ad5 product quality on medium and high ligand densities over extended binding durations of up to 24 min. Each ligand density produced several Ad5 populations over a single run with unique infective ratios (1-16.04 virus particle/infectious virus particle). Increasing Q amine ligand density improved resolution and separation of intact Ad5 capsids from host cell protein impurities and product-related impurities including free hexon (a major capsid coat protein) and replication-defective Ad5 capsids that contained DNA. Using 0.125mL adsorbent, flow rates in excess of 70 mL/min could be applied to nanofiber adsorbents for separation of Ad5, indicating efficiency in the purification workflow. Nanofiber nanofibers exhibited high dynamic binding capacity for Ad5 vectors in excess of 2.39 x 1010 virus particles. This study demonstrates the utility of Q amine functionalised nanofibre nanofibers for high recovery purification of infective Ad5. The bioprocess workflow devised for separation of Ad5 from crude cell lysate feed generated from an optimised upstream process can be scaled up for industrial manufacture of therapeutic Ad5 containing genetic payloads. Quaternary amine functionalised nanofibre nanofibers present features that clearly indicate their potential as next generation bioprocessing tools.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:763217 |
| Date | January 2018 |
| Creators | Turnbull, Jordan Philip |
| 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/10058286/ |
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