GlycoWorks® N-Glycan Sample Prep

Protein Glycosylation is Fundamental for Protein Function and Cell Physiology

Glycosylation is an essential form of post-translational modification (PTM) of human and eukaryotic proteins, which consists of the covalent attachment of a glycan moiety (saccharides or sugar chains) to the protein surface. Glycans are characterized by their composition and structural diversity and are found in both cell-surface and secreted proteins (Figure 1). They contribute to the conformation and biological function of proteins as well as to their binding characteristics, stability, and solubility. Glycosylated proteins, also known as glycoproteins, are involved in a variety of biological processes including intracellular trafficking, cell adhesion, receptor activation, signal transduction, and endocytosis. By regulating cellular functions, glycoproteins contribute to cellular homeostasis and the normal functioning of the living organism.

There is a growing interest in the study of protein glycosylation dynamics as aberrant glycosylation patterns have been associated with various human pathologies, such as congenital disorders, neurodegenerative diseases, autoimmune diseases, infectious and inflammatory diseases, as well as cancers (1). Therefore, the identification and quantification of protein glycans have become increasingly important in basic research to understand their role in protein regulation and function, both under normal and pathological conditions and in biotechnology to ensure efficient production of recombinant glycoproteins for therapeutic applications.

Glycosylation is Routinely Monitored for the Development of Therapeutically Safe and Effective Biologics

Many recombinant biopharmaceuticals such as monoclonal antibodies (mAbs) are glycoproteins. Bioprocessing requires precise control and monitoring of multiple parameters including cell line stability, product yield, protein folding, and PTMs. Among these parameters, the host cell’s biosynthesis of glycans is routinely monitored as a critical quality attribute (CQA). Glycan composition and structure are crucial for the biological or clinical activity of the drug. Slight changes in manufacturing conditions can alter the glycosylation patterns of recombinant proteins and consequently the biological activity, safety, stability, efficacy, and immunogenicity of the end drug product (2). Therefore, the manufacturing of therapeutic glycoproteins requires careful monitoring and characterization of their glycosylation profile (e.g. content, structure, abundance, etc...) to achieve consistent glycan composition and meet desired quality, clinical safety, and effectiveness targets.

Figure 1: Major forms of glycosylation in human cells (1). Glycans are covalently attached to proteins (and lipids) to form glycoconjugates. Glycans are classified according to the linkage to the protein, glycan (or lipid) moieties. Glycoproteins consist of glycans and branched glycan chains linked to nitrogen or oxygen atoms of amino acid residues. Glycans bound to the oxygen atom of the hydroxyl groups of Serine (Ser, S) or Threonine (Thr, T) are termed O-glycans. Glycans linked to the nitrogen atom of the amino group of Asparagine (Asn, N) are known as N-glycans. Fuc, Fucose; Gal, Galactose; GalNAc, N-acetylgalactosamine; Glc, Glucose; GlcA, Glucuronic acid; GlcN, Glucosamine; GlcNAc, N-acetylglucosamine; IdoA, iduronic acid; Man, Mannose; Xyl, Xylose; EGF, Epidermal growth factor; TSR, Thrombospondin type I repeats; GPI, Glycosylphosphatidylinositol; GAG, Glycosaminoglycan.

Analysis of Protein N-Glycans and Challenges

N-linked glycosylation is a very common protein modification in eukaryotic cells. It refers to the attachment of glycans to the amide group in the side chain of asparagine (Asn, N) residues of newly synthesized proteins. N-linked glycans or N-glycans are found in most living organisms and have a crucial role in regulating a spectrum of functions related to glycoprotein folding, protein solubility, protein protection against proteolytic degradation, and protein intracellular trafficking and secretion. They also serve as recognition ligands to modulate immune responses and mediate interactions with pathogens (3).

