Electrospray ionization quadrupole ion-mobility time-of-flight mass spectrometry as a tool to distinguish the lot-to-lot heterogeneity in N-glycosylation profile of the therapeutic monoclonal antibody trastuzumab

Monoclonal antibodies are typically glycosylated at asparagine residues in the Fc domain, and glycosylation heterogeneity at the Fc sites is well known. This paper presents a method for rapid analysis of glycosylation profile of the therapeutic monoclonal antibody trastuzumab from different production batches using electrospray quadrupole ion-mobility time-of-flight mass spectrometry (ESI-Q-IM-TOF). The global glycosylation profile for each production batch was obtained by a fast LC-MS analysis, and comparisons of the glycoprofiles of trastuzumab from different lots were made based on the deconvoluted intact mass spectra. Furthermore, the heterogeneity at each glycosylation site was characterized at the reduced antibody level and at the isolated glycopeptide level. The glycosylation site and glycan structures were confirmed by performing a time-aligned-parallel fragmentation approach using the unique dual-collision cell design of the instrument and the incorporated ion-mobility separation function. Four different production batches of trastuzumab were analyzed and compared in terms of global glycosylation profiles as well as the heterogeneity at each glycosylation site. The results show that each batch of trastuzumab shares the same types of glycoforms but relative abundance of each glycoforms is varied.     

Evaluation of glycosylation for quality assurance of antibody pharmaceuticals by capillary electrophoresis

The development of monoclonal antibody (mAb) pharmaceuticals has rapidly generated in this decade, and there are currently 23 FDA-approved mAb pharmaceuticals. Carbohydrate chains in mAb pharmaceuticals play important roles for the expression of their biological activities such as antibody-dependent cellular cytotoxicity, whereas the oligosaccharide profile is easily changed depending on the manufacturing conditions. Therefore, the evaluation of carbohydrate chains in detail is quite important for quality control in the development of mAb from early phases, but oligosaccharides in mAb have high heterogeneity and full characterization of oligosaccharide moiety in mAb has been a challenging target. Capillary electrophoresis (CE) is a powerful tool for the evaluation of glycan occupancy and oligosaccharide profile. This review focuses mainly on the application of CE to the analytical studies on glycosylation in mAb pharmaceuticals developed in our laboratory. The related techniques including the full-image microchip isoelectric focusing and the newly developed mass spectrometry technologies are also shown. The combination of the techniques described in this review will be a good guide for the evaluation of glycosylation for quality assurance of antibody pharmaceuticals by CE from the early stage of the development to the marketing stage.     

Fast Epitope Mapping for the Anti‐MUC1 Monoclonal Antibody by Combining a One‐Bead‐One‐Glycopeptide Library and a Microarray Platform

Anti‐MUC1 monoclonal antibodies (mAbs) are powerful tools that can be used to recognize cancer‐related MUC1 molecules, the O‐glycosylation status of which is believed to affect binding affinity. We demonstrate the feasibility of using a rapid screening methodology to elucidate those effects. The approach involves i) “one‐bead‐one‐compound”‐based preparation of bilayer resins carrying glycopeptides on the shell and mass‐tag tripeptides coding O‐glycan patterns in the core, ii) on‐resin screening with an anti‐MUC1 mAb, iii) separating positive resins by utilizing secondary antibody conjugation with magnetic beads, and (iv) decoding the mass‐tag that is detached from the positive resins pool by using mass spectrometric analysis. We tested a small library consisting of 27 MUC1 glycopeptides with different O‐glycosylations against anti‐MUC1 mAb clone VU‐3C6. Qualitative mass‐tag analysis showed that increasing the number of glycans leads to an increase in the binding affinity. Six glycopeptides selected from the library were validated by using a microarray‐based assay. Our screening provides valuable information on O‐glycosylations of epitopes leading to high affinity with mAb.     

Using optimized collision energies and high resolution,high accuracy fragment ion selection to improve glycopeptide detection by precursor ion scanning

Glycosylation is the most widespread protein modification and is known to modulate signal transduction and several biologically important interactions. In order to understand and evaluate the biological role of glycosylation it is important to identify the glycosylated protein and localize the site glycosylation under particular biological conditions. To identify glycosylated peptides from simple mixtures, i.e., in-gel digests from single SDS PAGE bands we performed high resolution, high accuracy precursor ion scanning using a quadrupole TOF instrument equipped with the Q(2) pulsing function. The high resolving power of the quadrupole TOF instrument results in the selective detection of glycan specific fragment ions minimizing the interference of peptide derived fragment ions with the same nominal mass. Precursor ion scanning has been previously described for these glycan derived ions. However the use of this method has been limited by the low specificity of the method. The analysis using precursor ion scanning can be applied to any peptide mixture from a protein digest without having previous knowledge of the glycosylation of the protein. In addition to the low femtomole (nanomolar) detection limits, this method has the advantage that no prior derivatization or enzymatic treatment of the peptide mixtures is required.     

