Department of Analytical Development, Biogen, Inc., 225 Binney St., Cambridge, MA, 02142, USA.
Anal Biochem. 2020 Dec 15;611:113842. doi: 10.1016/j.ab.2020.113842. Epub 2020 Aug 2.
FcγRIIa receptor binding is part of the mechanism of action for many therapeutic antibodies. AlphaScreen® technology and Biolayer Interferometry (BLI) are often used to assess protein-protein interactions. Recently we demonstrated that the presence of aggregates in samples significantly increased binding potency values in AlphaScreen®-based FcRn binding assays, sometimes masking the loss of potency. Even bigger effect of aggregates was observed in an AlphaScreen®-based FcγRIIa binding assay for a monoclonal antibody with strong effector function. To resolve this issue a novel BLI-based FcγRIIa binding assay was developed and qualified. The assay measures association binding responses and calculates the binding potency of the samples relative to the standard using Parallel Line Analysis. The method overcomes interference of aggregates present in the samples, distinguishes different Fc glycosylation patterns, and is stability-indicating. It can be used for sample characterization, drug product release and stability testing.
FcγRIIa 受体结合是许多治疗性抗体作用机制的一部分。AlphaScreen®技术和生物层干涉（BLI）常用于评估蛋白-蛋白相互作用。最近我们证明，样品中存在聚集物会显著增加 AlphaScreen® 基于 FcRn 结合测定中的结合效力值，有时会掩盖效力的丧失。在基于 AlphaScreen®的针对具有强效应功能的单克隆抗体的 FcγRIIa 结合测定中，聚集物的影响更大。为了解决这个问题，开发并验证了一种新的基于 BLI 的 FcγRIIa 结合测定法。该测定法测量结合的反应，并使用平行线分析相对于标准计算样品的结合效力。该方法克服了样品中存在的聚集物的干扰，区分了不同的 Fc 糖基化模式，并且具有稳定性指示性。它可用于样品特性描述、药物产品放行和稳定性测试。
