Nanoscale Biophotonics Laboratory, School of Chemistry, National University of Ireland - Galway, Galway, Ireland.
Appl Spectrosc. 2017 Jun;71(6):1085-1116. doi: 10.1177/0003702817703270.
The production of active pharmaceutical ingredients (APIs) is currently undergoing its biggest transformation in a century. The changes are based on the rapid and dramatic introduction of protein- and macromolecule-based drugs (collectively known as biopharmaceuticals) and can be traced back to the huge investment in biomedical science (in particular in genomics and proteomics) that has been ongoing since the 1970s. Biopharmaceuticals (or biologics) are manufactured using biological-expression systems (such as mammalian, bacterial, insect cells, etc.) and have spawned a large (>€35 billion sales annually in Europe) and growing biopharmaceutical industry (BioPharma). The structural and chemical complexity of biologics, combined with the intricacy of cell-based manufacturing, imposes a huge analytical burden to correctly characterize and quantify both processes (upstream) and products (downstream). In small molecule manufacturing, advances in analytical and computational methods have been extensively exploited to generate process analytical technologies (PAT) that are now used for routine process control, leading to more efficient processes and safer medicines. In the analytical domain, biologic manufacturing is considerably behind and there is both a huge scope and need to produce relevant PAT tools with which to better control processes, and better characterize product macromolecules. Raman spectroscopy, a vibrational spectroscopy with a number of useful properties (nondestructive, non-contact, robustness) has significant potential advantages in BioPharma. Key among them are intrinsically high molecular specificity, the ability to measure in water, the requirement for minimal (or no) sample pre-treatment, the flexibility of sampling configurations, and suitability for automation. Here, we review and discuss a representative selection of the more important Raman applications in BioPharma (with particular emphasis on mammalian cell culture). The review shows that the properties of Raman have been successfully exploited to deliver unique and useful analytical solutions, particularly for online process monitoring. However, it also shows that its inherent susceptibility to fluorescence interference and the weakness of the Raman effect mean that it can never be a panacea. In particular, Raman-based methods are intrinsically limited by the chemical complexity and wide analyte-concentration-profiles of cell culture media/bioprocessing broths which limit their use for quantitative analysis. Nevertheless, with appropriate foreknowledge of these limitations and good experimental design, robust analytical methods can be produced. In addition, new technological developments such as time-resolved detectors, advanced lasers, and plasmonics offer potential of new Raman-based methods to resolve existing limitations and/or provide new analytical insights.
原料药（API）的生产目前正在经历一个世纪以来最大的转型。这些变化基于蛋白质和大分子药物（统称为生物制药）的快速和显著引入，可以追溯到自 20 世纪 70 年代以来一直在进行的对生物医学科学（尤其是基因组学和蛋白质组学）的巨额投资。生物制药（或生物制剂）是使用生物表达系统（如哺乳动物、细菌、昆虫细胞等）制造的，并催生了一个庞大的（每年在欧洲的销售额超过 350 亿欧元）且不断增长的生物制药行业（BioPharma）。生物制剂的结构和化学复杂性，加上基于细胞的制造的复杂性，对正确表征和量化两个过程（上游）和产品（下游）都带来了巨大的分析负担。在小分子制造中，分析和计算方法的进步被广泛用于生成过程分析技术（PAT），这些技术现在用于常规过程控制，从而提高了过程的效率和药物的安全性。在分析领域，生物制造落后了很多，因此有很大的范围和需求来生产相关的 PAT 工具，以便更好地控制过程，并更好地表征产品大分子。拉曼光谱是一种具有许多有用特性（非破坏性、非接触、稳健性）的振动光谱，在生物制药领域具有显著的潜在优势。其中最重要的是固有的高分子特异性、在水中测量的能力、对最小（或无需）样品预处理的要求、采样配置的灵活性以及适用于自动化。在这里，我们回顾和讨论了拉曼在生物制药中的一些更重要的应用（特别强调哺乳动物细胞培养）的代表性选择。综述表明，拉曼的特性已成功地用于提供独特且有用的分析解决方案，特别是用于在线过程监测。然而，它也表明，它对荧光干扰的固有敏感性和拉曼效应的弱点意味着它永远不可能是万无一失的。特别是，基于拉曼的方法在本质上受到细胞培养物/生物加工培养基的化学复杂性和广泛的分析物浓度分布的限制，限制了它们用于定量分析。然而，通过对这些限制有适当的预先了解和良好的实验设计，可以生产出稳健的分析方法。此外，新的技术发展，如时间分辨探测器、先进的激光器和等离子体光学，为解决现有限制和/或提供新的分析见解提供了新的基于拉曼的方法的潜力。
