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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - PoreSelect (Macroporous Polymer Monoliths as a Low-Cost Bioanalytical Platform for Biopharmaceutical Glycoprotein)

Teaser

Summary

Glycoprotein therapeutics are widely seen as the next generation of drugs. 80% of new therapeutic drugs in phase 3 development are â€˜biologicsâ€™. However, the early detection of critical glycosylation changes in biopharma production (i.e. in the drug producing cells) and the rapid and sensitive identification and separation of closely related protein-drug glycoforms presents an industry wide problem.

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.

Work performed

First part of the project aimed at the production of porous polymers (polyHIPEs) and their functionalization to increase the amount of reactive groups in the material. To introduce the reactivity into the material for later bioconjugation with lectins, an amine protected hydrophobic monomer, 4-vinylbenzylphthalimide (VBP) was initially synthesised by reaction of 4-vinylbenzylchloride with potassium phthalimide. VBP was then introduced into the concentrated emulsion mixture. The internal aqueous phase consisted of water, a thermal initiator (potassium persulphate) and salt for the internal aqueous phase and a low hydrophobic lipophilic balance surfactant (Span 80), VBP, styrene and divinyl benzene for the external organic continuous phase of the emulsion. Internal phase volumes were typically around 80 % and the emulsion was thermally initiated at 60oC for 24h to produce a solid foam material. PolyHIPEs were then washed via soxlet extraction to remove any surfactants, unreacted monomers and also to remove water and then dried in vacuo. The materials were analysed by SEM and FTIR analysis.

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 Î±-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 â€˜greenerâ€™ 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 Oâ€™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.

Final results

The progress beyond the state of the art was that lectins were immobilized onto polymerized high internal phase emulsions and tested for separation of glycoproteins. We believe that the non-specific binding of the glycoproteins to the material was a result of the interaction of these biomolecules with the hydrophobic styrenic base material. Even though we grafted hydrophilic polymers from the surface, there was still some interaction from the proteins with the hydrophobic surface. In future, if this project was to be continues, I think that you would have to make a much more hydrophilic base material, possibly by making polyHIPEs from oil-in-water emulsion, before grafting functional groups from the surface, or graft block copolymers where the first block has a high hydrodynamic volume (such as polyethylene glycol (PEG) (meth)acrylate) and would prevent any non-specific binding with the surface of the polyHIPE and the second block could be functional which then could used for the immobilization of the lectin and subsequent separation of specific glycoproteins.