Polyethylene glycol (PEG) represents an effective strategy to improve the pharmacokinetic profile of a molecule as it extends the biotherapeutic's half-life, masks immunogenic epitopes or modifies its distribution. The addition of one or multiple PEG moieties, in either linear or branched form, is known to carry the risk of potentially inducing an immunogenic response against PEG. The importance of accurately quantifying anti-PEG antibodies during a clinical study is well recognized and stems from the fact that anti-PEG antibodies have been shown to negatively impact the efficacy of the biotherapeutic that the PEG is coupled to. As a consequence, sponsors are encouraged to develop immunogenicity assays to assess appropriately the presence of anti-drug antibodies (ADA) against the protein component as well as the PEG. However, detection of anti-PEG antibodies is complicated by a number of technical challenges, including the availability of appropriate positive control material. In addition, the fact that some anti-PEG antibodies are known to circulate as low-affinity IgM, drives the need for an assay able to detect low affinity anti-PEG ADA even in the presence of high concentrations of the biotherapeutic. To address this need, we developed and validated an Affinity Capture Elution (ACE)-AGL assay to detect anti-drug and anti-PEG antibodies. In this assay, which we call ACE-AGL, ADA are captured by biotin-PEG-drug, acid eluted and re-captured on a second plate coated with protein AGL. ADA are then detected using Ruthenium-PEG-drug. The new assay format described is highly sensitive to both anti-drug and anti-PEG antibodies and very drug-tolerant. The ACE-AGL assay is easy to perform and has been successfully validated at two separate CROs. We propose the ACE-AGL format as a valid and effective alternative to the currently available assay methods.

聚乙二醇（PEG）代表一种有效的策略，可改善分子的药代动力学特性，因为它可以延长生物治疗剂的半衰期，掩盖免疫原性表位或改变其分布。已知添加线性或支化形式的一个或多个PEG部分具有潜在地诱导针对PEG的免疫原性应答的风险。在临床研究中准确定量抗PEG抗体的重要性已广为人知，并且源于以下事实：已显示抗PEG抗体会对PEG偶联的生物治疗剂的功效产生负面影响。因此，鼓励申办者进行免疫原性测定，以适当评估针对蛋白质成分和PEG的抗药物抗体（ADA）的存在。然而，许多技术挑战使抗PEG抗体的检测变得复杂，其中包括适当的阳性对照材料的可用性。另外，已知一些抗PEG抗体以低亲和力IgM的形式流通的事实，驱使对即使在高浓度生物治疗剂存在的情况下也能够检测低亲和力抗PEG ADA的测定的需求。为了满足这一需求，我们开发并验证了亲和捕获洗脱（ACE）-AGL检测试剂盒来检测抗药物和抗PEG抗体。在这种称为ACE-AGL的测定中，ADA被生物素-PEG-药物捕获，被酸洗脱并重新捕获在第二个涂有蛋白AGL的板上。然后使用钌-PEG-药物检测ADA。所描述的新测定形式对抗药物和抗PEG抗体均高度敏感，并且具有很高的药物耐受性。ACE-AGL分析易于执行，并已在两个单独的CRO处成功验证。我们建议使用ACE-AGL格式作为当前可用测定方法的有效替代方法。