RESUMO
A recombinant IgG1 monoclonal antibody (mAb) showed multiple charge variants in a cation exchange chromatography profile. To better understand the correlation between charge heterogeneity and glycosylation, a rapid reversed phase ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method with integrated mass analysis has been developed for simultaneous determination of N-terminal pyroglutamate, C-terminal lysine truncation, and Fc glycosylation. The results show that various degrees and/or types of N-terminal pyroglutamate formation and C-terminal lysine (Lys) cleavage account for the majority of charge heterogeneity; and the charge variants showed Fc glycosylation patterns in relation to their terminal modifications. The amount of G1F decreased in the basic variants, whereas Man5 and G0F-GN increased. The complement-dependent cytotoxicity (CDC) activity of purified charge variants also suggested the potential impact of the charge differences on the glycosylation profile.
Assuntos
Cromatografia Líquida de Alta Pressão , Imunoglobulina G/análise , Espectrometria de Massas em Tandem , Animais , Células CHO , Sequência de Carboidratos , Cromatografia por Troca Iônica , Cricetinae , Cricetulus , Glicosilação , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Lisina/análise , Dados de Sequência Molecular , Mapeamento de Peptídeos , Polimorfismo Genético , Estrutura Terciária de Proteína , Ácido Pirrolidonocarboxílico/análise , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
OBJECTIVE: To explore the effect of Shenqi Fuzheng Injection (SFI) on gene expression profile of liver tissue with metastatic carcinoma in mice. METHODS: Twenty liver metastatic carcinoma model mice were established by splenectomy after their spleens were injected with 0.2 mL colon cancer C26 cell strain oncocyte liquid, then they were randomly divided into the model group and the SFI group. Starting from the 5th day after modeling, mice in the model group and the SFI group were given via intraperitoneal injection once every other day with physiological saline and SFI respectively. All the mice were sacrificed at the 15th day and the gene profile of metastatic liver carcinoma tissue in the two groups were screened by whole genome chip technique. RESULTS: (1) The model establishing successful rate reached 100%; (2) Gene expression showed that as compared with the model group, in the SFI group, 123 genes were up-regulated, with 52 of them registered to Ensemble, while only one gene was down-regulated and registered to Ensemble was none. CONCLUSION: SFI plays its role of anti-tumor mainly by upregulating several relative genes to promote apoptosis of tumor cells and stabilizing chromosomes.
