Freedom EVO Protein Chromatography workstations Automated parallel chromatography using MediaScout RoboColumn technology in 96 array format

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1 Freedom EVO Protein Chromatography workstations Automated parallel chromatography using MediaScout RoboColumn technology in 96 array format

2 Tecan and Atoll platforms combine to create automated parallel chromatography system Atoll s 96-array MediaScout RoboColumn system, together with the Tecan Freedom EVO liquid handling workstation, enables fully automated parallel column chromatography for the first time. Integrating the two technologies entirely removes all manual steps for buffer preparation, column equilibration, sample injection, elution, fraction collection, detection, and even column regeneration, allowing 96-array plates to be completely processed within a short period of time. For evaluation of the performance of RoboColumns, starter kits are available, either performing the chromatographic process manually using an electronic hand pipette, or by using a centrifuge. Column tube Inlet capillary Top filter Bottom filter Needle guide O-ring Resin chamber Capillary outlet The robotic manipulator arm transfers a UV plate containing collected fractions to the Tecan Infinite 200 reader.

3 Atoll MediaScout RoboColumn Sample application Collection of fractions Automated exchange of the RoboColumn plate MediaScout RoboColumns feature an array of individual MediaScout MiniColumns, installed on a base plate conforming to the standard 96-well microplate format. MediaScout RoboColumns are offered with any commercially available process separation resin, compression pre packed to defined density, and deliver the separation performance of regular ready-to-use LC columns with very high reproducibility. With inner dimensions of 2.5, 5, 10 or 30 mm bed height and 5 mm inner diameter (column volume 50, 100, 200 and 600 μl), the mini columns enable rapid parallel chromatographic separations with up to 96 columns, utilizing small sample and eluent volumes. The RoboColumn has a pressure tight inlet that seals to the fixed tips of Tecan s Freedom EVO l iquid handling workstation (patent pending). This adapter permits automated tight connection and disconnection of the fixed tips to the columns. Flow is controlled by the Tecan workstation into a 96 well plate for fully automated applications with high sample throughput and accurate control of transfer volumes and flow rates down to 16,2 cm/h. Chromatographic results may be gathered by normal detection methods, with individual fractions down to 25 μl collected in standard micro plates. These arrays introduce mini columns for parallel analytical and preparative chromatographic separations on Freedom EVO workstations.

4 Tecan Freedom EVO workstation The freely configurable Freedom EVO workstation, available in three deck sizes (100, 150 and 200 cm), provides precise and reliable liquid handling and accommodates a wide range of devices. In protein chromatography configuration, the Freedom EVO features a robotic manipulator (RoMa) arm that handles the MediaScout RoboColumns by its base plate, and a Te-Chrom holder to securely position the base plate during chromatography. Hotels/carriers provide storage space for the mini column arrays within the workstation. The liquid handling (LiHa) arm is equipped with eight fixed metal tips, and provides accurate, precise positive liquid displacement to achieve fractions as small as 25 μl, and is able to simulate concentration gradients. Eluted liquid from the MediaScout RoboColumns can be collected in standard 96-array plates for absorption readings which can be automated with an optional reader, such as the Tecan Infinite M200, for automated results analysis. An optional Te-Stack allows fraction collection by sliding the collection plate by one row after each fraction. Applications Using the MediaScout RoboColumns on the Freedom EVO workstation enables true parallel chromatography of up to eight columns, offering rapid screening and high productivity. Standardized chromatographic tests can be applied to compare a range of parameters in parallel, in the shortest time frame possible. The system offers a powerful new tool for fully automated high throughput process development and sample conditioning, by enabling users to use valid chromatographic conditions without using sophisticated LC systems. High throughput protein purification applications 1. Expression screening to select best producing clones, or cell culture screening, for mab selection. 2. Condition screening to determine optimum purification conditions. 3. Resin screening to test different resins in parallel. 4. Method development to optimize gradient elution, and transfer to large scale purification. 5. Sample conditioning/purification/concentration for downstream applications like protein crystallography or MS analysis. 6. Process analytics for bioreactor monitoring to determine the optimal harvesting point.

