Agilent Biocolumns Glycan Analysis Application Compendium Contents Background 2 Getting Started 3 How to Guide - Glycan Mapping 4 Featured Application Notes 12 Fast and Efficient HILIC Methods for Improved Analysis of Complex Glycan Structures - 5991-4896EN 12 N-Glycan Analysis of mAbs and Other Glycoproteins with UHPLC and Fluorescence Detection - 5991-5253EN 17 Agilent Solutions for High-throughput N-linked Glycan Profiling from Biotherapeutics - 5991-6904EN 25 Additional Application Notes 30 More Information 30 1 Glycan Analysis Background Glycosylation is an important post-translational modification as these sugar molecules play a key role in protein recognition and biotherapeutic efficacy. Human glycosylation profiles follow a typical antenna-like pattern and it is the goal of recombinant protein manufacturers to try to replicate that profile using cell culture. Mammalian cell lines are required, but the glycosylation pathway is complex, and not all clones will generate the necessary glycan profile. Regulatory authorities recognize this as a major challenge and provide instructions in how to determine the glycan fingerprint. This involves the use of a specific enzyme, PNGase F, to cleave N-linked glycans, labeling them to increase detection sensitivity, and then separating them using HILIC chromatography columns (often in combination with a fluorescence detector, although mass spectrometry may also be used). Agilent provides a kit containing all the components needed to manually perform the deglycosylation and labeling reaction with 2 aminobenzoic acid (2AB). Alternatively, much higher throughput can be obtained using a fully automated AssayMAP platform capable of handling a greater number of samples in a fraction of the time. This approach is illustrated in the application note featured on page 166. Return to Contents Section 2 Getting Started HILIC, or hydrophilic interaction chromatography, uses H2O reversed-phase type eluents with gradients starting at high H2O H2O Glycan Analysis organic solvent content. The mechanism of interaction of H O 2 analytes with the stationary phase is a partitioning from the high organic eluent into the aqueous layer present on the surface of the stationary phase. Water is also the strong eluting solvent. It is therefore important to minimize Hydrophilic interaction chromatography the amount of water present in the sample matrix and to allow sufficient time for the column to re-equilibrate Fast, high-resolution, reproducible and stabilize at the end of each gradient. Following the glycan separation protocol contained in the sample preparation kit should ensure the 2AB labeled N-glycans are ready for analysis. AdvanceBio Glycan Mapping columns are available in superficially porous 2.7 µm columns suitable for use on AdvanceBio Glycan Mapping all HPLC instruments, or in fully porous 1.8 µm columns An amide HILIC column designed for use on highly optimized UHPLC instruments. Appropriate method conditions to achieve the optimum Attribute Advantage resolution for closely related glycan structures can be 2.7 µm superficially High resolution at low found in the Workflow "How-To" guides detailed in this porous particle back pressure section. 1.8 µm totally porous Maximum resolution particles Fluorescence and MS Easy method transfer compatible 3 Return to Contents Section Glycan Mapping: A "How-To" Guide Introduction Post-translational modifications to the primary amino acid sequence, including glycosylation, have functional consequences and can impact efficacy and immunogenicity of a biopharmaceutical. The structure of the glycan also contributes to the half-life of the protein in plasma and the ability of the monoclonal antibody to trigger the immune response required for efficacy. Regulatory authorities consider glycosylation to be one of the critical quality attributes of biomolecules. Therefore, it must be characterized and quantified, with acceptable ranges determined, as part of the development process for a glycoprotein innovator, biosimilar, or biobetter pharmaceuticals. Agilent AdvanceBio Glycan Mapping solution provides an optimized workflow designed to deliver reproducibility in the analysis of 2-aminobenzamide (2AB) labeled glycans, for accurate identification and quantification. Return to Contents Section 4 Sample Preparation - AdvanceBio N-Glycan Column Selection Sample Preparation Kit Agilent AdvanceBio Glycan Mapping columns are The mapping of the N-linked glycan component of a designed and manufactured to deliver fast, high glycoprotein, including monoclonal antibodies, requires resolution, reproducible glycan identification using the N-glycans to be enzymatically cleaved, using HILIC chromatography. AdvanceBio Glycan Mapping PNGase F, from the protein amino acid