MabSelect SuRe. MabSelect. GE Healthcare

Transcription

1 GE Healthcare Instructions AC Affinity Chromatography MabSelect SuRe MabSelect SuRe is a novel, alkali-tolerant protein A-derived medium for capturing monoclonal antibodies from large volumes of feed by packed bed chromatography. Novel, alkali-tolerant protein A-derived ligand allows the use of M sodium hydroxide for cleaning-in-place (CIP). Improved product quality and reduction in overall costs. Eliminates the need for expensive and corrosive CIP reagents. High dynamic binding capacity (DBC) reduces process time and amount of medium used. High-flow agarose matrix allows processing of large volumes of feed. Simple scale-up to production-sized BPG, BioProcess and Chromaflow columns. MabSelect

2 Contents 1 Description Process development Recommended screening conditions Removal of leached ligand from final product Cleaning-in-place (CIP) Sanitization Storage Scaling up Troubleshooting Packing columns Evaluation of column packing Ordering information Description The protein A-derived MabSelect SuRe ligand is produced in Escherichia coli. Fermentation and subsequent purification are performed in the absence of animal products. The ligand has been specially engineered to create an affinity medium with enhanced alkali stability and high binding capacity for IgG. The specificity of binding to the Fc region of IgG is similar to that of conventional Protein A and provides excellent purification in one step. Alkali tolerance, high capacity and low ligand leakage plus the rigid base matrix, make MabSelect SuRe ideal for the purification of monoclonal antibodies for clinical applications. Figure 1 shows stability in alkaline conditions of MabSelect SuRe in terms of dynamic binding capacity. A pressure/flow curve is shown in Figure AC

3 The characteristics of the medium are summarized in Table Capacity (%) MabSelect SuRe 0.1 M NaOH MabSelect SuRe 0.5 M NaOH MabSelect 0.1 M NaOH CIP cycles Fig 1. Dynamic binding capacity for MabSelect and MabSelect SuRe after CIP with 0.1 or 0.5 M NaOH, 15 min contact time, for cycles. Each cycle in Figure 1 consisted of: 10 column volumes binding buffer, ph 7.4, 6 column volumes elution buffer, ph 3.0, 3 column volumes of binding buffer, ph 7.4, 0.1 or 0.5 M NaOH, 15 min contact time, and 6 column volumes binding buffer, ph 7.4. The dynamic binding capacity (DBC, 10% breakthrough) for MabSelect SuRe was measured every 20 cycles. The capacity of conventional Protein A media (MabSelect ) is also included in the figure for comparison AC 3

4 800 Velocity (cm/h) Pressure (MPa) Fig 2. Pressure/flow profile for MabSelect SuRe in a packed bed (bed height 20 cm) in BPG 300 column (i.d. 300 mm). Table 1. Characteristics of MabSelect SuRe. Composition Rigid, highly cross-linked agarose Average particle size (d 50v ) 1 85 µm Ligand Alkali-tolerant, protein A-derived (E. coli) Coupling chemistry Epoxy Dynamic binding capacity 2 Approx. 35 mg human IgG/ml medium Chemical stability Stable in all aqueous buffers commonly used in Protein A chromatography. ph working range 3 to 12 Cleaning-in-place stability M NaOH Maximum mobile 500 cm/h phase velocity 4 Temperature stability 5 4 C to 40 C Delivery conditions 20% ethanol Regulatory support Regulatory support file is available. No material of animal origin is used in the manufacturing process AC

5 1 d 50v is the medium particle size of the cumulative volume distribution. 2 Determined at 10% breakthrough by frontal analysis at a mobile phase velocity of 500 cm/h in a column with a bed height of 20 cm, i.e. residence time is 2.4 min.residence time is equal to bed height (cm) divided by nominal fluid velocity (cm/h) during sample loading. Nominal fluid velocity is equal to volumetric flow rate (ml/h) divided by column cross-sectional area (cm2). 3 See Figure 1 and section 5 Cleaning-in-place (CIP). 4 In BPG 300 column, bed height 20 cm, operating pressure < 2 bar, 25 C. 5 Recommended long-term storage conditions: +4 C to +8 C, 20% ethanol. 2 Process development For initial studies of MabSelect SuRe in small-scale columns, we recommend prepacked HiScreen columns or XK 16 columns with 10 cm bed height. Choose a residence time (see Table 1, footnote 2) that fulfills your demand on dynamic binding capacity and nominal fluid velocity according to Figure 3. Make sure the chosen bed height and velocity do not conflict with the large-scale pressure/flow limitations (Fig 3) Velocity (cm/h) Bed height (cm) Fig 3. Recommended operating window for MabSelect SuRe (white area). Choosing bed height and operating velocity in terms of residence time, pressure restrictions and large-scale column packing challenges. Figure 3 shows the possible combinations of bed height and operational nominal fluid velocity for MabSelect SuRe. The figure also displays the residence time in the interval 1 15 minutes for AC 5

