A platform for high-throughput molecular characterization of recombinant monoclonal antibodies
A platform for high-throughput molecular characterization of recombinant monoclonal antibodies
复制标题
DOI：
10.1016/j.jchromb.2005.08.021
复制
发表时间：
2005-11-05
影响因子：
3
通讯作者：
James, DC
中科院分区：
文献类型：
--
作者：
Bailey, MJ;Hooker, AD;James, DC
我们描述了用于重组单抗及其翻译后修饰的快速和高通量分析的样品制备平台的定量特征。MAb捕获、脱盐和原位还原/烷基化是通过将分析物顺序吸附到悬浮在微滴定板孔中的固相小球(蛋白质A，反相)上来完成的。在不稳定酸的表面活性剂(RapiGest(TM))存在下洗脱和快速胰酶消化后，从反相管尖端通过逐步洗脱的方式分离多肽，并分离含有Fc N-糖肽的部分。用MALDI-TOF MS在线性模式下直接定量分析多肽糖型的相对丰度，与PNGaseF释放的荧光团标记的N-糖链的正相高效液相色谱分析密切相关。(C)2005 Elsevier B.V.保留所有权利。
We describe quantitative characterization of a sample preparation platform for rapid and high-throughput analysis of recombinant monoclonal antibodies (MAbs) and their post-translational modifications. MAb capture, desalting and in situ reduction/alkylation were accomplished by sequential adsorption of analyte to solid phase beads (protein A, reverse-phase) suspended in microtiter plate wells. Following elution and rapid tryptic digestion in the presence of acid-labile surfactant (RapiGest (TM)), peptides were fractionated by stepwise elution from reverse-phase pipet tips and the fraction containing Fc N-glycopeptides isolated. Direct quantitative analysis of the relative abundance of peptide glycoforms by MALDI-TOF MS in linear mode closely correlated with normal phase HPLC analysis of fluorophore labeled N-glycans released by PNGaseF. (c) 2005 Elsevier B.V. All rights reserved.