蛋白质的N-糖基化是典型的翻译后修饰。与其他单克隆抗体相比,西妥昔单抗中的N-糖基化修饰更为复杂。因为西妥昔单抗含有两个N-糖基化位点,一个位于抗原结合片段(Fab)上,另一个位于重链(HC)的可结晶片段(Fc)上。在这两个人中,Fab片段的糖基化更复杂。由于该片段位于高变区(VH),它可能会影响抗体抗原的亲和力并引起其他问题。因此,有必要研究该位点的糖基化修饰。这种修改特别具有挑战性,需要开发特定的聚糖切割技术和稳定的聚糖比率分析方法。在这项研究中,以西妥昔单抗在中国仓鼠卵巢(CHO)细胞中表达为实验研究对象。基于内切-β-N-乙酰氨基葡萄糖苷酶F2(EndoF2)的消化,开发了一种可以快速释放Fab聚糖的实验方法。通过超高效液相色谱-高分辨率质谱(UPLC-HRMS)进行定性和聚糖比率分析。测试分为两个步骤:在第一步中,对CHO-西妥昔单抗药物进行非变性(天然状态)糖苷酶切除试验.通过添加超纯水将原料药稀释至1.0mg/mL,随后将1.0μL的EndoF2直接加入到100μL的药物中,在37℃下进行酶消化。通过HRMS,对数据进行解卷积,以获得原料药的准确质量。结果表明,当EndoF2的消化时间为5min时,Fab段中的聚糖可以被完全去除,而Fc段中的那些没有受到影响。实现了Fab聚糖的快速酶切割;同时,结论是该方法对于Fab聚糖的去除也是非常特异性的。第二步,对从CHO-西妥昔单抗中切除的Fab聚糖进行了准确的比率分析测试.释放的Fab聚糖用冰乙醇沉淀,将上清液离心并旋转干燥,然后用对氨基苄基(2-AB)标记。2-AB标记可以使聚糖具有荧光可检测信号,在70%乙腈水溶液中复原后,通过与荧光检测器(FLR)耦合的UPLC检测。使用亲水相互作用色谱(HILIC)柱获得良好的色谱峰分离。因此,该测试实现了稳定的聚糖比率分析。三个独立的EndoF2消化循环5分钟的分子量结果表明,消化后的质量相似;随后,基于HILIC进行聚糖比率分析。三个独立的聚糖比率分析实验的结果也相似,表明EndoF2的快速酶消化,然后在消化5分钟后进行聚糖比率分析具有良好的稳定性和可靠性。通过测量使用我们公司采用的两种不同工艺生产的样品获得的数据表明,这两种工艺的聚糖谱有明显的差异,特别是在唾液酸糖型方面。这些结果证明本研究中开发的方法可以准确地分析聚糖的比率。监测抗体生产过程对过程的评估具有重要意义。
The N-glycosylation of proteins is a typical post-translational modification. Compared with other monoclonal antibodies, N-glycosylation modification in cetuximab is more complicated. Because cetuximab contains two N-glycosylation sites, one is located on the antigen-binding fragment (Fab) and the other is on the crystallizable fragment (Fc) of the heavy chain (HC). Among the two, the glycosylation of the Fab segment is more complicated. As this segment is located in the hypervariable region (VH), it may affect the affinity of the antibody antigen and cause other issues. Therefore, it is necessary to study glycosylation modification at this site. This modification is particularly challenging, necessitating the development of specific glycan cutting technology and a stable glycan ratio analysis method. In this study, cetuximab expressed in Chinese hamster ovary (CHO) cell was used as the experimental research object. Based on the digestion with endo-β-N-acetylglucosaminidase F2 (Endo F2), an experimental method was developed that can quickly release Fab glycans. Qualitative and glycan ratio analyses were carried out by ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS). The test was divided into two steps: in the first step, a non-denaturing (native state) glycosidase excision test was performed on the CHO-cetuximab drug substance. The drug substance was diluted to 1.0 mg/mL by adding ultrapure water, following which 1.0 μL of Endo F2 was directly added to 100 μL of the drug substance for enzyme digestion at 37 ℃. Through HRMS, the data were deconvoluted to obtain the accurate mass of the drug substance. The results showed that when the digestion time of Endo F2 was 5 min, the glycans in the Fab segment could be completely removed, whereas those in the Fc segment were not affected. Rapid enzyme cutting of the Fab glycans was realized; simultaneously, it was concluded that this method was also very specific for the removal of Fab glycans. In the second step, an accurate ratio analysis test was performed on Fab glycans excised from CHO-cetuximab. The released Fab glycans were precipitated with ice ethanol, the supernatant was centrifuged and spin-dried, and then labeled with para-aminobenzyl (2-AB). 2-AB labeling could make glycans have fluorescent detectable signals, and after reconstitution in 70% acetonitrile aqueous solution, was detected by UPLC coupled with a fluorescence detector (FLR). Good chromatographic peak separation was obtained using a hydrophilic interaction chromatography (HILIC) column. Thus, the test enabled stable glycan ratio analysis. The molecular weight results for three independent Endo F2 digestion cycles for 5 min showed that the masses after digestion were similar; subsequently, glycan ratio analysis was performed based on HILIC. The results of three independent glycan ratio analysis experiments were also similar, indicating that the rapid enzyme digestion of Endo F2 followed by glycan ratio analysis after 5 min of digestion yielded good stability and reliability. Data obtained by measuring the samples produced using two different processes employed by our company showed that there were distinct differences in the glycan profiles of the two processes, especially in terms of the sialic acid glycoforms. These results prove that the method developed in this study can accurately analyze the ratio of glycans. Monitoring the antibody production process is important and meaningful for the evaluation of the process.