RESUMEN
Proteins are increasingly used as therapeutics. Their characterization is challenging due to their size and inherent heterogeneity notably caused by post-translational modifications, among which glycosylation is probably the most prominent. The glycosylation profile of therapeutic proteins must therefore be thoroughly analyzed. Here, we illustrate how the use of a combination of various cutting-edge LC or LC/MS(/MS) methods, and operating at different levels of analysis allows the comprehensive characterization of both the N- and O-glycosylations of therapeutic proteins without the need for other approaches (capillary electrophoresis, MALDI-TOF). This workflow does not call for the use of highly specialized/custom hardware and software nor an extensive knowledge of glycan analysis. Most notably, we present the point of view of a contract research organization, with the constraints associated to the work in a regulated environment (GxP). Two salient points of this work are i) the use of mixed-mode chromatography as a fast and straightforward mean of profiling N-glycans sialylation as well as an orthogonal method to separate N-glycans co-eluting in the HILIC mode; and ii) the use of widepore HILIC/MS to analyze challenging N/O-glycosylation profiles at both the peptide and subunit levels. A particular attention was given to the sample preparations in terms of duration, specificity, versatility, and robustness, as well as the ease of data processing.
Asunto(s)
Cromatografía Líquida de Alta Presión , Glicoproteínas/metabolismo , Polisacáridos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adalimumab/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/metabolismo , Secuencia de Carbohidratos , Cetuximab/metabolismo , Electroforesis Capilar , Etanercept/metabolismo , Glicopéptidos/análisis , Glicopéptidos/aislamiento & purificación , Glicosilación , Interacciones Hidrofóbicas e Hidrofílicas , Polisacáridos/aislamiento & purificaciónRESUMEN
Glycosylation of the Fc moiety of a monoclonal antibody is a heterogeneous posttranslational process considered as a critical quality attribute of the purified drug substance due to its major impact on safety and efficacy (i.e., immunogenicity, CDC or ADCC effector functions, etc.). Glycosylation should thus be addressed for batch-to-batch comparability and for drug substance characterization, in terms of identity and/or purity testing. We present below a set of efficient, performing and complementary analytical tests that can be used alone or in combination, depending on the information needed and available laboratory instrumentation. The results obtained using these techniques for "global" glycosylation profile, N-glycans profiling, monosaccharides, and sialic acids determination are presented for the Trastuzumab (Herceptin)-humanized mAb produced in CHO.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Glicopéptidos/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Líquida de Alta Presión/normas , Glucosa/química , Glucosa/aislamiento & purificación , Glicopéptidos/aislamiento & purificación , Glicosilación , Humanos , Datos de Secuencia Molecular , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/química , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Procesamiento Proteico-Postraduccional , Estándares de Referencia , Ácidos Siálicos/química , Ácidos Siálicos/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masa por Ionización de Electrospray/normas , Coloración y Etiquetado , TrastuzumabRESUMEN
New antibiotics were recently developed, among which are the (fluoro)quinolones. This paper presents an analytical method which allows the determination of 11 (fluoro)quinolones in swine kidneys: norfloxacin, ofloxacin, cinoxacin, oxolinic acid, nalidixic acid, flumequine, enrofloxacin, enoxacin, ciprofloxacin, danofloxacin and marbofloxacin. The procedure involves a rapid and efficient pre-treatment by solid-phase extraction (recoveries 83-98%), followed by the sensitive and selective determination of all compounds in a single run using LC-ESI-MS-MS. Multiple reaction monitoring (MRM) was used for selective detection of each (fluoro)quinolone. Quinine was selected as internal standard. The accuracy of the method, expressed as recovery, was between 89 and 109%; the repeatability had a maximum RSD lower than 15%. The limits of detection (LOD) were much lower than the respective Maximum Residue Limits (MRL)/4.