Characterization of lower molecular weight artifact bands of recombinant monoclonal IgG1 antibodies on non-reducing SDS-PAGE
Characterization of lower molecular weight artifact bands of recombinant monoclonal IgG1 antibodies on non-reducing SDS-PAGE
复制标题
DOI：
10.1007/s10529-007-9449-8
复制
发表时间：
2007-11-01
影响因子：
2.7
通讯作者：
Newby-Kew, Abigail
中科院分区：
文献类型：
--
作者：
Liu, Hongcheng;Gaza-Bulseco, Georgeen;Newby-Kew, Abigail
非还原条件下的SDS-PAGE是重组单克隆抗体纯度和稳定性指示试验最常用的技术之一。在非还原SDS-PAGE上，通常观察到分子量低于完整抗体的条带，这是IgG分子的常见特征。通过凝胶内消化，然后基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱，Western印迹和通过比较在还原剂存在下制备的样品的带型来分析这些片段。经鉴定，片段条带为缺失一条轻链、两条重链、一条轻链和一条重链、游离重链和游离轻链的抗体。片段化对样品缓冲液pH值、孵育时间、还原剂和烷基化试剂的灵敏度表明，在样品制备过程中形成了片段，但在分析的样品中不存在。二硫键错配和β-消除是抗体片段形成的两种主要机制。质谱分析表明，可以通过使用烷基化特异性修饰游离巯基来防止二硫键混乱，从而减少非还原SDS-PAGE上的伪影量。通过使用质谱法检测脱氢丙氨酸证明了通过β-消除的二硫键断裂。
SDS-PAGE under non-reducing conditions is one of the most commonly used techniques for recombinant monoclonal antibody purity and stability indicating assay. On non-reducing SDS-PAGE, bands with a lower molecular weight than the intact antibody are routinely observed and is a common feature of IgG molecules. These fragments were analyzed by in-gel digestion followed by matrix-assisted-laser-desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry, Western blot and by comparing the banding pattern of sample prepared in the presence of a reducing reagent. The fragments bands were identified as antibody lacking one light chain, two heavy chains, one light chain and one heavy chain, free heavy chain and free light chain. Sensitivity of fragmentation to sample buffer pH, incubation time, reducing reagent and alkylation reagents indicated that fragments were formed during sample preparation, but not present in the samples analyzed. Disulfide bond scrambling and beta-elimination are the two major mechanisms of the formation antibody fragments. Mass spectrometry analysis suggested that disulfide bond scrambling can be prevented by specifically modifying free sulhydryl using alkylation and thus reduced the amount of artifacts on non-reducing SDS-PAGE. Breakage of disulfide bonds by beta-elimination was evidenced by the detection of dehydroalanine using mass spectrometry.