High-throughput melting-temperature analysis of a monoclonal antibody by differential scanning fluorimetry in the presence of surfactants
High-throughput melting-temperature analysis of a monoclonal antibody by differential scanning fluorimetry in the presence of surfactants
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DOI：
10.1002/jps.23405
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发表时间：
2013-02-01
影响因子：
3.8
通讯作者：
Friess, Wolfgang
中科院分区：
文献类型：
--
作者：
Menzen, Tim;Friess, Wolfgang
差示扫描荧光法 (DSF) 已成功用作高通量筛选方法，用于分析治疗性单克隆抗体 (MAb) 制剂开发中的蛋白质熔解温度 (Tm)。通常，表面活性剂在 MAb 制剂中用作稳定剂，但常用的极性敏感染料 SYPRO (R) Orange 在胶束存在时显示出明亮的荧光，掩盖了蛋白质解折叠的信号。通过研究含有聚山梨酯 20、聚山梨酯 80 或泊洛沙姆 188 (PX 188) 的各种 MAb 制剂，研究了分子转子探针 4-(二氰乙烯基)久洛里定 (DCVJ)。尽管仅限于较高的 MAb 浓度，但 DCVJ 能够测定许多 SYPRO (R) Orange 无法测定的配方中的 Tm。值得注意的是，安慰剂制剂的仔细背景校正对于精确测定 Tm，尤其是 Tm 起始时间至关重要。 Tm1 的热位移（观察到的最低热转变）表明 pH 值或赋形剂的稳定或不稳定作用，在所有测试的制剂中都具有良好的一致性，并且与差示扫描量热法测量结果良好相关。此外，还确认了 PX 188 的胶束化温度，这会导致非蛋白质转变。通过这种新方法，可以在含表面活性剂配方中的治疗性蛋白质的开发过程中应用 DSF。 (C) 2012 年 Wiley periodicals, Inc. 和美国药剂师协会 J Pharm Sci 102:415428, 2013
Differential scanning fluorimetry (DSF) is successfully used as a high-throughput screening method for the analysis of the protein melting temperature (Tm) in the development of therapeutic monoclonal antibody (MAb) formulations. Typically, surfactants are utilized in MAb formulations as a stabilizer, but the commonly applied polarity-sensitive dye SYPRO (R) Orange shows bright fluorescence in the presence of micelles, concealing the signal of protein unfolding. Studying various MAb formulations containing polysorbate 20, polysorbate 80, or poloxamer 188 (PX 188), the molecular rotor probe 4-(dicyanovinyl)julolidine (DCVJ) was investigated. Although limited to higher MAb concentrations, DCVJ enabled the determination of Tm in many formulations where SYPRO (R) Orange failed. It is important to note that careful background correction of placebo formulations is essential for the precise determination of Tm and especially Tm onset. Thermal shifts of Tm1 (lowest observed thermal transition) indicating stabilizing or destabilizing effects of pH or excipient were in good agreement across all tested formulations and correlated well with differential scanning calorimetry measurements. Additionally, the micellization temperature of PX 188 was confirmed, which leads to a nonproteinous transition. With this new method, it is possible to apply DSF during the development of therapeutic proteins in surfactant-containing formulations. (C) 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:415428, 2013