Ludger BioQuant GPEP-A2G2S2 Standard A quantitative glycopeptide - - PowerPoint PPT Presentation

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Ludger BioQuant GPEP-A2G2S2 Standard A quantitative glycopeptide - - PowerPoint PPT Presentation

Aug 14, 2022 219 likes •379 views

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

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SLIDE 1

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
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SLIDE 2

BQ-GPEP-A2G2S2-10U

Highlights of BioQuant GPEP-A2G2S2 Standard

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Use as a Positive control

The GPEP-A2G2S2 standard can run in parallel with your sialic acid release or monosaccharide analysis

Well Characterised Sialylated Glycopeptide Standard

Identity and purity is assessed by HPLC, NMR and Mass Spectrometry techniques

Part of the BioQuant Standard Range

Quantity of GPEP-A2G2S2 standard accurately determined using qNMR. The dispensed quantity is checked by quantitative monosaccharide and sialic acid analysis

Regulatory Submissions

Supports drugs regulatory submission by demonstrating consistent and reproducible results

Quality Assurance

Monosaccharide and Sialic acid analysis is traceable to internationally accepted references from USP and dispensed using NIST traceable labware

System Suitability

Use to demonstrate efficiency of labelling, column efficiency and repeatability of glycoanalysis

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SLIDE 3

BQ-GPEP-A2G2S2-10U

The anatomy of Ludger BioQuant GPEP-A2G2S2 Standard

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N-linked Sialyated glycan (A2G2S2) Peptide sequence Lysine- Valine-Alanine-Asparagine- Lysine-Threonine (KVANKT)

Lys Val Ala Asn Lys Thr

NH2 COOH

Lys Val Ala Asn Lys Thr

NH2 COOH

CFG notation Text notation Oxford notation

Glycopeptide is comprised of an N-linked Sialyated glycan (A2G2S2. Alternative names: A2, G0 glycan) attached to the asparagine amino acid of a peptide

Lys Val Ala Asn Lys Thr

NH2 COOH

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SLIDE 4

BQ-GPEP-A2G2S2-10U

ɣ CH3 Val ɣ CH3 Thr ε CH2 Lys

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Accurate Quantity Determined by Quantitative NMR using BioQuant Metrology

1H-NMR (500 MHz) of BQ-GPEP-A2G2S2 in D2O

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

Method of quantification Amount of BQ-GPEP- A2G2S2-10U per vial (µg) Amount of BQ-GPEP- A2G2S2-10U per vial (nmol) Quantitiative Nuclear Magnetic Resonanace (qNMR) Based determination 10.00µg ± 0.48 3.49nmol

Table 1 – qNMR results for the BQ-GPEP-A2G2S2

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SLIDE 5

BQ-GPEP-A2G2S2-10U 5

The Purity is Assessed with LudgerTag™ V-Tag Glycan Labelling Kit

Glycan Purity determined as >95% by HILIC chromatography of fluorescently labelled glycopeptide

HILIC UHPLC profile of V-Tag (Cat. #: LT-VTAG-24) labelled BQ-GPEP-A2G2S2

V-Tag-KVANKT-A2G2S2

Glycopeptides

(BQ-GPEP-A2G2S2-10U)

Labelling+ Clean-up

V-Tag Dye LT-VTAG-24 Kit LC-A cartridges

V-Tag labelled glycopeptides

UHPLC Analysis

Click for a full presentation on our V-Tag technology

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SLIDE 6

Use of BQ-GPEP-A2G2S2 Standard as a positive control

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SLIDE 7

BQ-GPEP-A2G2S2-10U

Quantitative Analysis and Reliability in your Monosaccharide and Sialic Acid Analysis

BQ-GPEP-A2G2S2 is a system suitability standard compatible with these kits

Sialic Acid Release and labelling Kit

LudgerTagTM DMB (1,2-diamino-4,5-methylenedioxybenzene.2HCl)

Cat #. LT-KDMB-A1

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Monosaccharide Release and labelling Kit

LudgerTagTM 2-AA (2-aminobenzoic acid)

Cat #. LT-MONO-96

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SLIDE 8

BQ-GPEP-A2G2S2-10U

  • 2. Dry samples and resuspend

Labelling Procedure Using the BQ-GPEP-A2G2S2

and the LudgerTag Monosaccharide Release and Labelling Kit (LT-MONO-96)

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  • 1. Replicates of the glycopeptide standard are run in

parallel with your samples and standards, hydrolysed using TFA and HCL to release the monosaccharides

  • 3. Prepare 2-AA labelling

reagents and add to samples

  • 4. Incubate

samples

  • 5. Allow to cool and

centrifuge briefly to remove condensation

  • 6. Dilute samples
  • 7. Samples ready for

HPLC or UPLC analysis

Monosaccharide Release and Labelling Kit

Click to view product guide

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SLIDE 9

BQ-GPEP-A2G2S2-10U

Typical HPLC Data for Monosaccharide Analysis

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Standard Curve of HCl released GlcN from the serially diluted Monomix standard

