mab Glycopeptide Profiling with V-Tag Adding reliable glycoprofiling to your peptide mapping workflow with ease and simplicity

mab Glycopeptide Profiling with V-Tag Adding reliable glycoprofiling to your peptide mapping workflow with ease and simplicity

Who is the V-Tag Glycoprofiling Technology for? V = Velocity 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. ur 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 Integrate glycoprofiling into the peptide mapping workflow. The V-Tag labeled glycopeptides are analysed using orthogonal platforms, MALDI-MS and UHPLC *Glycoprofile = a map of the drug s glycosylation containing structural ID and relative abundance of each glycan species 2

ptions for Analysing mab Glycosylation Three types of mab-derived molecules can be analysed to gain information about mab glycosylation 1. Intact Glycoprotein Using lectin affinity, CE or MS 2. Glycopeptides Using MS and UHPLC 3. Glycans Using MS, UHPLC, CE Provides information on molecular weight and glycoform distribution Χ Poor structural information for the glycans. Insufficient for regulatory work or lot release Provides both glycan identity and quantitation as well as glycan attachment site and site occupancy V-Tag is designed for glycopeptide mapping 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) 3

Adding Glycoprofiling to the Traditional Peptide Mapping Workflow Incorporating glycopeptide mapping can reduce the time and costs of glycoprofiling work in drug production monitoring RP-HPLC peptide map of anti-cd4 IgG-1 * Intact mab Reduced mab Reduced, Alkylated mab Peptides + Glycopeptides Glycopeptide signals attenuated in a complex mixture Peptide Mapping Glycopeptide Mapping The V-Tag system labels and enriches mab glycopeptides efficiently, allowing deteration of their structures and relative quantities * J. Bongers et al. J. Pharm. Biomed. Anal. 2000, 21, 1099-1128. 4

Highlights of the V-Tag System Reliable mab Glycoprofiling Allows glycan identification and quantitation using the orthogonal analyses of MALDI-MS and UHPLC. Provides data comparable to gold-standard glycoprofiling methods based on 2-AB or 2-AA Minimal Sample Needed Excellent glycopeptide mapping using as little as 10 µg of mab glycoprotein Quick and Easy Labeling and enrichment is completed within 2 hours 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 Integrates Easily with Peptide Mapping Workflow Adds onto your existing peptide mapping workflow, without requiring extra steps for glycan release 5

Anatomy of V-Tag The molecular aspects that make V-Tag work Sulphate anion to improve analysis in negative ion mode on MALDI Ae reactive succinimidyl ester to react with the N-terus ae moiety of the peptide S 3 H N N Fluorescent group for detection in UHPLC λ ex = 250nm, λ em = 360nm Simple, non-reactive alkyl chain to link the fluorescent moiety to the reactive succinimidyl ester 6

Components of the Kit 1. Labeling 2. Enrichment Reaction buffer and solvent Ae reactive fluorescent labeling reagent HILIC resin cartridge Solvent involved in HILIC clean-up and enrichment PBS Buffer Tablet LT-PBS-TAB-0.01M V-Tag Labeling Dye LT-VTAG-01 LudgerClean A Cartridges (LC-A) LC-A-24 TFA 10% (aq.) LC-TFA10PC-01 7

Workflow for the V-Tag System: Stage 1 - Labeling S 3 H N HN S 3 H N HN S 3 H N HN S 3 H N N mab Glycopeptides + peptides V-Tag labeled peptides and glycopeptides Protease digestion Use your enzyme of choice e.g. sequencing grade trypsin Labeling V-Tag Dye + PBS Buffer Labels the ae on the N-terus of the peptide 8

