The effect of temperature and incubation time on the analysis of highly sialylated glycans from bovine fetuin

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1 APPLICATION NOTE GlycanAssure Glycan Analysis and Quantitation System The effect of temperature and incubation time on the analysis of highly sialylated glycans from bovine fetuin Abstract This application note demonstrates the analysis of highly sialylated glycans from bovine fetuin labeled with 1-aminopyrene-3,6,8-trisulfonate (APTS), using the Applied Biosystems 3500xL Genetic Analyzer for Protein Quality Analysis with 24-capillary array. The method exhibits excellent separation of glycans based on charge, isomeric structure, and size. This application note also illustrates the effects of incubation time and temperature on analysis of sialylated glycans. Introduction Glycans are involved in a variety of biological and physiological processes, including recognition and regulatory function, cellular immunity, communication, and development [1,2]. Glycans are often key biomarkers for human health and disease [3]. Complex, heterogeneous structures provide glycans with more structural diversity than any other class of biomolecules [4]. Variation in glycosylation patterns can result in dramatic differences in cell function. Therefore, structural characterization of glycans is essential during development and manufacturing of clinically useful proteins. Equally important is the development of an assay for biotherapeutic glycoproteins, for control of reproducible production [5 7]. Applied Biosystems GlycanAssure Glycan Analysis and Quantitation System When analyzing glycans, it is critical to be able to efficiently separate linkage and branch isomers, as well as charge and size variants in the sample to achieve comprehensive structural characterization. The most popular glycan analysis methods in use today require time-consuming sample preparation, including overnight N-glycan release steps and purification steps to remove excess salts and labeling reagents. Additionally, they require a large glycoprotein input, and have difficulty producing accurate data on highly sialylated glycans.

2 Late elution on liquid chromatographic (LC) separation of sialylated glycans impedes their separation, analysis, and accurate quantitation. Conversely, in capillary electrophoresis (CE), sialylated glycans migrate at the beginning of separation, providing an ideal platform for their analysis and accurate quantitation. CE also has a low requirement for glycoprotein input (<5 μg) compared to typical LC methods ( μg) [8]. The GlycanAssure glycan analysis system provides an integrated glycan solution that can generate data from 96 samples in 7 to 9 hours, comprising an easy magnetic bead based sample prep, a 24-capillary array CE instrument, and assay-specific software for analysis. The software allows the user to visualize glycan profiles as well as quantitate relative areas with ease (Figure 1). All CE separations were performed using the Applied Biosystems 3500xL Genetic Analyzer for Protein Quality Analysis (Cat. No. A30556), a 24-capillary system configured with a 505 nm solid-state laser and laser-induced fluorescence detection. Results were analyzed using Applied Biosystems GlycanAssure Data Analysis Software v1.0 (P/N A30751). Additional experimental details for CE separation were as follows: Applied Biosystems POP-7 Polymer for Protein Quality Analysis (Cat. No. A30936) Applied Biosystems Anode Buffer Container (Cat. No. A31278) and Cathode Buffer Container (Cat. No. A31279) 3500xL Genetic Analyzer 24-Capillary Array, 50 cm (Cat. No ) Applied Biosystems GlycanAssure APTS Kit (Cat. No. A28676) Applied Biosystems GeneScan LIZ Size Standard (Cat. No ), used in every injection Capillary length: total length = 61 cm, length to detector = 50 cm Capillary diameter: 50 μm I.D. Injection conditions: 1.6 kv for 24 sec Run voltage: 19.5 kv Figure 1. GlycanAssure glycan analysis workflow. A typical glycan analysis requires 3 hours of hands-on time, including sample incubation, and a total of 7 to 9 hours to process and analyze 96 samples, without the need for vacuum centrifugation steps. Capillary oven temperature: 60 C APTS dye Ex/Em 450/515 nm Materials and methods Preparations of 25 µg of bovine fetuin (Millipore Sigma, Cat. No. F2379) were processed through the GlycanAssure workflow (Figures 2 and 3) and labeled with APTS dye as described in the N-Linked Glycan Analysis user guide (P/N ), with variation in the time and temperature of the APTS labeling step. Dye labeling conditions were 2 hours at 50 C, 1 hour at 50 C, or overnight at 37 C. Independent triplicate preparations of bovine fetuin were prepared for each experimental condition. Each APTS-labeled sample was injected in triplicate for CE separation. Figure 2. Deglycosylation and purification workflow. Figure 3. APTS dye labeling and excess dye removal workflow.