6 any bed height and velocity. Included are also pressure drop limitations and packing limitations at large scale. The solid curved line shows the calculated large-scale column pressure restriction which is 2 bar according to specification (500 cm/h at 2 bar and 20 cm bed height). The dashed vertical line indicates that operating at below 10 cm bed height is not favorable. The reason for this is that large diameter columns have a very different aspect ratio, and that packing short wide beds is a greater challenge. Figure 3 can be used as a guide when determining suitable bed height and operating velocity in terms of residence time and thus capacity and pressure drop. Figure 4 shows the relation between dynamic binding capacity and residence time for MabSelect SuRe. DBC at 10% breakrough (mg/ml) Residence time (min) Fig 4. Relation between dynamic binding capacity and residence time for MabSelect SuRe for a purified monoclonal antibody AC

7 3 Recommended screening conditions Examples of suitable buffers: Buffer A: 20 mm sodium phosphate, 150 mm NaCl, ph 7.2 Buffer B: 0.1 M sodium citrate, ph Experimental conditions: Equilibrate the column with 10 column volumes of buffer A. Apply a small sample of antibody. Wash the column with 5 column volumes of buffer A. Elute the column with a linear gradient of 10 column volumes to 100% buffer B. Collect fractions into titrating diluent (e.g. 1.0 M Tris-HCl, ph 8.0 so that the diluent volume equals 5% of the programmed fraction volume). Regenerate the column with 5 10 column volumes of 100% buffer B. Wash the column with 3 column volumes of buffer A. Perform CIP with 5 column volumes of M NaOH. Re-equilibrate the column with buffer A. Due to the inherent properties of agarose, good contaminant clearance is achieved independent of choice of washing buffer (buffer A). To minimize the use of buffer, however, we recommend optimizing the washing procedure with respect to residence time, volumes, ph and conductivity. When optimizing elution conditions, determine the highest ph that allows efficient desorption of antibody from the column. This will prevent denaturing sensitive antibodies due to exposure to low ph. Step-wise elution (Fig 5) is often preferred in large-scale applications since it allows the target monoclonal antibody to be eluted in a more concentrated form, thus decreasing buffer consumption and shortening cycle times. It might be necessary to decrease the flow rate due to the high concentrations of protein in the eluate AC 7

8 Figure 5 shows an example of purification of a monoclonal antibody from a clarified mammalian cell culture on MabSelect SuRe. The load was 21 mg antibody/ml column volume (CV), and the yield was 94% of highly purified antibody. An XK 16/20 column with a CV of 20 ml and a bed height of 10 cm was used Sample application Elution CIP Absorbance (mau) UV 280 nm ph Volume (ml) Fig 5. Purification of a monoclonal antibody from a mammalian cell culture on MabSelect SuRe. The dynamic binding capacity for the target antibody should be determined by frontal analysis using real process feedstock. The dynamic binding capacity is a function of the sample residence time and should therefore be defined over a range of different sample residence times. 4 Removal of leached ligand from final product The ligand leakage from MabSelect SuRe is generally low. For example, the eluate from the purification run shown in Figure 5 contained 3 ppm (ng ligand/mg antibody) of leached ligand. However, in many monoclonal antibody applications it is a requirement to eliminate leached ligand from the final product. There are a number of chromatographic solutions, such as cation exchange chromatography, anion exchange chromatography, or size exclusion chromatography AC