N-glycosylation of biopharmaceuticals must be maintained during production as the N-glycan profile of recombinant proteins is a measure of efficacy, quality, bioactivity, and optimal manufacturing conditions. There are several analytical approaches for the analysis and characterization of protein N-glycans during process development. One commonly used approach involves the enzymatic release of N-glycan chains from the protein of interest and their derivatization with a fluorescent label. Subsequently, labeled N-glycans are analyzed using chromatographic methods and detected using fluorescence measurement and potentially mass spectrometry (MS). Hydrophilic interaction liquid chromatography (HILIC), ideally used for the separation of highly polar compounds, has emerged as a robust and reliable technique that effectively separates and quantitates fluorescently labeled N-glycans (4, 5). However, sample preparation approaches suitable for N-glycan HILIC analysis tended to be laborious and time-consuming and did not provide the required efficiency and sensitivity for N-glycan detection and characterization. For instance, conventional deglycosylation may require a long incubation time while labeling reactions are very cumbersome, sometimes resulting in labeled glycans that are challenging to detect using fluorescence or MS (5). Therefore, it would be beneficial for glycan analysis to implement a straightforward and rapid sample preparation workflow while improving sample throughput and ensuring selective and highly sensitive N-glycan profiling of recombinant glycoproteins. Accelerating the sample preparation of N-glycans and facilitating their detection is essential to perform routine monitoring of drug product consistency throughout biopharmaceutical process development and manufacturing. Precise N-glycan composition and structure data enables operators to perform interventions at critical control points in bioprocessing to reduce risks and ultimately ensure the delivery of safe and effective biotherapeutics.

Rapid Preparation of N-Glycans using Waters GlycoWorks RapiFluor-MS N-Glycan Kit

The GlycoWorks RFMS N-Glycan kit is the best combination of speed and simplicity, delivering enhanced fluorescence response (FLR) and mass spectrometric (MS) sensitivity for the detection of released N-linked glycans. The core technology of this sample preparation approach is the use of an innovative, proprietary labeling reagent, named RapiFluor-MS (or RFMS), that rapidly reacts with N-glycans upon their enzymatic release from glycoproteins (5, 6). RFMS is a highly reactive, primary/secondary amine labeling reagent. It is comprised of an N-hydroxysuccinimide (NHS)-carbamate reactive group, a quinoline fluorophore, and a basic tertiary amine (Figure 2). As a result, RFMS-labeled glycans can be detected both by fluorescence and positive ion mode electrospray ionization (ESI)-MS with high sensitivity.

Watch this short video to see how GlycoWorks RapiFluor-MS N-Glycan Kit enables unprecedented fluorescent and mass spectrometric performance for glycan detection while also improving the throughput of N-glycan sample preparation.

Reagents and reaction conditions used in this kit have been optimized to enable a fast and complete de-N-glycosylation of glycoproteins and subsequently rapid and efficient labeling of released N-glycans with RFMS. The acceleration of the entire N-glycan sample preparation workflow is a direct result of the integration of these two procedures with a HILIC solid-phase extraction (SPE) sample clean-up step. HILIC SPE purification provides quantitative recovery of RFMS-labeled N-glycans and allows for immediate analysis of prepared samples using hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-FLR) and potentially mass spectrometry for further characterization or mass confirmation.

Figure 2: Structure of the RapiFluor-MS (RFMS) labeling reagent. The features of the chemical structure that enable rapid tagging of N-glycans, efficient fluorescence response, and enhanced MS sensitivity are highlighted (6).

Experimental Procedure

The N-glycan sample preparation workflow comprises three steps: The release, labeling, and purification of N-glycans (Figure 3). To prepare N-glycans for labeling, glycoprotein samples (15 μg) are first denatured using RapiGest SF, an enzyme-friendly anionic surfactant, reconstituted in GlycoWorks Rapid Buffer. Mixtures are heated to approximately 90°C for 3 min, allowed to cool to room temperature, and subsequently mixed with the peptide-N-glycosidase F (GlycoWorks Rapid PNGase F). Heating samples to a temperature of at least 90 °C is crucial to ensuring that glycoproteins are sufficiently denatured and that N-glycans are readily accessible to the PNGase F enzyme (5). The de-N-glycosylation reaction is completed by incubating the samples at 50 °C for 5 min. In this approach, glycoproteins are efficiently deglycosylated in approximately 10 min to produce N-glycosylamines from a diverse set of glycoproteins. Released N-glycosylamines exhibit a relatively stable structure in the applied deglycosylation conditions (5).

Secondly, deglycosylation mixtures are allowed to cool to room temperature following their incubation at 50°C and then rapidly reacted with RFMS labeling reagent, dissolved in anhydrous dimethylformamide (DMF), without a protein depletion step. RFMS forms a highly stable urea linkage (NH−CO−NH) with released N-glycosylamines using its N-hydroxysuccinimide (NHS) carbamate rapid tagging group (5). This labeling procedure occurs at room temperature in 5 minutes, a major improvement over the multiple hours typically required for this process using traditional N-glycan labels (5, 7). The reaction was optimized to ensures a high yield of N-glycan labeling. Therefore, labeled N-glycosylamines bear both a fluorescent tag comprised of an efficient fluorophore and a basic tertiary amine motif to facilitate MS ionization. Labeling reactions are finally diluted with acetonitrile (ACN) in preparation for HILIC SPE clean-up.

