Ludger BioQuant™ GPEP-A2G2S2 Standard A quantitative glycopeptide standard Gives you reliability and confidence in your: drug analysis • glycan release techniques • sialic acid analysis • monosaccharide analysis •
Highlights of BioQuant GPEP-A2G2S2 Standard System Suitability Regulatory Submissions Use to demonstrate efficiency of labelling, Supports drugs regulatory submission by column efficiency and repeatability of glycoanalysis demonstrating consistent and reproducible results Well Characterised Part of the BioQuant Sialylated Glycopeptide Standard Range Quantity of GPEP-A2G2S2 standard Standard accurately determined using qNMR. Identity and purity is assessed by The dispensed quantity is checked by HPLC, NMR and Mass quantitative monosaccharide and Spectrometry techniques sialic acid analysis Quality Assurance Use as a Positive control Monosaccharide and Sialic acid analysis is traceable to The GPEP-A2G2S2 standard can run in parallel internationally accepted references from USP and with your sialic acid release or monosaccharide analysis dispensed using NIST traceable labware BQ-GPEP-A2G2S2-10U 2
The anatomy of Ludger BioQuant GPEP-A2G2S2 Standard N-linked Sialyated glycan Oxford notation NH2 Lys (A2G2S2) Val Ala Asn Lys Peptide sequence Lysine- Thr Valine-Alanine-Asparagine- COOH Lysine-Threonine (KVANKT) NH2 CFG notation Lys Val Ala Asn Lys Thr COOH NH2 Lys Text notation Val Ala Glycopeptide is comprised of an N-linked Sialyated Asn Lys glycan (A2G2S2. Alternative names: A2, G0 glycan) Thr attached to the asparagine amino acid of a peptide COOH BQ-GPEP-A2G2S2-10U 3
Accurate Quantity Determined by Quantitative NMR using BioQuant Metrology Concentration is determined by quantitative NMR (qNMR) The concentration of the BQ-GPEP-A2G2S2 is calculated by quantitative Nuclear Magnetic Resonance by comparison to an external certified NIST traceable quantitative standard ɣ CH 3 Val Method of quantification Amount of BQ-GPEP- Amount of BQ-GPEP- A2G2S2-10U per vial A2G2S2-10U per vial ( µg) ( nmol) ε CH 2 Lys ɣ CH 3 Thr Quantitiative Nuclear 10.00µg ± 0.48 3.49nmol Magnetic Resonanace (qNMR) Based determination Table 1 – qNMR results for the BQ-GPEP-A2G2S2 1 H-NMR (500 MHz) of BQ-GPEP-A2G2S2 in D2O BQ-GPEP-A2G2S2-10U 4
The Purity is Assessed with LudgerTag ™ V -Tag Glycan Labelling Kit Glycan Purity determined as >95% by HILIC chromatography of fluorescently labelled glycopeptide V-Tag-KVANKT-A2G2S2 Glycopeptides V-Tag labelled glycopeptides (BQ-GPEP-A2G2S2-10U) UHPLC Labelling+ Clean-up Analysis HILIC UHPLC profile of V-Tag (Cat. #: LT-VTAG-24) labelled BQ-GPEP-A2G2S2 V-Tag Dye LT-VTAG-24 Kit LC-A cartridges Click for a full presentation on our V-Tag technology BQ-GPEP-A2G2S2-10U 5
Use of BQ-GPEP-A2G2S2 Standard as a positive control
Quantitative Analysis and Reliability in your Monosaccharide and Sialic Acid Analysis BQ-GPEP-A2G2S2 is a system suitability standard compatible with these kits Monosaccharide Sialic Acid Release and labelling Kit Release and labelling Kit LudgerTag TM 2-AA LudgerTag TM DMB (2-aminobenzoic acid) (1,2-diamino-4,5-methylenedioxybenzene.2HCl) Cat #. LT-MONO-96 Cat #. LT-KDMB-A1 BQ-GPEP-A2G2S2-10U 7
Labelling Procedure Using the BQ-GPEP-A2G2S2 and the LudgerTag Monosaccharide Release and Labelling Kit (LT-MONO-96) 1. Replicates of the glycopeptide standard are run in 3. Prepare 2-AA labelling 2. Dry samples and resuspend parallel with your samples and standards, hydrolysed reagents and add to samples using TFA and HCL to release the monosaccharides Monosaccharide Release and Labelling Kit Click to view product guide 4. Incubate 5. Allow to cool and 7. Samples ready for 6. Dilute samples samples centrifuge briefly to remove HPLC or UPLC analysis condensation BQ-GPEP-A2G2S2-10U 8
Typical HPLC Data for Monosaccharide Analysis Standard Curve of HCl released GlcN from the serially diluted Monomix standard Quantitative monosaccharide analysis using the Ludger LT-MONO-96 Methods of Amount of BQ-GPEP- Amount of BQ-GPEP- kit was performed on 5 replicates of BQ-GPEP-A2G2S2 using 6M quantification A2G2S2-10U per vial A2G2S2-10U per vial hydrochloric acid hydrolysis (HCl) to release the N-acetylglucosamine ( µg) ( nmol) (GlcNAc – hydrolysed to GlcN) constituents of the glycopeptide. Monosaccharide 9.00µg ± 0.70 3.15nmol The GlcN monosaccharides were labelled with 2-aminobenzoic acid Based determination and chromatography was performed on a HPLC equipped with a LudgerSep R2 monosaccharide analysis column (LS-R2-4.6x150). A typical example of quantity of dispensed standard (Batch # B5B3-02) confirmed by Monosaccharide analysis BQ-GPEP-A2G2S2-10U 9
