Protein aggregation in solution: a colloidal approach

近年来，随着商业生产的治疗性蛋白质的数量和数量迅速增加，蛋白质错误折叠和聚集的问题得到了更广泛的认识。一种常见的生物药物表达系统是大肠杆菌，它在高产系统中经常以包涵体的形式表达重组蛋白。天然结构的恢复需要蛋白质的增溶和重新折叠。由于复性过程和聚集过程之间的竞争，这些过程可能是在高稀释度下进行的低产量过程。此外，在蛋白质加工和配方过程中，由于浓度和溶液条件的影响，蛋白质会明显聚集。虽然在这一领域已经有了很多工作，但由于实验的复杂性和个体的限制，目前报道的许多工作在很大程度上都是实证的，这使得很难得出一般的学习和新方法的范围尽管多肽链有大量的构型，但通常蛋白质折叠成天然状态可能是快速和稳健的。然而，溶剂(作为离子强度和pH的函数)与多肽主链及其侧链不同部分之间的作用力的细微变化和鲜为人知的变化会导致蛋白质的错误折叠和聚集。令人惊讶的微小变化可能会导致本质上不同的结果！多肽错误折叠和聚集的途径以及这些聚集相对于天然折叠蛋白质的稳定性具有相当大的工业相关性和学术重要性。这里提出的研究工作将基于一个实验聚焦计划，利用当前研究仪器的改进，使用动态光散射(DLS)、分析超速离心法(AUC)、动态表面张力/表面流变学(DSTSR)、傅里叶变换红外光谱(FTIR)和尺寸排除色谱(SEC)进行蛋白质聚集。特别是最近几年，DLS在性能上有了显著的改进，非常适合于研究稳态蛋白质大小以及蛋白质在溶液中聚集的动力学。作为CMO，Avecia有可能获得许多与模型和工业相关的蛋白质系统，该项目启动前的部分活动将是确定要包括在研究中的最有用的一组蛋白质。建议的初始模型是RPA(炭疽保护性抗原)，它是一种80kD的单亚单位蛋白质，具有已知的3D结构、折叠条件和已知的聚集倾向。该蛋白质和其他模型蛋白质将进行系统的实验研究，计划：(I)根据pH、离子强度、反离子、浓度、添加剂、表面活性剂和温度的变化确定聚集的敏感性；(Ii)确定推动聚集现象的常见机制，包括错误折叠和部分展开的重要性。(Iii)将成熟的胶体聚集(DVLO)和絮凝理论应用于蛋白质聚集现象中的问题。(4)考虑蛋白质的溶液水合/脱水行为，利用已知的水溶性聚合物的类似行为，包括蛋白质、反离子、水分子稳定剂之间的复杂竞争。(V)确定表面以及污染物颗粒在聚集现象中所起的作用(Vi)提出预测蛋白质聚集稳定性的方法和减轻聚集风险的策略。

As the number and volume of therapeutic proteins produced commercially has risen rapidly in recent years the problem of protein misfolding and aggregation have come to be more widely recognised. A common biopharmaceutical expression system is E coli which in high productivity systems can frequently express the recombinant protein as an inclusion body. Recovery of native structure requires solubilisation and then refolding of the protein. Due to competition between the processes of refolding and aggregation these can be low yielding processes carried out at high dilutions. Additionally during protein processing and in formulation protein aggregation can be manifest arising from concentration and solution conditions. While there has been much work in this area, much of the current work reported has a largely empirical emphasis due to both the experimental complexities and restricted to individual cases making it difficult to draw general learning and scope for new approaches Despite the enormous number of configurations of a polypeptide chain, often the protein folding into the native state can be rapid and robust. However, subtle and poorly understood variations in the forces between the solvent (as a function of ionic strength and pH) with the different part of the polypeptide backbone chain and it's side chains leads to misfolding and aggregated proteins. Surprising small variations can lead to substantially different outcomes! The pathways by which polypeptides misfold and aggregate, and the stability of these aggregates relative to the natively folded protein are of considerable industrial relevance and academic importance. The research work proposed here will be based on an experimentally focussed plan exploiting improvements in current research instrumentation for following protein aggregation using Dynamic Light Scattering (DLS), Analytical Ultracentrifugation (AUC), Dynamic Surface Tension/Surface Rheology (DSTSR), Fourier Transform Infrared Spectroscopy (FTIR) and Size Exclusion Chromatography (SEC). DLS especially has shown significant improvements in performance in recent years and is well suited for studying both steady state protein size as well as the kinetics of protein aggregation in solution. As a CMO Avecia potentially has access to a number of both model and industrially relevant protein systems and part of the pre-initiation activities of the project will be to determine the most useful set of proteins to include in the study. A suggested initial model is rPA (anthrax protective antigen) a 80kD single subunit protein with a known 3D structure, refolding conditions and known aggregation propensity. This and other model proteins will be experimentally investigated systematically with the plans to : (i) Determine the sensitivity to aggregation based on variations in pH, ionic strength, counter-ions, concentration, additives, surfactants and temperature (ii) Identify common mechanisms which drive aggregation phenomena including the importance of misfolding and partial unfolding. (iii) Apply well established colloidal aggregation (DVLO) and flocculation theories to problems in protein aggregation phenomena. (iv) Consider solution hydration/dehydration behaviour of proteins, exploiting the known like behaviour of water soluble polymers including the complex competition between proteins, counter ions, stabilisers for the water molecules. (v) Identify the role played by surfaces as well as contaminant particles in aggregation phenomena (vi) Propose methods for predicting protein stability to aggregation and well as strategies to mitigate the risks of aggregation.