mAb Glycopeptide Profiling with V-T ag Adding reliable - - PowerPoint PPT Presentation

mAb Glycopeptide Profiling with V-T ag

Adding reliable glycoprofiling to your peptide mapping workflow with ease and simplicity

LT-VTAG-24

Who is the V-Tag Glycoprofiling Technology for?

V = Velocity

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The Ludger V-Tag Glycoprofiling Technology is for mAb developers, both innovator and biosimilar companies, who need to glycoprofile* mAb samples reliably, at an affordable cost and with a fast turnaround time.

Our typical V-Tag clients are those who want to:

Monitor their mAb drug’s glycosylation during the product lifecycle. This includes showing the comparability of glycosylation throughout the drug lifecycle as well as biosimilarity to an innovator’s drug

*Glycoprofile = a map of the drug’s glycosylation containing structural ID and relative abundance of each glycan species

Integrate glycoprofiling into the peptide mapping workflow. The V-Tag labeled glycopeptides are analysed using orthogonal platforms, MALDI-MS and UHPLC

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Options for Analysing mAb Glycosylation

Three types of mAb-derived molecules can be analysed to gain information about mAb glycosylation

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• 1. Intact Glycoprotein

Using lectin affinity, CE or MS

✓ Provides information on molecular weight and glycoform distribution Χ Poor structural information for the glycans. Insufficient for regulatory work or lot release

• 2. Glycopeptides

Using MS and UHPLC

✓ Provides both glycan identity and quantitation as well as glycan attachment site and site occupancy V-Tag is designed for glycopeptide mapping

• 3. Glycans

Using MS, UHPLC, CE

✓ Provides glycan identity and quantitation Χ Need to release glycans – extra work and expense. No glycosylation site information (for mAbs with Fab and Fc glycosylation)

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Adding Glycoprofiling to the Traditional Peptide Mapping Workflow

Incorporating glycopeptide mapping can reduce the time and costs of glycoprofiling work in drug production monitoring

Intact mAb

Peptide Mapping

Peptides + Glycopeptides Reduced mAb Reduced, Alkylated mAb

Glycopeptide Mapping

The V-Tag system labels and enriches mAb glycopeptides efficiently, allowing determination of their structures and relative quantities

* J. Bongers et al. J. Pharm. Biomed. Anal. 2000, 21, 1099-1128. RP-HPLC peptide map of anti-CD4 IgG-1 * Glycopeptide signals attenuated in a complex mixture 4

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Highlights of the V-Tag System

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Minimal Sample Needed

Excellent glycopeptide mapping using as little as 10 mg of mAb glycoprotein

Integrates Easily with Peptide Mapping Workflow

Adds onto your existing peptide mapping workflow, without requiring extra steps for glycan release

Quick and Easy

Labeling and enrichment is completed within 2 hours

Reliable mAb Glycoprofiling

Allows glycan identification and quantitation using the orthogonal analyses

• f MALDI-MS and UHPLC. Provides data comparable to gold-standard

glycoprofiling methods based on 2-AB or 2-AA

Validated for GMP Labs

Validated to ICH Q2(R1) standards and tested in glycoprofiling labs

Automatable for High-Throughput Studies

The procedure is compatible with 96-well plate based assays, enabling high-throughput studies using a liquid handling robot

LT-VTAG-24

Anatomy of V-Tag

The molecular aspects that make V-Tag work

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Amine reactive succinimidyl ester

to react with the N-terminus amine moiety of the peptide

Fluorescent group for

detection in UHPLC lex = 250nm, lem = 360nm

Sulphate anion to improve analysis

in negative ion mode on MALDI Simple, non-reactive alkyl chain to link the fluorescent moiety to the reactive succinimidyl ester

LT-VTAG-24

Components of the LT-VTAG-24 Kit

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Reaction buffer and solvent Amine reactive fluorescent labeling reagent Solvent involved in HILIC clean-up and enrichment HILIC resin cartridge

PBS Buffer Tablet

LT-PBS-TAB-0.01M

V-Tag Labeling Dye

LT-VTAG-01

TFA 10% (aq.)

