Accelerate mab Characterization Using Automated Sample Prep David Knorr, Ph.D. Automation Solutions Ning Tang, Ph.D. LC/MS 15 February 2012 Page 1
Protein Sample Processing Workflows Glycan Profiling Biological system Crude Sample Prep Method Specific Peptide Mapping Clinical patient Oxidation Characterization Manufacturing process Sample Titer Page 2
Agilent AssayMAP Bravo Liquid Handling Platform Page 3
Agilent Bravo Liquid Handler Best-in-class liquid handling Accessorize deck for any application VWorks control software Page 4
AssayMAP Cartridges Chromatography columns on a micro scale Page 5
AssayMAP Bravo Head Different pipetting modes Page 6
AssayMAP Bravo vs. Pipette Tips Quantitative recovery in fewer steps / lower volume Single pass Accurate flow control No air bubbles Multi pass Variable flow control Air bubbles Page 7
Current AssayMAP BioPharma Portfolio General Ab Capture Protein A DIY Captures Streptavidin Protein Digestion Trypsin Sample Clean Up Reverse Phase In development Contaminant ELISA N-glycan sample prep 8
Form-Driven Protocols Page 9
Protein A for Ab Purification Sample Analysis 10
µg Eluted Protein A Performance Characteristics Quantitative Binding & Elution to 100 μg 200 160 0.7 120 80 40 0.6 0.5 0.4 0 0 50 100 150 200 250 µg higg Loaded No load volume effect over linear range A 280 0.3 0.2 0.1 0.0 0 20 40 60 80 100 µg higg loaded 10 µl 30 µl 90 µl 250 µl Page 11
Protein A Performance Characteristics Purification is consistent across a rack 0.8 0.6 %CV = 1.26 and across cartridges. A 280 0.4 0.2 0.0 0 20 40 60 80 100 Sample Number µg higg loaded 11 15 19 25 33 44 58 76 100 132 173 228 %CV 1.6 1.3 1.4 1.0 1.4 1.2 1.4 1.4 1.9 1.9 1.5 1.0 Page 12
User- Defined Affinity Purification with Streptavidin (SA-W) Anything you can biotinylate can be a capture ligand! 13
μg higg Eluted Streptavidin (SA) Custom affinity purification examples Elution of Hu IgG from biotinylated protein A 100 80 60 40 20 0 0 20 40 60 80 μg of Biotin-Protein A Immobilized Elution of FLAG-BAP from biotinylated αflag SA Page 14
Sample Fractionate, Clean up, Analyze On-Cartridge Trypsinization On Bravo Deck or Offline Page 15
mau mau mau mau Trypsin Cartridge (TR) TR-W comparisons 200 DAD: Signal A, 215 nm/bw:4 nm DAD: Signal A, 215 nm/bw:4 nm 200 180 160 140 120 Urea 1.11 pre-digest urea 1.11 pre-digest 9-29-2011 1-12-00 pm.dat Urea Urea 1.11 digest urea 1.11 digest 9-29-2011 5-20-09 pm.dat 180 160 140 120 Analyte: BSA Denaturant: 6M Urea (1.2M final) Flow Rate: 2 μl/min. Load: 67 μg RP-HPLC 100 100 80 80 60 60 TR-W comparisons 40 40 200 DAD: Signal A, 215 nm/bw:4 nm DAD: Signal A, 215 nm/bw:4 nm 200 TFE 18.1 pre-digest TFE 4.50 digest tfe 18.1 pre-digest 9-29-2011 4-18-06 pm.dat tfe 4.50 digest 9-29-2011 6-53-15 pm.dat 20 4 6 8 10 12 14 16 18 20 22 20 180 160 TFE 180 160 Minutes 140 140 120 120 Analyte: BSA Denaturant: 50% TFE (5% final) Flow Rate: 2 μl/min. Load: 67 μg RP-HPLC 100 80 60 40 100 80 60 40 20 20 0 4 6 8 10 12 14 16 18 20 22 0 Elution and coverage comparable to in-solution digestion. In Progress: antibody digestion with LC/MS characterization. Minutes Page 16
Peptide Digest Analyze Reverse Phase for Peptides Digest clean up and fractionation Page 17