9 The optimal conditions for removal of leached ligand must be evaluated for each individual antibody. Methods used for removal of leached recombinant Protein A from conventional recombinant Protein A media, e.g. MabSelect, MabSelect Xtra or rprotein A Sepharose Fast Flow, should also be applicable for removal of leached MabSelect SuRe ligand. Recombinant Protein A and the MabSelect SuRe ligand have similar antibody-binding characteristics and isoelectric points. Figures 6 and 7 show examples of separation by cation and anion exchange chromatography. An ELISA method was used to detect the small amounts of the ligand in the eluted fractions. The results are presented in ppm (ng ligand/mg antibody) in the chromatograms. Figure 6 shows an application of a MabSelect SuRe eluate on a cation exchange chromatography column (SP Sepharose Fast Flow, HR 10/10 column, bed height 10 cm, column volume 8 ml). The sample consisted of the MabSelect SuRe eluate from the run in Figure 5, adjusted to ph 5.0, and contained 160 mg of monoclonal antibody. The column was eluted with a gradient of M Naacetate, ph 5.0, followed by a step elution with 0.1 M Na-acetate, 1 M NaCl, ph 5.0. Absorbance (mau) UV 280 nm Ligand ppm Conductivity MabSelect SuRe ligand (ppm) Volume (ml) Fig 6. MabSelect SuRe eluate applied to SP Sepharose Fast Flow AC 9

10 Figure 7 shows an application of a MabSelect SuRe eluate on an anion exchange chromatography column (Q Sepharose Fast Flow, HR 10/10 column, bed height 10 cm, column volume 8 ml). The sample consisted of the MabSelect SuRe eluate from the run in Figure 5. It was prepared by addition of Na-phosphate, ph adjustment, and dilution with water. 160 mg of monoclonal antibody was applied to the column. 500 Absorbance (mau) UV 280 nm Ligand ppm Conductivity MabSelect Sure ligand (ppm) Volume (ml) Fig 7. MabSelect SuRe eluate applied to Q Sepharose Fast Flow. The chromatography step was performed in flowthrough mode, i. e., the antibody did not bind to the column and was present in the flowthrough. After the sample application, the column was washed with 10 mm Na-phoshpate, ph 7.4, followed by step elution with 10 mm Na-phosphate, 1 M NaCl, ph AC

11 5 Cleaning-in-place (CIP) Cleaning-in-place (CIP) is the removal of very tightly bound, precipitated or denatured substances from the purification system. If such contaminants are allowed to accumulate, they may affect the chromatographic properties of the column, reduce the capacity of the column and, potentially, come off in subsequent runs. If the fouling is severe, it may block the column, increase back pressure and reduce flow rate. Regular CIP prevents the build up of contaminants in the packed bed, and helps to maintain the capacity, flow properties and general performance of MabSelect SuRe. We recommend performing a blank run, including CIP, before the first run with antibody feed. MabSelect SuRe is an alkali-tolerant medium allowing the use of NaOH as CIP agent. NaOH is widely accepted for cleaning due to the low cost and the ability to dissolve proteins and saponify fats. CIP protocol 1 Wash the column with 3 column volumes of binding buffer. 2 Wash with at least 2 column volumes of M NaOH. Contact time minutes. 3 Wash immediately with at least 5 column volumes of sterile and filtered binding buffer at ph 7 8. CIP is usually performed immediately after the elution. Before applying the alkaline NaOH CIP solution, we recommend equilibrating the column with a solution of neutral ph in order to avoid the direct contact between low-ph elution buffer and highph NaOH solution on the column. Mixing acid and alkaline solutions might cause a rise in temperature in the column. NaOH concentration, contact time and frequency are typically the main parameters to vary during the optimization of the CIP. The nature of the feed material will ultimately determine the final CIP. However, the general recommendation is to clean the column at least every 5 cycles during normal use. Depending on the nature of the contaminants, different protocols may have to be combined, for example 0.1 M NaOH every cycle and 0.5 M NaOH every 10 cycles AC 11

12 6 Sanitization Sanitization reduces microbial contamination of the chromatographic bed to a minimum. MabSelect SuRe is alkalitolerant allowing the use of NaOH as sanitizing agent. NaOH is very effective for inactivating viruses, bacteria, yeasts, and endotoxins. In addition, NaOH is inexpensive compared with other sanitizing agents. Sanitization protocol 1 Wash the column with 3 column volumes of binding buffer. 2 Equilibrate the column with M NaOH. 3 Use a contact time of at least 15 minutes for 0.5 M NaOH or 30 minutes for 0.1 M NaOH (see also the note below). 4 Wash immediately with at least 5 column volumes of sterile and filtered binding buffer at ph 7 8. Note: Higher concentrations of NaOH and/or longer contact time inactivates microorganisms more effectively. However, these conditions might also lead to a decrease in the dynamic binding capacity. The conditions for sanitization should therefore be evaluated to maximize microbial killing and to minimize loss of capacity. 7 Storage Store unused media in its container at a temperature of +4 C to +8 C. Ensure that the screw top is fully tightened. Equilibrate packed columns in buffer containing 20% ethanol or 2% benzyl alcohol to prevent microbial growth. After storage, equilibrate with starting buffer and perform a blank run, including CIP, before use AC

