Belton Daniel J, Miller Aline F
Department of Chemical and Biological Sciences, University of Huddersfield, Queensgate, Huddersfield HD1 3DH, UK.
J Biophys. 2013;2013:751091. doi: 10.1155/2013/751091. Epub 2013 Feb 13.
The thermal aggregation of the biopharmaceutical protein recombinant protective antigen (rPA) has been explored, and the associated kinetics and thermodynamic parameters have been extracted using optical and environmental scanning electron microscopies (ESEMs) and ultraviolet light scattering spectroscopy (UV-LSS). Visual observations and turbidity measurements provided an overall picture of the aggregation process, suggesting a two-step mechanism. Microscopy was used to examine the structure of aggregates, revealing an open morphology formed by the clustering of the microscopic aggregate particles. UV-LSS was used and developed to elucidate the growth rate of these particles, which formed in the first stage of the aggregation process. Their growth rate is observed to be high initially, before falling to converge on a final size that correlates with the ESEM data. The results suggest that the particle growth rate is limited by rPA monomer concentration, and by obtaining data over a range of incubation temperatures, an approach was developed to model the aggregation kinetics and extract the rate constants and the temperature dependence of aggregation. In doing so, we quantified the susceptibility of rPA aggregation under different temperature and environmental conditions and moreover demonstrated a novel use of UV spectrometry to monitor the particle aggregation quantitatively, in situ, in a nondestructive and time-resolved manner.
对生物制药蛋白重组保护性抗原（rPA）的热聚集进行了研究，并使用光学和环境扫描电子显微镜（ESEM）以及紫外光散射光谱（UV-LSS）提取了相关的动力学和热力学参数。视觉观察和浊度测量提供了聚集过程的整体情况，表明存在两步机制。显微镜用于检查聚集体的结构，揭示了由微观聚集体颗粒聚集形成的开放形态。UV-LSS被用于并开发以阐明这些在聚集过程第一阶段形成的颗粒的生长速率。观察到它们的生长速率最初很高，然后下降并收敛到与ESEM数据相关的最终尺寸。结果表明颗粒生长速率受rPA单体浓度限制，并且通过在一系列孵育温度下获取数据，开发了一种方法来模拟聚集动力学并提取速率常数以及聚集的温度依赖性。通过这样做，我们量化了rPA在不同温度和环境条件下聚集的敏感性，此外还展示了紫外光谱法在原位、无损且时间分辨的情况下定量监测颗粒聚集的新用途。
