Iizuka Reiko, Kotch Frank, Xu April, Porter Thomas, Shion Henry, Birdsall Robert, Chen Weibin
Nihon Waters K.K.
Pfizer Inc.
Yakugaku Zasshi. 2017;137(5):525-528. doi: 10.1248/yakushi.16-00255-1.
Hydrophobic interaction chromatography (HIC) and MS are leading techniques for the characterization of the critical quality attributes (CQA) of antibody-drug conjugates (ADCs). This includes the average drug-to-antibody ratio (DAR) and drug loading distribution. A workflow that effectively utilizes the synergy between chromatography and detection technologies has been developed and was assessed using cysteine-conjugated ADCs. The DAR of low, moderate and high drug-loaded ADC samples were calculated from the chromatographic peak areas using LC(HIC)/UV or the deconvoluted mass spectra using native LC(SEC)/MS. The results of DAR by both technologies produced comparable results. In addition, the 2D-LC/MS system has been evaluated in combination with HIC and reversed-phase chromatography for structural identification. Individual peaks from the 1st dimension of the HIC separation were isolated online and re-directed to the 2nd dimension reversed-phase column. ADC was detected as the sub-units by MS and the conjugation site was identified via a middle down approach.
疏水作用色谱法（HIC）和质谱（MS）是用于表征抗体药物偶联物（ADC）关键质量属性（CQA）的主要技术。这包括平均药物与抗体比率（DAR）和药物负载分布。已开发出一种有效利用色谱法与检测技术之间协同作用的工作流程，并使用半胱氨酸偶联的ADC进行了评估。使用液相色谱（HIC）/紫外（UV）从色谱峰面积计算低、中、高药物负载ADC样品的DAR，或使用天然液相色谱（尺寸排阻色谱，SEC）/质谱的解卷积质谱计算DAR。两种技术得到的DAR结果具有可比性。此外，二维液相色谱/质谱系统已结合HIC和反相色谱进行结构鉴定评估。在线分离HIC分离第一维的各个峰，并将其重新导向第二维反相柱。通过质谱将ADC检测为亚基，并通过中间下切方法鉴定偶联位点。
