Application of Deep Uv Resonance Raman Spectroscopy to Bioprocessing

currently a relatively small percentage of conventional pharmaceuticals, continues to expand with predicted revenues of over USD 100 billion for 2010. With continual demand for high fidelity products, from recombinant therapeutic proteins to nucleic acid-based medicinal products, the need for reliable, cost effective and appropriate analytical techniques for assessing the conformity of the products is still paramount. Monitoring of these complex processes is important for accurate assessment of product yield and quality control, but is also necessary to gain a greater knowledge of both the chemical and physical changes that occur. Increased biological understanding of the cellular processes can further facilitate bioprocess design and ultimately improve both quality and quantity of the product, particularly if real-time and on-line information can be collected. The confocal nature of Raman spectroscopy and the fact that water has only a weak Raman scatter means that Raman measurements can be taken of solutions directly through transparent materials, such as the side of a reaction vessel. Furthermore, with continuing advancements in spectroscopic instrumentation reducing size and cost as well as improved software capabilities, Raman spectroscopy is extremely applicable to both on-line and real time monitoring. Just over a decade ago, Shaw et al. demonstrated that Raman spectroscopy could be successfully applied to the on-line monitoring of the biotransformation by yeast of glucose to ethanol. The results showed that with an appropriate on-line set-up and specifically designed software for data preprocessing and multivariate analysis, glucose and ethanol concentrations could be measured with over five per cent accuracy (compared to off-line chemical and enzymatic diagnostic methods). Further work, comparing a diverse range of unprocessed, industrial fed-batch fermentation broths containing the fungus Gibberella fujikuroi also successfully applied Raman spectroscopy and appropriate chemometric models to measure accurately the concentration of natural product gibberellic acid. Raman spectroscopy has also been used to evaluate the nature of particle contamination detecting micro-particles inside glass containers such as glass syringes, vials and test tubes. In recent years, Raman spectroscopy has been successfully applied to bioprocessing, including industrial processes. Raman studies have typically been aimed at measuring accurately both product yields and the presence of secondary products; including glucose and ethanol levels as well as secondary metabolites present in complex non-fractionated fermentation broths1,2. However, Raman spectroscopy as a tool for monitoring the complex structural changes occurring during protein production has to date been under utilised, particularly the use of the extremely sensitive structural technique of UV resonance Raman (UVRR) spectroscopy. APPLICATION OF DEEP UV RESONANCE RAMAN SPECTROSCOPY TO BIOPROCESSING