Abstract:
Advanced biopharmaceutical manufacturing requires novel process analytical technologies for the rapid and sensitive assessment of the higher-order structures of therapeutic proteins. However, conventional physicochemical analyses of denatured proteins have limitations in terms of sensitivity, throughput, analytical resolution, and real-time monitoring capacity. Although probe-based sensing can overcome these limitations, typical non-specific probes lack analytical resolution and provide little to no information regarding which parts of the protein structure have been collapsed. To meet these analytical demands, we generated biosensing probes derived from artificial proteins that could specifically recognize the higher-order structural changes in antibodies at the protein domain level. Biopanning of phage-displayed protein libraries generated artificial proteins that bound to a denatured antibody domain, but not its natively folded structure, with nanomolar affinity. The protein probes not only recognized the higher-order structural changes in intact IgGs but also distinguished between the denatured antibody domains. These domain-specific probes were used to generate response contour plots to visualize the antibody denaturation caused by various process parameters, such as pH, temperature, and holding time for acid elution and virus inactivation. These protein probes can be combined with established analytical techniques, such as surface plasmon resonance for real-time monitoring or plate-based assays for high-throughput analysis, to aid in the development of new analytical technologies for the process optimization and monitoring of antibody manufacturing.
摘要:
先进的生物制药制造需要新颖的工艺分析技术,以快速,灵敏地评估治疗性蛋白质的高阶结构。然而,变性蛋白质的常规物理化学分析在灵敏度方面有局限性,吞吐量,分析分辨率,和实时监控能力。尽管基于探针的传感可以克服这些限制,典型的非特异性探针缺乏分析分辨率,并且几乎没有提供有关蛋白质结构的哪些部分已崩溃的信息。为了满足这些分析需求,我们产生了来自人工蛋白质的生物传感探针,这些探针可以在蛋白质结构域水平上特异性识别抗体的高阶结构变化。噬菌体展示的蛋白质文库的生物淘选产生了与变性抗体结构域结合的人工蛋白质,但不是它的自然折叠结构,具有纳摩尔亲和力。蛋白质探针不仅识别完整IgG中的高阶结构变化,而且还区分了变性的抗体结构域。这些域特异性探针用于生成响应等高线图,以可视化由各种工艺参数引起的抗体变性,如pH值,温度,酸洗脱和病毒灭活的保持时间。这些蛋白质探针可以与已建立的分析技术相结合,例如用于实时监测的表面等离子体共振或用于高通量分析的基于板的测定,帮助开发新的分析技术,以优化抗体生产的过程和监测。