Application Update 180 Update Application Direct Determination of Sialic Acids in Glycoprotein Hydrolyzates by HPAE-PAD Thermo Fisher Scientific, Inc. INTRODUCTION In this work, sialic acids are determined in five Sialic acids are critical in determining glycoprotein representative glycoproteins by acid hydrolysis followed bioavailability, function, stability, and metabolism.1 by HPAE-PAD. Sialic acid determination by HPAE-PAD Although over 50 natural sialic acids have been identified,2 on a Thermo Scientific™ Dionex™ CarboPac™ PA20 two forms are commonly determined in glycoprotein column is specific and direct, eliminating the need for products: N-acetylneuraminic acid (Neu5Ac) and sample derivatization after sample preparation. The use N-glycolylneuraminic acid (Neu5Gc). Because humans of a disposable gold on polytetrafluoroethylene (Au on do not generally produce Neu5Gc and have been shown PTFE) working electrode simplifies system maintenance to possess antibodies against Neu5Gc, the presence of this compared to conventional gold electrodes while providing sialic acid in a therapeutic agent can potentially lead to an consistent response with a four-week lifetime. The rapid immune response.3 Consequently, glycoprotein sialylation, gradient method discussed separates Neu5Ac and Neu5Gc and the identity of the sialic acids, play important roles in under 10 min with a total analysis time of 16.5 min, in therapeutic protein efficacy, pharmacokinetics, and compared to 27 min using the Dionex CarboPac PA10 potential immunogenicity. column by a previously published method.6,7 By using the Dionex CarboPac PA20 column, the total analysis time is Sialic acid determination can be performed by many reduced, eluent consumption and waste generation are methods. Typically, sialic acids are released from glyco- reduced, and sample throughput is improved. proteins by acid hydrolysis or by enzymatic digestion before analysis. Once the sialic acids are liberated, there are many options for quantification. Numerous spectro- scopic methods exist, although interferences in these methods can cause overestimation of the concentration of sialic acids in many samples. Therefore, chromatographic methods that separate the sialic acids from potentially interfering compounds are preferred.4 Among the chromatographic methods, there are those that require further sample derivatization for analyte detection, such as fluorescent labeling followed by high-performance liquid chromatography (HPLC), and direct detection methods such as high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD).5 Of these methods, HPAE-PAD offers the advantage of direct analysis without sample derivatization. 2 EQUIPMENT CONDITIONS Thermo Scientific Dionex ICS-3000 or Thermo Scientific Columns: Dionex CarboPac PA20, 3 × 150 mm Dionex ICS-5000 Ion Chromatography (IC) system Dionex CarboPac PA20 Guard, including: 3 × 30 mm - SP Single Pump or DP Dual Pump module Eluent Gradient: 70–300 mM acetate in 100 mM NaOH from 0–7.5 min, 300 mM acetate in - DC Detector/Chromatography module 100 mM NaOH from 7.5–9.0 min, - Thermo Scientific Dionex AS Autosampler 70 mM acetate in 100 mM NaOH from 9.0–9.5 min, 7 min of equilibration at Dionex ICS-3000/5000 ED Electrochemical Detector 70 mM acetate in 100 mM NaOH. Electrochemical Cell Eluents: A: NaOH, 100 mM Disposable Gold Working Electrode, Au on PTFE B: Sodium acetate, 1.0 M, in 100 mM NaOH Reference Electrode (Ag/AgCl) Flow Rate: 0.5 mL/min Thermo Scientific™ Dionex™ Chromeleon™ Chromatography Data System, Workstation 7 Temperature: 30 °C (column and detector compartments) Polypropylene injection vials, 0.3 mL, with caps Inj. Volume: 10 μL Polypropylene injection vials, 1.5 mL, with caps Detection: Pulsed amperometric, disposable Thermo Scientific Nalgene 1000 mL 0.2 µm nylon Au on PTFE electrode filter units Background: 18–25 nC (using the carbohydrate waveform) Polypropylene microcentrifuge screw-cap tubes, 1.5 mL Noise: ~15–30 pC Dry block heater System REAGENTS AND STANDARDS Backpressure: ~3000 psi Deionized (DI) water, Type I reagent grade, 18 MΩ-cm resistivity or better Carbohydrate 4-Potential Waveform for the ED Sodium hydroxide, 50% (w/w) Time(s) Potential (V) Gain Region* Ramp* Integration 0.00 +0.1 Off On Off Sodium acetate, anhydrous 0.20 +0.1 On On On Acetic acid 0.40 +0.1 Off On Off N-Acetylneuraminic acid 0.41 -2.0 Off On Off N-Glycolylneuraminic acid (Neu5Gc, NGNA) 0.42 -2.0 Off On Off Micro BCA™ Protein Assay Kit 0.43 +0.6 Off On Off 0.44 -0.1 Off On Off α (2→3,6,8,9) Neuraminidase, proteomics grade from 0.50 -0.1 Off On Off Arthrobacter ureafaciens * Settings required in the Dionex ICS-3000/5000 IC system, SAMPLES but not used in older Dionex systems. Five glycoproteins were selected for analysis: Reference electrode in Ag mode (Ag/AgCl reference). - Calf fetuin See Application Update 141 for more information.6 - Bovine apo-transferrin (b. apo-transferrin) - Human transferrin (h. transferrin) (Sigma P/N T8158) - Sheep α1-acid glycoprotein (s. AGP) (Sigma P/N G6401) - Human α1-acid glycoprotein (h. AGP) (Sigma P/N G9885) PREPARATION OF SOLUTIONS Working Standard Solutions 3 AND REAGENTS Prepare calibration standards by diluting the standard Eluent Solutions stock solution as detailed in Table 1. For example, 10 µL Prepare 1 L of 100 mM sodium hydroxide by adding of the stock solution were added to 990 µL of DI water to 5.2 mL of 50% (w/w) NaOH to 994.8 mL of degassed prepare a calibration standard of 1.0 µM Neu5Ac, or DI water. 10 pmol/10 µL injection. Prepare standards daily from the stocks stored at -40 °C. Prepare 1 L of 1 M sodium acetate in 100 mM sodium hydroxide by dissolving 82.0 g of anhydrous sodium Protein Stock Solutions, 4.0 mg/mL Nominal acetate in ~800 mL of DI water. Filter and degas the Dissolve 3.44 mg of sheep α1-acid glycoprotein in 860 µL acetate solution through a 0.2 µm nylon filter unit. of DI water. Gently swirl to thoroughly mix the solution. Transfer the solution to a 1 L volumetric flask, add Prepare 200 µL aliquots of the solution in microcentrifuge 5.2 mL of 50% (w/w) NaOH, and fill the flask with vials to minimize freeze/thaw cycles when the stock is degassed DI water. See Dionex (now part of Thermo needed. Store all protein solutions at -40 °C. Repeat this Scientific) Technical Note 71 for detailed information process as follows. Dissolve 8.8 mg (b. apo-transferrin), on eluent preparation for HPAE-PAD applications.8 8.6 mg (h. transferrin), and 8.1 mg (fetuin) of the glycoprotein in individual aliquots of 2.0 mL DI water. Acetic Acid, 4 M Dissolve 2.2 mg of h. AGP in 0.56 mL of DI water. Transfer 22.5 mL of glacial acetic acid to a polyethylene Each glycoprotein will be at a nominal concentration of bottle containing 77.5 mL of DI water. 4 mg/mL. Sodium Acetate Buffer, 0.1 M, pH 5 for Working Stock Protein Solutions Neuraminidase Digestions Pipet 250 µL of a protein stock solution into Prepare a 0.3 M sodium acetate stock solution by 1750 µL DI water to prepare a working stock solution. dissolving 12.31 g of sodium acetate in 500 mL of DI Aliquot 400 µL of the working stock into individual water. Transfer 68.21 g (68.3 mL) of 0.3 M sodium microcentrifuge tubes and store the working stock acetate to a 250 mL polypropylene bottle. Add 1.8 mL solutions at -40 °C. Protein may be lost both during of 4 M actetic acid to the solution. Dilute to a total of freeze/thaw cycles and by adsorption to surfaces. There- 249.62 g (250 mL). fore, it is important to measure the working stock protein Stock Standard Solutions concentrations before hydrolysis by using a colorimetric Dissolve 149.8 mg of Neu5Ac in 50 mL DI water BCA protein assay kit. Values listed in Table 2 are results and 41.0 mg Neu5Gc in 50 mL of DI water. This results from BCA assay of the working stock solutions. in 9.68 mM and 2.52 mM stock solutions, respectively. Dilute 500 µL of 9.68 mM Neu5Ac and 130 µL of 2.52 mM Neu5Gc to 48.4 mL total with DI water. Aliquot this mixed stock of 0.10 mM Neu5Ac and 6.8 µM Neu5Gc into 1.5 mL cryogenic storage vials and store at -40 °C. Table 1. Sialic Acid Standards Used for Sample Analysis Volume of Neu5Ac Neu5Gc Neu5Ac Neu5Gc Combined Stock Concentration Concentration Amount Amount Standard (µL) (nM) (nM) (pmol/10µL) (pmol/10 µL) Diluted to 1000 µL 1.0 100 7.8 1.0 <LOQ* 2.5 250 20 2.5 <LOQ* 5.0 500 39 5.0 0.39 10 1000 78 10 0.78 25 2500 200 25 2.0 50 5000 390 50 3.9 75 7500 580 75 5.8 100 10000 780 100 7.8 *Not used for Neu5Gc calibration 4 Acetic Acid Hydrolysis of Proteins RESULTS AND DISCUSSION Add 14 µg (fetuin), 20 µg (h. transferrin), 25 µg Figure 1 shows the separation of Neu5Ac and Neu5Gc (b. apo-transferrin), 13 µg (h. AGP), and 7 µg (s. AGP) on the Dionex CarboPac PA20 column with a 70–300 mM of the glycoprotein to individual 1.5 mL microcentrifuge acetate gradient in 100 mM NaOH. The peaks are well vials with a total of 200 µL of 2 M acetic acid as detailed separated and easily quantified. Additionally, the Neu5Ac in Table 2. For example, pipet 50 µL of the working peak is well separated from the void, which is an impor- fetuin stock, 50 µL of DI water, and 100 µL of 4 M acetic tant consideration when analyzing acid hydrolyzed acid to prepare the solution for hydrolysis.