ÄKTAmicro. GE Healthcare. Modular design based on the proven ÄKTA design platform. ÄKTAmicro chromatography system offers:

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1 GE Healthcare Data file AA Chromatography systems ÄKTAmicro ÄKTAmicro chromatography system (Fig ) is designed for microscale liquid chromatography applications. Reliable, high-resolution purification of target molecules is achieved from extremely small amounts of starting material and from molecules as diverse as intact proteins, protein complexes, and nucleotides. Moreover, ÄKTAmicro is an excellent choice for characterization of target molecules and for rapid purity/ recovery analysis in method development and drug discovery applications. The system is built on the proven ÄKTA design platform and is controlled by UNICORN software. ÄKTAmicro chromatography system offers: High-performance purification and characterization using microbore to analytical scale columns, all controlled by UNICORN software High sample recovery and stability as the complete flowpath is manufactured from inert and biocompatible materials Optimized resolution by connection of columns directly to the UV cell and by minimized dead volumes in the tubing, valve, and flow cells Novel pump design for flexible flow and pressure range enabling high- as well as low-pressure separations Fast and reproducible purity analysis by gel filtration in method development and drug target characterization Absolute characterization of biomolecules in solution and on-line molecular weight analysis by the addition of light scattering equipment Modular design based on the proven ÄKTA design platform ÄKTAmicro is modular by design and is comprised of three main components: Pump P-95, Monitor ph/c-9, and Monitor UV-9. Autosampler A-95 and Fraction Collector Frac-95 are optional components. The system is based on the proven ÄKTA chromatography platform and is optimized for microbore-, narrowbore-, and analytical-scale purification of proteins and peptides (Table ). In combination with Fig. ÄKTAmicro chromatography system allows purification of extremely small sample volumes and is an excellent tool for rapid purity analysis in method development and drug target characterization. Fraction Collector Frac-95, ÄKTAmicro offers microliterscale fraction volumes and excellent chromatographic performance. Autosampler A-95 provides reliable and reproducible sample injection and unattended, multiple sample runs. Table. Column diameter scales suitable for use with ÄKTAmicro system Purification scale Column i.d. (mm) Microbore. Narrowbore 2. Analytical scale 4.6

2 Accurate and reproducible flow rates and gradients ÄKTAmicro generates highly accurate and consistent flow rates and gradients over the entire working range. Pump P-95 is incorporated in ÄKTAmicro and overcomes many of the problems associated with traditional high-performance pumps at low flow rates. The flexible flow and pressure range even enables low-pressure separations, making the system compatible with soft agarose-based media for fast, reproducible gel filtration. This low-pulsation pump is equipped with four pump heads in two binary modules, which allows for binary gradients with high pressure mixing enabling microbore- to analytical- scale purifications. Pump P-95 operates at flow rates of. to 2 ml/min in isocratic mode and.4 to 2 ml/min in gradient mode. Column: Sample: Injection: Buffer: Flow rate: System: A 28 (mau) 5 Superdex 2 5/5 GL µl of IgG4 (M r 5 ), 9.2 mg/ml Autosampler A-95, partial loopfill mode PBS.3 ml/min ÄKTAmicro The pump allows active mixing of gradients in a 9 μl or 2 μl mixing chamber to ensure reproducibility and accuracy. The low dead volume of ÄKTAmicro ensures that accurate gradients are generated. Performance reproducibility is illustrated (Fig 2) over 5 consecutive purification runs of IgG4 by gel filtration on ÄKTAmicro system. High, accurate reproducibility is maintained even in rapid purifications as demonstrated in Figure 3. Column: Sample: Buffer: Flow rate: System: Injection: A 28 (mau) 8 Ovalbumin Superdex 2 5/5 GL µl of a protein mixture containing 5 mg/ml ovalbumin (M r 43 ),.5 mg/ml ferritin (M r 44 ), 5 mg/ml bovine serum albumin (BSA, M r 66 ), 5 mg/ml aldolase (M r 58 ) PBS.3 ml/min ÄKTAmicro Autosampler A-95, partial loopfill mode Ferritin BSA Aldolase min ml Fig 2. Overlay curves of 5 consecutive purification runs of IgG4 on Superdex 2 5/5 GL connected to ÄKTAmicro system. The overlay curves demonstrate the reproducibility possible using a combination of short gel filtration column and ÄKTAmicro system. Fig 3. Four