High fidelity glycan sequencing using a combination of capillary electrophoresis and exoglycosidase digestion

High fidelity glycan sequencing using a combination of capillary electrophoresis and exoglycosidase digestion Andras Guttman Senior Manager Applications, Separations SCIEX, Brea, CA 92822 The rapidly increasing usage of glycoproteins as biopharmaceutical products has created a demand for fast, efficient and reliable bioanalytical techniques for glycosylation analysis [1]. One of the fastest growing groups of these new generation drugs are monoclonal antibodies (MAbs). MAbs in most instances possess a conserved N-linked glycosylation site in each of the C H2 domains of the Fc portion of the heavy chain of the molecule, but may also have additional attached sugar structures on the Fab domains [2]. Increasing evidence shows that the carbohydrate moieties of therapeutic antibodies play important roles in their biological activity, physicochemical properties, and effector functions [3]. Even minor changes in the carbohydrate structures (linkage, position and site occupancy) can influence the bioactivity of these products. The extremely high diversity of glycosylation makes their structural elucidation very difficult and in most instances only the combination of various methods can provide the desired information [4]. The most frequently used analytical methods for the structural analysis of complex carbohydrates include capillary electrophoresis (CE), mass spectrometry (MS) and high performance liquid chromatography (HPLC), often combined with exoglycosidase digestion techniques. Capillary electrophoresis with laser induced fluorescent detection (CE-LIF) is a high resolution and high sensitivity separation method applied for rapid profiling of complex carbohydrates [5]. CE-LIF is capable of discriminating between closely related positional and linkage isomers. Individual structures corresponding to peaks in the electropherogram can be proposed based on their glucose unit (GU) values [6] published in publicly available databases (https://glycobase.nibrt.ie/glycobase/about.action). However, there are instances when database-mediated structural proposition is ambiguous, calling for more comprehensive carbohydrate sequencing to achieve full structural elucidation. Unlike the commonly sequenced linear biopolymers of DNA and proteins, complex carbohydrates have branched structures and two sugar units can be connected through various positions and linkages making glycan sequencing a challenge. The sequencing process begins with the release of the N-linked carbohydrate moieties from the polypeptide backbone using an appropriate endoglycosidase, in most instances PNGase F. The released glycans are then labeled with a fluorophore (8- aminopyrene-1,3,6-trisulfonic acid, APTS), imparting charge and fluorescence properties, and separated by capillary electrophoresis. The resulting CE trace represents the N-linked glycan profile of the glycoprotein of interest. The sequencing strategy involves consecutive use of specific exoglycosidase enzymes to sequentially remove the sugar residues from the non-reductive end of the glycan structures [7]. The resulting glycan pools are analyzed by CE-LIF following each release step and peak migration shifts are recorded. This step is repeated using a series ofexoglycosidases in a consecutive manner, usually starting with sialidase, followed by fucosidase, galactosidase and hexoseaminidase until the characteristic trimannosyl chitobiose core structure of all N-linked sugars is reached. If antennary specification is needed, linkage specific mannosidases (1-3 or 1-6) can be used to attain such information [8]. This Technical Information Bulletin presents a high fidelity carbohydrate sequencing strategy for samples where more than one oligosaccharide is present in the released glycan pool. The method works without the need to isolate any of the individual structures. Published GU value data, or publicly available databases can be used to access the mobility shifts and accommodate structural elucidation. Figure 1: Glycan nomenclature. Left panel: graphical and alphabetic representation of a core fucosylated, disialo biantennary glycan with bisecting GlcNAc. Right panel: Symbols used for sugar units, linkage positions and linkage types. [9] p 1

