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.

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基于生物传感的治疗性抗体结构域高阶结构的质量控制监测

先进的生物制药制造需要新颖的过程分析技术来快速、灵敏地评估治疗性蛋白质的高阶结构。然而，变性蛋白质的传统理化分析在灵敏度、通量、分析分辨率和实时监测能力方面存在局限性。尽管基于探针的传感可以克服这些限制，但典型的非特异性探针缺乏分析分辨率，并且几乎无法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，我们生成了源自人工蛋白质的生物传感探针，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。对噬菌体展示蛋白文库进行生物淘选产生的人工蛋白能够以纳摩尔级亲和力与变性抗体结构域结合，但不与其天然折叠结构结合。蛋白质探针不仅可以识别完整 IgG 中的高级结构变化，还可以区分变性的抗体结构域。这些域特异性探针用于生成响应等值线图，以可视化由各种工艺参数（例如 pH、温度、酸洗脱和病毒灭活的保持时间）引起的抗体变性。这些蛋白质探针可以与现有的分析技术相结合，例如用于实时监测的表面等离振子共振或用于高通量分析的基于板的测定，以帮助开发用于工艺优化和监测抗体制造的新分析技术。