Karli R. Reiding

High-throughput profiling of protein N-glycosylation by MALDI-TOF-MS employing linkage-specific sialic acid esterification.

Protein glycosylation is an important post-translational modification associated, among others, with diseases and the efficacy of biopharmaceuticals. Matrix-assisted laser desorption/ionization (MALDI) time-of-fight (TOF) mass spectrometry (MS) can be performed to study glycosylation in a high-throughput manner, but is hampered by the instability and ionization bias experienced by sialylated glycan species. Stabilization and neutralization of these sialic acids can be achieved by permethylation or by specific carboxyl group derivatization with the possibility of discrimination between α2,3- and α2,6-linked sialic acids. However, these methods typically require relatively pure glycan samples, show sensitivity to side reactions, and need harsh conditions or long reaction times. We established a rapid, robust and linkage-specific high-throughput method for sialic acid stabilization and MALDI-TOF-MS analysis, to allow direct modification of impure glycan-containing mixtures such as PNGase F-released human plasma N-glycome. Using a combination of carboxylic acid activators in ethanol achieved near-complete ethyl esterification of α2,6-linked sialic acids and lactonization of α2,3-linked variants, in short time using mild conditions. Glycans were recovered by hydrophilic interaction liquid chromatography solid phase extraction and analyzed by MALDI-TOF-MS in reflectron positive mode with 2,5-dihydroxybenzoic acid as the matrix substance. Analysis of the human plasma N-glycome allowed high-throughput detection and relative quantitation of more than 100 distinct N-glycan compositions with varying sialic acid linkages.

Immunoglobulin G (IgG) Fab Glycosylation Analysis Using a New Mass Spectrometric High-throughput Profiling Method Reveals Pregnancy-associated Changes

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding.

Human plasma protein N-glycosylation.

Glycosylation is the most abundant and complex protein modification, and can have a profound structural and functional effect on the conjugate. The oligosaccharide fraction is recognized to be involved in multiple biological processes, and to affect proteins physical properties, and has consequentially been labeled a critical quality attribute of biopharmaceuticals. Additionally, due to recent advances in analytical methods and analysis software, glycosylation is targeted in the search for disease biomarkers for early diagnosis and patient stratification. Biofluids such as saliva, serum or plasma are of great use in this regard, as they are easily accessible and can provide relevant glycosylation information. Thus, as the assessment of protein glycosylation is becoming a major element in clinical and biopharmaceutical research, this review aims to convey the current state of knowledge on the N-glycosylation of the major plasma glycoproteins alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-1B-glycoprotein, alpha-2-HS-glycoprotein, alpha-2-macroglobulin, antithrombin-III, apolipoprotein B-100, apolipoprotein D, apolipoprotein F, beta-2-glycoprotein 1, ceruloplasmin, fibrinogen, immunoglobulin (Ig) A, IgG, IgM, haptoglobin, hemopexin, histidine-rich glycoprotein, kininogen-1, serotransferrin, vitronectin, and zinc-alpha-2-glycoprotein. In addition, the less abundant immunoglobulins D and E are included because of their major relevance in immunology and biopharmaceutical research. Where available, the glycosylation is described in a site-specific manner. In the discussion, we put the glycosylation of individual proteins into perspective and speculate how the individual proteins may contribute to a total plasma N-glycosylation profile determined at the released glycan level.

High-Throughput Analysis and Automation for Glycomics Studies

This review covers advances in analytical technologies for high-throughput (HTP) glycomics. Our focus is on structural studies of glycoprotein glycosylation to support biopharmaceutical realization and the discovery of glycan biomarkers for human disease. For biopharmaceuticals, there is increasing use of glycomics in Quality by Design studies to help optimize glycan profiles of drugs with a view to improving their clinical performance. Glycomics is also used in comparability studies to ensure consistency of glycosylation both throughout product development and between biosimilars and innovator drugs. In clinical studies there is as well an expanding interest in the use of glycomics—for example in Genome Wide Association Studies—to follow changes in glycosylation patterns of biological tissues and fluids with the progress of certain diseases. These include cancers, neurodegenerative disorders and inflammatory conditions. Despite rising activity in this field, there are significant challenges in performing large scale glycomics studies. The requirement is accurate identification and quantitation of individual glycan structures. However, glycoconjugate samples are often very complex and heterogeneous and contain many diverse branched glycan structures. In this article we cover HTP sample preparation and derivatization methods, sample purification, robotization, optimized glycan profiling by UHPLC, MS and multiplexed CE, as well as hyphenated techniques and automated data analysis tools. Throughout, we summarize the advantages and challenges with each of these technologies. The issues considered include reliability of the methods for glycan identification and quantitation, sample throughput, labor intensity, and affordability for large sample numbers.

