1. ����ҽѧԺ,a. ����ҽѧԺ; b. ����ҽѧ����ѧԺ; c. ������ѧѧԺ,���� ���� 453007; 2. ����ʡ���������ҽѧ���鼼��Эͬ��������,���� ���� 453007
Characterization and Biological Activity Analysis of Disulfide Isoforms of Panitumumab
YANG Yun1a,2, GUO Rui1b, HUANG Juan1c, LIU Peng1a, ZHANG Zi-heng1a, ZHANG Peng-fei1a, WANG Fan1c, L�� Xin-yan1a, LIU You-xun1a*
1a. School of Basic Medical Sciences; 1b. College of Biomedical Engineering; 1c. School of Life Sciences, Xinxiang Medical University, Xinxiang 453007, China; 2. Henan Collaborative Innovation Center of Molecular Diaghosis and Laboratory Medicine, Xinxiang 453007, China
Abstract��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 identified by reversed-phase chromatography. The isoform-A of panitumumab was enriched upon reduction-oxidation treatment when guanidine was added and isoform-B was enriched upon reduction-oxidation treatment without guanidine. Then the molecule structural difference 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 measurement showed an increased activity of IgG2-A compared with IgG2-B in inhibiting the growth of DiFi cells. The IC50s for IgG2-A and IgG2-B were 0.095 46 ��g��mL-1 (95% CI 0.079 86-0.114 1 ��g��mL-1 ) and 0.372 8 ��g��mL-1 (95% CI 0.306 7-0.453 1 ��g��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.
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2018, Vol. 53 
