A High Throughput Microchromatography Platform

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1 Agilent s NEW AssayMAP Bravo A High Throughput Microchromatography Platform for Quantitative Analytical Scale Protein Sample Preparation

2 The Agilent Bravo Liquid Handler Best in Class Compact Liquid Handling High accuracy pipetting from 300nL to 250μL into 96, 384 and 1536 Whole Plate, Column/Row-wise, Single Tip Transfer pipetting Can use for example Eppendorf Cups, 24 well cell culture plates etc... head change in 30s (96 to 384 or AssayMAP head for example) On deck plate stacking & tip nesting (with gripper) Re-use of tips up to 100 times (Washing station available) Adapt the Deck to Nearly Any Application On-deck barcode reading, plate heating & cooling, shaking. Auto-filling reservoir, vacuum locating of warped PCR plates. Magnetic bead separations, vacuum filtration. Applications All liquid handling tasks like: serial dilution, normalisation, hitpicking, cell culture, ELISA, screening, genomics (DNA/RNA-Extraction, NGS, PCR...), InGelDigest, many others more...

3 The AssayMAP Bravo The AssayMAP Bravo is a solution for quantitative, analytical scale protein sample preparation to deliver highly parallel microscale purification to biologics discovery & development. The AssayMAP Bravo enables workflows in biologics discovery & development for MAb titer and characterization including cell line selection & cell culture optimization rapid ELISA protein capture and purification chromatography method development. Page 3

4 What is the AssayMAP Microchromatography Technology? Page 4

5 One pot. Bottleneck in Protein Sample Processing Biological system Plate reader Clinical patient HPLC Manufacturing process Mass spectrometer Samples Instruments

6 AssayMAP Bravo Head & Syringe Bravo 96AM head with AssayMAP technology Direct displacement, ultra-low dead volume syringe with probe. Liquid handling via probes directly, through the cartridge. Unidirectional liquid handling through cartridges for fine control of separation and true, single pass, chromatography. Fully enabled for Bravo liquid handling pipette tips can be pressed on the probe, partial head, deck accessories,.

7 AssayMAP Cartridges Chromatography column on a micro scale. Various chromatographic resin as packed beds between two supports Can be affinity resins or immobilized enzymes IEX, RP, HIC, HILIC,. Streptavidin, protein A/G, lectin, metal chelates,. Trypsin, PNGase,.

8 AssayMAP vs. Pipett Tip Columns AssayMAP Cartridge Pipett Tip Column Separate entry & exit ports Single-pass contact ( true chromatography ) Controlled flow rate No air entrainment Fully quantitative binding & elution Single entry/exit port Multi-pass contact (equilibrium adsorption) Less controllable flow rate Air entrainment Non-quantitative extraction

9 AssayMAP Bravo VWorks Software VWorks VWorks Task Macros for AssayMAP Tasks Flow rate control. Page 9

10 AssayMAP Bravo - VWorks Data entry form VWorks Form VWorks Software features for AssayMAP protocols. VWorks forms & protocols packaged with each AssayMAP application. User specified flow rate control to fine tune AssayMAP parameters. flow to equilibrate, slow flow to bind sample. Fast Page 10

11 AssayMAP Bravo MAb Purification on Protein A

12 AssayMAP Bravo MAb Purification on Protein A Quantitative recovery, excellent purity & reproducibility with no detectable carryover.

13 AssayMAP Toolkit Surface Chemistries Available Now Protein A (PA) Streptavidin (SA) Wide Pore (W) Bead Types 40 µm polymer beads Perfusive wide pore ~100 µg IgG coating capacity Hydrophobic Binding Surface (RP-C 18 ) CaptureSelect Ligands (human Fc) Non-Porous (N) 40 µm glass beads Non-porous ~1 µg IgG coating capacity Still to come: IEX, other hydrophobic, IMAC/metal chelaters, other affinity (lectins, protein G, ), immobilized enzyme (proteases, glycosidases ) and custom resins.

14 Example: Rapid ELISA AssayMAP Microplate Micron scale diffusion path minutes turnaround Millimeter scale diffusion path 4 24 hours turnaround Coat Block Sample Conjugate Substrate Read

15 Protein Biomarker Quantitation for LC/MS Prefilter Digestion Purification Analysis In Dev In Dev Now! Complete SISCAPA sample preparation on the AssayMAP Bravo. Plasma Proteome Institute 15

16 AssayMAP Bravo MAb Purification on Protein A - literature Page 16

17 AssayMAP - Bravo High throughput microchromatography platform 5 µl packed bed cartridge in 96-channel ANSI/SBS microplate format Capacity per preparation up to ~100ug Single pass, fully quantitative binding & elution Positive displacement, bidirectional flow control Quantitative recovery, excellent purity & reproducibility with no detectable carryover Potential applications Affinity purification or bind/elute assays (e.g. cell culture titer, PK studies) ELISA (e.g. process impurity or biomarker assays) Sample preparation for characterization. Chromatographic method development

18 Question and answers? Page 18 This information is subject to change without notice Agilent Technologies, Inc Published in Germany, July 2011