separate and consecutive injections ( µl) of ovalbumin 5 mg/ml, ferritin.5 mg/ml, BSA 5 mg/ml, and aldolase 5 mg/ml, respectively using Autosampler A-95. The separations were performed on Superdex 2 5/5 GL short gel filtration column using ÄKTAmicro system. The flow rate was.3 ml/min and PBS was used as running buffer. Although the four proteins were analyzed in less than 5 min, excellent performance and resolution were maintained. System pump Mixer On-line filter Sample Column Monitoring (Fractionation) Injection UV-monitor Conductivity monitor Flow restrictor (optional) Switch valves Waste A A2 B B2 Fig 4. Standard flow configuration of ÄKTAmicro system. Waste Fraction Collector Frac-95 (optional) AA

3 Flow configuration The standard analytical flow configuration of ÄKTAmicro is shown in Figure 4. This flow configuration enables chromatography with mm i.d. columns or greater. Direct connection of the column to the UV- and conductivity monitors minimizes dead volumes, which ensures accurate gradient reproducibility and improves purification resolution. Accurate on-line gradient monitoring Monitor ph/c-9 (Fig 5) allows reliable on-line measurement of conductivity (range μs to ms/cm) and is an excellent complement to a UV monitor in liquid chromatography. The high precision conductivity measurement of Monitor ph/c-9 over a wide conductivity range makes it an excellent choice for all chromatography techniques, including RPC with very low conductivity eluents. The monitor consists of a control unit and a pressure-stable.2 μl flow cell. even for partial loopfill injections down to 5 μl. For small sample volumes, the microliter pick-up procedure allows injection with no sample loss while thorough wash routines efficiently eliminate cross-contamination. Fig 6. Autosampler A-95 increases system throughput. Fig 5. Monitor ph/c-9 allows simultaneous monitoring of ph and conductivity, which enables reliable monitoring of purifications Fraction Collector Frac-95 (Fig 5) enables fractionation over a wide flow-rate range. An optional Microfraction Collection Kit allows reliable collection of microliter fraction volumes as low as 4 µl directly into microplates. Collection is in volume or time mode and different fraction sizes can be collected during different stages of the purification. High sensitivity, multiwavelength detection Monitor UV-9 is a high sensitivity UV-Vis monitor, recording up to three wavelengths simultaneously for optimal detection in the 9 to 7 nm range. The low maintenance xenon lamp provides a significantly higher intensity compared to conventional deuterium lamps. Columns are connected directly to the low volume (.7 μl) UV flow cell, which ensures high sensitivity and resolution, with minimized dead volumes. The use of advanced fiber optics with the novel flow cell design ensures a high signal-to-noise ratio for maximum performance from microbore- to analytical-scale applications. Increased system throughput with automated sampling and fraction collection Autosampler A-95 (Fig 6) increases system throughput while a Peltier cooling element prevents sample degradation during long runs. The autosampler is optimized for sample volumes as low as nl and allows injections of up to 384 samples from vials and microplates. An optional Microfraction Collection Kit provides optimized sample tubing for collection of small sample volumes in microplates. A Dropsync function allows accurate fractionation and minimizes spillage between fractions. Three different injection methods can be selected: Microliter pick-up, flushed loop, or partial loopfill. Precision better than.5% relative standard deviation (RSD) is easily achieved Fig 7. Fraction Collector Frac-95 (left) and (right) Frac-95 modified for collection of extremely small fraction volumes using tubing provided in Microfraction Collection Kit. Built-in system control and evaluation UNICORN system control and evaluation (Fig 8) is a software control platform that has a proven track-record in ÄKTA design and BioProcess systems. UNICORN ensures fast and userfriendly interaction between ÄKTAmicro and the operator. Methods are built from a user-friendly method wizard for different applications. The method wizard is included in the default system software AA 3

