Otani Yuki, Yonezawa Atushi, Tsuda Masahiro, Imai Satoshi, Ikemi Yasuaki, Nakagawa Shunsaku, Omura Tomohiro, Nakagawa Takayuki, Yano Ikuko, Matsubara Kazuo
Department of Clinical Pharmacology and Therapeutics, Kyoto University Hospital, Kyoto, Japan.
Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
PLoS One. 2017 Jan 4;12(1):e0169588. doi: 10.1371/journal.pone.0169588. eCollection 2017.
Therapeutic monoclonal antibodies (mAbs) have heterogeneities in their structures. Multiple studies have reported that the variety of post-translational modifications could affect the pharmacokinetic profiles or pharmacological potencies of therapeutic mAbs. Taking into the account that the structural modification of mAbs would affect the efficacy, it is worth investigating the structural alteration of therapeutic mAbs in the blood and the relationship between their structures and pharmacological effects. Herein, we have developed the method to isolate rituximab from plasma in which endogenous IgGs interfere the detection of rituximab, and successfully developed the analytical method with a liquid chromatograph time-of-flight mass spectrometer to detect the structure of rituximab in plasma with errors less than 30 parts per millions. Eight types of carbohydrate chains in rituximab were detected by this method. Interestingly, time-dependent changes in carbohydrate chains such as AAF (G2F) and GnGn (G0) were observed in rats, although the amino acids were stable. Additionally, these structural changes were observed via incubation in plasma as in the rat experiment, suggesting that a certain type of enzyme in plasma caused the alterations of the carbohydrate chains. The present analytical methods could clarify the actual pharmacokinetics of therapeutic mAbs, and help to evaluate the interindividual variations in pharmacokinetics and efficacy.
治疗性单克隆抗体（mAb）在结构上存在异质性。多项研究报告称，多种翻译后修饰可能会影响治疗性mAb的药代动力学特征或药理活性。考虑到mAb的结构修饰会影响疗效，研究治疗性mAb在血液中的结构变化及其结构与药理作用之间的关系是很有必要的。在此，我们开发了一种从血浆中分离利妥昔单抗的方法，其中内源性免疫球蛋白会干扰利妥昔单抗的检测，并成功开发了一种液相色谱-飞行时间质谱分析方法，用于检测血浆中利妥昔单抗的结构，误差小于百万分之30。通过该方法检测到了利妥昔单抗中的八种糖链类型。有趣的是，在大鼠体内观察到了糖链如AAF（G2F）和GnGn（G0）的时间依赖性变化，尽管氨基酸是稳定的。此外，正如在大鼠实验中一样，在血浆中孵育也观察到了这些结构变化，这表明血浆中的某种酶导致了糖链的改变。目前的分析方法可以阐明治疗性mAb的实际药代动力学，并有助于评估药代动力学和疗效的个体间差异。
