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

用于生物过程中蛋白质在线分析的多模态荧光光谱

• 批准号: BB/K011162/1
• 负责人: Paul Dalby
• 金额: $ 54.82万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK011162%2F1
• 关键词: Multi; modal; fluorescence; spectroscopy; online

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.

生物制药蛋白质通常使用多个色谱步骤从澄清发酵液中纯化，每个步骤根据一种或多种物理化学特征（例如净电荷、大小、疏水性和生物亲和力）分离蛋白质。在每个步骤中含有天然蛋白质产物的洗脱峰通常被宿主细胞蛋白质污染，或被产物的轻微修饰形式污染，所述产物几乎无法与天然形式区分开，因此非常难以去除。因此，每个步骤的精确产品特征对上游变异性、缓冲液组成和pH值的微小变化以及通过重复使用影响性能的色谱树脂逐渐污染非常敏感。因此，必须能够监测产物分布，优选在线或在线，以便能够真实的调整工艺参数，或者做出关于何时开始和停止收集洗脱峰内的产物级分的响应决定。适用于色谱在线监测的蛋白质和蛋白质混合物的真实的实时、低成本和低体积分析通常限于简单的吸光度、折射率和电导率测量，其仅提供基本的峰检测，而不提供蛋白质产物分布的详细表征。多角度激光散射（MALLS）具有在线表征近似分子质量的潜力，而精确的在线质谱法太昂贵且技术要求高。我们将利用我们最近的进展，在生物物理分析和快速激光诱导的温度扰动的未标记的蛋白质在微毛细管流动，以及国家的最先进的光学元件，建立一个低容量的流量检测器用于色谱，可以评估的异质性蛋白质产品的档案在真实的时间。将建立一套用于蛋白质内源荧光的光学器件，以测量荧光强度（FLI）、时间分辨荧光（TRF）和荧光相关光谱（FCS），从而同时测量蛋白质产物谱的正交特征。这些将测量蛋白质量（峰检测），并检测溶液构象、低聚状态和粒度（包括可溶性聚集体）的潜在变异性。FCS比DLS或MALLS对相对颗粒浓度更敏感和定量，DLS或MALLS对较大颗粒不成比例地敏感。为了进一步解决产品的异质性，流动的样品（从主洗脱流中连续分离）也将使用我们最近展示的微流体红外诱导加热技术进行高达70摄氏度的快速（12 ms）温度跃升。这将诱导蛋白质结构域的部分结构解折叠，以及可溶性低聚物的解离，其动力学和幅度是样品中每种不同蛋白质种类的特征，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。作为一个额外的奖励，检测器也将适用于独立的样品分析，如用于分析剂量制剂及其粘度。每种新的光谱模式的好处将被证明为广泛的相关蛋白质从我们的实验室和其他金砖四国成员，包括IgG，Fab，GCSF。为了测试可以检测到的异质性范围，将这些异质性泵入检测器中，并以部分和完全纯化的形式进行分析，以及在通过部分蛋白水解、部分解折叠和低pH下聚集、氧化和剪切损伤进行故意修饰后进行分析。然后将演示在线应用于色谱分析，使用分流器将连续流引入检测器，与馏分收集并行。

项目成果

Optically induced thermal gradients for protein characterization in nanolitre-scale samples in microfluidic devices.

• DOI: 10.1038/srep02130
• 发表时间: 2013
• 期刊: SCIENTIFIC REPORTS
• 影响因子: 4.6
• 作者: Sagar, D. M.;Aoudjane, Samir;Gaudet, Matthieu;Aeppli, Gabriel;Dalby, Paul A.
• 通讯作者: Dalby, Paul A.

Proof-of-concept analytical instrument for label-free optical deconvolution of protein species in a mixture.

用于混合物中蛋白质种类的无标记光学解卷积的概念验证分析仪器。

• DOI: 10.1016/j.chroma.2021.461968
• 发表时间: 2021
• 期刊: Journal of chromatography. A
• 作者: Hales JE

Machine learning reveals hidden stability code in protein native fluorescence.

• DOI: 10.1016/j.csbj.2021.04.047
• 发表时间: 2021
• 期刊: Computational and structural biotechnology journal
• 影响因子: 6
• 作者: Zhang H;Yang Y;Zhang C;Farid SS;Dalby PA
• 通讯作者: Dalby PA

Column-free optical deconvolution of intrinsic fluorescence for a monoclonal antibody and its product-related impurities

单克隆抗体及其产品相关杂质的内在荧光的无柱光学解卷积

• DOI: 10.1016/j.chroma.2023.464463
• 发表时间: 2023
• 期刊: Journal of Chromatography A
• 影响因子: 4.1
• 作者: Uçan D