利用β-N-乙酰氨基葡萄糖苷酶快速酶切分析中国仓鼠卵巢细胞表达的西妥昔单抗抗原结合区的聚糖比例

Analysis of glycan ratio of Chinese hamster ovary cell-cetuximab antigen-binding segment via rapid enzyme digestion with endo-β-N-acetylglucosaminidase F

西妥昔单抗具有较复杂的糖基化修饰,在抗原结合片段(Fab)和可结晶片段(Fc)的重链上都含有2个N-糖基化位点,其中Fab段的糖基化最为复杂,要研究清楚该位点的糖基化修饰,开发专一性切糖技术和稳定的聚糖比例分析方法是当前迫切需要解决的难题。以中国仓鼠卵巢(CHO)细胞表达的西妥昔单抗为研究对象,使用β-N-乙酰氨基葡萄糖苷酶(Endo F2)开发了一种快速Fab段聚糖释放的方法,利用超高效液相色谱-高分辨质谱(UPLC-HRMS)进行了定性和聚糖比例分析。第一步对抗体原液进行非变性酶切,抗体原液经超纯水稀释后,加入糖苷酶Endo F2进行酶切,通过质谱对质量数的解析,结果表明Endo F2酶切时间5 min,Fab段的聚糖就能完全切除,而Fc段的聚糖不受影响,实现了快速酶切,而且切糖具有很好的专一性。第二步对Fab段聚糖进行比例分析,将释放的聚糖经对氨基苯甲酰胺(2-AB)荧光标记后使用超高效液相色谱联用荧光检测器(FLR)进行检测,在亲水作用色谱(HILIC)柱上得到良好的分离并可以进行稳定地聚糖比例分析。3次独立试验结果表明,酶切后的质谱图基本一致,且聚糖的比例结果也基本一致,表明Endo F2酶切方法和聚糖比例分析方法都具有较好的稳定性和可靠性。此外,通过测定来自两个不同工艺生产的样品,数据显示两者的糖谱上具有非常明显的差异,表明利用开发的方法可以实现对抗体生产工艺进行监测研究,对抗体生产工艺的评估具有非常重要的意义。

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.