13 8 Scaling up After optimizing the antibody fractionation at laboratory scale, the process can be scaled up to pilot and process scales. Keep the residence time constant in order to maintain the dynamic binding capacity. Select bed volume according to required binding capacity. Verify the purification step with the new bed height, if it is changed. Select column diameter according to your volume throughput requirements. Then determine the bed height to give the desired residence time. Bed heights of cm are generally considered appropriate. Note that the backpressure increases proportionally with increasing bed height at constant nominal velocity. Keep sample concentration and elution conditions constant. See also Figure 3 for appropriate windows of operation for MabSelect SuRe AC 13

14 9 Troubleshooting The list describes faults observed from the monitor curves. Fault Possible cause/corrective action High backpressure during the run Unstable pressure curve during sample application Gradual broadening of the eluate peak Change the in-line filter. The column is clogged. Perform CIP. The adaptor net/filter is clogged. Clean or replace the net/filter. Remove air bubbles that might have been trapped in the sample pump. Degas the sample using a vacuum degasser or an air trap. Might be due to insufficient elution and CIP caused by contaminants accumulating in the column. Optimize the elution conditions, the CIP protocol and/or perform CIP more frequently. Gradual decrease in yield Too high sample load. Decrease the sample load. Precipitation during elution. Optimize the elution conditions. Might be due to insufficient elution and CIP. Optimize the elution conditions, the CIP protocol and/or perform CIP more frequently. Gradual increase in CIP peaks High ligand leakage during the first purification cycle Might be due to insufficient elution or CIP. Optimize the elution conditions, the CIP protocol and/or perform CIP more frequently. Perform a blank run, including CIP, before the first purification cycle on a new column AC

15 10 Packing columns Recommended columns Table 2. Recommended columns for MabSelect SuRe. Column Inner diameter Bed volume 1 (mm) Lab scale Bed height (cm) XK 16/ ml max 30 Production scale 2 AxiChrom liters max 40 AxiChrom liters max 40 BPG liters max 40 Chromaflow standard liters max 30 Chromaflow custom liters max 30 1 Bed volume range calculated from 10 cm bed height to maximum bed height. 2 Packing instructions for MabSelect in Chromaflow columns are described in Application Note All large-scale columns can be supplied as variable bed height columns. Do not choose large diameter columns if the bed height is low. Packing lab-scale columns (XK 16/40) Preparing the suspension The following instruction is for packing MabSelect SuRe in XK 16/40 to a bed height of 20 cm. Materials needed 45 ml MabSelect SuRe Glass filter funnel Plastic spoon Filtering flask 20% EtOH 20% EtOH M NaCl Equilibrate all material to room temperature AC 15

16 Washing the medium Mount the glass filter funnel onto the filtering flask. Pour the medium into the funnel and wash with 200 ml 20% EtOH M NaCl. Preparation of the slurry Move the sedimented medium from the funnel into a beaker. Add 20% EtOH M NaCl to obtain a 50% slurry concentration. Note: The packing solution is 20% EtOH. Equipment needed An XK 16/40 column, two AK 16 adaptors, an XK 16/20 column used as packing tube, and an XK 16 connector. An ÄKTAdesign system or a stand alone pump, such as Pump P 900 that can deliver 25 ml/min. Packing the column 1 Assemble the column. Make sure that all parts of the column are clean and intact. 2 Attach the packing tube to the column with a packing connector. 3 Wet the bottom and top filter by putting the adaptor/end piece in a beaker with 20% EtOH and draw the solution through the outlet tubing with the aid of a syringe. Make sure that there are no air bubbles trapped under the net. Close the tubing with an end plug. 4 Mount the bottom adaptor a few centimeters up the tube to be able to obtain 20 cm bed height. Tighten the sealing. 5 Mount the column vertically on a lab stand. 6 Add some packing solution to the column, open the outlet and let some liquid drain through. Leave about 2 cm of solution and close the outlet. 7 Pour the slurry into the column. Avoid formation of air bubbles in the medium by pouring it along a glass rod AC