Figure 3: Workflow for the rapid preparation of N-glycans samples using the GlycoWorks RapiFluor-MS N-glycan kit.

The final step of the workflow is the purification and enrichment of the RFMS-labeled N-glycans through a robust HILIC SPE method. The ACN-diluted samples of labeled N-glycosylamines are subjected to SPE using vacuum aspiration and a silica-based amino-propyl sorbent (GlycoWorks μElution plate) with slight ion exchange properties (5). Wells containing 5 mg sorbent are conditioned with water and equilibrated with 85% (v/v) ACN before loading samples. Adsorbed samples were subsequently washed twice with a solution containing 1% formic acid in 90% ACN to remove potential interferences, such as labeling reaction byproducts from RFMS-labeled N-glycans as well as excess labeling reagent. Finally, after replacing the waste collection tray with the sample collection plate, enriched, RFMS-labeled N-glycosylamines are eluted from the SPE sorbent with three 30 μL volumes of GlycoWorks SPE Elution Buffer composed of 200 mM ammonium acetate in 5% ACN. This elution buffer delivers optimal, unbiased recovery of labeled N-glycosylamines from the HILIC μElution plate. In preparation for analysis by HILIC separation combined with fluorescence detection (HILIC-FLR), the SPE eluate is diluted with 310 μL of the GlycoWorks Sample Diluent (DMF/ACN mix, 32/68%). DMF/ACN diluted samples can be immediately analyzed with the ACQUITY UPLC Glycan BEH Amide 130 Å, 1.7 μm column (p/n 186004742, Waters) on a well-configured ACQUITY UPLC system (Waters).

The procedure used in the GlycoWorks RapiFluor-MS N-Glycan Kit, when compared with other N-glycan labeling methods, reduces preparation time from multiple hours or days to between 1 and 2 hours, depending on the number of samples processed (5). This approach will considerably facilitate studies of N-glycosylation by allowing samples to be more quickly analyzed and consequently more easily characterized at a higher level of sensitivity. Overall, the improvement of N-glycan sample preparation and profiling will help to accelerate the process development and quality control of biopharmaceuticals.

Automation of the GlycoWorks RFMS Protocol on the Andrew+ Pipetting Robot

During the process development and manufacturing of biopharmaceutical products, researchers may require screening many samples to identify those likely to have a desired, precise glycan profile. Consequently, glycan sample preparation can be time-consuming and monotonous. Users usually aim to achieve reproducible results when samples are prepared manually. Additionally, robust and reproducible methods for glycan preparation and analysis are a prerequisite for highly regulated glycoprotein-based biotherapeutics to ensure accuracy and consistency.

Standardization of glycan sample preparation is essential to minimize user variations and pipetting errors, and to ensure that glycan data is accurate and reproducible. Consequently, glycan sample preparation and analysis methods become easily transferable between experiments and laboratories, enhancing quality, efficiency, and productivity. One approach to achieving this is through the integration of automation to streamline routine sample preparation, either using a fully automated liquid handling system such as the Andrew+ pipetting robot, improving both reproducibility and sample throughput or the Pipette+ guided pipetting system, providing real-time step-by-step guidance throughout the experiment to minimize pipetting errors. The two systems use highly accurate Andrew Alliance electronic pipettes, which are manufactured by Sartorius and based upon their award-winning Picus design, and OneLab software, which enables full traceability as well as easy and secure sharing of validated protocols between laboratory members so that the same protocol is executed among them.

The End-to-end RapiFluor-MS N-glycan sample preparation using the Andrew+ pipetting robot provides scientists with the required components to successfully perform fast and efficient N-glycan release, labeling, and clean-up while reducing manipulation time for multiple samples with the use of Andrew Alliance electronic 8-channel pipettes. The GlycoWorks Andrew+ workstation is a robust solution enabling accurate and precise liquid handling throughout the experiment thus relieving the analyst of repetitive pipetting tasks and expediting the overall laboratory efficiency. All protocol steps are carried out on OneLab, which records the actual execution of the protocol, facilitating the identification of potential errors.

Watch this short video to see how Andrew+ simplifies the GlycoWorks workflow through automation.

