Optimization of a reversed-phase high-performance liquid chromatography/mass spectrometry method for characterizing recombinant antibody heterogeneity and stability
Optimization of a reversed-phase high-performance liquid chromatography/mass spectrometry method for characterizing recombinant antibody heterogeneity and stability
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DOI：
10.1016/j.chroma.2006.01.016
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发表时间：
2006-07-07
影响因子：
4.1
通讯作者：
Ricci, MS
中科院分区：
文献类型：
--
作者：
Dillon, TM;Bondarenko, PV;Ricci, MS
开发了用于监测完整和片段化单克隆抗体（MAb）的稳定性和生产的增强型分析RP-HPLC/MS方法。使用高柱温（70-80 ℃）、具有高洗脱强度系数的有机溶剂（异丙醇和正丙醇）和Zorbax StableBond色谱柱对于免疫球蛋白GI（IgG 1）和IgG 2单克隆抗体的良好回收率和分离度至关重要。使用该方法，通过在线电喷雾电离飞行时间（ESI-TOF）质谱法明确分离和鉴定降解的IgG 1抗体的裂解产物，生成精确的质量和独特的末端梯状序列。通过完整MAb的质谱法测定糖基化谱，包括N-连接糖的末端半乳糖和岩藻糖异质性的图谱。此外，我们发现，几个IgG 2单克隆抗体表现出更大的结构异质性相比，IgG 1。质谱表征数据和还原数据表明，异质性与二硫键相关。这种反相LC/MS方法代表了监测完整MAb生产和稳定性的关键进步。(c)2006 Elsevier B. V.保留所有权利。
An enhanced analytical RP-HPLC/MS method was developed for monitoring the stability and production of intact and fragmented monoclonal antibodies (MAbs). The use of high column temperatures (70-80 degrees C), organic solvents with high eluotropic strength coefficients (isopropyl and n-propyl alcohols), and Zorbax StableBond columns, were critical for good recovery and resolution of immunoglobulin GI (IgG1) and IgG2 monoclonal antibodies. Using this method, cleavage products of a degraded IgG1 antibody were clearly separated and identified by in-line electrospray ionization time-of-flight (ESI-TOF) mass spectrometry generating exact masses and unique terminal ladder sequences. The glycosylation profile, including mapping of the terminal galactose and fucose heterogeneity of the N-linked sugars, was determined by mass spectrometry of intact MAbs. In addition, we discovered that several IgG2 MAbs exhibited greater structural heterogeneity compared to IgG1s. Mass spectral characterization data and reduction data suggested that the heterogeneity is disulfide related. This reversed-phase LC/MS method represents a key advancement in monitoring intact MAb production and stability. (c) 2006 Elsevier B.V. All rights reserved.