Clinical Immunogenicity Analysis, Non-clinical and Clinical Assay Sciences, Novo Nordisk A/S, Novo Nordisk Park, Måløv, Denmark.
Immunogenicity Assay Development, Non-clinical and Clinical Assay Sciences, Novo Nordisk A/S, Novo Nordisk Park, Måløv, Denmark.
J Immunol Methods. 2021 Oct;497:113002. doi: 10.1016/j.jim.2021.113002. Epub 2021 Feb 25.
Highly sensitive assays for anti-drug antibodies (ADAs) are both a regulatory requirement and requisite for proper evaluation of the effects of immunogenicity on clinical efficacy and safety. Determination of ADA assay sensitivity depends on positive control antibodies to represent naturally occurring or treatment-induced ADA responses. An accurate determination of the proportion of drug-specific antibodies in these polyclonal positive control batches is critical for correct evaluation of assay sensitivity. Target purification of positive control antibodies is commonly applied but infers the risk to lose a proportion of the antibodies. This may lead to an incorrect estimate of the ADA assay sensitivity, especially if high-affinity antibodies are lost that may be representative of natural ADAs with clinical implication. The Surface Plasmon Resonance platform on the Biacore™ systems offers methods for real-time analysis of biomolecular interactions without introducing any modifications to the analysed material. Calibration-free concentration analysis (CFCA) is such an application for determination of the proportion of drug-specific antibodies, which allows direct determination of active antibody concentrations, as defined by the ligand, in a flow-based system. Here, we present a novel CFCA method for ADA quantification developed and validated using polyclonal positive control antibodies against endogenous human insulin, insulin degludec (Tresiba®) and turoctocog alfa (NovoEight®). We find that CFCA precisely and accurately measures concentrations of drug-specific IgG antibodies with a precision of ±10% and 90%-112% recovery of expected values of monoclonal positive control antibodies. Additionally, we have achieved a more accurate measure of the sensitivity of a cell-based bioassay for in vitro neutralising ADAs using the specific concentration determined with CFCA. Moreover, we effectively quantified serum anti-insulin antibodies in high-titre clinical samples from individuals with diabetes mellitus. This application extends the relevance of the CFCA technology to analysis of immunogenicity for accurate quantification of ADAs in both the polyclonal positive control and in clinical samples.
高灵敏度的抗药物抗体（ADA）检测方法既是监管要求，也是评估免疫原性对临床疗效和安全性影响的必要条件。ADA 检测方法的灵敏度取决于阳性对照抗体，以代表自然发生或治疗诱导的 ADA 反应。准确确定这些多克隆阳性对照批次中药物特异性抗体的比例对于正确评估检测方法的灵敏度至关重要。阳性对照抗体的靶向纯化通常被应用，但会带来失去一部分抗体的风险。这可能导致 ADA 检测方法灵敏度的不正确估计，特别是如果失去了具有临床意义的天然 ADA 代表性的高亲和力抗体。Biacore™系统上的表面等离子体共振（SPR）平台提供了实时分析生物分子相互作用的方法，而不会对分析材料进行任何修饰。无标曲浓度分析（CFCA）就是这样一种应用，可用于确定药物特异性抗体的比例，该方法允许在基于流的系统中直接测定配体定义的活性抗体浓度。在这里，我们提出了一种新的 CFCA 方法，用于针对内源性人胰岛素、胰岛素德古鲁德（Tresiba®）和特立帕肽（NovoEight®）的多克隆阳性对照抗体进行 ADA 定量，该方法已得到开发和验证。我们发现 CFCA 能够精确和准确地测量药物特异性 IgG 抗体的浓度，其精度为±10%，并且对单克隆阳性对照抗体的预期值的回收率为 90%-112%。此外，我们使用 CFCA 确定的特异性浓度，实现了对体外中和 ADA 的细胞生物测定灵敏度的更准确测量。此外，我们有效地量化了来自糖尿病患者的高滴度临床样本中的血清抗胰岛素抗体。这项应用扩展了 CFCA 技术的相关性，可用于分析免疫原性，以准确地定量多克隆阳性对照和临床样本中的 ADA。