Experimental Design Chemicals: The N-CHO Carbohydrate Analysis Assay Kit (Beckman Coulter, sold through Sciex, Brea, CA; pn 477600) was used in all experiments and contained all chemicals necessary for glycan labeling and CE separation, including the charged fluorophore (APTS), the N-CHO Carbohydrate Separation Buffer and the maltooligosaccharide ladder. IgG was purchased from Sigma Aldrich (St. Louis, MO). The PNGase F enzyme and the exoglycosydase enzymes of Arthrobacter ureafaciens sialidase (ABS), Bovine kidney fucosidase (BKF), Jack bean galactosidase (JBG) and Jack Bean hexosaminidase (JBH) were from ProZyme (Hayward, CA) and the reaction mixtures were prepared following the manufacturer s protocol. The Agencourt CleanSEQ magnetic beads were from Beckman Coulter (Brea, CA; pn A29151 Sample Preparation: Release of the N-linked IgG glycans was accomplished by the addition of 2 U of recombinant PNGase F (Prozyme) and incubation at 50 C for 1 hour. This was followed by magnetic bead based partitioning of the released glycans from the remaining polypeptide chains and the endoglycosidase using 200 μl Agencourt CleanSEQ magnetic bead suspension in 87.5% final acetonitrile concentration [10]. Following thorough mixing, the tube was placed on a strong magnet for fast and efficient partitioning. The supernatant was discarded and the captured glycans were eluted from the beads into the same tube by the addition of 21 μl of 40 mm APTS in 20 % acetic acid. The elution step was immediately followed by initiating the reductive amination reaction with the addition of 7 μl sodium cyanoborohydrate (1 M in THF). The reaction mixture was incubated at 37 C for 2 hours, then the excess labeling dye was removed by the same Agencourt CleanSEQ magnetic beads, which were used after the digestion reaction, again in 87.5% final acetonitrile concentration. The captured APTS-labeled glycans were eluted from the magnetic beads by adding 25 μl water and then analyzed by CE-LIF. Exoglycosidase based carbohydrate sequencing: Consecutive exoglycosidase digestions included Arthrobacter ureafaciens sialidase (ABS) to remove α2-3, 6 and 8 linked sialic acids; Bovine kidney fucosidase (BKF) to release α1-6 corelinked fucoses; Jack bean galactosidase (JBG) to remove β1-4 and 6 linked galactoses; and Jack bean hexosaminidase (JBH) to remove the β1-2, 4 and 6 linked N-acetyl-glucosamines. Briefly, the APTS-labeled released glycan pool was first analyzed by CE-LIF, then sequentially digested using the above listed enzymes (0.5 U each) or using an array of those at 37 C overnight in 50 mm ammonium acetate buffer (ph 5.5). Samples were dried in a centrifugal vacuum evaporator after each digestion step for pre-concentration and to remove the salt (ammonium acetate) content from the reaction mixture prior to the next digestion step. Capillary Electrophoresis: All capillary electrophoresis separations were performed in a Beckman Coulter PA 800 plus Pharmaceutical Analysis System (sold through SCIEX Separations, a part of SCIEX), equipped with a solid state laser and fluorescence detector (excitation: 488 nm, emission: 520 nm). The effective length of the N-CHO separation capillary was 50 cm (60 cm total length, 50 μm i.d.), filled with N-CHO Carbohydrate Separation Buffer (both from SCIEX). The applied electric field strength was 500 V/cm in reversed polarity mode (cathode at the injection side). Samples were pressure injected by applying 1 psi (6.89 kpa) for 5 sec. The 32 Karat version 9.1 software package (SCIEX) was used for data acquisition and analysis. APTS labeled maltose (G2, lower bracketing standard) and 2-aminoacridone (AMAC) labeled glucuronic acid (upper bracketing standard) were co-injected with all samples for migration time normalization [6]. The normalized migration times were converted to glucose unit (GU) values by the application of a fifth order polynomial time based standardization against the maltooligosaccharide ladder. Preliminary identification of the glycan structures corresponding to the peaks in the electropherogram was aided by Glycobase ver 3.2 from NIBRT (Dublin, Ireland) based on their GU values measured using identical CE-LIF separation conditions. N-glycan nomenclature and symbolic representations used the notation previously described by Harvey et al [9]. Results and Discussion Sequencing of complex glycan pools usually preceded by monosaccharide composition analysis to gain information about the type of sugar building blocks aiding to pick the relevant exoglycosidases to be used [11]. Commonly occurring monosaccharides in IgG molecules include sialic acid, galactose, N-acetylglucosamine, fucose and mannose. As the first step, the N-linked glycans were enzymatically removed from the sample by PNGase F digestion, labeled by APTS, purified with magnetic bead technology and analyzed by CE-LIF as shown in Figure 1, trace A. This step was followed by consecutive application of the corresponding exoglycosidases to begin sequencing of the APTS labeled glycan. As the first step, the sample was treated with sialidase to remove all sialic acids from the non-reductive end of the sugar structures (Figure 1B). The arrows between trace A and B depict the shift of peaks from the higher mobility regime (GU = 4 6, sialylated glycans) to the neutral regime (GU = 7 10). Please note that cutting off the sialic acids decreased the hydrodynamic volume of the structures, but also removed the extra charges the sialic acids p 2