Quantitative monosaccharide analysis using the Ludger LT-MONO-96 kit was performed on 5 replicates of BQ-GPEP-A2G2S2 using 6M hydrochloric acid hydrolysis (HCl) to release the N-acetylglucosamine (GlcNAc – hydrolysed to GlcN) constituents of the glycopeptide. The GlcN monosaccharides were labelled with 2-aminobenzoic acid 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 Methods of quantification Amount of BQ-GPEP- A2G2S2-10U per vial (µg) Amount of BQ-GPEP- A2G2S2-10U per vial (nmol) Monosaccharide Based determination 9.00µg ± 0.70 3.15nmol

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SLIDE 10

BQ-GPEP-A2G2S2-10U

Data analysis of BQ-GPEP-A2G2S2 with Monosaccharide Release and Labelling Kit

HPLC conditions: Column: LudgerSep R2 (Cat. #: LS-R2-4.6x150) Flow: 0.8 ml/min. Temperature: 35 °C 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

LudgerSepTM R2 HPLC profile of 2-aminobenzoic acid (2-AA) labelled monosaccharides of HCl hydrolysed BQ-GPEP-A2G2S2-10U. Peak between 10 and 15 mins is the free dye peak.

Oxford notation Monosaccharide

  • No. Of

Monosaccharides/molecule of BQ-GPEP-A2G2S2-10U Theoretical Value nmols * N-acetylglucosamine 4 13.96 Mannose 3 10.47 Galactose 2 6.98

Lys Val Ala Asn Lys Thr

NH2 COOH

LudgerTagTM BQ-GPEP-A2G2S2-10U Molecular Weight 2865.17 Da Size; 10ug 3.49nmol

GlcN Gal Man 10

*3.49nmols of the lyophilised standard

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SLIDE 11

BQ-GPEP-A2G2S2-10U

  • 3. Prepare DMB labelling

solution and add to samples

  • 6. Dilute samples

Labelling Procedure Using the BQ-GPEP-A2G2S2

and the LudgerTag Sialic Acid Release and Labelling Kit (LT-KDMB-A1)

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Sialic Acid Release and Labelling Kit

  • 2. Samples are incubated

and allowed to cool 1. Replicates of the glycopeptide standard are run in parallel with your samples and Sialic acids of the glycopeptides are released using 2M acetic acid

  • 5. Terminate reaction by adding

water to standard and samples

  • 4. Incubate

samples

  • 7. Samples ready for

HPLC or UPLC analysis

Click to view product guide

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SLIDE 12

BQ-GPEP-A2G2S2-10U

Typical HPLC Data for Sialic Acid Release Analysis

LudgerSep-uR2 HPLC profile of 1,2-diamino-4,5-methylenedioxybenzene.2HCl (DMB) labelled Neu5Ac of acetic acid hydrolysed BQ-GPEP-A2G2S2-10U

NeuAc DMB labelled sialic acid standards eluted under the following HPLC conditions: Column: LudgerSep R1 (Cat. #: LS-R1-4.6x150) Flow: 0.5 ml/min. Temperature: 30 °C Solvent A: methanol:acetonitrile:water (7:9:84) Solvent B: acetonitrile Gradient: 0-15 min Excitation wavelength: 373 nm Emission wavelength: 448 nm 12 A typical example of quantity of dispensed standard (Batch # B5B3-02) confirmed by Sialic acid analysis

Methods of quantification Amount of BQ-GPEP- A2G2S2-10U per vial (µg) Amount of BQ-GPEP- A2G2S2-10U per vial (nmol) Sialic acid Based determination 10.52µg ± 0.74 3.67nmol

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SLIDE 13

BQ-GPEP-A2G2S2-10U 13

Data Analysis of BQ-GPEP-A2G2S2 Labelled with the LudgerTag DMB Kit

Standard Curve of DMB labelled NeuAc standard BQ-GPEP-A2G2S2 NeuAC nmols in sample Rep1 6.5944 Rep 2 6.3694 Rep 3 6.3216 Ave 6.43 Sd 0.15 CV 2.27 GPEP-A2G2S2 Value obtained (nmol) 6.43 Theoretical Value (nmol) 6.98 % 92.1 GPEP-A2G2S2 analysis using the LT-KDMB-A1 Kit. GPEP-A2G2S2 has 2 NeuAC molecules that are released during the procedure.

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.

Lys Val Ala Asn Lys Thr

NH2 COOH

Oxford notation Monosaccharide

  • No. of

Monosaccharides /molecule of BQ-GPEP-A2G2S2 Theoretical Value nmols * N-acetyl neuraminic acid 2 6.98

*3.49nmols of the lyophilised standard

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SLIDE 14

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

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SLIDE 15

BQ-GPEP-A2G2S2-10U

Next Steps…

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CLICK to contact Simon

Simon Peel

Head of Glycan Production

simon.peel@ludger.com

If you have a question

Contact us to discuss BioQuant™ GPEP-A2G2S2 Standard

  • r any of our BioQuant™ product range

Request a quotation

Contact us for a quotation and place your order Catalogue # BQ-GPEP-A2G2S2-10U

CLICK to contact Sales

Sales Team

Quotations: info@ludger.com Orders: sales@ludger.com