Monitoring of the glycosylation status of proteins using raman spectroscopy

Abstract: The project will develop the application of Raman spectroscopy to the monitoring of post translational modifications of proteins in situ in fermentation broths as well as other metrics of the fermentation process. Motivation: Protein based biopharmaceuticals are becoming increasingly widely used and important therapeutic agents with existing products treating a range of auto-immune diseases and many more in the pipeline. However, manufacture of protein based pharmaceuticals in cell based expression systems is a complicated and relatively poorly understood and poorly controlled process, and characterisation of the product is also very challenging. As a consequence, biopharmaceutical manufacturers face significant technical challenges in the efficient manufacture of consistently safe and efficacious products and in confirming the safety and quality of the material generated. From the consumers' and health care providers' perspectives this results in biopharmaceuticals being very expensive, indeed sometimes prohibitively so. Overcoming these issues is critical to the further development of the industry and the wider application of these therapeutics. General Approach: Applying innovative analytical technologies to bio-processing has the potential to contribute significantly to the improvement of production efficiency, decrease costs and improve the quality of the final product. Examples include process development to improve understanding and allow optimisation of processes prior to implementation, real time monitoring of processes to allow for improved and adaptive control and in quality assurance and control of the final product. Optical spectroscopic approaches to analysis are well suited to bio-processing applications due to the flexibility of implementation either in- or at- line and rapid data acquisition. Raman spectroscopy is particularly promising in this regard due to the richness of information contained in the spectra and relative immunity to interference from water in the sample. Focus of Project: One particular area of need in bio-processing of therapeutic proteins is the ability to monitor post-translational modifications (PTM's) such as glycosolation. The details of a protein's PTM can significantly affect its biopharmaceutical properties such as binding to the desired receptor, serum half life and unwanted immunogenicity. Currently, however, in situ monitoring of glycosylation of proteins in mammalian culture is not possible and samples need to be taken off-line for mass spectrometric (MS) analysis. Earlier work in Professor Goodacre's laboratory has demonstrated the potential of Raman spectroscopy to monitor bioprocesses for metabolite (giberllic acid, ethanol, glucose) and recombinant protein levels (cytochrome b5, alpha2-interferon and TNF-alpha) and for assessing protein PTM (in particular phosphorylation). The proposed project would build on this earlier work to obtain Raman measurements directly from cultures and supernatants. These spectra would then be associated with the glycosylation status of the proteins (obtained using MS) using multivariate data analysis/chemometric approaches. In addition the project would also attempt to use the same data analysis methods to extract other relevant information about the fermentation process from the recorded spectra such as metabolite levels and protein concentration with a view to developing Raman spectroscopy as a versatile tool for multi-component monitoring of mammalian bioprocesses. This project will combine the strengths of the industrial and academic partners and will provide high quality training for a post-graduate student who will complete the project with a highly sought after skill-set in what has become an immensely important area for the UK pharmaceutical industry, as exemplified by BBSRC's Bioprocessing Research Industry Club (BRIC; http://www.bbsrc.ac.uk/science/initiatives/bric/Welcome.html).