4 AD-9 analog/digital converter If external instruments are to be connected to UNICORN, the AD-9 analog/digital converter is needed. AD-9 has an analog input for monitoring signals received as voltage outputs from external sources, such as fluorescence detectors, radioactivity monitors, refractive index monitors, or diode arrays. Valve INV-97 This seven-port valve can be used as an additional valve together with the optional software strategy for customized applications. Fig 8. UNICORN provides fast and user-friendly interaction between the user and ÄKTAmicro system. When ÄKTAmicro is connected to external equipment or when an extra valve is needed, additional method templates are available to accommodate these options. To facilitate routine runs, the Method scouting function may be used. All data, including running conditions, are stored permanently by UNICORN for every run (e.g., flow, pressure, UV, conductivity, and temperature). A Flow-between-run option keeps the column pressurized after or between runs to increase reproducibility of the system, which is particularly important for gel filtration applications. In addition to standard evaluation, an optional UNICORN Analysis Module allows approximate quantitation using internal or external standards, measurement of sample amount and concentration, as well as determination of molecular size in gel filtration and recovery calculations (Fig 9). Fig 9. UNICORN Analysis Module user interface showing molecular weight calibration of standard proteins. High-resolution columns for reliable target molecule purification For high-performance purification of target molecules, support from GE Healthcare is provided in our range of PC columns (Precision Columns) for microscale purification by RPC, ion exchange chromatography, or gel filtration. Superdex 5/5 GL high-performance gel filtration columns provide excellent results when determining aggregate and contaminant levels in method development and drug test characterization. These columns have a short bed height of only 5 mm, which gives short purification times with retained function of the target molecule. These columns are also cost-effective since they operate with smaller volumes of buffers and other reagents. Inert and fully biocompatible materials throughout All wetted materials in ÄKTAmicro are inert, fully biocompatible, and resistant to all commonly used solvents. These features ensure high recovery, sample stability, and easy cleaning of the system when using almost any chromatography technique. The biocompatibility is especially important in protecting labile samples and preventing adsorption of samples to component surfaces (e.g., phosphorylated peptides that would otherwise adhere to metal surfaces). On-line light scattering analysis for sample characterization ÄKTAmicro is designed for easy connection to light scattering equipment. Absolute characterization of proteins in solution is critical in a variety of industries. In pharmaceutical or biotechnology applications, for example, protein-based products must be made precisely and without the presence of aggregates. Multi-angle light scattering (MALS) is an excellent tool to accurately measure the molar mass of proteins in solution independently from the elution. Light scattering response is directly proportional to the weight-averaged molar mass of the sample being measured multiplied by the concentration. Light scattering therefore provides an extremely sensitive method for detection and quantitation of aggregates in solution AA

5 Reliable results, whatever the application Micropurification There are three main reasons to perform purification in microscale: ) only small amounts of sample are available, 2) low abundance of target protein in the sample, 3) to keep consumption of reagent and sample down to minimize cost. Figure shows the micropurification of PEP-9 (Purkinjecell protein 4; PCP 4, M r 67), a brain protein involved in calmodulin-dependent signal transduction. To demonstrate the suitability for micropreparative protein purification using ÄKTAmicro, a protein extract from mouse brain tissue was prepared and PEP-9 was purified using a two-step procedure consisting of anion exchange chromatography and gel filtration. The presence of PEP-9 in the various fractions was confirmed by nanoscale LC-MS/MS. As PEP-9 contains no aromatic amino acid residues, it can be readily detected in the gel filtration step by the absence of UV absorbance at 28 nm. This feature highlights the benefit of multiple wavelength detection in ÄKTAmicro. Purity analysis and characterization by gel filtration ÄKTAmicro can perform a range of characterization applications including protein and peptide purity analysis. Purity assessment is a key evaluation step in method development. The high sensitivity and precision of the system ensures that accurate results are obtained. Analysis of sample homogeneity after purification of antibodies