A new Raman instrument for polarized spectroscopy of biomacromolecular systems

复杂环境（如生物细胞、膜或制剂载体（用于生物制药产品））中生物大分子的结构表征要求越来越高的细节水平，并且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）向“设计质量”的转变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、建造和验证一个新的仪器，它将收集拉曼光谱、拉曼光学活性（ROA）和拉曼线性差（RLD）光谱。ROA已经建立，但相对较少用于探测蛋白质和其他生物大分子的二级和三级结构。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合的亚基的相对取向。拉曼光谱提供了对振动光谱中可用的丰富信息的访问，而没有水信号占主导地位的红外吸收所面临的挑战。该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方面公认的专业知识的基础上。在此之前，他们成功地使UV-LD成为一种可用的技术（通过他们过去10年的工作引发的出版物和线性二色（LD）仪器销售的增加来衡量）。开发一种新的光谱学形式的动机是，在生物大分子的结构和功能中起关键作用的分子（包括糖和脂类）的结构和排列对许多技术来说是不可见的。此外，现有技术不能为许多应用提供足够的信息。包括晶体学和核磁共振在内的原子水平技术并不适合于大型不规则分子组装，因为大分子和周围分子的结构都对组件的功能有贡献。圆二色法是目前应用最广泛的测定蛋白质溶液相二级结构的方法，但其信息量相对较低，可用浓度范围较小。因此，我们需要其他方法来提供所需的信息。我们相信不同形式的拉曼光谱可以有助于解决这些问题，但所需的仪器尚未发明。我们的目标是：1。了解细胞组装中生物大分子的原子水平结构和功能，如果我们希望控制生物过程，如细胞分裂，用于疾病控制和生物技术应用，这是必不可少的。2. 通过改进过程分析技术（PAT）方法和帮助实现“设计质量”（QbD），提高制药（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。药品QbD的基本假设是，生产中的质量是可以计划的，大多数质量危机和问题都与最初计划（或未计划）质量的方式有关。QbD在制药行业的运作相当有效。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，可能对药物足够的分析方法显然不适用于生物制药。新兴的“生物类似药”市场也带来了新的挑战：最简单地说，什么是“高度类似药”？

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'?