A new Raman instrument for polarized spectroscopy of biomacromolecular systems

Lead Research Organisation: University of Warwick
Department Name: Chemistry


Structural characterization of biomacromolecules in complex environments such as a biological cell, in membranes, or in formulation vehicles (for biopharmaceutical products) is being demanded at ever increasing levels of detail and remains an extremely challenging task. In addition to the research drivers, the moves towards 'Quality by Design' for biopharmaceuticals (proteins, nucleic acids, viruses, bacteria) means that the biopharmaceutical industry needs new methods. In this project we shall design, build and validate a new instrument that will collect Raman, Raman Optical Acitity (ROA), and Raman Linear Difference (RLD) spectra. ROA is well-established, but comparatively underused as a means of probing secondary and tertiary structures of proteins and other biomacromolecules. RLD is a newly invented technique (Rodger et al. Analytical Chemistry, 2012) that can be used to give relative orientations of subunits of complex molecular assemblies. Raman spectroscopy provides access to the wealth of information available in vibrational spectroscopy without the challenges which confront infra red absorbance where water signals dominate. This project builds on the investigators' acknowledged expertise in developing novel spectroscopies for the study of biomolecules. It follows their success (measured by the increase in publications and linear dichroism (LD) instrument sales triggered by their work over the past 10 years) in making UV-LD an available technology.

The motivation for developing a new form of spectroscopy is that the structures and arrangements of molecules, including sugars and lipids, that play key roles in the structures and functions of biomacromolecules are invisible to many techniques. Further, existing techniques do not provide sufficient information for many applications. Atomic-level techniques including crystallography and NMR are not well-suited to large irregular molecular assemblies where the structures of both the macromolecule and surrounding molecules contribute to the function of the components. Circular dichroism, which is currently the most widely used method for determining solution-phase secondary structures of proteins, has comparatively low information content and usable concentration ranges. Thus we need alternative approaches to provide the required information. We believe different forms of Raman spectroscopy can contribute to addressing these issues, but the required instrumentation has not yet been invented.

In particular we aim to:

1. Understand atomic-level structures and functions of biomacromolecules in cellular assemblies which is essential if we wish to control biological processes, such as cell division, for disease control and biotechnology applications.

2. Enhance efficiency in the development and production of pharmaceutical (small molecule) and biopharmaceutical (proteins, nucleic acids, viruses, bacteria) products by improving the approach to Process Analytical Technology (PAT) and helping to enable 'Quality by Design' (QbD). The hypothesis underlying QbD for pharmaceutical drugs, is that quality in production can be planned, and that most quality crises and problems relate to the way in which quality was (or was not) planned in the first place. QbD operates fairly effectively in the pharmaceutical industry. Regulators such as the European Medicines Agency are looking to expand the concept and process of QbD from pharmaceutical products to biopharmaceuticals. However, the analytical methodologies that are possibly sufficient for pharmaceuticals are clearly not adequate for biopharmaceuticals. New challenges are also being brought by the emerging 'Biosimilars' market: most simply, what is 'highly similar'?

Planned Impact

Analytical science is a key feature of the success of any fundamental or applied research programme and underpins industrial progress and production. Therefore to achieve the next level of innovation in European research and industry we need new techniques and scientists trained in new ways to use them. The goal of this project is to design, build, validate and encourage use of a new instrument for biomacromolecule characterization. Within the lifetime of the project our aim is to have the Raman Linear Difference (RLD) and Raman Optical Activity (ROA) instrument being part of the biomacromolecule characterisation armoury of ~12 academic research groups and one industrial group by the end of the project. We shall achieve this by the following.
(i) Involve SGS M-Scan Ltd in the project producing data on their samples in the first instance, then involving then in developing new methods for biopharamceuticals
(ii) Include the new methodology of RLD and also ROA in the UK/EU Circular Dichroism/Linear Dichroism workshop that has been designed to take place mid-term in the project. We shall invite collaborators L. Nafie of Syracuse and BioTools Inc. and J. Cheeseman of Gaussian Inc. to contribute to the workshop.
(iii) Encourage every user of the unique linear dichroism facilities at Warwick to make samples available for RLD data collection. This will help establish a library of useful data and also indicate where scientists will benefit from using RLD.
(iv) Publications in high quality journals and conference presentations.

The variety of researchers who will measure spectra on the new instrument will benefit from learning a new technique and expanding their structural characterization possibilities.

This is a short project, without any postdoctoral research associates. The direct opportunities for Outreach will be limited to laboratory tours, student placements, and University open days.

At the conclusion of the project we shall have a new instrument up and running and available to the wider community. We anticipate that the research groups of the applicants will use ~1/3 of the available time; other Warwick users a further ~1/3; and external users ~1/3. The balance between industrial and academic users is anticipated to evolve as a function of time with initial dominance by researchers in academia followed by increasing use by biopharmaceutical users. The drive towards 'Quality by Design' of pharmaceutical and biopharmaceutical products is motivated by the need to increase the efficiency of the European manufacturing platform in order to increase its economic viability. A step-change in how processes are controlled is required to proceed to the next level. Quality by Design is a driver for the development of the new instrument as it requires more understanding of the nature of a product and production process than has previously been possible. The European approach to QbD has been developed together with the FDA and Japan and is summarized in International Conference on Harmonisation (ICH) guidelines. However, these developments have made it clear that we do not have the technologies required either for the pre-production phases of QbD or for the production phases where continuous, fast, non-peturbative in- or on-line testing at the point of manufacture is required to allow correction or at least to avoid expensive further processing.

We anticipate that both the new spectrometer and the new Couette flow cell will become products for respectively BioTools Europe Ltd and Crystal Precision Optics.
Description We have been developing a new technique for polarised Raman spectroscopy. This involved redesigning an existing instrument for new techniques. So far we have worked with molecules oriented on films and got some nice data. The instrument was a challenge to get working and we spent a lot of time awaiting an engineer to come to fix it. Since the completion of the grant we have been working to collect the data base of spectra we had hoped to collect during the grant. Thiswork is still in progress.
Exploitation Route When the techniques are up and running they will be a great asset to those working on structure and function of biomacromolecular assemblies. We have agreed to work with two pharmaceutical companies to develop the methods for characterising biosimilars. Thsi work has started.
Sectors Agriculture, Food and Drink,Chemicals,Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

Description We are working with biopharmaceutical companies to develop new methos of characterising biosimilar products
First Year Of Impact 2017
Sector Chemicals,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

Description Industry contract
Amount £5,000 (GBP)
Organisation Syngenta International AG 
Sector Private
Country Switzerland
Start 12/2016 
End 12/2016
Description Jasco (U K) Ltd 
Organisation JASCO UK Ltd
Country United Kingdom 
Sector Private 
Start Year 2005
Description Raman Optical Activity of lipid systems 
Organisation University of Manchester
Department School of Computer Science
Country United Kingdom 
Sector Academic/University 
PI Contribution Instrument support, data collection, data analysis
Collaborator Contribution Sample preparation, data collection, data analysis
Impact Paper in preparation
Start Year 2014