Wang Deyun, Wynne Colin, Gu Flora, Becker Chris, Zhao Jia, Mueller Hans-Martin, Li Huijuan, Shameem Mohammed, Liu Yan-Hui
Protein Mass Spectrometry, Sterile Product and Analytical Development, Bioprocess Development, Merck Research Laboratories , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.
Anal Chem. 2015 Jan 20;87(2):914-21. doi: 10.1021/ac503158g. Epub 2015 Jan 7.
Mass spectrometry (MS) characterization of recombinant monoclonal antibody (mAb) drugs and their degraded and/or post-translationally modified counterparts, drug-product-related impurities and variants, is critical for successful development of biotherapeutics. Specifically in this study, drug-product-related impurities of an anti-Clostridium difficile IgG1 mAb drug substance were profiled by cation-exchange liquid chromatography (CEX) followed by the CEX peaks being fraction-collected for MS characterization. A reversed-phase liquid chromatography/mass spectrometry (LC/MS) methodology was developed on a Thermo Q-Exactive orbitrap mass spectrometer for (1) accurate mass measurements of the mAb, its CEX fractionated impurities, and their respective heavy chains and light chains and (2) middle-down LC/MS/MS of the light chains and the heavy chains using higher energy C-trap dissociation (HCD). The accurate mass measurements and the HCD middle-down MS/MS experiments identify that major impurities and variants of the anti-C. difficile mAb are degradation species of the heavy chains at residue Asn101 as well as at the hinge region amino acids, including Cys222, Lys224, His226, and Thr227, with levels ranging from 0.3% to 6.2% of the total drug substance. Additional impurities were identified as light chain C-terminal truncation at Gly93 and oxidized heavy chains at Met40, Met93, and Met430. Our impurity characterization results demonstrate that the middle-down MS method allows direct and accurate identification of drug-product-related impurities of therapeutic mAbs. It is particularly useful for those low-level impurities and variants that are not suitable for further fractionation and characterization by bottom-up MS.
重组单克隆抗体（mAb）药物及其降解和/或翻译后修饰的对应物、药物产品相关杂质和变体的质谱（MS）表征对于生物治疗药物的成功开发至关重要。具体而言，在本研究中，通过阳离子交换液相色谱（CEX）对一种抗艰难梭菌IgG1单克隆抗体原料药的药物产品相关杂质进行了分析，随后对CEX峰进行分馏收集以进行MS表征。在Thermo Q-Exactive轨道阱质谱仪上开发了一种反相液相色谱/质谱（LC/MS）方法，用于（1）对单克隆抗体、其CEX分馏杂质及其各自的重链和轻链进行精确质量测量，以及（2）使用高能C阱解离（HCD）对轻链和重链进行自上而下的LC/MS/MS分析。精确质量测量和HCD自上而下的MS/MS实验表明，抗艰难梭菌单克隆抗体的主要杂质和变体是重链在Asn101残基以及铰链区氨基酸（包括Cys222、Lys224、His226和Thr227）处的降解产物，其含量占原料药总量的0.3%至6.2%。其他杂质被鉴定为轻链在Gly93处的C末端截短以及重链在Met40、Met93和Met430处的氧化。我们的杂质表征结果表明，自上而下的MS方法能够直接、准确地鉴定治疗性单克隆抗体的药物产品相关杂质。对于那些不适合通过自下而上的MS进行进一步分馏和表征的低水平杂质和变体而言，该方法尤其有用。
