RESUMO
Monoclonal antibodies have been used increasingly as therapeutic agents to target various diseases. Although most monoclonal antibodies have only one N-linked glycosylation site in the Fc region, N-linked glycosylation sites in the Fab region have also been observed. Because glycosylation of a monoclonal antibody can have a significant impact on its effector function, efficacy, clearance, and immunogenicity, it is essential to assess the glycosylation profile during cell line and clone selection studies and to assess the impact of cell culture conditions on the glycoform distribution during process optimization studies to ensure that the antibody is being produced with appropriate and consistent glycosylation. This article describes a liquid chromatography-mass spectrometry-based approach, in combination with papain digestion and partial reduction, to obtain site-specific glycosylation profile information for a therapeutic monoclonal antibody with two N-linked glycosylation sites in the heavy chain.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Anticorpos Monoclonais/uso terapêutico , Sítios de Ligação , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Glicosilação , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos Fc das Imunoglobulinas/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Lisina/metabolismo , Oxirredução , Papaína/metabolismo , Proteínas Recombinantes/uso terapêutico , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Purified capsular polysaccharide preparations from Streptococcus pneumoniae that are used for vaccine production typically contain residual levels of C-polysaccharide (C-Ps). Residual C-Ps is typically found in one of two forms, either chemically linked to the capsular polysaccharide (bound) or present by itself (free). Two analytical methods have been developed and applied to determine the relative percentages of the two C-Ps forms present in various capsular polysaccharide preparations. Both methods differentiate the two forms of C-Ps according to the difference of their hydrodynamic sizes. One method is based on labeling C-Ps with a fluorescent tag and separating the two forms of C-Ps by high-performance size exclusion chromatography with on-line refractive index and fluorescence detection, and the other method is based on measuring self-diffusion rates of the two forms of C-Ps by nuclear magnetic resonance (NMR) and quantifying each form with deconvolution. Both methods were evaluated for relative accuracy, precision, and ease of application, and they were found to provide comparable results for a large number of pneumococcal polysaccharide preparations. These analyses, combined with other quantitative NMR measurement of total C-Ps in the polysaccharide powder, provide a more refined means of evaluating the amount of each form of C-Ps in polysaccharide preparations targeted for vaccine production.