Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

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The primary goal of this study was to develop a method to study the N-glycosylation of IgG from swine in order to detect epitopes containing N-glycolylneuraminic acid (Neu5Gc) and/or terminal galactose residues linked in α1-3 susceptible to cause xenograft-related problems. Samples of immunoglobulin were isolated from porcine serum using protein-A affinity chromatography. The eluate was then separated on electrophoretic gel, and bands corresponding to the N-glycosylated heavy chains were cut off the gel and subjected to tryptic digestion. Peptides and glycopeptides were separated by reversed phase liquid chromatography and fractions were collected for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOF-MS) analysis. Overall no α1-3 galactose was detected, as demonstrated by complete susceptibility of terminal galactose residues to β-galactosidase digestion. Neu5Gc was detected on singly sialylated structures. Two major N-glycopeptides were found, EEQFNSTYR and EAQFNSTYR as determined by tandem MS (MS/MS), as previously reported by Butler et al. (Immunogenetics, 61, 2009, 209-230), who found 11 subclasses for porcine IgG. Out of the 11, ten include the sequence corresponding to EEQFNSTYR, and only one codes for EAQFNSTYR. In this study, glycosylation patterns associated with both chains were slightly different, in that EEQFNSTYR had a higher content of galactose. The last step of this study consisted of peptide-mapping the 11 reported porcine IgG sequences. Although there was considerable overlap, at least one unique tryptic peptide was found per IgG sequence. The workflow presented in this manuscript constitutes the first study to use MALDI-TOF-MS in the investigation of porcine IgG structural features.

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For antibody analysis, MALDI-MS ion abundances give a better semi-quantitative estimate of sialylation levels for esterified than for unreacted glycopeptides. The method is simple to use and helps to differentiate the branching patterns of sialic acids in antibodies.

Glycoproteomics represent the field of study of the dynamic changes occurring among glycoconjugates within the cellular compartments. Changes in glycosylation have been linked to various diseases, including metastatic carcinomas in which the 9 carbon sialic acid moiety has been shown to play a prominent role. The common method used to study these aberrant changes most often includes a mass spectrometer at some stage in the workflow. However, serum samples contain many proteins which inhibit the analysis of these glycosylation changes, and ergo, enrichment steps are employed as a measure to help alleviate this ailment. Routinely, this is accomplished using lectins, either alone or in combination, to retrieve proteins with specific sugar linkages within the serum sample. This methodology, although known to be very specific, requires many washing steps, making it a cumbersome addition to a high throughput workflow. Presented here is an alternative protocol using custom-made amine functionalized magnetic nanoparticles (MNP) which are nearly 4× smaller than those used before for similar purposes. The developed protocol is based on both hydrophilic interaction and weak anion exchange principles, allowing it to target glycopeptides but, more specifically, those which contain sialylation. For quantification purposes, tandem mass tags from Thermo Scientific were utilized to compare the enrichment efficiencies between the magnetic nanoparticle method and a commercially available glycopeptide enrichment kit offered through EMD Millipore. The MNP method is fast (~10 min) and simple and can quantitatively and qualitatively enrich sialylated glycopeptides more than the commercially available kit.

The characterization of the N-glycan portion of antibodies has been the subject of several studies involving mass spectrometry. In this article, a workflow is presented that starts with the expression of a monoclonal antibody (EG2-hFc) in Chinese hamster ovary cells and continues with Protein A purification of the antibody. Then the protocol continues with gel electrophoresis. Bands containing the heavy chain are cut and isolated from the gel followed by tryptic digestion to obtain peptides and glycopeptides. The enrichment of glycopeptides by C18 chromatography is described followed by characterization using positive and negative modes MALDI-MS and MS/MS. An exoglycosidase, beta-galactosidase, is used to verify anomericity of linkages in the glycan portion of glycopeptides. In the last step, glycans are detached from glycopeptides using PNGase F labelled with phehylhydrazine and characterized by MALDI-MS/MS. This workflow is reported for the first time for this particular antibody and presents a valuable approach for the analysis of N-glycans on most antibodies and glycoproteins.Résumé : La caractérisation de la portion N-glycane des anticorps a fait l'objet de nombreuses études faisant appel à la spectrométrie de masse. Dans le présent article, nous présentons un protocole méthodologique qui débute par l'expression d'un anticorps monoclonal (EG2-hFc) dans des cellules ovariennes de hamster et se poursuit avec la purification de la protéine A de l'anticorps suivie de l'électrophorèse en gel. Les bandes contenant les chaînes de masse élevée sont découpées et isolées du gel, et une digestion tryptique est effectuée en vue d'obtenir des peptides et des glycopeptides. Nous décrivons l'enrichissement des glycopeptides par chromatographie sur colonne C18, puis la caractérisation au moyen de la MALDI-MS et SM/SM en modes positif et négatif. Nous avons utilisé la bêta-galactosidase, une exoglycosidase, afin de vérifier l'anoméricité des liaisons dans la portion glycane des glycopeptides. En dernière étape, nous avons détaché les glycanes des glycopeptides à l'aide de la PNGase F, les avons marqués à l'aide de la phénylhydrazine et les avons caractérisés par spectroscopie de masse MALDI-SM/SM. Ce protocole pour cet anticorps précis est publié ici pour la première fois et présente une approche intéressante permettant d'effectuer l'analyse des N-glycanes pour la plupart des anticorps et des glycoprotéines. [Traduit par la Rédaction]