Workflow for the V-Tag System: Stage 2 Enrichment Analysis by MALDI-MS and UHPLC S 3 H N HN S 3 H N HN S 3 H N HN S 3 H N HN Intens. [a.u.] x10 4 2.0 1.5 MALDI-MS For glycan Identification 1.0 V-Tag labeled peptides and glycopeptides V-Tag labeled glycopeptides 0.5 0.0 2.0e7 1.5e7 1500 2000 2500 3000 3500 m/z UHPLC Quantitation 1.0e7 5.0e6 Enrichment Kit LC-A cartridges with TFA solutions Separation of mixture using HILIC cartridge. Conditions optimised for recovery of glycopeptides with glycosylation patterns preserved rthogonal Analyses MALDI-MS and UHPLC 0.0e0 14.0 16.0 18.0 20.0 22.0 24.0 IgG-1 V-Tag labeled glycopeptides 9

V-Tag Workflow: Simple and Easy Enrichment Labeling Buffering the mab digest Label the digest Remove peptides Elute glycopeptides 10

Set-up of rthogonal Glycoanalytical Platforms Typical setup for analysis of V-Tag labeled glycopeptides by UHPLC and MALDI-MS 2.0e7 1.5e7 1.0e7 5.0e6 UHPLC Thermo Scientific Dionex U3000 (150mm x 2.1mm) Temperature: 60 C FLD Fluorescence Detector 16.0 18.0 20.0 22.0 24.0 Typical data V-Tag labeled glycopeptide map of IgG 1 λex = 250 nm λem = 360 nm Intens. [a.u.] 30 ute gradient 25 µl injection Waters UHPLC Glycan BEH Amide Column (HILIC) 0.0e0 14.0 x105 1.50 1.25 1.00 0.75 0.50 MALDI 0.25 0.00 3000 Spot Sample Matrix: 2,5-dihydroxybenzoic acid (DHB) Load Plate Bruker Autoflex MALDI-MS instrument Collect Data Mode: reflectron negative ion 3200 3400 3600 3800 m/z Typical Data V-Tag labeled glycopeptide map of IgG 1 11

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 2.0e7 1.8e7 2.0e7 1.8e7 1.5e7 1.8e7 2.0e7 1.0e7 2.0e7 2.2e7 5.0e6 2.2e7 1.5e7-3.2e6 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0 24.0 Stacked IgG 1 V-Tag labeled glycopeptide UHPLC profiles (9 replicates) 0.0e0 14.0 16.0 18.0 20.0 22.0 24.0 verlaid IgG 1 V-Tag labeled glycopeptide UHPLC profiles (9 replicates) Validation studies typically show repeatability with CVs for relative abundances < 4% 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 Average relative % area, SD and CVs for V-Tag labeled IgG 1 glycopeptides 12

Studies using V-Tag at Ludger

Comparability of 2-AB and V-Tag V-Tag labeling of glycopeptides gives comparable results to the gold standard 2-AB labeling of glycans 9,000,000 7,500,000 6,250,000 5,000,000 3,750,000 2,500,000 1,250,000 0 2,000,000-7.5 8.8 10.0 11.3 12.5 13.8 15.0 16.3 17.5 18.8 20.0 2.0e7 1.8e7 1.5e7 1.3e7 1.0e7 7.5e6 5.0e6 2.5e6 0.0e0 FA2/G0F (1) 1 FA2G1/G1F (3) 2 (4) UHPLC Chromatogram of 2-AB labeled glycans from IgG-1 mab (PNGase F release) FA2/G0F (1) FA2B/G0FN (2) FA2B/G0FN (2) FA2G1/G1F FA2BG1/G1FN FA2G1/G1F (3) 3 4 FA2G2/G2F (5) FA2G1/G1F + FA2BG1/G1FN (4) -1.4e6 14.45 15.00 16.25 17.50 18.75 20.00 21.25 22.50 23.75 24.30 FA2G1S1/G1FS1 (6) FA2G2/G2F (5) 5 FA2G1S1/G1FS1 (6) FA2G2S1/G2FS1 (7) 2-AB FA2G2S1/G2FS1 (7) 6 7 V-Tag UHPLC chromatogram of V-Tag labeled glycopeptides from IgG-1 mab (tryptic digestion) Comparable in quantitation and in reliability, using a Waters UHPLC Glycan BEH Amide Column (HILIC) AVERAGE REL. % AREA (N=3) 1 2 3 4 5 6 7 PEAK NUMBER 2-AB V-Tag 14