5 High reproducibility low volume column chromatography Simultaneous automated step-gradient elution for the separation of two proteins (1 mg/ml each of lysozyme and cytochrome c) on cation ex change resin (Toyopearl SP-650S) was performed in eight 200 μl MediaScout RoboColumns on a Freedom EVO workstation. The resulting eight elution profiles were superimposed to show the excellent reproducibility achieved by automated low volume parallel chromatography (Figure 1). Subsequently, using same experimental conditions, one Robo- Column with appropriate adapter fittings was used in a common LC instrumentation (Figure 2). The results demonstrate that Robocolumns allow parallel, automated chromatography on Freedom EVO platforms. Figure 1 Chromatograms for the separation of two proteins on cation exchangers packed in 200μl Atoll columns for 8 RoboColumns using 0.1 M NaCl or 1 M NaCl respectively. Rapid sample preparation for in-process monitoring Rapid in-process monitoring of monoclonal antibodies in production scale fermentation is one of several industrial applications of MediaScout RoboColumns on the Freedom EVO workstation. A sample from each of eight parallel fermentation reactors was applied to eight MediaScout Robo- Columns with cation exchange resin (ProSep va Ultra). The columns were rinsed to remove host cell proteins and the anti bodies were rapidly eluted, neutralized and analyzed by high performance cation exchange chroma tography. The normally time consuming sample preparation step was shortened by one order of magnitude (Figure 3). Figure 2 Chromatograms for the separation of two proteins on cation exchangers packed in one 200μl Atoll column on a common LC instrumentation using 0.1 M NaCl or 1 M NaCl respectively. Tecan s Protein Chromatography Workstation has proven reliability in Laboratories of leading pharmaceutical companies. Let Tecan Freedom EVO become the workhorse of your laboratory. Figure 3 Second dimension of In-process monitoring by CIEX of mab samples from fermentation broth, after purification on 8 parallel operated RoboColumns, showing excellent reproducibility.

6 MediaScout Robocolumns Inner dimensions Bed volume Packing Flow rate on Freedom EVO Working temperature Storage conditions Materials Filter plates Chemical stability 5mm x 2.5, 5, 10 or 30 mm (diameter x length) 50, 100, 200 and 600 μl Pre-packed with any one from a large selection of commercially available chromatographic media, at defined compression set to manufacturers specifications Between 16.2 and 1500 cm/h (depending on chromatography media) Range 5 60 C Equilibrated with 20 % ethanol (ion exchangers with the addition of 150 mm NaCl) Polypropylene (column body, filter holder and plates) Polypropylene/polyethylene 7 μm pore size ph 1 14, organic solvents (except for halogenated), mineral and organic salts, usual eluents and additives (urea, antioxidants, thiols, detergents) Note: Resin stability must be also considered A robotic manipulator compatible spacer allows for using both, deepwell blocks as well as UV-plates in the same run. For contact information for your local office Please visit: ATOLL GmbH Ettishofer Straße 10, WEINGARTEN, GERMANY Phone , Fax info@atoll-bio.com, For contact information for your local office Please visit: Tecan Switzerland Seestrasse 103, 8408 Männedorf ZH, Switzerland Phone , Fax info@tecan.com, Scientific instrumentation. Not for use in human clinical or diagnostic procedures. Research application, not for clinical diagnostic use. Not all applications discussed in this brochure have been cleared for use in all countries. Contact your local sales office for specific information. Tecan Group Ltd. makes every effort to include accurate and up-to-date information within this publication, however, it is possible that omissions or errors might have occurred. Tecan Group Ltd. cannot, therefore, make any representations or warranties, expressed or implied, as to the accuracy or completeness of the information provided in this publication. Changes in this publication can be made at any time without notice. All mentioned trademarks are protected by law. For technical details and detailed procedures of the specifications provided in this document please contact your Tecan representative. This brochure may contain reference to applications and products which are not available in all markets. Please check with your local sales representative. 2008, Tecan Trading AG, Switzerland, all rights reserved. Tecan is in major countries a registered trademark of Tecan Group Ltd., Männedorf, Switzerland Freedom EVO, Te-Chrom, Te-Stack are either trademarks or registered trademarks in major countries of Tecan Group Ltd., Männedorf, Switzerland MediaScout, RoboColumn are trademarks of Atoll GmbH, Weingarten, Germany Austria Belgium China Denmark France Germany Italy Japan Netherlands Portugal Singapore Spain Sweden Switzerland UK USA ROW V2.0,