Global and site-specific detection of human integrin alpha 5 beta 1 glycosylation using tandem mass spectrometry and the StrOligo algorithm

Glycans are oligosaccharides associated with proteins, and are known to confer specific functions and conformations on glycoproteins. As protein tridimensional structures are related to function, the study of glycans and their impact on protein folding can provide important information to the field of proteomics. The subdiscipline of glycomics (or glycoproteomics) is rapidly growing in importance as glycans in proteins have shown to be involved in protein-protein or protein-(drug, virus, antibody) interactions. Glycomics studies most often aim at identifying glycosylation sites, and thus are performed on deglycosylated proteins resulting in loss of site-specific details concerning the glycosylation. In order to obtain such details by mass spectrometry (MS), either whole glycoproteins must be digested and analyzed as mixtures of peptides and glycopeptides, or glycans must be isolated from glycopeptide fractions and analyzed as pools. This article describes parallel experiments involving both approaches, designed to take advantage of the StrOligo algorithm functionalities with the aim of characterizing glycosylation microheterogeneity on a specific site. A hybrid quadrupole-quadrupole-time-of-flight (QqTOF) instrument equipped with a matrix-assisted laser desorption/ionization (MALDI) source was used. Glycosylation of alpha 5 beta 1 subunits of human integrin was studied to test the methodology. The sample was divided in two aliquots, and glycans from the first aliquot were released enzymatically, labelled with 2-aminobenzamide, and identified using tandem mass spectrometry (MS/MS) and the StrOligo program. The other aliquot was digested with trypsin and the resulting peptides separated by reversed-phase high-performance liquid chromatography (HPLC). A specific collected fraction was then analyzed by MS before and after glycan release. These spectra allowed, by comparison, detection of a glycopeptide (several glycoforms) and elucidation of peptide sequence. Compositions of glycans present were proposed, and identification of possible glycan structures was conducted using MS/MS and StrOligo.     

Mass spectrometry characterization for chemoenzymatic glycoprotein synthesis

The current project describes the chemoenzymatic modification of bovine ribonuclease B (RNase B) to contain a single glycosylation site with a known glycan. A reactive disaccharide oxazoline derivative was synthesized and stereospecifically added to deglycosylated RNase B through endo-β-N-acetylglucosaminidase M catalyzed chemoenzymatic transglycosylation. Oxazoline formation conditions were optimized using mass spectrometry, and the product verified based on its collision-induced dissociation (CID) mass spectrum. Enzymatic removal of native glycans as well as formation of the desired homogeneous product was also monitored using mass spectrometry. LC-MS(n) using four sequential rounds of CID was used to verify that the original glycosylation site had been reorganized to contain the new glycan. The techniques described herein are not limited to this analyte or glycan and should be amenable to the synthesis of numerous homogeneous glycoconjugates with judicious choice of enzyme/substrate combinations. The combined use of chemoenzymatic synthesis and mass spectrometry-based characterization shows promise for the development of homogeneous glycoprotein reference materials. A well-defined glycoprotein standard containing a single glycan of known composition, linkage and stereochemistry would be of great value for the comparison and evaluation of glycoprotein analysis techniques.     

LC-MS/MS Peptide Mapping with Automated Data Processing for Routine Profiling of N-Glycans in Immunoglobulins

Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.

Glycosylation analysis of interleukin-23 receptor: elucidation of glycosylation sites and characterization of attached glycan structures