The V-Tag protocol is much shorter than 2-AB 2-AB labeling of N-Glycans Total: 8-21 hrs 3-16 hr 4 hr 1 hr PNGase F release 2-AB Labeling + Reduction Clean-up IgG 1 2-AB labeled glycans V-Tag labeling of glycopeptides Total: 3 hrs 1hr 1 hr 1 hr Trypsin Digest V-tag Labeling Clean-up and Enrichment 2.0e7 1.8e7 1.5e7 1.3e7 1.0e7 7.5e6 5.0e6 2.5e6 1 2 3 4 5 6 7 0.0e0-1.4e6 14.45 15.00 16.25 17.50 18.75 20.00 21.25 22.50 23.75 24.30 IgG 1 V-tag labeled glycopeptides 15

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 Before V-Tag labeling and enrichment IgG 1 tryptic peptides and glycopeptides After V-Tag labeling and enrichment IgG 1 tryptic glycopeptides Intens. [a.u.] x10 4 2.5 A Attenuation of glycopeptide signals Intens. [a.u.] 5 x10 1.0 B Increased signal intensity Clean data with good enrichment of glycopeptides 2.0 0.8 1.5 0.6 1.0 0.4 0.5 0.2 0.0 1000 1500 2000 2500 3000 3500 m/z 0.0 1000 1500 2000 2500 3000 3500 m/z 16

V-Tag Allows Identification of Glycopeptides using MALDI-MS Example of V-Tag labeled IgG-1 mab glycopeptide analysis by negative mode MALDI-MS S 3 H N HN Intens. [a.u.] x10 5 1.50 1.25 2951.224 Vtag pep 3154.254 Vtag pep 3270.16 (Vtag) 2 pep 3275.221 Vtag pep 3316.21 Vtag pep 3404.208 Vtag pep 3432.028 (Vtag) 2 pep 3566.145 Vtag pep 1.00 Intens. [a.u.] 5000 M= V-Tag + EEQYNSTYR + Glycan M = 319.33 + 1189.52 + 1444.53 (G0F) 0.75 3113.257 Vtag pep 4000 3000 M = 2952.11 0.50 2000 0.25 1000 0 3150 3200 3250 3300 3350 3400 3450 3500 3550 3600 m/z 0.00 3000 3200 3400 3600 3800 MALDI-MS ion signals for V-Tag labeled IgG-1 glycopeptides (negative ion mode; Bruker Autoflex) m/z 17

Incorporating V-Tag into your drug programme

QbD Study: The impact of cell culture conditions on glycoform patterns Variations are reliably detected with V-tag system 70.00 60.00 Small analytical variation for reliable trend identification UHPLC Data Glycan Quantitation Relative % Area 50.00 40.00 30.00 20.00 10.00 0.00 G0F / FA2 G1F / FA2G1 G2F / FA2G2 Si membrane aeration Direct gas aeration (low flow) Direct gas aeration (high flow) V-tag-PEP V-tag-PEP V-tag-PEP Chinese hamster ovary (CH) 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 teral 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) 19

Automated High Throughput Studies using V-Tag Adapted to 96-well plate system to use with a liquid handling robot Morning Afternoon or vernight Protease Digestion V-Tag Labeling Clean-up and Enrichment Sample Preparation for MALDI-MS and UHPLC Data Acquisition Data Analysis The workflow can be completed in 1 day making this technology a good candidate for high throughput analysis of mabs 20

Next Steps If you have a question Request a quotation CLICK to contact Jenifer Dr Jenifer Hendel Senior Scientist jenifer.hendel@ludger.com CLICK to contact Karen Karen akes Sales Manager karen.oakes@ludger.com 21