backbone. columns apply technology that optimizes results for MS The cleaved N-glycans can be analyzed by hydrophilic and fluorescence detection. Choose from two UHPLC interaction chromatography (HILIC) with MS detection. configurations: 2.7 µm superficially porous, for high Alternatively, N-glycans can labeled with the fluorophore, resolution and lower backpressure, or 1.8 µm for highest 2-aminobenzamide (2AB), and analyzed using HILIC resolution. chromatography followed by either fluorescence or MS Concentration of Final AA solution (pmol/µL) detection. AdvanceBio Amide bonded phase for rapid equilibration and AdvanceBio glycan sample preparation kits provide Glycan Mapping enhanced selectivity for glycans. all the reagents needed to prepare samples. The kit is 1.8 µm Based on a fully porous particle for high speed composed of four components that perform the steps separations and high throughput applications. Stability to 1200 bar for use with the Agilent 1290 Infinity II LC. of N-glycan release, N-glycan purification, labeling 2.7 µm Based on Poroshell technology to give a superficially with 2-aminobenzamide, and labeled glycan cleanup. porous particle with reduced diffusion distances to In addition, kits for each of the separate steps of the give high resolution separations at lower pressures workflow have separate part numbers for flexibility. The and enable the use of longer column lengths for increased separation efficiency. workflow yields samples that are suitable for analysis by liquid chromatography, typically by hydrophilic interaction chromatography (HILIC). Figure 1. Glycan mapping sample preparation workflow. 5 Return to Contents Section Speed of Analysis The AdvanceBio Glycan Mapping 1.8 µm columns provide high throughput N-glycan analysis where speed is the primary concern either due to the number of samples or to the immediate requirement for data. These columns deliver superior results in 40 % less time than the competition. Conditions Parameter Value Column A AdvanceBio Glycan Mapping, 2.1 × 150 mm, 1.8 μm Column B Competitor sub-2 μm glycan column Instrument Agilent 1290 Infinity LC with 1260 Infinity Flourescence Detection Column Temperature 55 °C Sample thermostat 105 °C Mobile Phase A: 100 mM NH4Formate. pH 4.5 B: ACN FLD Excitation = 260 Emission = 430 Injection Volume 2 μl in 70:30 ACN: 100mM NH4Formate Sample Agilent 2-AB labeled N-linked Human IgG glycan library (p/n5190-6996) Figure 2. The Agilent AdvanceBio Glycan Mapping column delivers better resolution, narrower bands, and higher peak capacity than the Resolution non-Agilent sub-2 µm column in a 2.1 x 150 mm configuration. High resolution separations are achieved using the AdvanceBio glycan mapping 2.7 µm media, in longer 250 mm columns. This increased resolution enables accurate quantitation of target glycans and changes to the protein glycosylation profile, which may have occurred during expression. Return to Contents Section 6 Ensure consistent results with glycan standards Flexible, high-performance LC instruments for performance testing and retention mapping Robust and easy to use: Missed information in the early stages of development The 100 % bio-inert Agilent 1260 Infinity II Bio-inert LC can cause major setbacks downstream. What’s delivers outstanding results with its low surface activity, more, production process inconsistency may lead to corrosion resistance, active seal wash, and quaternary changes in glycosylation, which can negatively impact buffer mixing. immunogenicity and efficacy. Agilent reference standards New Benchmarks in Efficiency: help you make sure that critical data have been captured, The Agilent 1290 Infinity II LC is the next generation in and that every workflow component is working optimally. UHPLC, providing maximum analytical, instrument, and Choose from two options: laboratory efficiency, with pressures up to 1300 bar and – Dextran ladder standard facilitates data analysis and flows up to 5 mL/min. reporting Better efficiency and interaction-free results: Agilent – IgG N-linked glycan standard confirms workflow bio-inert LC supplies improve chromatographic reliability efficiency with sharper peaks and high reproducibility. The two standards are available with the 2AB label attached and also without the 2AB label for use as sample preparation reference materials. Separation of a 2-AB labeled dextran ladder Conditions Parameter Value Column AdvanceBio Glycan Mapping, 859700-913 2.1 × 150 mm, 1.8 μm Mobile Phase A: 100 mM NH4Formate. pH 4.5 B: ACN FLD Excitation = 260 Emission = 430 Injection Volume 2 μl (10pmol total glycan/1 μl 75:25 ACN:water) Sample Agilent 2-AB labeled dextran ladder (p/n5190-6998) Figure 3. This analysis uses the agilent dextran ladder standard, together with an AdvanceBio Glycan