Multi-modal fluorescence spectroscopy for online analysis of proteins in bioprocesses

生物制药蛋白质通常是通过多个层析步骤从澄清的发酵液中纯化出来的，其中每个步骤都基于一个或多个物理化学特征(如净电荷、大小、疏水性和生物亲和力)来分离蛋白质。每一步含有天然蛋白质产物的洗脱峰往往被宿主细胞蛋白质污染，或者被产物的轻微修改版本污染，这些版本几乎与天然形式难以区分，因此去除起来非常困难。因此，每一步的精确产品轮廓对上游变化性、缓冲液成分和pH的微小变化以及通过重复使用而影响性能的层析树脂的逐渐污染非常敏感。因此，必须能够监测产品概况，最好是在线或在线，以便能够实时调整过程参数，或者就何时开始和停止收集洗脱峰内的产品组分做出响应决定。实时、低成本和低容量的蛋白质和蛋白质混合物分析适合在线监测层析，通常仅限于简单的吸光度、折射率和电导率测量，这些测量只提供基本的峰检测，而不提供蛋白质产品轮廓的详细表征。多角度激光光散射(MALLS)具有在线表征近似分子质量的一些潜力，而准确的在线质谱分析过于昂贵和技术要求高。我们将利用我们在微毛细管流动中非标记蛋白质的生物物理分析和快速激光诱导温度扰动方面的最新进展，以及最先进的光学元件，建立用于层析的小体积流动检测器，该检测器可以实时评估蛋白质产物图谱的异质性。将建立一套用于蛋白质本征荧光的单一光学装置，以测量荧光强度(FLI)、时间分辨荧光(TRF)和荧光相关光谱(FCS)，从而同时表征蛋白质产品轮廓的正交特征。它们将测量蛋白质数量(峰值检测)，并检测溶液构象、低聚状态和颗粒大小(包括可溶聚集体)的潜在变异性。与DLS或MALL相比，FCS对相对颗粒物浓度更敏感，也更定量，后者对较大颗粒物的敏感性不成比例。为了进一步解决产品的异质性，流动样品(从主洗脱流中连续分离)还将利用我们最近展示的微流控红外诱导加热技术，经历高达70℃的快速(12ms)温度跃升。这将导致蛋白质结构域的部分结构展开和可溶性低聚物的解离，具有样品中每个不同蛋白质物种特有的动力学和幅度，从而提供相对于已知参考标准或先前洗脱曲线的样品复杂性的进一步在线解析。作为一个额外的好处，该检测器还将适用于独立的样品分析，例如用于剂量配方及其粘度的剖析。每一种新的光谱模式的好处将在我们实验室和其他金砖四国成员的广泛相关蛋白质中得到证明，包括免疫球蛋白、Fab、GCSF。为了测试可以检测到的异质性范围，这些物质将被注射器泵入检测器，以部分和完全纯化的形式进行分析，并在经过刻意修改后，在低pH、氧化和剪切损伤下进行部分蛋白质分解、部分展开和聚集。然后，将使用分流装置演示在线应用于层析，以使连续流动与馏分收集平行地进入检测器。

Biopharmaceutical proteins are typically purified from clarified fermentation broths using multiple chromatographic steps where each separates the proteins based on one or more physico-chemical feature such as net charge, size, hydrophobicity and biological affinity. The elution peak containing the native protein product at each step is often contaminated by host cell proteins, or with slightly modified versions of the product which can be almost indistinguishable from the native form and therefore very challenging to remove. The precise product profile at each step is therefore very sensitive to small changes in upstream variability, buffer composition and pH, and the gradual fouling of chromatographic resins that affects performance through repeated re-use. It is therefore imperative to be able to monitor the product profile, preferably in-line or on-line, to be able to adjust the process parameters in real time, or to make a responsive decision as to when to start and stop collecting the product fraction within the elution peak. Real time, low cost and low volume analysis of proteins and protein mixtures suitable for online monitoring of chromatography, is generally limited to simple absorbance, refractive index and conductivity measurements which only provide basic peak detection and no detailed characterisation of the protein product profile. Multi-angle laser light scattering (MALLS) has some potential for online characterisation of approximate molecular masses, whereas accurate online mass-spectrometry is too expensive and technically demanding. We will take advantage of our recent advances in both the biophysical analysis and rapid laser-induced temperature perturbations of unlabelled proteins in microcapillary flow, as well as state-of-the-art optical components, to establish a low volume flow-detector for use in chromatography, that can evaluate the heterogeneity of the protein product profile in real time. A single set of optics for the intrinsic fluorescence of proteins will be set up to measure fluorescence intensity (FLI), time-resolved fluorescence (TRF) and fluorescence correlation spectroscopy (FCS) and so simultaneously characterise orthogonal features of the protein product profile. These will measure protein quantity (peak detection), and detect underlying variability in solution conformation, oligomeric state, and particle sizes, including the soluble aggregates. FCS is more sensitive and quantitative for relative particle concentrations than DLS or MALLS which are disproportionately sensitive to larger particles. To further resolve product heterogeneity, the flowing sample (continuously split from the main elution stream) will also be subjected to a rapid (12 ms) temperature jump of up to 70degC using our recently demonstrated microfluidic IR-induced heating technology. This will induce partial structural unfolding of protein domains, and the dissociation of soluble oligomers, with kinetics and amplitudes that are characteristic to each different protein species in the sample, and thus provide further online resolution of the sample complexity relative to known reference standards or previous elution profiles. As an additional bonus, the detector will also be suitable for standalone sample analysis, such as for the profiling of dosage formulations and their viscosities. The benefits of each new spectroscopic mode will be demonstrated for a wide range of relevant proteins from our lab and from other BRIC members, including IgG, Fab, GCSF. To test the range of heterogeneity that can be detected, these will be syringe-pumped into the detector and analysed in partially and fully purified forms, and also after deliberate modification by partial proteolysis, partial unfolding and aggregation at low pH, oxidation, and shear damage. Online application to chromatography will then be demonstrated using a flow splitter to give a continuous flow into the detector in parallel to fraction collection.