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N-Glycan Analysis of Monoclonal Antibodies and Other Glycoproteins

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N-Glycan Analysis of Monoclonal Antibodies and Other Glycoproteins N-Glycan analysis of monoclonal antibodies and other glycoproteins using UHPLC with fl uorescence detection Agilent 1260 Infi nity Bio-inert Quaternary LC System with Agilent 1260 Infi nity Fluorescence Detector Application Note Biopharmaceuticals LU G0 2000 0A 0G Authors 8 1317.49 Sonja Schneider, Edgar Nägele 6 Agilent Technologies, Inc. G0F 4 2100 0A 0G 1463.55 (1,6) G1F Waldbronn, Germany (1,3) G1F 2110 0A 0G 2 G2FS2 Man5 1625.60 G2F G2FS1 2120 0A 0G 2121 1A 0G 2122 2A 0G Man5 2488.910 1235.44 1787.6574 2197.814 0 15 20 25 30 35 40 45 min Abstract This Application Note shows the analysis of N-linked glycans with hydrophilic interaction chromatography (HILIC) separation using the Agilent 1260 Infi nity Bio- inert Quaternary LC System together with the Agilent 1260 Infi nity Fluorescence Detector. Enzymatic glycan release with PNGase F followed by derivatization by 2-aminobenzamide (2-AB) was conducted on monoclonal antibodies (mAbs) and two other glycoproteins from avian egg white: ovalbumin and conalbumin. The glycan pattern of the monoclonal antibody showed very good resolution and all major N-glycans occurring in mAbs could be detected with good signal-to-noise ratios. In addition, the complex glycan patterns of the two major types of avian egg glycoproteins were well resolved, resulting in over 30 to 35 detected peaks. The 1260 Infi nity Bio-inert Quaternary LC System together with the 1260 Infi nity Fluorescence Detector is an ideal solution for sensitive and high resolution analysis of 2-AB derivatized glycans released from mAbs and other glycoproteins. Verified for Agilentlent 1260 Infinity IIII LCLC Bio-inert System Introduction Protein glycosylation is one of the most frequently observed post trans- lational modifi cations. Mammalian VH VL glycoproteins contain three major Heavy chain types of glycans: N-linked, O-linked, Light chain and glycosylphosphatidyinositol (GPI) Fab 1 lipid anchors, which consist of one or CH more monosaccharide units. A single CL glycosylation site can generate consid- _ _ _ erable heterogeneity regarding mass _S_S_ and charge of glycoproteins. These S S oligosaccharides are involved in many Asn-297 biological regulation and recognition processes, for example, protein sort- CH2 ing, immune and receptor recognition, infl ammation, pathogenicity, metas- Fc tasis, and other cellular processes. Therefore, certain glycosylation pat- CH3 terns can be associated to the diseased or healthy state of a patient1, 2. In addi- tion, properties like safety, effi cacy and the serum half-life of therapeutic proteins can be affected by their Figure 1 glycosylation pattern. IgG antibody structure. Recombinant monoclonal antibody therapeutics (mAbs) represent the largest group of therapeutic proteins as a major new class of drug. The effi cacy of these therapeutics is highly dependent on the correct glycosyla- tion patterns of the mAbs and so far, all licensed therapeutic mAbs are immunoglobulines G (IgGs)3. Human IgG has a single conserved N-linked glycosylation site located on the Fc region of each heavy chain at Asn-2974 (Figure 1). This fact results in two sugar moieties per IgG, which are highly het- erogeneous and contain up to 30 differ- ent glycan types5. The combination of glycans at each of the two glycosyla- tion sites on the Fc region leads to large numbers of different glycoforms in each batch of mAb production. 2 The glycan structure on this glyco- sylation site plays a critical role in complement activation and receptor G0 6 affi nity , which affects the effi cacy of Monosaccharides the therapeutic mAbs. Moreover, non- human glycans are a safety issue due GlcNAc to induced immune responses. G0F Gal Therefore, analysis of the glycan Man pattern is an important part of charac- terization of therapeutic glycoproteins, Fuc G1 especially mAbs. Figure 2a shows the general nomenclature used to describe NeuNAc sugar residues of different glycan GalNAc structures on proteins. Figure 2b shows the predominant glycan structures G2 present on the Asn-297 site in IgG. In general, N-glycans have a core struc- a b ture, containing two b-D-N-acetylglu- cosamine (GlcNac) and three mannose Figure 2 (Man) units. IgG Fc N-glycans are pre- Glycan structure and isoforms. a) General nomenclature for glycans b) Predominant glycan structures of IgGs. G = Galactose units, F = Fucose units. Modifi ed after Arnold et al4. dominantly biantennary complex-type structures, partially core-fucosylated (for example, G0F). Different strategies for the analysis of 2-aminobenzamide, glycans have been described. A large 136 Da Glycan number of methods are based on pro- tein-released and subsequently derivat- O HO 7 HO R ized glycans due to the lack of chromo- NH2 O phores needed for optical detection + O OH NH2 methods, for example, UV detection. NHAc We have presented a combination of enzymatic release of N-glycans using _ PNGase F with subsequent derivatiza- H O 2 _17 Da tion with 2-aminobenzamide (2-AB) for + H+ fl uorescence detection. 