Abstract：
Drug product development of therapeutic antibody formulations is still dictated by the risk of protein particle formation during processing or storage, which can lead to loss of potency and potential immunogenic reactions. Since structural perturbations are the main driver for irreversible protein aggregation, the conformational integrity of antibodies should be closely monitored. The present study evaluated the applicability of a plate reader-based high throughput method for Intrinsic Tryptophan Fluorescence Emission (ITFE) spectroscopy to detect protein aggregation due to protein unfolding in high-concentrated therapeutic antibody samples. The impact of fluorophore concentration on the ITFE signal in microplate readers was investigated by analysis of dilution series of two therapeutic antibodies and pure tryptophan. At low antibody concentrations (< 5 mg/mL, equivalent to 0.8 mM tryptophan), the low inner filter effect suggests a quasi-linear relationship between antibody concentration and ITFE intensity. In contrast, the constant ITFE intensity at high protein concentrations (> 40 mg/mL, equivalent to 6.1 mM tryptophan) indicate that ITFE spectroscopy measurements of IgG1 antibodies are feasible in therapeutically relevant concentrations (up to 223 mg/mL). Furthermore, the capability of the method to detect low levels of unfolding (around 1 %) was confirmed by limit of detection (LOD) determination with temperature-stressed antibody samples as degradation standards. Change of fluorescence intensity at the maximum (ΔIaM) was identified as sensitive descriptor for protein degradation, providing the lowest LOD values. The results demonstrate that ITFE spectroscopy performed in a microplate reader is a valuable tool for high-throughput monitoring of protein degradation in therapeutic antibody formulations.
摘要：
治疗性抗体制剂的药物产品开发仍然取决于加工或储存过程中蛋白质颗粒形成的风险，这可能导致效力丧失和潜在的免疫原性反应。由于结构扰动是不可逆蛋白质聚集的主要驱动因素，应密切监测抗体的构象完整性。本研究评估了基于读板仪的高通量方法用于固有色氨酸荧光发射（ITFE）光谱法检测由于高浓度治疗性抗体样品中蛋白质解折叠而导致的蛋白质聚集的适用性。通过分析两种治疗性抗体和纯色氨酸的稀释系列研究了荧光团浓度对微孔板读取器中ITFE信号的影响。在低抗体浓度（<5mg/mL，相当于0.8mM色氨酸)，较低的内滤效应表明抗体浓度与ITFE强度之间存在准线性关系。相比之下,在高蛋白质浓度下恒定的ITFE强度（>40mg/mL，相当于6.1mM色氨酸）表明IgG1抗体的ITFE光谱测量在治疗相关浓度（高达223mg/mL）下是可行的。此外，通过以温度应激抗体样品作为降解标准品的检测限(LOD)测定,证实了该方法检测低水平解折叠(约1%)的能力.最大荧光强度的变化（ΔIaM）被鉴定为蛋白质降解的敏感描述符，提供最低LOD值。结果表明,在酶标仪中进行的ITFE光谱学是用于高通量监测治疗性抗体制剂中的蛋白质降解的有价值的工具。