Interleukin-23 (IL-23) is a heterodimeric cytokine, a central factor in chronic/autoimmune inflammation. It signals through a heterodimeric receptor consisting of IL-23r, which is heavily glycosylated. The structural characterization of IL-23r has not been reported. In this work, glycosylation profiles of soluble recombinant human IL-23r (rhIL-23r) were established using mass spectrometry (MS), which included defining glycosylation sites, degree of glycosylation occupancy of each site and structure of attached oligosaccharides. Specifically, precursor ion scan of oxonium ion protonated N-acetylglucosamine (GlcNAc(+)) (m/z 204) was performed using a triple quadrupole MS instrument to locate the retention time of glycopeptides. Both the glycopeptides and their corresponding deglycosylated forms in each collected HPLC fraction were studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (LTQ-Orbitrap) for glycosylation site profiling. The attached glycan structures were elucidated by collision-induced dissociation (CID) fragmentation of target glycopeptides in combination with accurate mass measurement. Eight glycosylation sites were identified on IL-23r (Asn24, Asn209, Asn239, Asn157, Asn118, Asn250, Asn58 and Asn6). Most of the glycosylation sites were > 95% occupied except Asn250 and Asn6. Those two sites were 88% and 45% occupied by estimation from trypsin digestion and were 55% and 42% occupied from LysC digestion. Multiple glycoforms were observed in IL-23r. Most of them were bi-, tri- or tetra-antennary complex type structures with fucose and sialic acid. High mannose and hybrid type glycans were only observed on Asn157. The structural characterization on IL-23r glycosylation provides useful information for better understanding of the biological function of IL-23r.     

利用β-N-乙酰氨基葡萄糖苷酶快速酶切分析中国仓鼠卵巢细胞表达的西妥昔单抗抗原结合区的聚糖比例

西妥昔单抗具有较复杂的糖基化修饰,在抗原结合片段(Fab)和可结晶片段(Fc)的重链上都含有2个N-糖基化位点,其中Fab段的糖基化最为复杂,要研究清楚该位点的糖基化修饰,开发专一性切糖技术和稳定的聚糖比例分析方法是当前迫切需要解决的难题。以中国仓鼠卵巢(CHO)细胞表达的西妥昔单抗为研究对象,使用β-N-乙酰氨基葡萄糖苷酶(Endo F2)开发了一种快速Fab段聚糖释放的方法,利用超高效液相色谱-高分辨质谱(UPLC-HRMS)进行了定性和聚糖比例分析。第一步对抗体原液进行非变性酶切,抗体原液经超纯水稀释后,加入糖苷酶Endo F2进行酶切,通过质谱对质量数的解析,结果表明Endo F2酶切时间5 min, Fab段的聚糖就能完全切除,而Fc段的聚糖不受影响,实现了快速酶切,而且切糖具有很好的专一性。第二步对Fab段聚糖进行比例分析,将释放的聚糖经对氨基苯甲酰胺(2-AB)荧光标记后使用超高效液相色谱联用荧光检测器(FLR)进行检测,在亲水作用色谱(HILIC)柱上得到良好的分离并可以进行稳定地聚糖比例分析。3次独立试验结果表明,酶切后的质谱图基本一致,且聚糖的比例结果也基本一致,表明Endo F2酶切方法和聚糖比例分析方法都具有较好的稳定性和可靠性。此外,通过测定来自两个不同工艺生产的样品,数据显示两者的糖谱上具有非常明显的差异,表明利用开发的方法可以实现对抗体生产工艺进行监测研究,对抗体生产工艺的评估具有非常重要的意义。     

HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals

In contrast with conventional drugs, biopharmaceuticals are highly complex molecules with remarkable heterogeneity. Protein glycosylation is an inherent source of this heterogeneity and also affects the safety, efficacy, and serum half-life of therapeutic glycoproteins. Therefore analysis of the glycan pattern is an important issue for characterization and quality control in the biopharmaceutical industry. In this publication we describe a complete workflow for the analysis of protein N-glycans. The sample-preparation procedure, consisting of the release of the N-glycans by PNGase-F, followed by fluorescence labeling with 2-aminobenzamide and removal of excess label, was optimized to avoid alteration of the glycan sample. Subsequently, labeled glycans were analyzed by hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection. The developed method was validated for analysis of antibody N-glycans. To demonstrate the accuracy of the method an antibody sample was additionally analyzed by an orthogonal method. The antibody was digested with lysyl endopeptidase and the (glyco-)peptides were analyzed by RP-HPLC–MS. The consistency of the results between these two methods demonstrates the reliability of the glycan analysis method introduced herein.     

