Gadgil Himanshu S, Bondarenko Pavel V, Pipes Gary D, Dillon Thomas M, Banks Douglas, Abel Jeffrey, Kleemann Gerd R, Treuheit Michael J
Amgen Inc., Thousand Oaks, CA 91320, USA.
Anal Biochem. 2006 Aug 15;355(2):165-74. doi: 10.1016/j.ab.2006.05.037. Epub 2006 Jun 15.
MAB007, an IgG1 monoclonal antibody, is unique because of the presence of a free cysteine residue in the Fab region at position 104 on the heavy chain in the CDR3 region. Mass spectrometric analysis of intact MAB007 showed multiple peaks varying in mass by 120-140 Da that cannot be fully attributed to glycosylation isoforms typically present in IgG molecules. Limited proteolysis of MAB007 with Lys-C led to a single cleavage at the C-terminus of a lysine residue in the hinge region of the heavy chain at position 222, generating free Fab and Fc fragments. Reversed-phase liquid chromatography/mass spectrometry analysis of the Fab and Fc fragments revealed several modifications. The Fab fraction showed cysteinylation of a free cysteine in the CDR3 region resulting in a mass shift of 119 Da. Using limited proteolysis, we also identified modifications resulting in a mass increase of 127 Da in the Fc region, corresponding to C-terminal lysine variants in the heavy chain. Other modifications, such as oxidation (+16 Da) and succinimide formation (-17 Da), were also detected in the Fab fragment. The cysteinylation observed after limited proteolysis was confirmed by peptide mapping coupled with tandem mass spectrometry analysis.
MAB007是一种IgG1单克隆抗体，因其在互补决定区3（CDR3）重链第104位的Fab区域存在一个游离半胱氨酸残基而独具特色。对完整的MAB007进行质谱分析显示，存在多个质量相差120 - 140 Da的峰，这些峰不能完全归因于IgG分子中通常存在的糖基化异构体。用Lys-C对MAB007进行有限蛋白酶解，导致重链铰链区第222位赖氨酸残基的C末端发生单次切割，产生游离的Fab和Fc片段。对Fab和Fc片段进行反相液相色谱/质谱分析揭示了几种修饰。Fab组分显示CDR3区域的游离半胱氨酸发生了半胱氨酸化，导致质量偏移119 Da。通过有限蛋白酶解，我们还鉴定出Fc区域存在导致质量增加127 Da的修饰，这与重链中的C末端赖氨酸变体相对应。在Fab片段中还检测到其他修饰，如氧化（+16 Da）和琥珀酰亚胺形成（-17 Da）。有限蛋白酶解后观察到的半胱氨酸化通过肽图分析结合串联质谱分析得到了证实。
