In biopharmaceutical processing, the glycosylation of a therapeutic protein must be stringently monitored as this can affect the efficacy and safety of the final formulation. Current methods of analysis are time consuming, expensive, and require skilled personnel. Therefore, it would be advantageous to have rapid, cheap, and user-friendly assays to sensitively assess glycan composition. This research has made progress in the development of such assays.The first objective was to create sensing beacons which could be easily detected when binding to a target glycan occurred. Gold nanoparticles were investigated for this role due to their ease of functionalisation and optical properties. These were conjugated with a Raman-active molecule to act as a beacon, and a carbohydrate-binding protein via a heterobifunctional ligand. The conjugation procedure was shown to be successful and versatile, allowing for the attachment of various lectins using two different linking molecules.Next, a solution-based assay was investigated to characterise the sensing capabilities of the functionalised gold nanoparticles. This could allow for the development of a glycosylation evaluation kit whereby a library of nanoparticle conjugates could be mixed with protein samples and analysed to assess glycan composition. Originally, the drive was to use surface enhanced resonance Raman scattering (SERRS) as a sensitive method of analysis. However, this was shown to be less useful in this setting, but localised surface plasmon resonance measurements proved to be a simple and rapid avenue for glycan characterisation. Detection of mannose was shown to have a limit of detection of 29.2 nM using the lectin from Galanthus nivalis. This technique was also shown to be versatile using various lectins for the detection of different glycan targets, and could potentially be more sensitive.A second route for glycan detection was investigated to provide a rapid and user-friendly method of analysis. Lateral flow assays were used to show the binding capabilities of the prepared nanoparticle conjugates, but had significantdisadvantages arising from the application of the target glycoprotein to the nitrocellulose surface as a spot rather than a line spanning the width of the strip. This led to an unsatisfactory assay sensitivity. To overcome this, a simpler and more sensitive format was investigated to create a high-throughput glycan analysis platform. For this to remain user-friendly, evaluation methods involved the use of a simple flatbed scanner to provide rapid colorimetric results, but this assay was also coupled with SERRS to investigate the capabilities of producing highly sensitive glycan detection. This paper-based analytical device still requires research, but has shown excellent potential for use as a valuable tool in biopharmaceutical glycan analysis.Finally, the development of an enzyme-linked immunosorbent assay was investigated. This relied on the production of hydrogen peroxide by glucose oxidase when the target glycan was present. Hydrogen peroxide would then act as a reducing agent and induce the growth of gold nanoparticles in the presence of Au3+ cations. Although still in the early stages of development, this assay produced interesting results with many issues still to consider, but showed that it could have the capability of becoming a sensitive method of glycan detection. The common use of enzyme-linked immunosorbent assays in biochemical detection means that a glycosylation analysis kit based on this assay format could be an attractive alternative to current industrial analysis methods.
|Date of Award||3 Oct 2019|
- University Of Strathclyde
|Sponsors||University of Strathclyde & EPSRC (Engineering and Physical Sciences Research Council)|
|Supervisor||Duncan Graham (Supervisor) & Karen Faulds (Supervisor)|