2-AB is a neutral and stable bonded O label, numerously used in glycan HO analysis7, 8, 10. Figure 3 shows the NH2 HO R 2-AB-labeling by reductive amina- O NH tion (Schiff’s base intermediate not OH shown). The 2-AB labeling of the NHAc glycans results in the mass of the gly- 2-AB labeled cans +119 Da. Due to protonation, the Glycan, + 119 Da resulting mass shift in the MS is 120 Da (Tables 1, 2, 3). Figure 3 Labeling of a glycan with 2-aminobenzamide (2-AB). 3 Due to the hydrophilic properties Software one glycosylation site, whereas the of glycans, subsequent purifi ca- Agilent OpenLAB CDS, ChemStation mAb contains two glycosylation sites. tion using Hydrophilic Interaction Edition for LC & LC/MS Systems, The amount of PNGase F was adjusted Chromatography – Solid Phase Rev. C.01.02 [14] to the amount of glycans. The proteins Extraction (HILIC-SPE) is added for were deglycosylated according to removal of excess label8. Agilent MassHunter Workstation instructions from Sigma-Aldrich for Software, Version B.04.00, 3 hours at 37 °C. The reaction was then Separation using HILIC with fl uores- Build 4.0.479.0 stopped and the sample was vacuum cence detection is a robust method dried for further processing. for glycan analysis9. Pauline Rudd and Sample preparation coworkers established a database (http://glycobase.nibrt.ie/) based Deglycosylation procedure AB-labeling for fl uorescence on HIILIC retention properties, a Deglycosylation of the monoclonal detection and sample cleanup frequently used technology for the antibody and the glycoproteins was The dried glycan samples were labeled analysis of protein glycosylation8, 10. performed using PNGase F to release with the fl uorophore 2-AB (2-amin- To identify the monosaccharide compo- asparagine-linked oligosaccharides obenzamide) using the GlycoProfi l sition of the glycans within a chromato- (N-glycans) from the glycoproteins. 2-AB Labeling Kit according to the graphic peak, HILIC-LC can be coupled PNGase cleaves asparagine-linked high preparing protocol from Sigma-Aldrich to ESI-QToF-MS for mass and structure mannose as well as hybrid and complex for 3 hours at 65 °C. After the labe- information. oligosaccharides from the glycopro- ling procedure, the samples were teins and leaves the glycans intact. purifi ed using the GlycoProfi l Glycan Experimental The average glycosylation of proteins is Cleanup Cartridges from Sigma-Aldrich 2–5 % with 1,000 Da as average according to the instructions manual. The Agilent 1260 Infi nity Bio-inert molecular weight. Therefore, to After the HILIC cleanup procedure, Quaternary LC System consisted of the release approximately 20 µg glycans the samples were vacuum dried and following modules: 400 µg of glycoproteins were used. reconstituted in 15 µL ultrapure water Ovalbumin and conalbumin have only for LC analysis. • Agilent 1260 Infi nity Bio-inert Quaternary Pump (G5611A) Chromatographic conditions • Agilent 1260 Infi nity High Performance Bio-inert Autosampler Gradient antibody standard Gradient glycoproteins (G5667A) Starting fl ow rate: 0.5 mL/min 0.5 mL/min • Agilent 1290 Infi nity Thermostatted Gradient: 0 minutes – 85% B 0–6 minutes – 85% B Column Compartment (G1316C) 5 minutes – 75% B 10 minutes – 80%B • Agilent 1260 Infi nity Diode Array 35 minutes – 64% 60 minutes – 64% B Detector VL (G1315D), with bio-inert 40 minutes – 50% 65 minutes – 50% B standard fl ow cell, 10 mm 42 minutes - Flow 0.25 mL/min 67 – Flow 0.25 mL/min • Agilent 1200 Series Fluorescence 43 minutes – 0% B 68 minutes – 0% B Detector (G1321A), with standard 48 minutes – 0% B 73 – 0% B fl o w c e l l 50 minutes – 85% B 75 minutes – 85% B MS system 55 minutes – Flow 0.5 mL/min 80 minutes – Flow 0.5 mL/min Agilent 6530 Accurate Mass QTOF Stop time: 55.01 minutes 80.01 minutes LC/MS system Post time: 20 minutes 20 minutes Injection volume: 10 µL 1 µL Column Column temperature: 60 °C HILIC Glycan Amide column, FLD: Ex. 260 nm 2.1 × 150 mm, < 2 µm Em. 430 nm Peak width: > 0.025 minutes (18.52 Hz) 4 Solvents and samples LU Buffer A: 100 mM ammonium G0 formate, pH 4.5 2000 0A 0G 8 1317.49 Buffer B: acetonitrile 6 All solvents used were LC grade. Fresh ultrapure water was obtained G0F from a Milli-Q Integral system equipped 4 2100 0A 0G 1463.55 with a 0.22 µm membrane point-of- (1,6) G1F use cartridge (Millipak). The mono- (1,3) G1F 2 2110 0A 0G G2FS2 1625.60 G2F G2FS1 clonal antibody standard, that was Man5 2122 2A 0G used, is part of the mAb-Glyco Chip Man5 2120 0A 0G 2121 1A 0G 2488.910 1235.44 1787.6574 2197.814 Kit (p/n G4240-64026).
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