RESUMEN
Minor N-linked glycans containing N-glycolylneuraminic acid residues and/or α-Gal epitopes (i.e., galactose-α1,3-galactose residues) have been reported to be present in recombinant monoclonal antibody (mAb) therapeutics. These contaminations are due to their production processes using nonhuman mammalian cell lines in culture media containing animal-derived materials. In case of the treatment of tumors, we inevitably use such mAbs by careful risk-benefit considerations to prolong patients' lives. However, expanding their clinical applications such as for rheumatism, asthma, and analgesia demands more careful evaluation of the product characteristics. The present work for detailed evaluations of N-glycans demonstrates the methods using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) and a combination of high-performance liquid chromatography and electrospray ionization time-of-flight mass spectrometry. The CE-LIF method provides excellent separation of both major and minor N-glycans from six commercial mAb pharmaceuticals within 30 min and clearly indicates that a possible trigger of immunogenicity in humans due to the presence of nonhuman N-glycans is present. We strongly believe that the proposed method will be a powerful tool for the analysis of N-glycans of recombinant mAb products in various development stages, such as clone selection, process control, and routine release testing to ensure safety and efficacy of the products.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Electroforesis Capilar , Polisacáridos/análisis , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión , Galactosa/análisis , Glicosilación , Rayos Láser , Datos de Secuencia Molecular , Ácidos Neuramínicos/análisis , Polisacáridos/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
An incomplete elongation of O-glycans in mucins has been found in epithelial cancers, leading to the expression of shorter carbohydrate structures such as Tn antigen (GalNAc-O-Ser/Thr), which has been reported to be one of the most specific human cancer-associated structures. However, there have been no appropriate physicochemical methods for the determination of Tn antigen in biological samples. In the present paper, we developed a capillary electrophoresis method for the determination of Tn antigen, and applied the method to the analysis of the expressed Tn antigen on some leukemia and epithelial cancer cells.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/análisis , Electroforesis Capilar/métodos , Acetilgalactosamina/aislamiento & purificación , Humanos , Células Tumorales Cultivadas , ortoaminobenzoatos/metabolismoRESUMEN
Antibody pharmaceuticals are becoming more and more prevalent due to their excellent effectiveness in clinical medications, and are expected to allow tailor-made medical treatment for rheumatic diseases, immunosuppression in cardiac transplantation, and cancer. Antibody-type pharmaceuticals of immunoglobulin G (IgG) commonly have N-glycosylated carbohydrate chains attached to heavy chains. The carbohydrate chains play important roles in the effectiveness of antibodies. Therefore evaluation of a glycosylated species is important in the first step of quality control of antibody pharmaceuticals. In the present work, we examined capillary electrophoresis with a newly developed, chemically modified capillary, the inner surface of which is modified with carboxyl groups, for evaluation of IgG molecular species which have carbohydrate chains; tocilizumab was used as a model. The analytical system developed in the present study is useful for determining the content of non-glycosylated peptides. In the analysis of tocilizumab, the ratio of non-glycosylated peptide was estimated to be 1.23% with a relative standard deviation of 3.05%. The method affords high reproducibility with simple operation, and analysis can be completed within 6 min.
Asunto(s)
Anticuerpos Monoclonales/análisis , Ácidos Carboxílicos/química , Química Farmacéutica , Electroforesis Capilar/métodos , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales Humanizados , Concentración de Iones de Hidrógeno , Inmunoglobulina G/análisis , Inmunoglobulina G/química , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Carboxymethyl cellulose (CMC) is one of the most important cellulose derivatives and used in the fields of food, pharmaceuticals, cosmetics, and paint. Fibrous CMC is used an antiadhesive material to prevent postoperative wound adhesions. The degree of substitution and distribution of the substituent (i.e., the carboxymethyl group) are the most important parameters for the function of CMC. Thus, CMC used for antiadhesive material must be carefully evaluated, because the CMC product is retained in patients' bodies over the long term. Although identification tests of CMC are defined in the Japanese Pharmacopoeia, it is difficult to evaluate its structure using those tests. In the present study, we propose improved methods for evaluating CMC products by analyzing monosaccharides after hydrolysis.