Data analysis of BQ-GPEP-A2G2S2 with Monosaccharide Release and Labelling Kit LudgerSep TM R2 HPLC profile of 2-aminobenzoic acid (2-AA) labelled GlcN monosaccharides of HCl hydrolysed BQ-GPEP-A2G2S2-10U. Peak between 10 and 15 mins is the free dye peak. HPLC conditions: Column: LudgerSep R2 (Cat. #: LS-R2-4.6x150) Flow: 0.8 ml/min. Temperature: 35 °C Man Gal Solvent A: LudgerSep BPT Solvent x10 (Cat #. LS-R-BPTX10) Solvent B: Acetonitrile Gradient: 0-35 min Excitation wavelength: 360 nm Emission wavelength: 425 nm LudgerTag TM BQ-GPEP-A2G2S2-10U Oxford Monosaccharide No. Of Theoretical Value NH2 notation Monosaccharides/molecule of nmols * Lys Molecular Weight 2865.17 Da BQ-GPEP-A2G2S2-10U Size; 10ug Val N-acetylglucosamine 4 13.96 3.49nmol Ala Asn Mannose 3 10.47 Lys Galactose 2 6.98 Thr COOH *3.49nmols of the lyophilised standard BQ-GPEP-A2G2S2-10U 10
Labelling Procedure Using the BQ-GPEP-A2G2S2 and the LudgerTag Sialic Acid Release and Labelling Kit (LT-KDMB-A1) 1. Replicates of the glycopeptide standard are run in 2. Samples are incubated 3. Prepare DMB labelling parallel with your samples and Sialic acids of the and allowed to cool solution and add to samples glycopeptides are released using 2M acetic acid Sialic Acid Release and Labelling Kit Click to view product guide 4. Incubate 5. Terminate reaction by adding 7. Samples ready for 6. Dilute samples samples water to standard and samples HPLC or UPLC analysis BQ-GPEP-A2G2S2-10U 11
Typical HPLC Data for Sialic Acid Release Analysis NeuAc LudgerSep-uR2 HPLC profile of 1,2-diamino-4,5-methylenedioxybenzene.2HCl (DMB) labelled Neu5Ac of acetic acid hydrolysed BQ-GPEP-A2G2S2-10U DMB labelled sialic acid standards eluted under the following HPLC conditions: Methods of Amount of BQ-GPEP- Amount of BQ-GPEP- Column: LudgerSep R1 (Cat. #: LS-R1-4.6x150) quantification A2G2S2-10U per vial A2G2S2-10U per vial Flow: 0.5 ml/min. ( µg) ( nmol) Temperature: 30 °C Solvent A: methanol:acetonitrile:water (7:9:84) Solvent B: acetonitrile Sialic acid 10.52µg ± 0.74 3.67nmol Gradient: 0-15 min Based determination Excitation wavelength: 373 nm Emission wavelength: 448 nm A typical example of quantity of dispensed standard (Batch # B5B3-02) confirmed by Sialic acid analysis BQ-GPEP-A2G2S2-10U 12
Data Analysis of BQ-GPEP-A2G2S2 Labelled with the LudgerTag DMB Kit The NeuAC standard in the LT-KDMB-A1 Kit for sialic acid release is diluted serially to generate a standard curve. The quantity of released sialic acids from the BQ-GPEP-A2G2S2 is then derived from the standard curve. 3 replicates of BQ-GPEP-A2G2S2 are used. BQ-GPEP-A2G2S2 NeuAC nmols in sample Rep1 6.5944 Rep 2 6.3694 Standard Curve of DMB labelled NeuAc standard Rep 3 6.3216 Ave 6.43 Sd 0.15 NH2 CV 2.27 Oxford Monosaccharide No. of Theoretical Lys notation Monosaccharides Value GPEP-A2G2S2 Val /molecule of nmols * Value obtained (nmol) 6.43 BQ-GPEP-A2G2S2 Ala Theoretical Value (nmol) 6.98 N-acetyl 2 6.98 Asn neuraminic acid % 92.1 Lys Thr *3.49nmols of the lyophilised standard GPEP-A2G2S2 analysis using the LT-KDMB-A1 Kit. GPEP-A2G2S2 has 2 NeuAC COOH molecules that are released during the procedure. BQ-GPEP-A2G2S2-10U 13
Benefits of the BioQuant GPEP-A2G2S2 Standard Confidence in your glycan release, • recovery and labelling techniques Reliability in your sialic acid or • monosaccharide analysis Use as a positive control and system • suitability standard
Next Steps… If you have a question Request a quotation Contact us for a quotation and place your order Contact us to discuss BioQuant™ GPEP-A2G2S2 Standard Catalogue # BQ-GPEP-A2G2S2-10U or any of our BioQuant ™ product range Simon Peel Sales Team Head of Glycan Production Quotations: info@ludger.com CLICK CLICK Orders: sales@ludger.com to contact simon.peel@ludger.com to contact Simon Sales BQ-GPEP-A2G2S2-10U 15
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