Linkage-specific sialic acid derivatization for MALDI-TOF-MS profiling of IgG glycopeptides.

Glycosylation is a common co- and post-translational protein modification, having a large influence on protein properties like conformation and solubility. Furthermore, glycosylation is an important determinant of efficacy and clearance of biopharmaceuticals such as immunoglobulin G (IgG). Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) shows potential for the site-specific glycosylation analysis of IgG at the glycopeptide level. With this approach, however, important information about glycopeptide sialylation is not duly covered because of in-source and metastable decay of the sialylated species. Here, we present a highly repeatable sialic acid derivatization method to allow subclass-specific MALDI-TOF-MS analysis of tryptic IgG glycopeptides. The method, employing dimethylamidation with the carboxylic acid activator 1-ethyl-3-(3-dimethylamino)propyl)carbodiimide (EDC) and the catalyst 1-hydroxybenzotriazole (HOBt), results in different masses for the functionally divergent α2,3- and α2,6-linked sialic acids. Respective lactonization and dimethylamidation leads to their direct discrimination in MS and importantly, both glycan and peptide moieties reacted in a controlled manner. In addition, stabilization allowed the acquisition of fragmentation spectra informative with respect to glycosylation and peptide sequence. This was in contrast to fragmentation spectra of underivatized samples, which were dominated by sialic acid loss. The method allowed the facile discrimination and relative quantitation of IgG Fc sialylation in therapeutic IgG samples. The method has considerable potential for future site- and sialic acid linkage-specific glycosylation profiling of therapeutic antibodies, as well as for subclass-specific biomarker discovery in clinical IgG samples derived from plasma.

Automation of High-Throughput Mass Spectrometry-Based Plasma N-Glycome Analysis with Linkage-Specific Sialic Acid Esterification

Glycosylation is a post-translational modification of key importance with heterogeneous structural characteristics. Previously, we have developed a robust, high-throughput MALDI-TOF-MS method for the comprehensive profiling of human plasma N-glycans. In this approach, sialic acid residues are derivatized with linkage-specificity, namely the ethylation of α2,6-linked sialic acid residues with parallel lactone formation of α2,3-linked sialic acids. In the current study, this procedure was used as a starting point for the automation of all steps on a liquid-handling robot system. This resulted in a time-efficient and fully standardized procedure with throughput times of 2.5 h for a first set of 96 samples and approximately 1 h extra for each additional sample plate. The mass analysis of the thus-obtained glycans was highly reproducible in terms of relative quantification, with improved interday repeatability as compared to that of manual processing.

Human plasma N-glycosylation as analyzed by MALDI-FTICR-MS associates with markers of inflammation and metabolic health

Glycosylation is an abundant co- and post-translational protein modification of importance to protein processing and activity. Although not template-defined, glycosylation does reflect the biological state of an organism and is a high-potential biomarker for disease and patient stratification. However, to interpret a complex but informative sample like the total plasma N-glycome, it is important to establish its baseline association with plasma protein levels and systemic processes. Thus far, large-scale studies (n >200) of the total plasma N-glycome have been performed with methods of chromatographic and electrophoretic separation, which, although being informative, are limited in resolving the structural complexity of plasma N-glycans. MS has the opportunity to contribute additional information on, among others, antennarity, sialylation, and the identity of high-mannose type species. Here, we have used matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR)-MS to study the total plasma N-glycome of 2144 healthy middle-aged individuals from the Leiden Longevity Study, to allow association analysis with markers of metabolic health and inflammation. To achieve this, N-glycans were enzymatically released from their protein backbones, labeled at the reducing end with 2-aminobenzoic acid, and following purification analyzed by negative ion mode intermediate pressure MALDI-FTICR-MS. In doing so, we achieved the relative quantification of 61 glycan compositions, ranging from Hex4HexNAc2 to Hex7HexNAc6dHex1Neu5Ac4, as well as that of 39 glycosylation traits derived thereof. Next to confirming known associations of glycosylation with age and sex by MALDI-FTICR-MS, we report novel associations with C-reactive protein (CRP), interleukin 6 (IL-6), body mass index (BMI), leptin, adiponectin, HDL cholesterol, triglycerides (TG), insulin, gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), and smoking. Overall, the bisection, galactosylation, and sialylation of diantennary species, the sialylation of tetraantennary species, and the size of high-mannose species proved to be important plasma characteristics associated with inflammation and metabolic health.

