Periodic Reporting for period 1 - PoreSelect (Macroporous Polymer Monoliths as a Low-Cost Bioanalytical Platform for Biopharmaceutical Glycoprotein)
Berichtszeitraum: 2016-02-15 bis 2018-02-14
Current technology: Traditional chromatographic methods, such as ion exchange chromatography, size exclusion chromatography and hydrophobic interaction chromatography are used to separate glycoforms but all of them lack the selectivity required to separate and identify closely related glycoproteins or glycoforms. These critical variations are usually a matter of small differences in neutral glycans or alpha- or beta- forms of sugars. Presently, such glycoprotein analyses and separations is difficult, and is carried out by reverse phase high performance liquid chromatography (RP-HPLC) followed by high-end mass spectrometry, which is relatively expensive, time consuming and requires a technical expert specialist. The entire industry has a requirement for a relatively easy, fast and sensitive system for glycoproprotein analysis and separation.
This project aimed to develop a low-cost and easy-to-produce glycopeptide separation platform. This was based on producing highly porous materials that are obtained from the fast and scalable polymerisation from high internal phase emulsions (PolyHIPEs) and the subsequent functionalisation of reactive functional groups. The latter is employed for the conjugation with novel recombinant proteins, called lectins that have very selective carbohydrate-binding properties. The separation of closely related glycoproteins will be investigated. Moreover, the integration of the lectin conjugated porous polymers into a device format will be investigated. This is a highly interdisciplinary project at the interface of polymer and material science, biotechnology and separation science.
Amine functional porous materials were produced by deprotecting the phthalimide group by mixing pieces of polyHIPE in a solution of refluxing ethanol containing terbutyl cathecol and hydrazine for 24 h.
An atom transfer radical polymerisation (ATRP) initiator, was immobilized onto the surface of the material via the reaction of α-bromoisobutyryl bromide with the amine functional polyHIPE. Poly(tert-butyl (t-Bu) acrylate) was grafted from the surface via activator regenerated by electron transfer ATRP (ARGET ATRP), this technique is a ‘greener’ method than ATRP as it uses much lower quantities of copper in the system by the in-situ generation of the active copper species. Quantification of the grafting of the polymer was undertaken by thermogravametic analysis (TGA). The t-Bu group was removed with a strong acid resulting in a highly porous material coated in functional carboxylic groups.
The project was multidisciplinary and also involved the biotechnology school in Dublin City University collaborating with Dr. Brendan O’Connor. It was in his group where they have expertise in engineering prokaryotic lectins for the selective binding to glycoproteins. These lectins were synthesised in this group and purified prior to starting the second stage of the project which focused on the development of methods to bioconjugate these lectins onto these materials. A general strategy was to use EDC/NHS chemistry to conjugate the lectins onto the polymeric material in a two stage process where the polymer was activated then the lectin was then added to the material. The lectin immobilized porous materials were then tested for the separation of different glycoprotein types, however it was observed that there were issues with non-specific binding of glycoproteins, which led to unfortunately no manufacture of any device.
So far, this research has been presented in a poster format at one conference in the USA, and twice orally at two conferences in the USA and in Europe.