17 8 Fill the remainder of the packing tube with 0.25 M NaCl in 20% EtOH. Mount the top adaptor to the packing tube, tighten the adaptor sealing and press the adaptor down until the air in the inlet tubing is removed. Connect the column inlet to the pump. 9 Open the column outlet and start the packing by pumping 20% EtOH through the column at a flow rate of 25 ml/min (750 cm/ h) until the bed height stabilizes (approximately 5 min). Note: Make sure that the backpressure does not exceed the pressure limits of the column (5 bar) during packing. 10 Stop the flow and close the column outlet. Disconnect the pump. 11 Remove the packing tube. 12 Add 20% EtOH to the upper edge of the column. 13 Insert the adaptor into the top of the column, making sure not to trap any air under the net. 14 Mount the top adaptor approximately 1 cm above the medium surface. Tighten the adaptor sealing. Air in the adaptor tubing can be displaced by pressing the adaptor down a few millimeters. 15 Connect the column to the pump and open the outlet. 16 Pack the medium for an additional 3 minutes at 25 ml/min. Mark the bed height. 17 Stop the flow, close the column outlet and reposition the adaptor to approximately 2 mm below the mark. Note: Make sure that the QuickLock is in position (it should click into position). This is easier if the adaptor is pushed straight down instead of screwed into the column. 18 Close the column inlet AC 17

18 Packing large-scale columns (BPG 300) The following instruction is for packing MabSelect SuRe in BPG 300. Definitions The bed height of a gravity settled bed differs from the bed height of a bed settled at low flow (consolidated). Therefore, the compression factor (CF), used in the media preparation step to calculate the medium volume, has to be separated from the packing factor (PF) which is used in the packing procedure to calculate the packed bed height after the consolidation step. Symbol Description Equation L settled : Gravity settled bed height Bed height measured after settling by gravity L cons : Consolidated bed height Bed height measured after settling the medium at a given flow velocity L packed : Packed bed height CF: Compression factor CF = L settled L packed PF: A C : V C : c slurry : Packing factor PF = L cons L packed Cross sectional area of the column Column volume V c = L packed A c Concentration of the slurry Slurry preparation Start by calculating the medium volume, (V), needed to pack the desired bed height. In this step use the compression factor, (CF = 1.07), which is the factor in 20% EtOH for gravity settled AC

19 MabSelect SuRe. This corresponds to the right packing factor in the method. V = A c L packed CF c slurry Column and system preparation A detailed description of column preparation is available in the BPG instructions ( ). The packing pump should be as pulsation free as possible. Screw or rotary lobe pumps are the most suitable for this task and multiheaded diaphragm pumps are satisfactory. 1 Place a new 23 µm net on both adapter and bottom end piece. 2 Level the column with the aid of a spirit level. 3 A pressure relief valve should be used for safety reasons, especially against pressure spikes. Position this valve on the pump outlet and add a pressure gauge on the adapter. 4 Mount one 4-port-2-way valve on bottom inlet and one on top of the pressure gauge, 10 mm inner diameter for BPG 300. Medium Preparation Equilibration to the packing solution can be performed by using the BPG column as a filter. Pour the medium into the column (amount calculated above), mount the adapter, tighten the adapter O-ring, move the adapter down and compress the bed slightly, connect the pump and wash the medium with the packing solution for at least 3 V C. For details, use step 1-6 in the packing instruction below but continue with step 6 until 10 cm bed has built up against the adaptor net. Unpack and re-suspend the slurry and pack according to the method. MabSelect SuRe is packed with water. Packing the column 1 Set the pressure alarm or pressure relief valve according to the pressure specification (4 bar). Purge the system and tubing from air AC 19

20 2 Purge the net of trapped air by draining some packing solution through the column outlet. Leave about 2 cm of solution in the column and close the bottom valve. If air is still trapped under the end-piece net add more packing solution and connect a tube to the suction side of a pump. Start the pump and place the pump inlet tube on the bottom net and extract the remaining air. 3 Add the slurry to the column and, if needed, additional packing solution to about 40 cm. Mix the medium and the packing solution to a homogeneous slurry. 4 Rinse the wall from particles and let the medium settle until there is about 1 cm clear liquid on top of the slurry. This reduces the risk of particles sticking between the O-ring and the column wall, which can cause the column to leak. 5 Insert the adapter and secure it to the column tube. Lower the adapter to the surface of the slurry, allow some clear liquid to pass the O-ring. Tighten the adapter O-ring. 6 Make sure the column top valve is open. Slowly move the adapter down until no air bubbles can be seen leaving the top valve. 7 Start the pump and adjust the settling velocity to 60 cm/h (42 l/h). Shift the top valve into the column and immediately open the bottom valve and lead the liquid to waste. 8 Run the settling flow until the bed is completely consolidated. Note the consolidated bed height and calculate the packed bed height using a packing factor (PF) = The packed bed height is the ratio between the consolidated bed height and the packing factor. Use a marker pen to indicate the packed bed height on the column. 9 Stop the flow and close the bottom valve. Loosen the O-ring and lower the adapter down to 1 cm above the settled bed and seal the adapter O-ring. Shift the top valve to waste and use the adapter to mechanically compress the bed to the mark on the column (step 8). Excessive packing solution is removed through the adapter tube. The column is now ready for use AC