Pregnancy-associated serum N-glycome changes studied by high-throughput MALDI-TOF-MS

Pregnancy requires partial suppression of the immune system to ensure maternal-foetal tolerance. Protein glycosylation, and especially terminal sialic acid linkages, are of prime importance in regulating the pro- and anti-inflammatory immune responses. However, little is known about pregnancy-associated changes of the serum N-glycome and sialic acid linkages. Using a combination of recently developed methods, i.e. derivatisation that allows the distinction between α2,3- and α2,6-linked sialic acids by high-throughput MALDI-TOF-MS and software-assisted data processing, we analysed the serum N-glycome of a cohort of 29 healthy women at 6 time points during and after pregnancy. A total of 77 N-glycans were followed over time, confirming in part previous findings while also revealing novel associations (e.g. an increase of FA2BG1S1(6), FA2G1S1(6) and A2BG2S2(6) with delivery). From the individual glycans we calculated 42 derived traits. With these, an increase during pregnancy and decrease after delivery was observed for both α2,3- and α2,6-linked sialylation. Additionally, a difference in the recovery speed after delivery was observed for α2,3- and α2,6-linked sialylation of triantennary glycans. In conclusion, our new high-throughput workflow allowed the identification of novel plasma glycosylation changes with pregnancy.

Automated High-Throughput Permethylation for Glycosylation Analysis of Biologics Using MALDI-TOF-MS

Monitoring glycoprotein therapeutics for changes in glycosylation throughout the drugs life cycle is vital, as glycans significantly modulate the stability, biological activity, serum half-life, safety, and immunogenicity. Biopharma companies are increasingly adopting Quality by Design (QbD) frameworks for measuring, optimizing, and controlling drug glycosylation. Permethylation of glycans prior to analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a valuable tool for glycan characterization and for screening of large numbers of samples in QbD drug realization. However, the existing protocols for manual permethylation and liquid-liquid extraction (LLE) steps are labor intensive and are thus not practical for high-throughput (HT) studies. Here we present a glycan permethylation protocol, based on 96-well microplates, that has been developed into a kit suitable for HT work. The workflow is largely automated using a liquid handling robot and includes N-glycan release, enrichment of N-glycans, permethylation, and LLE. The kit has been validated according to industry analytical performance guidelines and applied to characterize biopharmaceutical samples, including IgG4 monoclonal antibodies (mAbs) and recombinant human erythropoietin (rhEPO). The HT permethylation enabled glycan characterization and relative quantitation with minimal side reactions: the MALDI-TOF-MS profiles obtained were in good agreement with hydrophilic liquid interaction chromatography (HILIC) and ultrahigh performance liquid chromatography (UHPLC) data. Automated permethylation and extraction of 96 glycan samples was achieved in less than 5 h and automated data acquisition on MALDI-TOF-MS took on average less than 1 min per sample. This automated and HT glycan preparation and permethylation showed to be convenient, fast, and reliable and can be applied for drug glycan profiling and clinical glycan biomarker studies.

Subclass-specific IgG glycosylation is associated with markers of inflammation and metabolic health

This study indicates that glycosylation of immunoglobulin G, the most abundant antibody in human blood, may convey useful information with regard to inflammation and metabolic health. IgG occurs in the form of different subclasses, of which the effector functions show significant variation. Our method provides subclass-specific IgG glycosylation profiling, while previous large-scale studies neglected to measure IgG2-specific glycosylation. We analysed the plasma Fc glycosylation profiles of IgG1, IgG2 and IgG4 in a cohort of 1826 individuals by liquid chromatography-mass spectrometry. For all subclasses, a low level of galactosylation and sialylation and a high degree of core fucosylation associated with poor metabolic health, i.e. increased inflammation as assessed by C-reactive protein, low serum high-density lipoprotein cholesterol and high triglycerides, which are all known to indicate increased risk of cardiovascular disease. IgG2 consistently showed weaker associations of its galactosylation and sialylation with the metabolic markers, compared to IgG1 and IgG4, while the direction of the associations were overall similar for the different IgG subclasses. These findings demonstrate the potential of IgG glycosylation as a biomarker for inflammation and metabolic health, and further research is required to determine the additive value of IgG glycosylation on top of biomarkers which are currently used.