LC-TFA10PC-01

LudgerClean A Cartridges (LC-A)

LC-A-24

• 2. Enrichment
• 1. Labeling

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Workflow for the V-Tag System: Stage 1 - Labeling

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Labeling

V-Tag Dye + PBS Buffer Labels the amine on the N-terminus of the peptide

V-Tag labeled peptides and glycopeptides

Protease digestion

Use your enzyme of choice e.g. sequencing grade trypsin

Glycopeptides + peptides mAb

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• 2.0e6

Workflow for the V-Tag System: Stage 2 – Enrichment

Analysis by MALDI-MS and UHPLC

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Orthogonal Analyses Enrichment LT-VTAG-24 Kit

LC-A cartridges with TFA solutions

V-Tag labeled glycopeptides

UHPLC

Quantitation

MALDI-MS

For glycan Identification

• Intens. [a.u.]

Separation of mixture using HILIC

• cartridge. Conditions optimised for

recovery of glycopeptides with glycosylation patterns preserved

IgG-1 V-Tag labeled glycopeptides

MALDI-MS and UHPLC

V-Tag labeled peptides and glycopeptides

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V-Tag Workflow: Simple and Easy

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Buffering the mAb digest Label the digest Remove peptides Elute glycopeptides Enrichment Labeling

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14.0 16.0 18.0 20.0 22.0 24.0

• 2.0e6

0.0e0 5.0e6 1.0e7 1.5e7 2.0e7 min counts

Set-up of Orthogonal Glycoanalytical Platforms

Typical setup for analysis of V-Tag labeled glycopeptides by UHPLC and MALDI-MS

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Thermo Scientific Dionex U3000

30 minute gradient 25 µl injection

Waters UHPLC Glycan BEH Amide Column (HILIC)

(150mm x 2.1mm) Temperature: 60 °C

FLD Fluorescence Detector

λex= 250 nm λem = 360 nm

Spot Sample

Matrix: 2,5-dihydroxybenzoic acid (DHB)

Collect Data

Mode: reflectron negative ion

Load Plate

Bruker Autoflex MALDI-MS instrument

• Intens. [a.u.]

Typical Data

V-Tag labeled glycopeptide map of IgG 1

Typical data

V-Tag labeled glycopeptide map of IgG 1

MALDI UHPLC

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14.0 16.0 18.0 20.0 22.0 24.0

• 2.0e6

0.0e0 5.0e6 1.0e7 1.5e7 2.0e7 min counts

• 3.2e6

• 2.0e6

• 2.0e6

• 2.0e6

• 2.0e6

• 2.0e6

• 2.0e6

• 2.0e6

• 2.0e6

V-Tag System Has Been Validated to ICH Q2(R1) Level

V-Tag is reliable and robust and can be used for GMP level glycoprofiling of monoclonal antibodies

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Validation studies typically show repeatability with CVs for relative abundances < 4%

Stacked IgG 1 V-Tag labeled glycopeptide UHPLC profiles (9 replicates) Overlaid IgG 1 V-Tag labeled glycopeptide UHPLC profiles (9 replicates) Average relative % area, SD and CVs for V-Tag labeled IgG 1 glycopeptides

Peak Number 1 2 3 4 5 6 7 Glycopeptide G0F (FA2) G0FB (FA2B) G1F (FA2G1) G1F + G1FB (FA2G1 + FA2BG1) G2F (FA2G2) G1FS1 (FA2G1S1) A1F (FA2G2S1) Relative % Area Av. 50.2 4.2 25.8 12.6 3.8 1.8 1.7

• Std. Dev.

0.35 0.14 0.18 0.26 0.10 0.02 0.04 CV 0.70 3.20 0.69 2.05 2.54 0.92 2.40

Studies using V-T ag at Ludger

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14.45 15.00 16.25 17.50 18.75 20.00 21.25 22.50 23.75 24.30

• 1.4e6

0.0e0 2.5e6 5.0e6 7.5e6 1.0e7 1.3e7 1.5e7 1.8e7 2.0e7 1 2 3 4 5 6 7

Comparability of 2-AB and V-Tag

V-Tag labeling of glycopeptides gives comparable results to the gold standard 2-AB labeling of glycans

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7.5 8.8 10.0 11.3 12.5 13.8 15.0 16.3 17.5 18.8 20.0

• 2,000,000

1,250,000 2,500,000 3,750,000 5,000,000 6,250,000 7,500,000 9,000,000 min

FA2/G0F (1) FA2G1/G1F (3) (4)

UHPLC Chromatogram of 2-AB labeled glycans from IgG-1 mAb (PNGase F release) UHPLC chromatogram of V-Tag labeled glycopeptides from IgG-1 mAb (tryptic digestion) 1 2 3 4 5 6 7