基于固相萃取的微量糖蛋白糖基化修饰表征技术的建立

结合自制亲水固相萃取富集柱和生物质谱鉴定技术,实现了糖基化蛋白质核糖核酸酶B的糖含量测定、糖基化位点确认、聚糖富集及结构表征,以及不同糖型相对丰度分析。结果表明:其糖含量8.47%,糖基化位点为34位的Asn,糖链主要为5种高甘露糖型结构(Man5-9GlcNAc2)。所建立的HILIC富集技术,有利于针对微量生物样本,如生物工程药物糖蛋白及重要功能糖蛋白,开展位点特异性糖链结构解析,为糖蛋白质的药效或功能研究提供线索。     

Improvement of mass spectrometry analysis of glycoproteins by MALDI-MS using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid

Protein glycosylation analysis is important for elucidating protein function and molecular mechanisms in various biological processes. We previously developed a glycan analysis method using a 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix (3-AQ/CHCA LM) and applied it to the quantitative glycan profiling of glycoproteins. However, information concerning glycosylation sites is lost; glycopeptide analysis is therefore required to identify the glycosylation sites in glycoproteins. Human epidermal growth factor receptor 2 (HER2) is a glycoprotein that plays a role in the regulation of cell proliferation, differentiation, and migration. Several reports have described the structure of HER2, but the structures of N-glycans attached to this protein remain to be fully elucidated. In this study, 3-AQ/CHCA LM was applied to tryptic digests of HER2 to reveal its N-glycosylation state and to evaluate the utility of this LM in characterizing glycopeptides. Peptide sequence coverage was considerably improved compared to analysis of HER2 using α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. Most of the peaks observed using only this LM were localized at the inner or outer regions of sample spots. Furthermore, five of the peptide peaks that were enriched within the inner region were confirmed to be glycosylated by MS/MS analysis. Three glycosylation sites were identified and their glycan structures were elucidated. The reduction in sample complexity by on-target separation allowed for higher sequence coverage, resulting in effective detection and characterization of glycopeptides. In conclusion, these results demonstrate that MS-based glycoprotein analysis using 3-AQ/CHCA is an effective method to identify glycosylation sites in proteins and to elucidate the glycan structures of glycoproteins in complex samples.     

Structure-Guided Design of a Group B Streptococcus Type III Synthetic Glycan–Conjugate Vaccine

Identification of glycan functional epitopes is of paramount importance for rational design of glycoconjugate vaccines. We recently mapped the structural epitope of the capsular polysaccharide from type III Group B Streptococcus (GBSIII), a major cause of invasive disease in newborns, by using a dimer fragment (composed of two pentasaccharide repeating units) obtained by depolymerization complexed with a protective mAb. Although reported data had suggested a highly complex epitope contained in a helical structure composed of more than four repeating units, we showed that such dimer conjugated to a carrier protein with a proper glycosylation degree elicited functional antibodies comparably to the full-length conjugated polysaccharide. Here, starting from the X-ray crystallographic structure of the polysaccharide fragment–mAb complex, we synthesized a hexasaccharide comprising exclusively the relevant positions involved in binding. Combining competitive surface plasmon resonance and saturation transfer difference NMR spectroscopy as well as in-silico modeling, we demonstrated that this synthetic glycan was recognized by the mAb similarly to the dimer. The hexasaccharide conjugated to CRM₁₉₇, a mutant of diphtheria toxin, elicited a robust functional immune response that was not inferior to the polysaccharide conjugate, indicating that it may suffice as a vaccine antigen. This is the first evidence of an X-ray crystallography-guided design of a synthetic carbohydrate-based conjugate vaccine.     

Site-specific characterisation of densely O-glycosylated mucin-type peptides using electron transfer dissociation ESI-MS/MS

Site-specific characterisation of mucin-type O-linked glycosylation is an analytical challenge due to glycan heterogeneity, lack of glycosylation site consensus sequence and high density of occupied glycosylation sites. Here, we report the use of electron transfer dissociation (ETD) for the site-specific characterisation of densely glycosylated mucin-type O-linked glycopeptides using ESI-IT-MS/MS. Synthetic glycopeptides from the human mucin-1 (MUC-1) tandem repeat region containing a range of O-linked, tumour-associated carbohydrate antigens, namely Tn, T and sialyl T, with different glycosylation site occupancies and an increasing number of tandem repeats were studied. In addition, a glycopeptide from the anti-freeze glycoprotein of Antarctic and Arctic notothenoids, bearing four O-linked, per-acetylated T antigens was characterised. ETD MS/MS of infused or capillary LC-separated glycopeptides provided broad peptide sequence coverage (c/z·-type fragment ions) with intact glycans still attached to the Ser/Thr residues. Thus, the glycosylation sites were unambiguously determined, while simultaneously obtaining information about the attached glycan mass and peptide identity. Highly sialylated O-glycopeptides showed less efficient peptide fragmentation, but some sequence and glycosylation site information was still obtained. This study demonstrates the capabilities of ETD MS/MS for site-specific characterisation of mucin-type glycopeptides containing high-density O-linked glycan clusters, using accessible and relative low-resolution/low-mass accuracy IT MS instrumentation.     