The GlycoWorks RFMS workflow offers two different methodologies: the standard, variable volume method (VV) (also called flexible volume), and the alternative, quality con¬trol/automation-friendly method (QC) (8). The protocol used for the implementation of the GlycoWorks RFMS kit on Andrew+ is the QC/automation-friendly sample preparation method. While the VV method offers maximum flexibility concerning starting sample concentrations, it involves several low volume liquid transfers (1.2 μL – 7μL). This may result in pipetting volume inaccuracies, which makes the VV method not suitable for use with liquid handling automation (8). Looking to improve volume accuracy and precision, the standard, VV RFMS sample preparation method was revised to include larger pipetting volumes (≥ 10 μL) thereby mitigating the effect of volume inaccuracy on absolute quantities of analytes and reagents required for achieving optimal results. The QC protocol is an adaptation of the VV method, in which starting sample concentration and reagent concentrations used during protein denaturation, deglycosyl¬ation, and N-glycan labeling have been carefully modified to allow pipetting volumes of 10 μL or more (Figure 4), thus improving pipetting accuracy and generating results comparable to those produced by the standard VV method (8).

Figure 4: Aliquoted volumes for the quality control (QC) and automation-friendly GlycoWorks RapiFluor-MS protocol in 200 μL tube (8). ¹RapiGest SF surfactant reconstitution = 10 mg with 200 μL buffer + 135 μL water OR 3 mg with 60 μL buffer + 40 μL water. ²GlycoWorks Rapid PNGase F dilution = contents of vial 30 μL + 220 μL water. ³GlycoWorks RFMS labeling reagent reconstitution = 23 mg with 280 μL DMF or 9 mg with 110 μL DMF.

The QC GlycoWorks RFMS protocol (Figure 4) was validated by Waters on Andrew+. Experimental conditions and parameters were fine-tuned to fit the hardware configuration of the Andrew+ robot, i.e. labware Dominos and required, additional, connected devices (Figure 5), and produce reliable results, comparable to target scores and reproducibility among users. The Andrew+ robot can also greatly simplify the process of normalizing the concentrations of protein samples to meet the requirements of the GlycoWorks RFMS protocol.

Figure 5: Andrew+ Domino configuration for the automated GlycoWorks RapiFluor-MS protocol. Domino/Device positions on the Andrew+ working deck: [1, 2], Tip Insertion Systems; [3] Storage Plate Domino; [4] Deep-Well Plate Domino; [5] Microplate Vacuum+; [6] Conical Microtube Domino; [7] 96-PCR Peltier+.

Considerations

1- Recommended Starting Glycoprotein Quantity:

• Each reaction in The GlycoWorks RFMS N-glycan protocol is designed to produce optimal results from 15 μg of glycoprotein. The QC method uses 10 μL of glycoprotein sample at a concentration of 1.5 mg/mL (Figure 4). Samples with a concentration slightly higher or lower than 1.5 mg/mL can still potentially produce an appropriate output. Significant changes to the optimal glycoprotein quantity, i.e. < 5 μg and > 30 μg, can affect the PNGase F enzyme to substrate ratio as well as the molar excess of RFMS labeling reagent, which will potentially result in undesirable labeling artifacts or low yield (8).

2- Buffer/Formulation Considerations:

• The rapid deglycosylation reaction, facilitated by the RapiGest SF surfactant, can be compromised by the presence of sodium dodecyl sulfate (SDS) in the sample. Moreover, the presence of nucleophiles at high concentrations can compromise the N-glycan labeling reaction. Therefore, the concentration of amine and/or thiol-containing compounds should be diluted to a final concentration < 0.1 mM. On the other hand, a buffer exchange step could be necessary for some samples before enzymatic deglycosylation. In this case, it is recommended to exchange the protein sample into a neutral sodium phosphate, citrate or ideally a 50 mM HEPES buffer (free acid, titrated to pH 7.9 with sodium hydroxide).
• To perform a buffer exchange or to prepare samples out of complex matrices (lysates/biofluids), consider the use of molecular weight cut-off (MWCO) filtration, dialysis, or protein precipitation (i.e., ethanol precipitation). With these techniques, it is recommended to exchange a protein sample into a neutral sodium phosphate, citrate, or HEPES buffer. A matching formulation to the GlycoWorks Rapid Buffer would be 50 mM HEPES free acid titrated to pH 7.9 with sodium hydroxide.

3- Ensuring Complete Denaturation & Deglycosylation:

• It is imperative that the glycoprotein be subjected to heat denaturation prior to the addition of PNGase F. In the heat denaturation step, ensure that the glycoprotein is subjected to a temperature of at least 90 °C. Some challenging samples may require such a high temperature and possibly even near-boiling conditions (100 °C) in order for complete deglycosylation to be achieved.