is essential, especially if the substance is to be used for therapeutic purposes. Homogeneity analysis of a monoclonal antibody was performed by gel filtration on ÄKTAmicro system (Fig ). In this case, homogeneity analysis was employed downstream of an affinity chromatography purification step revealing the presence of a contaminating antibody dimer. Protein prefractionation of complex samples Simplification of complex protein samples is often needed prior to analysis, particularly if protein expression profiling and relative quantitation will be employed. Common techniques used are ion exchange, gel filtration, reversed phase, and combinations of these. Figure 2 shows a typical result for the prefractionation of a complex protein sample. Strong anion exchange chromatography with Mono Q PC.6/5 column on ÄKTAmicro system was used to reduce the complexity of proteins derived from immortalized lymphatic endothelial cell culture, with or without treatment with the vascular endothelial growth factors, VEGF-A and VEGF-C. Aliquots from each fraction from the prefractionation step were trypsin digested and analyzed by LC-MS/MS. Further analysis of the data revealed that this prefractionation approach was an effective first step to ultimately allow identification of 42 proteins by LC-MS/MS (data not shown, request application note for details, or download a pdf from Anion exchange chromatography Column: Mono Q PC.6/5 Sample: 5 µl mouse brain extract corresponding to 5 mg brain tissue Eluent A: 2 mm Tris-HCl, ph 8. Eluent B: 2 mm Tris-HCl, M NaCl, ph 8., Flow rate:. ml/min Gradient: % to 5% B in 4 ml, % B in.5 ml Fraction size: µl Detection: UV 25 and 28 nm System: ÄKTAmicro Gel filtration Column: Superdex Peptide PC 3.2/3 Sample: Fraction C2 from Mono Q PC.6/5 containing PEP-9 concentrated to a volume of µl Elution buffer: 2 mm Tris-HCl, mm NaCl, ph 8. Flow rate:.25 ml/min Fraction size: 4 µl Detection: UV 25 and 28 nm System: ÄKTAmicro A) B) A 25, A 28 (mau) A 25, A 28 (mau) A 25 A 28 Conductivity A A9 A B7 B9 C5 C7 Volume (ml) A3 A5 A7 B B3 B5 B C C3 C9 C PEP A9 A B9 B C9 C Volume (ml) A A3 A5 A7 B B3 B5 B7 C C3 C5 C7 Fig. (A) Anion exchange chromatography on Mono Q PC.6/5 column connected to ÄKTAmicro system of a small sample of clarified mouse brain tissue. (B) Gel filtration of the fraction shown in (A) was performed on Superdex Peptide PC 3.2/3, demonstrating the possibility of collecting 4 µl fractions with high precision using ÄKTAmicro system AA 5

6 Column: Superdex 2 PC 3.2/3 Sample: Monoclonal IgG4, fractions from purification step using MabSelect SuRe affinity chromatography medium Elution buffer: Phosphate Buffered Saline (PBS) Flow rate:.4 ml/min Fraction size: 4 µl Detection: UV 28 nm System: ÄKTAmicro A 28 (mau) Fig. Purity analysis of IgG4 on Superdex 2 PC 3.2/3 gel filtration column and ÄKTAmicro system. The purity analysis was performed after initial affinity purification on a column packed with MabSelect SuRe chromatography medium. Column: Mono Q PC.6/5 Sample: Total protein extract from immortalized lymphatic endothelial cells left untreated (controls) or treated with growth factors VEGF-A or VEGF-C Buffer A: 2 mm Tris-HCl, 8 M urea, 6% isopropanol, ph 8. Buffer B: Buffer A + M NaCl Flow rate:.2 ml/min Gradient: Linear salt gradient, % to 5% B Detection: UV 25 nm System: ÄKTAmicro VEGF-A A 28 (mau) Dimer Monomer Volume (ml) A 28 Conductivity Volume (ml) Fig 2. Chromatograms resulting from strong anion exchange chromatography of total protein extracts prepared from VEGF-A treated cells using Mono Q PC.6/5 on ÄKTAmicro system. Data for prefractionation of control and VEGF-C treated cells are not shown. Fractions of.4 ml were collected throughout the separations. Specifications System specifications Flow rate range. to 2 ml/min Pressure range to 35 MPa Conductivity range μs/cm to ms/cm ph range 2 to 2 Temperature range 4 C to 4 C Solvent compatibility All commonly used chromatographic solvents Peak tubing dimension.5 mm i.d. Dimensions (W H D) mm Weight 55 kg Pump P-95 Flow rate range Isocratic mode. to 2. ml/min Gradient mode.4 to 2. ml/min During PumpWash 6 ml/min Flow rate accuracy MPa ±2%, or 2 μl/min, whichever is greater with compression compensation activated Flow rate reproducibility >.4 ml/min, RSD <.5% Increment. ml/min Pressure range 35 MPa (35 bar, 575 psi) Pressure limits Programmable upper and lower Internal volume < 6 μl/pump module Viscosity maximum 3cP Peak tubing dimension.5 mm i.d. Dimensions (W H D) mm Weight 55 kg Monitor UV-9 Wavelength range Bandwidth Wavelength accuracy Wavelength reproducibility Linearity Noise at 23 nm Drift at 254 nm Flow cell Maximum flow rate Maximum pressure Path length Internal volume Typical values at room temperatue after warm-up 9 to 7 nm in steps of nm, three wavelengths simultaneously 4 nm ± 2 nm ±. nm < 2% deviation up to 2 AU at 26 nm with uracil at ph 2 < 6-5 AU, with 3 mm cell, H 2 O at ml/min < 2-4 AU/h 2 ml/min 2 MPa (2 bar, 29 psi) 3 mm.7 μl AA

