title: Orthogonal liquid chromatography-mass spectrometry methods for the comprehensive characterization of therapeutic glycoproteins, from released glycans to intact protein level.
abstract: 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.
authors.affiliation: Quality Assistance sa, Technoparc de Thudinie 2, 6536, Donstiennes, Belgium.
authors.forename: Eric
authors.initials: E
authors.lastname: Largy
authors.affiliation: Quality Assistance sa, Technoparc de Thudinie 2, 6536, Donstiennes, Belgium.
authors.forename: Fabrice
authors.initials: F
authors.lastname: Cantais
authors.affiliation: Quality Assistance sa, Technoparc de Thudinie 2, 6536, Donstiennes, Belgium.
authors.forename: Géry
authors.initials: G
authors.lastname: Van Vyncht
authors.affiliation: Centre d'Immunologie Pierre Fabre (CIPF), 5 Av. Napoléon III, BP 60497, 74164, Saint-Julien-en-Genevois, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Beck
authors.affiliation: Quality Assistance sa, Technoparc de Thudinie 2, 6536, Donstiennes, Belgium. Electronic address: arnaud.delobel@quality-assistance.be.
authors.forename: Arnaud
authors.initials: A
authors.lastname: Delobel
mesh_terms: D000068879:Adalimumab / Q000378:metabolism; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002240:Carbohydrate Sequence; D000068818:Cetuximab / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary; D000068800:Etanercept / Q000378:metabolism; D006020:Glycopeptides / Q000032:analysis / Q000302:isolation & purification; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation; D057927:Hydrophobic and Hydrophilic Interactions; D011134:Polysaccharides / Q000032:analysis* / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000911:Antibodies, Monoclonal; D006020:Glycopeptides; D006023:Glycoproteins; D011134:Polysaccharides; D000068879:Adalimumab; D000068800:Etanercept; D000068818:Cetuximab
keywords: High-resolution mass spectrometry; Hydrophilic interaction chromatography; Mixed-mode chromatography; Porous graphitic carbon chromatography; Protein glycosylation; Therapeutic proteins