PEAK NUMBER AVERAGE REL. % AREA (N=3) 2-AB V-Tag

Comparable in quantitation and in reliability, using a Waters UHPLC Glycan BEH Amide Column (HILIC)

V-Tag 2-AB

FA2B/G0FN (2) FA2G1/G1F FA2BG1/G1FN FA2G2/G2F (5) FA2G1S1/G1FS1 (6) FA2G2S1/G2FS1 (7) FA2/G0F (1) FA2G1/G1F (3) FA2B/G0FN (2) FA2G1/G1F + FA2BG1/G1FN (4) FA2G2/G2F (5) FA2G1S1/G1FS1 (6) FA2G2S1/G2FS1 (7)

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Trypsin Digest V-tag Labeling Total: 3 hrs Total: 8 -21 hrs

The V-Tag protocol is much shorter than 2-AB

IgG 1 V-tag labeled glycopeptides IgG 1 2-AB labeled glycans

1hr 1 hr 1 hr

Clean-up and Enrichment PNGase F release 2-AB Labeling + Reduction

3-16 hr 4 hr 1 hr

Clean-up

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• 1.4e6

V-Tag labeling of glycopeptides 2-AB labeling of N-Glycans

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V-Tag Greatly Enhances MALDI-MS Analysis of Glycopeptides

The signal for underivatised glycopeptides is suppressed in MALDI-MS but is enhanced after V-tag labeling and enrichment

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After V-Tag labeling and enrichment

IgG 1 tryptic glycopeptides

Before V-Tag labeling and enrichment

IgG 1 tryptic peptides and glycopeptides

• Increased signal intensity
• Clean data with good

enrichment of glycopeptides

• Attenuation of

glycopeptide signals

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V-Tag Allows Identification of Glycopeptides using MALDI-MS

Example of V-Tag labeled IgG-1 mAb glycopeptide analysis by negative mode MALDI-MS

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0.00 0.25 0.50 0.75 1.00 1.25 1.50 5 x10

• Intens. [a.u.]

3000 3200 3400 3600 3800 m/z

1000 2000 3000 4000 5000

• Intens. [a.u.]

3150 3200 3250 3300 3350 3400 3450 3500 3550 3600 m/z

2951.224 3113.257 3154.254 3270.16 3275.221 3316.21 3404.208 3432.028 3566.145

Vtag pep Vtag pep (Vtag)2 pep Vtag pep Vtag pep Vtag pep Vtag pep (Vtag)2 pep Vtag pep

M= V-Tag + EEQYNSTYR + Glycan M = 319.33 + 1189.52 + 1444.53 (G0F) M = 2952.11

MALDI-MS ion signals for V-Tag labeled IgG-1 glycopeptides (negative ion mode; Bruker Autoflex)

Incorporating V-T ag into your drug programme

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0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 G0F / FA2 G1F / FA2G1 G2F / FA2G2

QbD Study: The impact of cell culture conditions on glycoform patterns

Variations are reliably detected with V-tag system

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• Chinese hamster ovary (CHO) cell line GS-CY01 expressing a mAb was grown in bio-reactors using different aeration conditions
• Fc galactosylation patterns analysed (i.e. the ratios of the G0F, G1F and G2F glycans) for different aeration conditions.
• Increasing the levels of terminal galactose are known to positively correlate with complement dependent cytotoxicity (CDC) activity
• The cells grown under silicon membrane aeration showed the highest degree of Fc galactosylation (higher abundance of G2F)

Direct gas aeration (high flow) Si membrane aeration Relative % Area

V-tag-PEP V-tag-PEP V-tag-PEP

UHPLC Data Glycan Quantitation

Direct gas aeration (low flow)

Small analytical variation for reliable trend identification

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Automated High Throughput Studies using V-Tag

Adapted to 96-well plate system to use with a liquid handling robot

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Protease Digestion Sample Preparation for MALDI-MS and UHPLC V-Tag Labeling Clean-up and Enrichment Data Acquisition Data Analysis

Afternoon or Overnight Morning

The workflow can be completed in 1 day making this technology a good candidate for high throughput analysis of mAbs

LT-VTAG-24

Next Steps…

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CLICK to contact Sales CLICK to contact Jenifer

Request a quotation

Sales Team

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

Dr Jenifer Hendel

Senior Scientist

jenifer.hendel@ludger.com

If you have a question