Harvey, Joseph Gregory Havelock;
(2023)
Using a microfluidic approach to test for critical quality attributes of viral vectors for gene therapy applications.
Doctoral thesis (Eng.D), UCL (University College London).
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Abstract
Viral vector based gene therapy treatments offer a lifeline to patients suffering from previously untreatable diseases; however, single treatment courses can cost millions of pounds. Viral vectors are complex products which require specialised analytical technologies and methods to characterise the product’s critical quality attributes, reduce costs and improve the safety and efficacy of the treatments. The gold standard for viral vector analytics is the cell based transduction assay which allows quantitative measurements of functional viral vectors. This thesis presents a microfluidic platform specially designed for low volume, automated transduction assays. First, the assay conditions were optimised, including the cell seeding density, and dilution ranges. Next, a method for calculating functional titre by predicting the multiplicity of infection was shown. A transient transfection method was selected for lentiviral vector production, and a transduction assay was compared to qPCR. A cyclic olefin polymer (COP) microdevice was designed and fabricated, and flow characterisation was carried out with highly homogeneous sample injection demonstrated by automated fluorescein injection. Cell seeding and perfusion hardware were developed and shown to improve the robustness of cell culture operations. The cell growth properties of COP were compared to tissue culture polystyrene by the culture of HEK293T cells. Following optimisation of cleaning and plasma treatment conditions, similar growth was observed on the two materials with similar maximal growth rates of 0.22 and 0.21 h-1, respectively. Biocompatibility studies showed cell expansion profiles consistent with tissue culture polystyrene (TCPS) controls in the presence of each material required for microdevice cell culture. An image analysis tool for non-invasive measurements of transduction efficiency was shown to be highly correlative to standard flow cytometry analysis. The microdevice and a standard well plate were compared. Comparable growth of HEK293T cells and improved cell seeding homogeneity was observed in the microdevice. Finally, a transduction assay on-chip was compared to a standard well plate method. Both approaches led to titre estimations in the order of 107 TU mL-1, with an 85 % reduction of sample volume required for microdevice analysis. The microdevice and image analysis tool combined offered a quicker, small volume, automated alternative to current transduction assay methods.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Eng.D |
| Title: | Using a microfluidic approach to test for critical quality attributes of viral vectors for gene therapy applications |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10168866 |
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