Design and manufacture of specific recombinant protein glycoforms in yeast

The vast majority of proteins are glycoproteins, which means that they have been enzymatically decorated with sugar molecules in a series of co- and post-translational steps. The consequence of this is that in most cases, what one would regard as a pure protein is actually a heterogeneous mix of defined 'glycoforms', where the sugar attached to each glycosylation site differs according to the available pool of glycosylating enzymes in the cell. Many commercially-available therapeutic proteins are glycoproteins (such as hormones and antibodies), their function being modulated by the mix of different glycoforms that comprise the 'pure protein'. One of the major challenges in producing therapeutic glycoproteins in a consistent manner is controlling this mix of glycoforms and hence the effectiveness of the therapeutic glycoprotein itself. This collaborative project aims to directly address this challenge by providing solutions for the design and manufacture of specific, single glycoforms of any therapeutic glycoprotein. Defining glycoprotein function by attaching specific sugars offers the opportunity to improve pharmacokinetic properties and increase efficacy of potential glycoprotein drugs. GlycoForm Ltd has acquired technology (GlycoLigase) that allows an activated sugar (an oxazoline) to be conjugated to a N-acetylglucosamine sugar residue, which is the first sugar residue attached to all human glycoproteins. The GlycoLigase process uses an engineered enzyme, Endo-A, for which the backwards hydrolysis reaction has been suppressed. By using the activated sugar oxazoline intermediate, only the forward reaction can proceed as normal. The result of this is that a synthetic glycoform can now be produced with an entirely natural saccharide linkage. Building on the GlycoLigase Technology, GlycoForm is in a strong position to develop a range of appropriate oligosaccharides for conjugation. Roslyn Bill's team at Aston have preliminary data to suggest that a convenient and cheap source of proteins with N-acetylglucosamine sugars attached is yeast-produced glycoproteins that have been enzymatically 'trimmed'. This leaves only the initial N-acetylglucosamine sugar conjugated to its native asparagine glycosylation site. To develop innovation in yeast expression and subsequent purification leading to a predictable, reproducible and reliable technology platform to produce trimmed glycoprotein samples, mere trial and error is not likely to be successful. A systematic approach, based on scientific rationale is essential to tackle the complexity of this problem. Therefore we will approach the challenges of producing recombinant proteins as a scientific problem requiring an understanding of underlying biological bottlenecks. This is in stark contrast to approaches used by the majority of laboratories which still rely on trial and error without ever understanding why most attempts fail, and builds on our knowledge base in this key area of systems biotechnology. The objectives of the PhD project are therefore the: (1) Design and enzymatic synthesis of suitable sugars; (2) Production and purification of therapeutic protein targets in a glycosylatable form; (3) Glycosylation to yield defined glycoforms with specific functional properties and native sugar linkages. In summary, GlycoForm will draw on its expertise in oligosaccharide production and combine this with Aston University's ability to produce target proteins in an efficient and cost-effective manner. By conjugating these elements via GlycoLigase, the resultant novel, pure, single glycoform glycoproteins will be used to develop structure activity relationships (SAR) and hence produce optimised drug candidates. This work will therefore open new avenues in rational drug development that have not been possible to date.