4- Regarding the RapiFluor-MS Reagent:

• RapiFluor-MS is purified as a 1:1 complex with NHS. The formula weight for the reagent as provided is 542.41 g/mol.
• RapiFluor-MS is a highly reactive, primary/secondary amine labeling reagent. It hydrolyzes in water with a half-life on the order of 10–100 seconds (see Figure 4). It is, therefore, important that the reagent be dissolved in the provided anhydrous DMF, a non-nucleophilic, polar aprotic solvent. Reagent solution can be used across the span of a day if care has been taken to limit exposing the solution to atmospheric moisture.
• Glycans are released from glycoproteins as glycosylamines, an important fact for an analyst to consider when using this labeling chemistry.

Application Kits and Labware

Kit components for the GlycoWorks 8 sample protocol

• GlycoWorks Rapid Deglycosylation Kit (3 x 8 Samples) by Waters | p/n 186008841
• GlycoWorks RapiFluor-MS Labeling Module (3 x 8 Samples) by Waters | p/n 186008091
• GlycoWorks HILIC μElution Plate by Waters | p/n 186002780
• GlycoWorks SPE Reagents – Automation by Waters | p/n 186008747

Kit components for the GlycoWorks 24 sample protocol

• GlycoWorks Rapid Deglycosylation Kit (4 x 24 Samples) by Waters | p/n 186008840
• GlycoWorks RapiFluor-MS Labeling Module (4 x 24 Samples) by Waters | p/n 186007989
• GlycoWorks HILIC μElution Plate by Waters | p/n 186002780
• GlycoWorks SPE Reagents – Automation by Waters | p/n 186008747

Kit components for the GlycoWorks 48 sample protocol

• GlycoWorks Rapid Deglycosylation Kit (2 x 48 Samples) by Waters | p/n 186004579
• GlycoWorks RapiFluor-MS Labeling Module (2 x 48 Samples) by Waters | p/n 186008822
• GlycoWorks HILIC μElution Plate by Waters | p/n 186002780
• GlycoWorks SPE Reagents – Automation by Waters | p/n 186008747

Recommended labware & consumables (Not provided in GlycoWorks kits)

• QuanRecovery with MaxPeak, 700 μL Plate by Waters | p/n 186009185
• Eppendorf twin.tec® 96-well skirted LoBind PCR Plate by Eppendorf | p/n 0030129555
• Axygen® 12-well reservoir with 12-channel trough by Corning/Axygen | p/n RES-MW12-HP

(1) Reily C, Stewart TJ, Renfrow MB et al. Glycosylation in health and disease. Nat Rev Nephrol. 2019; 15, 346–366.

(2) Fournier J. A Review of Glycan Analysis Requirements. BioPharm International. 2015; 28(10): 32–37.

(3) Helenius A, Aebi M. Roles of N-Linked Glycans in the Endoplasmic Reticulum. Annual Review of Biochemistry. 2004; 73(1):1019–1049.

(4) Tharmalingam T, Adamczyk B, Doherty MA, Royle L, Rudd PM. Strategies for the profiling, characterisation and detailed structural analysis of N-linked oligosaccharides. Glycoconjugate Journal. 2013; 30(2):137–146.

(5) Lauber MA, Yu Y-Q, Brousmiche DW, Hua Z, Koza SM, Magnelli P, Guthrie E, Taron CH, Fountain KJ. Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Labeling Reagent that Facilitates Sensitive Fluorescence and ESI-MS Detection. Analytical Chemistry. 2015; 87(10):5401–5409.

(6) Lauber MA, Brousmiche DW, Hua Z, Koza SM, Guthrie E, Magneli P, Taron CH, Fountain KJ. Rapid Preparation of Released N-Glycans for HILIC Analysis Using a Novel Fluorescence and MS-Active Labeling Reagent. Application Note. Waters Corporation: Milford, MA, 2015. (p/n 720005275EN).

(7) Ruhaak LR, Zauner G, Huhn C, Bruggink C, Deelder AM, Wuhrer M. Glycan labeling strategies and their use in identification and quantification. Analytical and Bioanalytical Chemistry. 2010; 397(8):3457–3481.

(8) Koza SM, McCall SA, Lauber MA, Chambers EE. Quality Control and Automation Friendly GlycoWorks RapiFluor-MS N-Glycan Sample Preparation. Application Note. Waters Corporation: Milford, MA, 2016. (p/n 720005506EN).

Explore the GlycoWorks RapiFluor-MS N-Glycan Kit

Find more information about the Quality Control (QC) and Automation-Friendly GlycoWorks RapiFluor-MS Protocol and Instructions for Reagent Reconstitution and Dilution

Learn more about the GlycoWorks RapiFluor-MS N-Glycan kit – Standard VV Method

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