Comprehensive assessment of N-glycans derived from a murine monoclonal antibody: a case for multimethodological approach

Highly efficient separation techniques, laser-induced fluorescence (LIF) detection, and different mass-spectrometric (MS) measurements were combined in a multimethodological scheme to perform a comprehensive structural characterization of N-linked oligosaccharides in a murine monoclonal antibody (immunoglobulin G (IgG(kappa))). Monosaccharide compositional analysis was carried out through a capillary electrophoresis (CE)-LIF method, in which the chemically and enzymatically released sugars were fluorescently labeled. This analysis provides a preliminary assessment of certain structures, being followed by CE-LIF and matrix-assisted laser desorption/ionization (MALDI)-MS profiling of the intact glycan structures. Linkages and monosaccharide residues were confirmed by MALDI-MS in conjunction with exoglycosidase digestion. MALDI-MS and CE data were effectively combined to reveal the overall structural diversity of both acidic and neutral glycans. Finally, the sites of glycosylation and site occupancies were deduced through the measurements performed with microcolumn liquid chromatography coupled via electrospray to a quadrupole/time-of-flight instrument.     

Determination of glycopeptide structures by multistage mass spectrometry with low-energy collision-induced dissociation: comparison of electrospray ionization quadrupole ion trap and matrix-assisted laser desorption/ionization quadrupole ion trap reflectron time-of-flight approaches

Multistage mass spectrometry, as implemented using low-energy collision-induced dissociation (CID) analysis in three-dimensional (3D) quadrupole ion traps (QITs), has become a powerful tool for the investigation of protein glycosylation. In addition to the well-known combination of QITs with electrospray ionization (ESI), also a matrix-assisted laser desorption/ionization--quadrupole ion trap--reflectron time-of-flight (MALDI-QIT-rTOF) mass spectrometer has recently become available. This study systematically investigates the differences between these types of instrument, as applied to characterization of glycopeptides from human antithrombin. The glycopeptides were obtained by tryptic digestion followed by lectin-affinity purification. Some significant differences between the ESI-QIT and MALDI-QIT-rTOF approaches appeared, most of them are causally related to the desorption/ionization process. The combination of a vacuum MALDI source with an ion-trap analyzer accentuates some characteristic differences between MALDI and ESI due the longer time frame needed for the trapping process. In contrast to ESI, MALDI generated ions that exhibited considerable metastable fragmentation during trapping. The long time span of the QIT process (ms range) compared with that for conventional rTOF experiments (micros range) significantly magnified the extent of this metastable fragmentation. With the investigated glycopeptides, a complete depletion of the terminal sialic acids of the glycopeptides as well as a variety of other fragment ions was already found in the MS1 spectra from the MALDI-QIT-rTOF instrument. The positive ion low-energy CID spectra (MS2) of the selected glycopeptides obtained using the two different QIT equipped instruments were found to be quite similar. In both approaches, fragmentation of the glycan and peptide structures occurred sequentially, allowing unambiguous sequence determination. In the case of ESI-QIT-MS, fragmentation of the glycan structure occurred at the MS2 stage and fragmentation of the peptide structure was obtained only at the MS3 stage, which indicates the necessity of multistage CID experiments for complete structure elucidation. The MALDI-QIT-rTOF instrument yielded both kinds of fragments at the MS2 stage but without mutual interference.     

Characterization of N- and O-glycopeptides of recombinant human erythropoietins as potential biomarkers for doping analysis by means of microscale sample purification combined with MALDI-TOF and quadrupole IT/RTOF mass spectrometry

The structural characterization of the O- and N-glycan structures of three different commercially available recombinant human erythropoietins (rhEPOs) is represented by means of a microscale sample purification using ZipTip technology and MALDI-TOF and MALDI low-energy CID MS. Glycopeptides were released from rhEPO samples by a differential endoproteolytic digestion to obtain site-specific glycosylation patterns. Mass accuracies in the range of +/- 0.04% obtained by the high-resolution TOF instrument allowed an unambiguous assignment of N-glycan structures via glycan database software. Furthermore, the O-glycan structures were directly analyzed on the glycopeptide level by MS/MS experiments. Principally, site-specific glycosylation was found to be very similar for the three different rhEPOs (EPO-alpha, EPO-beta, and novel erythropoiesis stimulating protein (NESP)) but exhibiting quantitative differences in distinct O- and N-glycan moieties. Significant differences were found in the degree of sialylation and acetylation. Especially, a considerable degree of variation of the O-acetylation of sialic acid residues could be realized on the glycan structures of O- and N-glycopeptides, whereas EPO-alpha and EPO-beta could be clearly differentiated from NESP solely on the O-glycopeptide level.     

