Accelerate mab Characterization Using Automated Sample Prep

Transcription

1 Accelerate mab Characterization Using Automated Sample Prep David Knorr, Ph.D. Automation Solutions Ning Tang, Ph.D. LC/MS 15 February 2012 Page 1

2 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

3 Agilent AssayMAP Bravo Liquid Handling Platform Page 3

4 Agilent Bravo Liquid Handler Best-in-class liquid handling Accessorize deck for any application VWorks control software Page 4

5 AssayMAP Cartridges Chromatography columns on a micro scale Page 5

6 AssayMAP Bravo Head Different pipetting modes Page 6

7 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

8 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

9 Form-Driven Protocols Page 9

10 Protein A for Ab Purification Sample Analysis 10

11 µg Eluted Protein A Performance Characteristics Quantitative Binding & Elution to 100 μg µg higg Loaded No load volume effect over linear range A µg higg loaded 10 µl 30 µl 90 µl 250 µl Page 11

12 Protein A Performance Characteristics Purification is consistent across a rack %CV = 1.26 and across cartridges. A Sample Number µg higg loaded %CV Page 12

13 User- Defined Affinity Purification with Streptavidin (SA-W) Anything you can biotinylate can be a capture ligand! 13

14 μg higg Eluted Streptavidin (SA) Custom affinity purification examples Elution of Hu IgG from biotinylated protein A μg of Biotin-Protein A Immobilized Elution of FLAG-BAP from biotinylated αflag SA Page 14

15 Sample Fractionate, Clean up, Analyze On-Cartridge Trypsinization On Bravo Deck or Offline Page 15

16 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 Urea 1.11 pre-digest urea 1.11 pre-digest pm.dat Urea Urea 1.11 digest urea 1.11 digest pm.dat Analyte: BSA Denaturant: 6M Urea (1.2M final) Flow Rate: 2 μl/min. Load: 67 μg RP-HPLC TR-W comparisons 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 pm.dat tfe 4.50 digest pm.dat TFE Minutes Analyte: BSA Denaturant: 50% TFE (5% final) Flow Rate: 2 μl/min. Load: 67 μg RP-HPLC Elution and coverage comparable to in-solution digestion. In Progress: antibody digestion with LC/MS characterization. Minutes Page 16

17 Peptide Digest Analyze Reverse Phase for Peptides Digest clean up and fractionation Page 17

18 A280 Reverse Phase (RP) Quantitative binding and elution to ~400 μg µL/min 5µL/min 10µL/min 20µL/min Complete elution in 2 column volumes Cumulative Elution Volume (µl) Page 18

19 Reverse Phase Cleanup of BSA Cleanup with a leading pipette tip Page 19

20 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

21 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

22 N-Glycan Profile Analysis Conventional method Manual only 3 days samples/run AssayMAP method Manual or automated No drying steps 3 hours Up to 192 samples/run 22

23 % of Total Peak Area GlykoPrep System Results- (3 hr manual mode) Comparison to Standard Methods Reproducibility consecutive runs α-1 Acid Glycoprotein CV s = 2-5 % Sample Peak

24 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

25 AssayMAP Cartridge Performance Different loads and sample backgrounds Page 25

26 mab Characterization Using LC/MS Intact molecular weight Glycan Analysis 26 Agilent Confidential February 17, 2012

27 Agilent s LC/MS Systems for Biopharma Applications HPLC-Chip QTOF/TOF Applications Intact protein analysis Peptide mapping PTM analysis Glycan analysis

28 Intact Protein Analysis Zorbax SB300-C8 HPLC-Chip Agilent Confidential February 17, 2012

29 Intact mab Analysis Control mab Sample 1 +CCS Sample 2 -CCS Sample 3 PBS

30 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 // 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

31 mab-glyco Chip-LC/MS Solution Total analysis time of 12 min with great chromatographic resolution

32 mab-glyco Chip-LC/MS Solution PCDL Personal Compound Database and Library

33 Relative molar distribution [mol%] Comparison of mab-glyco Chip with CE and MALDI MS Results 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) Man-5 G0-GlcNAc G0-F G0 G1 G2

34 mab-glyco Chip-LC/MS Solution Chip Reproducibility

35 Glycan Analysis G0F G1F G2F control sample 1 sample 2 sample 3

36 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