mAb Glycopeptide Profiling with V-T ag 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. Our typical V-Tag clients are those who want to: Monitor their mAb drug’s glycosylation Integrate glycoprofiling into the during the product lifecycle. This includes peptide mapping workflow. The V-Tag showing the comparability of glycosylation labeled glycopeptides are analysed throughout the drug lifecycle as well as using orthogonal platforms, MALDI-MS biosimilarity to an innovator’s drug and UHPLC *Glycoprofile = a map of the drug’s glycosylation containing structural ID and relative abundance of each glycan species LT-VTAG-24 2
Options for Analysing mAb Glycosylation Three types of mAb-derived molecules can be analysed to gain information about mAb glycosylation 1. Intact Glycoprotein 2. Glycopeptides 3. Glycans Using lectin affinity, CE or MS Using MS and UHPLC Using MS, UHPLC, CE ✓ Provides information on ✓ Provides both glycan identity ✓ Provides glycan molecular weight and and quantitation as well as identity and quantitation glycoform distribution glycan attachment site and site occupancy Χ Need to release glycans – extra Χ Poor structural information work and expense. No for the glycans. Insufficient for V-Tag is designed for glycosylation site information regulatory work or lot release glycopeptide mapping (for mAbs with Fab and Fc glycosylation) LT-VTAG-24 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 * Glycopeptide signals attenuated in a complex mixture Reduced Reduced, Peptides + Peptide Mapping Intact mAb mAb Alkylated mAb Glycopeptides 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. LT-VTAG-24 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 Quick and Easy Excellent glycopeptide mapping using as Labeling and enrichment little as 10 m g of mAb glycoprotein is completed within 2 hours Integrates Easily with Validated for GMP Labs Peptide Mapping Workflow Validated to ICH Q2(R1) standards and Adds onto your existing peptide mapping tested in glycoprofiling labs workflow, without requiring extra steps for glycan release 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 5
Anatomy of V-Tag The molecular aspects that make V-Tag work Sulphate anion to improve analysis Amine reactive succinimidyl ester in negative ion mode on MALDI to react with the N-terminus amine moiety of the peptide Fluorescent group for detection in UHPLC Simple, non-reactive alkyl chain to l ex = 250nm, l em = 360nm link the fluorescent moiety to the reactive succinimidyl ester LT-VTAG-24 6
Components of the LT-VTAG-24 Kit 1. Labeling 2. Enrichment Reaction buffer Amine reactive Solvent HILIC resin and solvent fluorescent labeling involved in HILIC cartridge reagent clean-up and enrichment LudgerClean A PBS Buffer Tablet V-Tag Labeling Dye TFA 10% (aq.) Cartridges (LC-A) LT-PBS-TAB-0.01M LT-VTAG-01 LC-TFA10PC-01 LC-A-24 LT-VTAG-24 7
Workflow for the V-Tag System: Stage 1 - Labeling Glycopeptides + V-Tag labeled peptides mAb peptides and glycopeptides Protease digestion Labeling V-Tag Dye Use your enzyme of choice + PBS Buffer e.g. sequencing grade trypsin Labels the amine on the N-terminus of the peptide LT-VTAG-24 8
Workflow for the V-Tag System: Stage 2 – Enrichment Analysis by MALDI-MS and UHPLC MALDI-MS Intens. [a.u.] 4 x10 2.0 For glycan 1.5 Identification 1.0 UHPLC 0.5 V-Tag labeled V-Tag labeled peptides Quantitation 0.0 1500 2000 2500 3000 3500 m/z glycopeptides and glycopeptides 2.0e7 counts 1.5e7 1.0e7 5.0e6 0.0e0 Enrichment Orthogonal min -2.0e6 14.0 16.0 18.0 20.0 22.0 24.0 IgG-1 V-Tag labeled glycopeptides LT-VTAG-24 Kit Analyses LC-A cartridges MALDI-MS with TFA solutions and UHPLC Separation of mixture using HILIC cartridge. Conditions optimised for recovery of glycopeptides with glycosylation patterns preserved LT-VTAG-24 9