Glycosylation Profiling of Therapeutic Antibodies in Serum Samples Using a Microfluidic CD Platform and MALDI-MS

The serum clearance rate of therapeutic antibodies is important as it affects the clinical efficacy, required dose, and dose frequency. The glycosylation of antibodies has in some studies been shown to have an impact on the elimination rates in vivo. Monitoring changes to the glycan profiles in pharmacokinetics studies can reveal whether the clearance rates of the therapeutic antibodies depend on the different glycoforms, thereby providing useful information for improvement of the drugs. In this paper, a novel method for glycosylation analysis of therapeutic antibodies in serum samples is presented. A microfluidic compact-disc (CD) platform in combination with MALDI-MS was used to monitor changes to the glycosylation profiles of samples incubated in vitro. Antibodies were selectively purified from serum using immunoaffinity capture on immobilized target antigens. The glycans were enzymatically released, purified, and finally analyzed by MALDI-TOF-MS. To simulate changes to glycan profiles after administration in vivo, a therapeutic antibody was incubated in serum with the enzyme α1-2,3 mannosidase to artificially reduce the amount of the high mannose glycoforms. Glycan profiles were monitored at specific intervals during the incubation. The relative abundance of the high mannose 5 glycoform was clearly found to decrease and, simultaneously, that of high mannose 4 increased over the incubation period. The method can be performed in a rapid, parallel, and automated fashion for glycosylation profiling consuming low amounts of samples and reagents. This can contribute to less labor work and reduced cost of the studies of therapeutic antibodies glycosylation in vitro and in vivo.

Analysis of native and APTS-labeled N-glycans by capillary electrophoresis/time-of-flight mass spectrometry

The glycosylation of proteins is of particular interest in biopharmaceutical applications. The detailed characterization of glycosylation based on the released carbohydrates is mandatory since the protein stability, folding, and efficacy are strongly dependent on the structural diversity inherent in the glycan moieties of a glycoprotein. For glycan pattern analysis, capillary electrophoresis with laser-induced fluorescence using 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans is used frequently. In this paper, a robust capillary electrophoresis–mass spectroscopy method both for the analysis of APTS-labeled glycans and unlabeled charged glycans is presented. The background electrolyte consists of 0.7 M ammonia and 0.1 M ε-aminocaproic acid in water/methanol 30:70 (v/v). High separation efficiency including separation of structural isomers was obtained. The method was validated in terms of reproducibility and linearity. Submicromolar sensitivity is achieved with linearity up to 24 μM. The ability to analyze APTS-labeled, as well as unlabeled, charged glycans enables the determination of labeling and ionization efficiency: APTS-labeled glycans show a factor of three better ionization efficiency compared to non-labeled native glycans. The presented method is applied to the analysis of pharmaceutical products. Furthermore, the system can be applied to the analysis of 2-ANSA-labeled glycans, though separation efficiency is limited.

Characterization of the N-linked glycosylation site of recombinant pectate lyase

Recombinant pectate lyase from Aspergillus niger was overexpressed in Aspergillus nidulans. The two recombinant proteins produced differed in molecular mass by 1200 Da, which suggested that the larger molecular weight protein was glycosylated. The deduced amino acid sequence was searched for potential N-linked glycosylation sites, and one potential site was identified at residue 64. The proteins were analyzed for their ability to bind various lectins as an assay for the presence of carbohydrates. The proteins were then digested with trypsin to facilitate the isolation of the potential glycosylation site. The resulting digestion products were subsequently analyzed by liquid chromatography/mass spectrometry using in-source collision induced dissociation to detect glycopeptides. Once the glycopeptide had been identified, treatment with an endoglycosidase both verified the location of glycosylation and identified the mass of the glycan. The Complex Carbohydrate Structural Database was searched for possible N-linked structures with the same mass, and the suggested primary sequence was confirmed by an exoglycosidase digestion. The data demonstrated that the larger recombinant protein contained a high mannose N-linked structure (Man(5)GlcNAc(2)) attached to N-64, while this site was not occupied in the smaller protein.