A platform for the optimisation of metabolic pathways for glycosylation to achieve a narrow and targeted glycoform distribution

Recently, the development of treatments for new disease has shifted away from traditional chemical compounds and towards protein therapeutics (biopharmaceuticals) like antibodies for the treatment of cancer and hormones for chronic diseases. Nearly 70% of these protein therapeutics have sugar molecules attached to them naturally which affect their function and how long they remain in the body. Because the sugars are so important for the drug function, one of the biggest problems in their manufacture is how to control what sugars are added (glycoform) and to ensure that all the proteins produced have the same sugars on them (homogeneous glycoform profile). Current production methods yield a non-homogeneous mix of glycoforms. Also, different glycoforms interact with the immune system in different ways, so it would be of benefit to be able to produce certain glycoforms over others depending on what the drug is and how it is meant to function. Our goal is to develop technology to rapidly determine the effects of different production methods on which glycoforms are produced and how homogeneous the glycoform profile is. To do this we will develop proteins which are produced inside the cells that are also producing the biopharmaceutical that report the concentrations of nutrients that are already known to influence glycoforms. Alongside, we will develop a computer model of the metabolism of the cells which can predict which glycoforms are produced. Using these two together, we should be able to design new media for the cells to use that result in a more homogeneous glycoform profile which we can change based on what the cells are fed with. We can also suggest genetic changes to the cells that would further help us produce a single, designed glycoform. This could lead to the production of drugs that are safer and require lower doses because they have a single glycoform attached which is the most appropriate for the function of that drug.

Most biopharmaceuticals are glycoproteins and the composition of the glycans has impacts on their activity, stability, and immunogenicity. The ability to produce glycoproteins of a single, defined glycoform would allow designer drugs with maximum stability and reduced immunogenicity that interact with the immune system in the most appropriate way for the disease which they are designed to treat. However, current production methods, most of which involved mammalian cell culture, produce a heterogeneous mix of glycoforms and result in the need for higher doses to ensure efficacy. In theory, both medium design and cellular engineering could be used to manipulate the glycoform profile of a biopharmaceutical on a case-by-case basis. However, this would require a high throughput screening method in which various combinations of media and production strains could be tested to determine which produces the most narrow and appropriate glycoform. The goal of this project is to utilise a combination of genetically-encoded FRET biosensors for primary metabolites whose concentrations are known to affect glycoform profile and a full mathematical model of the production of nucleotide sugar donors, protein secretion and glycosylation in order to accurately predict glycoform profile in a miniaturised assay. In the long term, the platform could be transformed into a fully automated system that takes real-time readings of the fluorescent signal from a culture plate with various cell lines or media to be screened and transmits the results to the model, which calculates (a) the cell growth and metabolic profiles and (b) the expected glycomic profiles for each cell line or condition.

The proposed work will develop a platform for glycosylation engineering that will allow for testing of conditions to produce glycoproteins with a narrow profile in high throughput. We expect that the work will have economic and societal impacts as well as benefiting the academic community. Biopharmaceutical production has been expanding rapidly in recent years and this trend is expected to continue. Attracting further investment from industrial biotechnology companies has been highlighted as a priority for the government with the formation of the Bioscience Innovation and Growth Team (BIGT). We will engage with the BRIC industrial members at the annual dissemination meeting to ensure that we pursue the correct model systems and targets to ensure that they get the most benefit out of the results. In addition, the platform could equally be applied to other expression systems, particularly microbial ones which are gaining prominence. This could be the target of future applications to the research councils, CASE studentships, or industrial partnership awards using the contacts which are gained through our involvement in BRIC. The proposed work also has significant potential for training researchers for future employment in the industrial biotechnology industries, another goal of the BIGT. We expect that the two PDRAs employed on the project, as well as students associated with satellite projects will gain a significant number marketable skills. The interdisciplinary environment, supported by biannual dissemination meetings within the project investigation group as well as contact with other researchers working on BRIC supported grants will also further enhance their training. For instance, in BRIC Phase 1, several of the PDRAs formed networks which met outside of the dissemination meetings to discuss research techniques. We will ensure that the PDRAs gain valuable transferable skills in scientific writing through preparation of publications and patents and in public speaking through conference presentations. We have included funds for conference travel for the PDRA in our budget. The ability to produce glycoproteins of a single, designed glycoform would greatly increase the utility of these as medications by increasing their stability within the patient, decreasing their immunogenicity, and allowing us to stimulate specifically the part of the immune system that would be most useful in treating the disease. This would result in the need for less glycoprotein in each formulated dose, which in turn could ease problems in downstream purification and storage. All of these would combine to decrease the cost of production and therefore the cost to the consumer and/or the NHS. This will, in turn, increase the accessibility of these types of medications. We will ensure that our results are disseminated widely through active public engagement and through engagement with policy makers and patient lobby groups. For more information on ensuring impact in the scientific community please see academic beneficiaries (above) and the dissemination and exploitation section of the case for support.