Zhang Wei, Czupryn Marta J
Wyeth BioPharma, Genetics Institute Campus, One Burtt Road, Andover, MA 01810, USA.
J Pharm Biomed Anal. 2003 Jan 1;30(5):1479-90. doi: 10.1016/s0731-7085(02)00479-x.
Monoclonal antibody (mAb) therapy applications have been growing rapidly in recent years. Like other proteins, therapeutic mAbs can under go various enzymatic and non-enzymatic reactions that can affect their structural integrity and stability. Among the degradation reactions, isoaspartate (isoAsp) formation is one of the major sources of charge heterogeneity of mAbs. This paper reports the detection and quantification of isoAsp in a recombinant mAb and its charge isoforms resolved by cation exchange high performance liquid chromatography. The assay utilizes the enzyme protein isoaspartyl methyltransferase in conjunction with strong cation exchange separation and UV detection (at 260 nm) of S-adenosyl-L-homocysteine, which is produced stoichiometrically in the enzymatic reaction. The mAb is found to contain an average 0.2 mol of isoAsp per mol of protein, however, various charge isoforms were found to contain different levels of isoAsp. The most acidic isoforms contain approximately 0.7 mol of isoAsp per mol of protein, and no isoAsp is detected in the most basic isoform. It appears that the majority of isoAsp in the mAb is formed as a result of asparagine deamidation.
近年来，单克隆抗体（mAb）治疗应用发展迅速。与其他蛋白质一样，治疗性单克隆抗体可能会经历各种酶促和非酶促反应，这些反应会影响其结构完整性和稳定性。在降解反应中，异天冬氨酸（isoAsp）的形成是单克隆抗体电荷异质性的主要来源之一。本文报道了通过阳离子交换高效液相色谱法对重组单克隆抗体中的异天冬氨酸及其电荷异构体进行检测和定量。该测定方法利用酶蛋白异天冬氨酰甲基转移酶，结合强阳离子交换分离和对S-腺苷-L-高半胱氨酸的紫外检测（在260nm处），S-腺苷-L-高半胱氨酸是在酶促反应中按化学计量产生的。发现该单克隆抗体每摩尔蛋白质平均含有0.2摩尔异天冬氨酸，然而，发现各种电荷异构体含有不同水平的异天冬氨酸。酸性最强的异构体每摩尔蛋白质含有约0.7摩尔异天冬氨酸，而在碱性最强的异构体中未检测到异天冬氨酸。看来该单克隆抗体中的大多数异天冬氨酸是天冬酰胺脱酰胺作用的结果。