held. After sialidase treatment, the desialylated glycan pool was treated using fucosidase to remove the core fucose residues (Figure 1 C). This step again resulted in a mobility shift, however, towards the higher mobility region as shown by the arrows between traces B and C. In this instance, the total charge of the glycans did not change but their hydrodynamic volume decreased. The next step of the sequencing process was the removal of the galactose residues (Figure 1 D). Finally, hexosaminidase was added to the reaction mixture to remove all antennary and bisecting GlcNAc residues, resulting in only one large remaining peak in trace E, corresponding the N-linked core of trimannosil chitobiose. respectively. The shift for the digalactosylated A2G2 structure was the sum of these two (2.44 GU). A B C D E Figure 1. CE-LIF traces of top-down sequential digestion of the APTS labeled IgG glycan pool. Symbols: sialidase: ; galactosidase: and hexosaminidase:. ; fucosidase: At this stage, identification of the structures started in a bottom up fashion as shown in Figure 2, considering the GU values and GU shifts of the peaks identified in Figure 1. Here we start with trace E, the trimannosil chitobiose structure with GU=4.83. The GU values of the two peaks in trace D were 6.67 and 7.18, corresponding to the shift caused by the removal of two antennary GlcNAc residues (0.92 each) and an additional bisecting GlcNac (0.51). Trace C shows the glycan structure profile after sialidase and fucosidase treatment featuring several additional major peaks in the neutral region possessing GU values of 7.74, 8.04 and 9.11. Based on the GU shifts corresponding the galactose residues, these structures were identified as monogalactosylated biantennary structures with 1-6 (GU=7.4) and 1-3 (GU=8.04) linkages as well as a digalacto biantennary structure (GE=9.11). Interesting to note that the galactoses on the 1-6 and 1-3 antennas contribute different GU shifts values of 1.07 and 1.37, Figure 2. Bottom-up identification of the IgG N-glycan structures. Trace B shows the electropherogram of the reaction mixture before fucosidase treatment with peaks corresponding to GU values of 7.63, 8.72, 9.06 and 10.10 matching database entries for core fucosylated agalacto- (FA2G2), monogalacto- with 1-6 (FA2(6)G1) and 1-3 (FA2(3)G1) linkages and digalactobiantennary (FA2G2) structures, respectively. The average contribution of the core fucose residue to the total GU values was 0.99. Finally, the GU shifts between the original IgG glycan pool were compared with the sialidase treated traces suggesting the presence of several monosialo and disialo structures with GU values between 4 and 5, as well as between 6 and 7 representing the core fucosylated biantennary structures with one or two sialic acid residues containing 2-3 or 2-6 linkages. Figure 3. Identified IgG glycan structures using the combination of CE-LIF and consecutive exoglycosidase digestions. p 3

Following the above explained scheme, the structures of all IgG glycans in Figure 1, trace A was identified as shown in Figure 3. For structural elucidation, GU values listed in Table 1 were used. Table 1. Abbreviated names, structures and CE glucose unit (GU) values of IgG N-glycans. Conclusions A high fidelity glycan sequencing scheme was demonstrated using capillary electrophoresis-laser induced fluorescence detection in combination with exoglycosidase digestion steps. This method allowed multi-structure glycan sequencing without the necessity for isolation of the individual components for analysis. The PNGase F released and APTS labeled N-glycan pool was subject to sequential exoglycosidase digestion using sialidase, fucosidase, galactosidase and finally hexosaminidase. Following enzymatic cleavage, the resulting reaction products were analyzed using CE-LIF and corresponding GU values and shifts were determined. Through the analysis of all cleavage data, bottom up identification of all individual structures was accomplished using the corresponding GU database values. Capillary electrophoresis provided excellent resolving power to separate positional and linkage isomers, making sequence determination easier. The high sensitivity of laser induced fluorescence (LIF) detection enabled trace level analysis, i.e., even minor glycan structures were detected and structurally elucidated. Acknowledgement The authors also acknowledge the support of the MTA-PE Translation Glycomics project (#97101). p 4

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