Sinha Sandipan, Pipes Gary, Topp Elizabeth M, Bondarenko Pavel V, Treuheit Michael J, Gadgil Himanshu S
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas, USA.
J Am Soc Mass Spectrom. 2008 Nov;19(11):1643-54. doi: 10.1016/j.jasms.2008.07.004. Epub 2008 Jul 16.
High-performance liquid chromatography (LC) and liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-MS) methods with various sample preparation schemes were compared for their ability to identify and quantify glycoforms in two different production lots of a recombinant monoclonal IgG1 antibody. IgG1s contain a conserved N-glycosylation site in the fragment crystallizable (Fc) subunit. Six methods were compared: (1) LC/ESI-MS analysis of intact IgG, (2) LC/ESI-MS analysis of the Fc fragment produced by limited proteolysis with Lys-C, (3) LC/ESI-MS analysis of the IgG heavy chain produced by reduction, (4) LC/ESI-MS analysis of Fc/2 fragment produced by limited proteolysis and reduction, (5) LC/MS analysis of the glycosylated tryptic fragment (293EEQYNSTYR301) using extracted ion chromatograms, and (6) normal phase HPLC analysis of N-glycans cleaved from the IgG using PNGase F. The results suggest that MS quantitation based on the analysis of Fc/2 (4) is accurate and gives results that are comparable to normal phase HPLC analysis of N-glycans (6).
采用多种样品制备方案的高效液相色谱（LC）和液相色谱/电喷雾电离飞行时间质谱（LC/ESI-MS）方法，针对两种不同生产批次的重组单克隆IgG1抗体中糖型的鉴定和定量能力进行了比较。IgG1在可结晶片段（Fc）亚基中含有一个保守的N-糖基化位点。比较了六种方法：（1）完整IgG的LC/ESI-MS分析；（2）用Lys-C进行有限蛋白酶解产生的Fc片段的LC/ESI-MS分析；（3）还原产生的IgG重链的LC/ESI-MS分析；（4）有限蛋白酶解和还原产生的Fc/2片段的LC/ESI-MS分析；（5）使用提取离子色谱图对糖基化胰蛋白酶片段（293EEQYNSTYR301）进行LC/MS分析；（6）使用PNGase F对从IgG上切下的N-聚糖进行正相HPLC分析。结果表明，基于Fc/2分析（4）的质谱定量是准确的，其结果与N-聚糖的正相HPLC分析（6）相当。
