IBET, Apartado 12, P-2781-901 Oeiras, Portugal.
J Biotechnol. 2010 Aug 2;148(4):171-81. doi: 10.1016/j.jbiotec.2010.05.005. Epub 2010 May 26.
The binding and elution of the key components of a bioreaction bulk for the production of recombinant baculoviruses-a promising viral vector for gene therapy and vaccination-on a model ion-exchange surface have been successfully measured and interpreted by surface plasmon resonance (SPR) spectroscopy. The micro-scaled, ion-exchange surface was produced by immobilizing a typical ion-exchange ligand, diethylaminoethyl, onto commercially available planar gold sensor chip surfaces, which were pre-derivatized with a self-assembled monolayer of 11-mercaptoundecanoic acid. Each isolated analyte was injected into the SPR cell at defined operating conditions of salt and solute concentrations to determine the adsorption equilibrium plateau, and then eluted at the same salt concentration, upon which a well-defined, residual desorption equilibrium plateau was observed. From the analysis of the binding and elution curves and equilibrium plateaus for seven key biomolecules, it is possible to determine the adsorption isotherms over a broad range of equilibrium conditions for the three main cuts of the baculovirus bioreaction bulk: the product (the infective baculovirus), the main product-related impurities, and the main process-related impurities. A model that quantitatively explains the SPR-derived adsorption/desorption data was successfully developed for this complex biological system.
通过表面等离子体共振（SPR）光谱技术，成功测量并解释了生物反应基质中关键成分在模型离子交换表面上的结合和洗脱情况，该生物反应基质用于生产重组杆状病毒——一种用于基因治疗和疫苗接种的有前途的病毒载体。该微尺度离子交换表面是通过将典型的离子交换配体二乙氨基乙基固定在商业上可获得的平面金传感器芯片表面上来制备的，这些芯片表面先用 11-巯基十一酸自组装单层进行了衍生化处理。每个分离的分析物都在定义的盐和溶质浓度操作条件下注入 SPR 细胞，以确定吸附平衡平台，然后在相同的盐浓度下洗脱，此时可以观察到定义明确的、残留的解吸平衡平台。通过对七种关键生物分子的结合和洗脱曲线以及平衡平台的分析，可以确定杆状病毒生物反应基质三个主要部分（产物（感染性杆状病毒）、主要产物相关杂质和主要工艺相关杂质）在广泛的平衡条件下的吸附等温线。成功为这个复杂的生物系统开发了一个定量解释 SPR 衍生吸附/解吸数据的模型。
