Agilent Technologies, Inc. · NYSE:A
Agilent Technologies — Technical Presentation (September 2011)
26 slides from Agilent Technologies's technical presentation, Sep 21, 2011
Slide 1: A High Throughput Microchromatography Platform for Quantitative Analytical Scale Protein Sample Preparation
A High Throughput Microchromatography Platform for Quantitative Analytical Scale Protein Sample Preparation Scott Fulton BioSystem Development, LLC Madison, WI USA www.biosystemdevelopment.com
Slide 2: Protein Sample Processing Bottleneck
Protein Sample Processing Bottleneck Biological Plate reader system Clinical patient HPLC Manufacturing process Mass spectrometer Samples Instruments
Slide 3: AssayMAP Cartridges
AssayMAP® Cartridges g os | q | \| i packed vd resin bed ty 4 sample cup | i. si \ q \ if inlet seal ‘ thy bed support 5 ul packed resin bed bed support
Slide 4: Two Operating Modes
Two Operating Modes Spin Format Probe Syringe • Fully quantitative binding & elution • Fully quantitative binding & elution • Compatible with microplate systems • Compatible with microplate systems • Uses standard liquid handling • Precise, bidirectional flow control • Limited flow control & resin o
Slide 5: Probe Syringe Liquid Handling
Probe Syringe Liquid Handling
Slide 6: AssayMAP Bravo 96-Channel Workstation
AssayMAP Bravo 96-Channel Workstation 96 probe syringe head
Slide 7: AssayMAP vs. Pipet Tip Columns
AssayMAP vs. Pipet Tip Columns AssayMAP Cartridge Pipet Tip Column • Separate entry & exit ports • Single entry/exit port • Single-pass contact • Multi-pass contact (“true chromatography”) (equilibrium adsorption) • Controlled flow rate • Highly variable flow rate • No air entrainment • Air entrainm
Slide 8: Ab Purification on Protein A (PA-W)
Ab Purification on Protein A (PA-W)
Slide 9: PA-W: Effect of Flow Rate
PA-W: Effect of Flow Rate Binding (Dynamic Capacity) Elution
Slide 10: PA-W: Reproducibility & Precision
PA-W: Reproducibility & Precision 1.2 1.0 100 µg load 0.8 N = 96 A280 0.6 %CV = 1.26 0.4 0.2 0.0 0 50 100 150 200 250 µg hIgG Loaded N=8 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
Slide 11: Streptavidin (SA-W) Cartridge
Streptavidin (SA-W) Cartridge Ligand Antigen Specific Antibody Immobilization Purification Purification Anti-FLAG Ab immobilized on SA-W Cartridges Washed 1X in PBS
Slide 12: Human IgG Purified from Rat Serum
Human IgG Purified from Rat Serum Protein A CaptureSelect® hFc Ligand PBS Rat Serum PBS Rat Serum 20 – 0 µg hIgG spiked into PBS or rat serum, purified using the ligands shown • Camelid VHH domain ligands (14 kD, manufactured in yeast) • Available ligands for antibodies – Human Fc, all-species Fc –
Slide 13: Fluorescent Ligand Binding Assay
Fluorescent Ligand Binding Assay CaptureSelect hFc Neutralize Protein A µg hIgG 20 10 5 2.5 1.3 0.63 0.31 0.16
Slide 14: AssayMAP ELISA vs. Microplate ELISA
AssayMAP ELISA vs. Microplate ELISA Coat Block Sample Conjugate Substrate Read Micron scale IA diffusion path AssayMAP 15 – 30 minutes turnaround Millimeter scale diffusion path Microplate 4 – 24 hours turnaround
Slide 15: Leached Protein A ELISA
Leached Protein A ELISA 3.0 ng/mL Protein A Run 1 Run 2 Mean St Dev %CV 2.5 0 0.083 0.077 R² = 0.992 0 0.090 0.083 0.083 0.005 6.4 2.0 0.1 0.103 0.112 A450 0.1 0.098 0.099 0.103 0.006 6.2 0.6 0.183 0.211 1.5 0.6 0.199 0.194 0.197 0.012 5.9 Run 1 1.5 0.468 0.478 1.0 Run 2 1.5 0.473 0.508 0.482 0.018
Slide 16: Non-Porous Bead Technology
Non-Porous Bead Technology Non-Porous (N) Wide Pore (W) Loading capacity ~1 µg ~100 µg Elution volume 3 µL 20 µL 1000 fmol load
Slide 17: Peptide Mapping Analysis Workflow
Peptide Mapping Analysis Workflow Denature Reduce Enzyme Peptide Purify Alkylate Digest Cleanup LC/MS • Integrated automation of purification, digestion & cleanup • Rapid digestion with immobilized trypsin cartridge • High throughput (up to 96 samples/run) • Excellent reproducibility
Slide 18: Automated Tryptic Digestion
Automated Tryptic Digestion Protein: bSA Mass load: 125 µg Digestion volume: 50 µL Flow rate: 2 µL/min Digestion time: 25 min 2 separate digestions run side-by-side
Slide 19: Reversed-Phase Peptide Cleanup
Reversed-Phase Peptide Cleanup Dynamic Capacity Elution Profiles
Slide 20: N-Glycan Profile Analysis
N-Glycan Profile Analysis • Current method – Manual only – 3 days to complete – 10 - 30 samples/run • AssayMAP method – Manual or automated – No drying steps – 3 hours to complete – Up to 192 samples/run
Slide 21: GlykoPrep System Results
GlykoPrep System Results Comparison to Standard Methods HILIC HPLC chromatograms with fluorescence detection Reproducibility 25 12 consecutive runs α-1 Acid Glycoprotein 20 % of Total Peak Area Peak 15 1 2 10 3 4 5 5 6 0 0 20 40 60 80 100 Sample
Slide 22: Parallel Micro-Chromatography
Parallel Micro-Chromatography Cytochrome C/ribonuclease A on cation exchange resin Columns 1 – 12 = 0 – 100 mM NaCl wash
Slide 23: Cation Exchange Gradient Scouting
Cation Exchange Gradient Scouting Equilibrate Load pH 4.5 pH 6.0 cartridge sample Wash Elute 50% RNase A 50% RNase A Cytochrome C 40% 40% Cytochrome C % of Feed % of Feed 30% 30% 20% 20% 10% 10% 0% 0% 0 100 200 300 400 500 0 100 200 300 400 500 mM NaCl mM NaCl pH 5.0 pH 6.5 50% 50% RNase A RNase A 4
Slide 24: Purification Method Development
Purification Method Development System Method Development Multi-Dimensional Separation Conventional First Column Second Column AssayMAP
Slide 25: AssayMAP Cartridge Pipeline
AssayMAP Cartridge Pipeline • Affinity purification • Cleanup/Extraction – Protein A – Reversed-phase – Streptavidin (porous) – Cation exchange – Streptavidin (non-porous) – Anion exchange – Protein G, L, etc. – Hydrophilic interaction (HILIC) – BAC Vhh ligands – Porous graphitic carbon – IMAC – Spe
Slide 26: Summary of AssayMAP Bravo
Summary of AssayMAP Bravo • High throughput microchromatography platform – 5 µL packed bed cartridge with any 15 – 100 µm particle resin – 96-channel ANSI/SBS microplate format – Single pass, fully quantitative binding & elution – Positive displacement, bidirectional flow control – Integrated workfl
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