A new Raman instrument for polarized spectroscopy of biomacromolecular systems

对生物大分子在生物细胞、膜或制剂载体(生物制药产品)等复杂环境中的结构表征的要求越来越高，这仍然是一项极具挑战性的任务。除了研究驱动因素外，生物制药(蛋白质、核酸、病毒、细菌)“按设计质量”的趋势意味着生物制药行业需要新的方法。在这个项目中，我们将设计、建造和验证一种新的仪器，它将收集拉曼光谱、拉曼光学强度(ROA)和拉曼线性差(RLD)光谱。ROA已经得到了很好的证实，但作为探测蛋白质和其他生物大分子的二级和三级结构的一种手段，它的使用相对较少。RLD是一种新发明的技术(Rodger等人)。分析化学，2012)，可用于给出复杂分子组件的亚基的相对取向。拉曼光谱提供了获取振动光谱中丰富的信息的途径，而不需要面对水信号占主导地位的红外吸收所面临的挑战。这个项目建立在研究人员在开发用于生物分子研究的新型光谱学方面公认的专业知识的基础上。在此之前，他们在使UV-LD成为一项可用的技术方面取得了成功(通过过去10年来他们的工作引发的出版物和线性二色性(LD)仪器销售的增加来衡量)。开发一种新形式的光谱学的动机是，在生物大分子的结构和功能中发挥关键作用的分子的结构和排列，包括糖和脂，对于许多技术来说是看不见的。此外，现有技术不能为许多应用提供足够的信息。包括结晶学和核磁共振在内的原子级技术不太适合于大的不规则分子组装，在这种组装中，大分子结构和周围分子的结构对组成成分的功能都有贡献。圆二色谱是目前应用最广泛的测定蛋白质溶液相二级结构的方法，但信息量较低，适用的浓度范围较小。因此，我们需要其他办法来提供所需的信息。我们相信不同形式的拉曼光谱可以帮助解决这些问题，但所需的仪器尚未发明。我们的目标尤其是：1.了解细胞组装中生物大分子的原子级结构和功能，这对于我们希望控制细胞分裂等生物过程，用于疾病控制和生物技术应用是必不可少的。2.提高药物(小分子)和生物制药(蛋白质、核酸、病毒、细菌)产品的开发和生产效率，方法是改进过程分析技术(PAT)，并帮助实现“按设计质量”(QbD)。药品QbD背后的假设是，生产中的质量是可以规划的，而且大多数质量危机和问题都与最初规划(或不规划)质量的方式有关。QBD在制药行业的运营相当有效。欧洲药品管理局(European Medicines Agency)等监管机构正寻求将QbD的概念和流程从制药产品扩展到生物制药。然而，可能足以用于制药的分析方法显然不足以用于生物制药。新兴的“生物仿制药”市场也带来了新的挑战：最简单的是，什么是“高度相似”？

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'?