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.

There is a growing interest in the study of protein glycosylation dynamics as aberrant glycosylation patterns have been associated with various human pathologies (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. The 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). The Hydrophilic interaction liquid chromatography (HILIC), ideally used for the separation of highly polar compounds, has proven to be a robust and reliable technique that effectively separates and quantitates fluorescently labeled N-glycans (3, 4).

Conventional workflows for N-glycan HILIC analysis can be laborious and time-consuming including a long incubation time and cumbersome labeling reactions, sometimes resulting in labeled glycans that are challenging to detect using fluorescence or MS (4). Therefore, there is a need for a more rapid sample preparation method allowing for improved sample throughput while 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 following their enzymatic release from glycoproteins (4, 5). 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 (5).

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 (4). 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 (4).

The 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. 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 (4, 6). To facilitate MS ionization, labeled N-glycosylamines bear both a fluorescent tag comprised of an efficient fluorophore and a basic tertiary amine motif. 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. Wells containing 5 mg sorbent are conditioned with water and equilibrated with 85% (v/v) ACN before loading samples. Adsorbed samples are 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. In preparation for analysis, 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).

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

During the process of development and manufacturing of biopharmaceutical products, it may be necessary to screen large numbers of samples, which can be time-consuming and monotonous. 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. 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.

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

The protocol used for the implementation of the GlycoWorks RFMS kit on Andrew+ is the quality con­trol (QC)/automation-friendly sample preparation method (7). The QC protocol is an adaptation of the standard variable volume (VV) method, in which the starting sample concentration and reagent concentrations used during protein denaturation, deglycosyl­ation, and N-glycan labeling, have been carefully modified to allow pipetting volumes of 10 μL or more thus improving pipetting accuracy and generating results comparable to those produced by the standard VV method (7).

The QC GlycoWorks RFMS protocol 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 4), 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 4: Andrew+ Domino configuration for the automated GlycoWorks RapiFluor-MS 8-sample protocol. Domino/Device positions on the Andrew+ working deck: [1, 2] Tip Insertion Systems; [3] Storage Plate Domino; [4] Deepwell Microplate 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 (7).

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 (Not provided in GlycoWorks kits)

• Waters QuanRecovery™ 700 µL 96-well plate by Waters Corporation| p/n 186009185
• Eppendorf twin.tec® 96-well skirted LoBind® PCR plate by Eppendorf | p/n 0030129555
• Axygen® 12-well reservoir, 12-channel trough by Corning Inc. | 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) 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.

(4) 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.

(5) 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).

(6) 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.

(7) 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

Check out the Application Note "GlycoWorks RapiFluor-MS Automation Using the Andrew+ Pipetting Robot" (Waters, p/n 720006971EN)

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