3 Results and discussion The effectiveness of CE-based methods like GlycanAssure analysis to accurately quantitate glycans with high sialylation, and the effects of incubation time and temperature on the quantitation of sialylated glycans, were evaluated. Cleaved glycans were purified using magnetic beads (Figure 2) labeled with APTS, and excess dye was removed (Figure 3). CE separation was performed using optimized conditions. Figures 4 and 5 show the results from the standard 2-hour and shortened 1-hour APTS labeling, respectively, compared to the traditional overnight labeling at 37 C (Figure 6). Increased incubation time adversely affects the stability of highly sialylated (penta-, tetra-, and trisialylated) species. On the other hand, shorter incubation time negatively impacts peak intensity, but provides sufficient labeling efficiency and minimizes desialylation. Average (%) SD CV (%) Average (%) SD CV (%) Figure 4. CE separation of APTS-labeled bovine fetuin N-glycans following the GlycanAssure workflow with APTS labeling at 50 C for 2 hours. Average (%) SD CV (%) Average (%) SD CV (%) Figure 5. CE separation of APTS-labeled bovine fetuin N-glycans following the GlycanAssure workflow with APTS labeling at 50 C for 1 hour. Average (%) SD CV (%) Average (%) SD CV (%) Figure 6. CE separation of APTS-labeled bovine fetuin N-glycans following the GlycanAssure workflow with APTS labeling at 37 C, overnight.

4 Conclusions The effects of variation in APTS labeling conditions of the GlycanAssure workflow on the quantitation of highly sialyated glycans were investigated. Preparations of bovine fetuin were labeled with APTS following the GlycanAssure workflow with three labeling conditions: 2 hours at 50 C, 1 hour at 50 C, or overnight at 37 C. CE separations were performed using the 3500xL Genetic Analyzer for Protein Quality Analysis, and results were analyzed using GlycanAssure Data Analysis Software v1.0. The results illustrate the advantage of a fully integrated GlycanAssure workflow to analyze and quantitate highly sialylated glycans. The GlycanAssure CE system with its full-length capillary array offers simultaneous analysis of labeled glycans in a high-throughput manner, without compromising on data quality. Significantly higher peak areas for highly sialylated (penta-, tetra-, and trisialylated) glycans, compared with lower peak areas for less sialylated (di- and monosialylated) species, suggest preservation of highly sialylated species with a shorter labeling time (Figure 7). While a shorter labeling time resulted in overall decreased signal intensity (data not shown), sufficient labeling efficiency was obtained, and importantly, desialylation was minimized. Figure 7. Average relative peak areas of APTS-labeled bovine fetuin N-linked glycans from 3 different labeling conditions. For each condition, bovine fetuin was labeled with APTS in triplicate. Each labeled sample was injected in triplicate for a total of 9 samples per experimental condition. Glycans have been grouped by their degree of sialylation, and displayed relative area pecent values are the percentage of the total glycan peak area. The critical importance of sialylated glycans in biotherapeutic functions necessitates accurate quantitation of these species. Increased labeling time can increase desialylation, negatively impacting accurate quantitation. Thus, it is important to optimize sample preparation conditions when highly sialylated glycan species are present in the sample. The GlycanAssure Glycan Analysis and Quantitation System offers a valuable, fully integrated tool to analyze glycoproteins with high sialylation.

5 References 1. National Research Council (2012) Transforming Glycoscience: A Roadmap to the Future. Washington, DC: National Academies Press. pp Arnold JN, Wormald MR, Sim RB, Rudd PM, Dwek RA (2007) The impact of glycosylation on the biological function and structure of human immunoglobulins. Annu Rev Immunol 25: Rademacher TW, Parekh RB, Dwek RA (1998) Glycobiology. Ann Rev Biochem 57: Khan SH, Hindsgaul O (1994) Chemical Synthesis of Oligosaccharides. In: Fukuda M, Hindsgaul O (editors), Frontiers in Molecular Biology. Oxford: IRL Press. pp Higgins E (2010) Carbohydrate analysis throughout the development of a protein therapeutic. Glycoconj J 27: Duke R, Taron CH (2015) N-glycan composition profiling for quality testing of biotherapeutics. BioPharm Int 28: Zhang L, Luo S, Zhang B (2016) Glycan analysis of therapeutic glycoproteins. MAbs 8: Reusch D, Haberger M, Maier B et al. (2015) Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles Part 1: Separation-based methods. MAbs 7: Find out more at thermofisher.com/glycanassure For Research Use Only. Not for use in diagnostic procedures Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. COL