Merck & Co., Inc, Rahway, NJ, 07065, USA.
AAPS J. 2023 Jan 25;25(1):18. doi: 10.1208/s12248-023-00783-9.
Immunogenicity testing to detect and characterize anti-drug antibody (ADA) is required for almost all biotherapeutics. Monoclonal antibody biotherapeutics usually have long half-lives and for high-dose indications such as oncology, high level of drug will be present in the testing samples and interfere with ADA and/or neutralization antibody (NAb) measurement. To overcome this drug interference, acid-dissociation-based sample pre-treatment such as Bead-Extraction and Acid Dissociation (BEAD) has been successfully applied. The main concern for these acid-dissociation-based methods, however, is that harsh acid treatment could denature positive control Abs as well as NAb species in testing samples. In addition, high amount of biotinylated drug is needed in order to have effective competition with high level of drug in the samples, which in turn requires expensive magnetic beads. And the whole process of magnetic beads handling is tedious if doing manually and often causes trouble during assay transfer. Here, we describe a novel method which we named as Precipitation, Acid Dissociation and Biotin-drug as Assay Drug (PABAD). This novel method will need only one step of acid dissociation, with much milder and shorter acid treatment to maximally preserve NAb activity. In addition, only a fraction of biotinylated-drug is needed and there is no need to use additional streptavidin (SA)-plate or SA-magnetic beads for extraction. Compared to a BEAD-based assay, PABAD demonstrates significantly improved recovery of acid-sensitive NAb positive controls (PCs) and similar recovery of acid-resistant NAb PCs.
免疫原性检测用于检测和鉴定抗药物抗体（ADA），几乎所有的治疗性生物制剂都需要进行该检测。单克隆抗体治疗性生物制剂通常具有较长的半衰期，并且对于高剂量的适应症，如肿瘤学，药物将在测试样品中存在较高水平，从而干扰 ADA 和/或中和抗体（NAb）的检测。为了克服这种药物干扰，已经成功应用了基于酸解离的样品预处理方法，如珠子提取和酸解离（BEAD）。然而，这些基于酸解离的方法主要存在两个问题，即苛刻的酸处理会使阳性对照抗体以及测试样品中的 NAb 物种发生变性。此外，为了与样品中高水平的药物进行有效竞争，需要使用大量的生物素化药物，这反过来又需要昂贵的磁珠。如果手动进行，磁珠处理的整个过程繁琐，并且在进行测定转移时常会引起问题。在这里，我们描述了一种新方法，我们将其命名为沉淀、酸解离和生物素化药物作为检测药物（PABAD）。这种新方法仅需要一步酸解离，酸处理更为温和且时间更短，从而最大限度地保留 NAb 活性。此外，只需要使用少量的生物素化药物，并且不需要使用额外的链霉亲和素（SA）板或 SA-磁珠进行提取。与 BEAD 测定相比，PABAD 显著提高了对酸敏感的 NAb 阳性对照（PC）的回收率，并且对酸抗性 NAb PC 的回收率也相似。