21 11 Evaluation of column packing Test column efficiency to check the quality of the packing. Tests should be made directly after packing and at regular intervals during the working life of the column and also when separation performance is seen to deteriorate. The best method of expressing the efficiency of a packed column is in terms of the height equivalent to a theoretical plate (HETP) and the asymmetry factor (A s ). These values are easily determined by applying a sample such as 1% acetone solution to the column. Sodium chloride can also be used as a test substance. Use a concentration of 0.8 M NaCl in water with 0.4 M NaCl in water as eluent. Note: The calculated plate number will vary according to the test conditions and it should only be used as a reference value. It is important that test conditions and equipment are kept constant so that results are comparable. Changes of solute, solvent, eluent, sample volume, flow velocity, liquid pathway, temperature, etc. will influence the results. For optimal results, the sample volume should be at maximum 2.5% of the column volume and the fluid velocity 30 cm/h. If an acceptance limit is defined in relation to column performance, the column plate number can be used as one of the acceptance criteria for column use. Method for measuring HETP and A s Calculate HETP and A S from the UV curve (or conductivity curve) as follows: L = bed height (cm L HETP = N 2 V N = 5.54 R W h N = number of theoretical plates V R = volume eluted from the start of sample application to the peak maximum W h = peak width measured as the width of the recorded peak at half of the peak height V R and W h are in the same units The concept of reduced plate height is often used for comparing column performance AC 21

22 The reduced plate height, h, is calculated as follows: HETP h = d 50v d 50v = mean diameter (m) of the beads As a guideline, a value of < 3 is very good. The peak should be symmetrical, and the asymmetry factor as close to 1 as possible (values between 0.8 and 1.6 are usually acceptable). A change in the shape of the peak is usually the first indication of bed deterioration due to excessive use. Peak asymmetry factor calculation: A s = b a a = ascending part of the peak width at 10% of peak height b = descending part of the peak width at 10% of peak height Figure 8 shows a UV trace for acetone in a typical test chromatogram from which the HETP and A s values are calculated. Absorbance V R W h 50% a b 10% Volume Fig 8. A typical test chromatogram showing the parameters used for HETP and A s calculations AC

23 12 Ordering information Product Quantity Code No MabSelect SuRe 25 ml ml l l l Related product Quantity Code No HiTrap MabSelect SuRe 5 1 ml ml ml HiScreen MabSelect SuRe ml XK16/20 column XK 16/40 column XK 16 packing connector AK 16 adapter Related literature Code No. Data Files MabSelect SuRe BPG columns AxiChrom Columns Chromaflow columns Application notes MabSelect SuRe Leakage and Toxicity MabSelect Column packing AC 23

24 For contact information for your local office, please visit: contact GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5, D Freiburg, Germany GE Healthcare UK Ltd Amersham Place Little Chalfont Buckinghamshire, HP7 9NA UK GE Healthcare Bio-Sciences Corp 800 Centennial Avenue P.O. Box 1327 Piscataway, NJ USA GE Healthcare Bio-Sciences KK Sanken Bldg , Hyakunincho Shinjuku-ku, Tokyo Japan GE, imagination at work and GE monogram are trademarks of General Electric Company. ÄKTAdesign, AxiChrom, BPG, BioProcess, Drop Design, Chromaflow, HiScreen, HiTrap, MabSelect, MabSelect SuRe, and Sepharose are trademarks of GE Healthcare companies. MabSelect SuRe Ligand: MabSelect SuRe Ligand Restricted License and Cys-rProtein A Ligand Restricted License are protected by the following patents and equivalent patents and patent applications in other countries: US 5,151,350, US 5,143,844, US 6,399,750, WO 03/00475 and EP A free, non-transferable limited license to use this product for internal analytical purposes only accompanies the purchase of the product from a GE Healthcare company and its licensed distributors. Any other use will require a separate license from a GE Healthcare company. All third party trademarks are the property of their respective owners General Electric Company All rights reserved. First published June 2004 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. imagination at work AC 09/2008