7 Monitor ph/c-9 Conductivity unit Conductivity range Deviation from theoretical conductivity Noise Flow cell Maximum flow rate Maximum pressure Internal volume Autosampler A-95 Sample capacity μs/cm to ms/cm Maximum ± 2% of full-scale calibration range or ± μs/cm, whichever is greater in range Maximum ±.5% of full-scale calibration range 2 ml/min 5 MPa (5 bar, 725 psi).2 μl Plate with 48 wells for standard,.5 ml vials Low plate with 96 wells High plate with 96 wells Low plate with 384 wells Injection volume range Flushed loop 2 or μl loop volume Partial loop % to 5% of loop volume Microliter pick-up 2 μl loop. to 2.5 μl in. μl increments μl loop. to 42.5 μl in. μl increments Dispenser syringes and 25 μl Cooling specification Sample cooling Built-in Peltier cooling processing unit Programmable range 4ºC to 4ºC Dimensions (W H D) mm Cooling capacity Max. 2ºC below ambient temperature for working temperatures between 6ºC and 4ºC Accessories for Autosampler A-95 Syringe, ml Peek loop, ml 8-4- Peek sample needle Buffer tubing, 2 ml To support larger injection volumes than A-95 standard Fraction Collector Frac-95 Flow rate range ph stability range Fraction size Volume mode Time mode No spillage range Dropsync. range Accumulator range From 4 µl using Microfraction Collection Kit. to ml/min to 3, to 4 (< day exposure).4 to ml.4 to ml to 3 ml/min 5 to ml/min Accessories for Frac-95 Microfraction Collection Kit Rack A (2 8 and 8 3 mm tubes) Rack B (24 2 mm tubes), supports Eppendorf tubes Rack C (4 microtiter plates and 8 3 mm tubes) Microplate, 96-well ( pieces) Delivered with Frac-95 as standard Ordering information System and components Code no. ÄKTAmicro system includes ÄKTAmicro, software, and installation accessories Autosampler A-95 for ÄKTAmicro Autosampler A-9 with cooling Fraction Collector Frac UNICORN v UNICORN Analysis Module, v Special strategy for ÄKTAmicro (for use with one additional valve and/or external equipment) A/D converter AD Valve INV Column holders Short column holder Long column holder Small column clamp ÄKTA extension equipment holder Columns Code no. μrpc C2/C8 ST 4.6/ SOURCE 5RPC ST 4.6/ SOURCE 5RPC ST 4.6/ RESOURCE RPC ( ml) 7-8- RESOURCE RPC (3 ml) Mini Q PC 3.2/ Mini Q 4.6/5 PE Mini S PC 3.2/ Mini S 4.6/5 PE Mono Q PC.6/ Mono Q 5/5 GL Mono S PC.6/ Mono S 5/5 GL Superose 6 PC 3.2/ Superose 2 PC 3.2/ Superdex Peptide PC 3.2/ Superdex 75 PC 3.2/ Superdex 2 PC 3.2/ Superdex 75 5/5 GL Superdex 2 5/5 GL AA 7

8 Accessories for columns Code no. Precision Column Holder for 3 5 cm and 3 cm columns for cm columns Required for all PC columns. Related product literature Literature Application note: Micropreparative prefractionation using ÄKTAmicro for protein expression profiling in endothelial cells Data file: Fraction Collectors Frac-95 and Frac Data file: Autosampler A Handbook: Ion exchange Chromatography and -4-2 Chromatofocusing, Principles and Methods Handbook: Hydrophobic Interaction Reversed Phase Chromatography, Principles and Methods Selection Guide: Prepacked Chromatography columns for ÄKTAdesign and Ettan LC For local office contact information, visit GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden imagination at work GE, imagination at work, and GE monogram are trademarks of General Electric Company. ÄKTA, ÄKTAmicro, BioProcess, Drop design, Ettan, MabSelect SuRe, Mini Q, Mini S, Mono Q, Mono S, RESOURCE, SOURCE, Superdex, Superose, and UNICORN are trademarks of GE Healthcare companies. Purification and preparation of fusion proteins and affinity peptides comprising at least two adjacent histidine residues may require a license under US patent numbers 5,284,933 and 5,3,663, and equivalent patents and patent applications in other countries (assignee: Hoffman La Roche, Inc). All third party trademarks are the property of their respective owners. 29 General Electric Company All rights reserved. First published Apr. 29 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA UK GE Healthcare Europe, GmbH Munzinger Strasse 5, D-79 Freiburg Germany GE Healthcare Bio-Sciences Corp. 8 Centennial Avenue, P.O. Box 327, Piscataway, NJ USA GE Healthcare Bio-Sciences KK Sanken Bldg., 3-25-, Hyakunincho, Shinjuku-ku, Tokyo Japan AA 4/29