A280 Reverse Phase (RP) Quantitative binding and elution to ~400 μg 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 2µL/min 5µL/min 10µL/min 20µL/min Complete elution in 2 column volumes 0.2 0.0 5 10 15 20 25 30 35 40 45 50 Cumulative Elution Volume (µl) Page 18
Reverse Phase Cleanup of BSA Cleanup with a leading pipette tip Page 19
Example Workflow from Sample to Analysis (in progress) Protein A or Streptavidin On or Off Bravo Trypsin Reverse Phase Agilent HPLC or LCMS Automated Sample preparation with AssayMAP Bravo Page 20
GlykoPrep N-Glycan Kit Collaboration with Crude Ab sample Purified glycoprotein Purify with Protein A Denature & immobilize Digest with N-Glycanase (PNGaseF) Fluorescent label Cleanup & elute Analysis by HPLC, CE, LC/MS 21
N-Glycan Profile Analysis Conventional method Manual only 3 days 10-30 samples/run AssayMAP method Manual or automated No drying steps 3 hours Up to 192 samples/run 22
% of Total Peak Area GlykoPrep System Results- (3 hr manual mode) Comparison to Standard Methods Reproducibility 25 12 consecutive runs α-1 Acid Glycoprotein 20 15 10 5 CV s = 2-5 % 0 0 20 40 60 80 100 Sample Peak 1 2 3 4 5 6 23
Antibody Characterization Example Monoclonal antibody production in CHO cells Hu IgG / mab purification from CHO culture supernatants (CSS) Effect of high amounts of FBS Different initial antibody concentrations 10, 50, 100, 500 µg/ml (triplicate samples) Mimic screening to scale-up MW determination N-Glycan profiling (mab-glyco Chip) Page 24
AssayMAP Cartridge Performance Different loads and sample backgrounds Page 25
mab Characterization Using LC/MS Intact molecular weight Glycan Analysis 26 Agilent Confidential February 17, 2012
Agilent s LC/MS Systems for Biopharma Applications HPLC-Chip QTOF/TOF Applications Intact protein analysis Peptide mapping PTM analysis Glycan analysis
Intact Protein Analysis Zorbax SB300-C8 HPLC-Chip 52+ 28 Agilent Confidential February 17, 2012
Intact mab Analysis Control mab Sample 1 +CCS Sample 2 -CCS Sample 3 PBS
HPLC-Chip/MS Solution for Glycan Analysis mab-glyco Chip Workflow mab sample LC- or CE- Fluorescence MALDI MS mab-glyco Chip MS Experimental Time // 10-30 minutes PNGase F, enzymatic N-glycan release N-Glycan concentration 4 minutes 1 minute // 3-8 hours N-Glycan separation 2 minutes 2-4 days TOF MS Detector 30 Agilent Confidential February 17, 2012
mab-glyco Chip-LC/MS Solution Total analysis time of 12 min with great chromatographic resolution
mab-glyco Chip-LC/MS Solution PCDL Personal Compound Database and Library
Relative molar distribution [mol%] Comparison of mab-glyco Chip with CE and MALDI MS Results 70 60 50 40 30 MALDI-MS (N=34) CE-LIF (N=43) Agilent Glycan Workflow Chip (N=50) Agilent Glycan Workflow Chip (N=50) Agilent Glycan Workflow Chip (N=50) 20 10 0 Man-5 G0-GlcNAc G0-F G0 G1 G2
mab-glyco Chip-LC/MS Solution Chip Reproducibility
Glycan Analysis G0F G1F G2F control sample 1 sample 2 sample 3
Conclusions The Agilent AssayMap Bravo platform enables automated sample preparation for biologic characterization. Agilent HPLC-Chip LC/MS system provides fast analysis of therapeutic proteins. Thank You! Page 36