RESUMEN
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.
Asunto(s)
Cromatografía Líquida de Alta Presión , Inmunoglobulina G/análisis , Espectrometría de Masas en Tándem , Animales , Células CHO , Secuencia de Carbohidratos , Cromatografía por Intercambio Iónico , Cricetinae , Cricetulus , Glicosilación , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Lisina/análisis , Datos de Secuencia Molecular , Mapeo Peptídico , Polimorfismo Genético , Estructura Terciaria de Proteína , Ácido Pirrolidona Carboxílico/análisis , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
A bioassay was developed to assess the potency of TGFbeta antagonists by measuring IL-11 production in TGFbeta-1 treated human lung epithelial cells (A549). The production of IL-11 by A549 cells, measured by ELISA, was shown to be proportional to the TGFbeta-1 concentration. The A549 cells were responsive to all three isoforms of TGFbeta in the range of 3.0 ng/mL to 1.4 pg/mL, with an 18 to 24 h exposure time found to be within the linear portion of the bioassay response range. The Effective Dose at 80% of the maximal response (ED80) of TGFbeta-1 determined for the assay was 0.3 ng/mL. With this level of TGFbeta-1, a human anti-TGFbeta-1 antibody (CAT-192) yielded an approximate median Inhibitory Concentration (IC50) value of 3 microg/mL. To investigate assay specificity, alternate members of the TGFbeta superfamily were evaluated. Recombinant human activin B, inhibin A and BMP-2 (Bone Morphogenetic Protein-2) did not elicit a significant IL-11 response from the A549 cell line. Bioassay qualification was performed to obtain estimates of precision and accuracy, as well as to establish plate validity criteria. Assay precision was estimated at 30%, while the accuracy was +/-13%. Additionally, the ability of the A549 cell potency assay to detect potency differences in structurally modified samples was investigated.