Lack of simple and robust methods to determine complement activation in human serum induced by antigen-antibody complexes is a major hurdle for monitoring therapeutic antibody drug quality and stability. Dezamizumab is a humanized IgG1 monoclonal antibody that binds to serum amyloid P component (SAP) for potential treatment of systemic amyloidosis. The mechanism of action of Dezamizumab includes the binding of SAP, complement activation through classical pathway, and phagocytosis; however, the steps in this process cannot be easily monitored. We developed two novel methods to determine Dezamizumab-SAP complex-induced complement activation. Complement component 3 (C3) depletion was detected by homogeneous time-resolved fluorescence (HTRF), and C3a desArg fragment, formed after the cleavage of C3 to yield C3a followed by removal of its C-terminal arginine residue, was determined using Meso Scale Discovery (MSD) technology. We found that the presence of both Dezamizumab and SAP was required for complement activation via both methods. The optimal molar ratio of Dezamizumab:SAP was 6:1 in order to obtain maximal complement activation. The relative potency from both methods showed a good correlation to Dezamizumab-SAP-dependent complement component 1q (C1q) binding activity in Dezamizumab thermal-stressed samples. Both SAP and C1q binding, as determined by surface plasmon resonance and the two complement activation potency methods described here, reflect the mechanism of action of Dezamizumab. We conclude that these methods can be used to monitor Dezamizumab quality for drug release and stability testing, and the novel potency methods reported here can be potentially used to evaluate complement activity induced by other antigen-antibody complexes.

中文翻译：

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确定由 Dezamizumab 和血清淀粉样蛋白 P 复合物诱导的人血清中补体激活的新方法。

缺乏确定抗原-抗体复合物诱导的人血清中补体激活的简单而可靠的方法是监测治疗性抗体药物质量和稳定性的主要障碍。Dezamizumab 是一种人源化 IgG1 单克隆抗体，可与血清淀粉样蛋白 P 成分 (SAP) 结合，用于治疗系统性淀粉样变性。Dezamizumab的作用机制包括结合SAP、通过经典途径激活补体、吞噬作用；然而，这个过程中的步骤不容易监控。我们开发了两种新方法来确定 Dezamizumab-SAP 复合物诱导的补体激活。通过均相时间分辨荧光 (HTRF) 和 C3a desArg 片段检测到补体成分 3 (C3) 消耗，使用 Meso Scale Discovery (MSD) 技术测定 C3 裂解后形成的 C3a，然后去除其 C 端精氨酸残基。我们发现通过这两种方法激活补体都需要 Dezamizumab 和 SAP 的存在。Dezamizumab:SAP 的最佳摩尔比为 6:1，以获得最大的补体激活。两种方法的相对效力显示出与 Dezamizumab 热应激样品中 Dezamizumab-SAP 依赖性补体成分 1q (C1q) 结合活性的良好相关性。SAP 和 C1q 结合，由表面等离子体共振和此处描述的两种补体激活效力方法确定，反映了 Dezamizumab 的作用机制。我们得出结论，这些方法可用于监测 Dezamizumab 的质量以进行药物释放和稳定性测试，