V-Tag Workflow: Simple and Easy Buffering the mAb digest Labeling Label the digest Remove peptides Enrichment Elute glycopeptides LT-VTAG-24 10
Set-up of Orthogonal Glycoanalytical Platforms Typical setup for analysis of V-Tag labeled glycopeptides by UHPLC and MALDI-MS 2.0e7 counts 1.5e7 1.0e7 5.0e6 UHPLC 0.0e0 min -2.0e6 14.0 16.0 18.0 20.0 22.0 24.0 FLD Thermo Scientific Waters UHPLC Glycan Typical data Fluorescence Detector Dionex U3000 BEH Amide Column (HILIC) V-Tag labeled glycopeptide map of IgG 1 λ ex = 250 nm 30 minute gradient (150mm x 2.1mm) λ em = 360 nm 25 µl injection Temperature: 60 °C Intens. [a.u.] 5 x10 1.50 1.25 1.00 0.75 0.50 MALDI 0.25 0.00 3000 3200 3400 3600 3800 m/z Spot Sample Load Plate Collect Data Typical Data Matrix: 2,5-dihydroxybenzoic Bruker Autoflex Mode: reflectron V-Tag labeled glycopeptide map of IgG 1 acid (DHB) MALDI-MS instrument negative ion 11 LT-VTAG-24
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 2.0e7 counts counts min -2.0e6 1.8e7 counts min -2.0e6 1.8e7 1.5e7 counts min -2.0e6 1.8e7 counts min -2.0e6 2.0e7 1.0e7 counts min -2.0e6 2.0e7 counts min -2.0e6 5.0e6 2.2e7 counts min -2.0e6 2.2e7 counts min -2.0e6 0.0e0 1.5e7 counts min -2.0e6 min -3.2e6 14.0 16.0 18.0 20.0 22.0 24.0 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) Overlaid IgG 1 V-Tag labeled glycopeptide UHPLC profiles (9 replicates) Validation studies typically 1 2 3 4 5 6 7 Peak Number show repeatability with CVs G0F G0FB G1F G1F + G1FB G2F G1FS1 A1F for relative abundances < 4% Glycopeptide (FA2) (FA2B) (FA2G1) (FA2G1 + FA2BG1) (FA2G2) (FA2G1S1) (FA2G2S1) Av. 50.2 4.2 25.8 12.6 3.8 1.8 1.7 Relative Std. Dev. 0.35 0.14 0.18 0.26 0.10 0.02 0.04 % Area 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 LT-VTAG-24 12
Studies using V-T ag 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 Comparable in quantitation and in reliability, using a FA2G1/G1F (3) 2-AB FA2/G0F (1) 7,500,000 Waters UHPLC Glycan BEH Amide Column (HILIC) (4) 6,250,000 FA2G1S1/G1FS1 (6) FA2G2S1/G2FS1 (7) 5,000,000 FA2G1/G1F FA2G2/G2F (5) FA2B/G0FN (2) FA2BG1/G1FN 3,750,000 2,500,000 1,250,000 AVERAGE REL. % AREA (N=3) 0 min 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 UHPLC Chromatogram of 2-AB labeled glycans from IgG-1 mAb (PNGase F release) 2.0e7 FA2G1/G1F (3) 1 counts FA2/G0F (1) 1.8e7 V-Tag FA2BG1/G1FN (4) 1.5e7 FA2G1/G1F + FA2G2S1/G2FS1 (7) FA2G1S1/G1FS1 (6) 1.3e7 FA2B/G0FN (2) FA2G2/G2F (5) 3 1.0e7 1 2 3 4 5 6 7 7.5e6 PEAK NUMBER 2-AB V-Tag 4 5.0e6 2.5e6 2 5 6 7 0.0e0 min -1.4e6 14.45 15.00 16.25 17.50 18.75 20.00 21.25 22.50 23.75 24.30 UHPLC chromatogram of V-Tag labeled glycopeptides from IgG-1 mAb (tryptic digestion) LT-VTAG-24 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 2-AB Labeling Clean-up release + Reduction IgG 1 2-AB labeled glycans V-Tag labeling of glycopeptides Total: 3 hrs 2.0e7 counts 1 1.8e7 1 hr 1 hr 1hr 1.5e7 1.3e7 V-tag Clean-up and Trypsin 3 1.0e7 7.5e6 Labeling Enrichment 4 Digest 5.0e6 2.5e6 2 5 6 7 0.0e0 min -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 LT-VTAG-24
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 After V-Tag labeling and enrichment IgG 1 tryptic peptides and glycopeptides IgG 1 tryptic glycopeptides • • Attenuation of Increased signal intensity • glycopeptide signals Clean data with good enrichment of glycopeptides LT-VTAG-24 16
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