帕尼单抗中二硫键异构体的鉴定及生物活性分析

Characterization and Biological Activity Analysis of Disulfide Isoforms of Panitumumab

目的帕尼单抗是一种IgG2亚类抗体,已用于转移性结肠癌的临床治疗。然而尚没有研究对帕尼单抗中二硫键异构体的构成和活性进行分析鉴定。本实验将建立对帕尼单抗二硫键异构体鉴定并富集的方法,进一步分析异构体间的生物学活性差异。方法利用反相色谱分析帕尼单抗中二硫键异构体的构成;通过氧化还原处理富集A型和B型,并利用分子筛色谱对分离得到的异构体进行分析鉴定;进一步利用细胞杀伤实验对异构体的生物学活性进行评估。结果反相色谱结果表明,帕尼单抗中A型、B型和A/B型比例分别为38%、32%及30%;分子筛色谱结果显示A型的分子半径较之B型更大;细胞杀伤实验表明帕尼单抗的A型具有较B型更强的抑制肿瘤生长效能。结论本实验建立了对帕尼单抗二硫键异构体进行鉴定、富集及分析的方法,有助于进一步增进对IgG2亚类二硫键异构体结构和功能的理解,为IgG2亚类治疗性抗体的生产工艺优化和质量控制提供有益参考。

OBJECTIVE To establish the methods of characterizing and enriching the disulfide isoforms of panitumumab which is a human IgG2 mAb that has been used in the treatment of metastatic colorectal cancer （mCRC） and evaluate the biological activity difference among the isoforms, ie, IgG2-A, IgG2-B, and IgG2-A/B. METHODS The disulfide isoforms of panitumumab were iden- tified by reversed-phase chromatography. The isoform-A of panitumumab was enriched upon reduction-oxidation treatment when guani- dine was added and isoform-B was enriched upon reduction-oxidation treatment without guanidine. Then the molecule structural differ- ence of the isoform-A and isoform-B was analyzed by size-exclusion chromatography. At last, the biological activities of these isoforms were further investigated by cell-killing assay. RESULTS The component proportions of isoform A, B and A/B in panitumumab were 38%, 32% and 30%, respectively. Under reduction-oxidation conditions, the disulfide isoforms converted to isoform-A when 0. 9 mol · L- 1 guanidine was used, whereas isoform-B was enriched in the absence of guanidine. And isoform-A was eluted earlier in SEC- HPLC, suggesting a larger apparent molecular size as compared with isoform-B. Furthermore, the in vitro biological activity measure- ment showed an increased activity of IgG2-A compared with lgG2-B in inhibiting the growth of DiFi cells. The ICs0 s for IgG2-A and IgG2-B were 0. 095 46 μg · mL-1（95% CI 0. 079 86 -0. 114 1μg ·mL 1） and 0. 372 8 μg · mL-1（95% CI 0. 306 7 -0. 453 1 μg · mL-1）, respectively. CONCLUSION The methods for characterizing and enriching the disulfide isoforms are established. Difference in the biological activity between isform-A and isoform-B is observed. The methods will provide technical assist to the process optimization and quality control of panitumumab.