Operating Instructions

ID Self Pack Packing Device for ImmunoDetection Cartridges Operating Instructions In This Document This document describes how to pack your own ImmunoDetection (ID ) Sensor Cartridges for use in Perfusion Immunoassays, automated assays that selectively capture biomolecules in a rapid, reproducible, flow-through format. You can immobilize the ligand before or after packing the sensor If you are immobilizing ligand before packing, refer to the Immobilizing Ligand for Perfusion Immunoassays document provided before continuing. Packing Device Parts The Self Pack packing device is made of corrosion-resistant 316L stainless steel that can be used with salt buffers. The packing device consists of these parts: Cap (center cover and lock nut) O-ring Chamber A backpressure regulator is included with the packing device. Figure 1 shows the parts of the packing device and the backpressure regulator. Related documents You receive two documents with the POROS Self Pack media: Outlet End Inlet End O-ring Cap Immobilizing Ligand for Perfusion Immunoassays Refer to this document before packing the cartridge, if you want to immobilize ligand on a batch of media. Refer to this document after packing if you want to immobilize on the packed Performing Unlabeled Perfusion Immunoassays Refer to this document after immobilizing ligand and packing the Backpressure regulator PB1000362 Chamber Lock nut Center cover 1 Product Description The ID Self Pack Packing Device for ImmunoDetection Sensor Cartridges lets you pack and repack empty PEEK 2.1 mmd/30 mml cartridges with POROS affinity Self Pack media. By packing your own ID cartridges, you get increased reproducibility between cartridges, flexibility in immobilization chemistry, and unsurpassed affordability. Figure 1 Packing Device Parts Flat surfaces on the packing device chamber allow you to grasp the chamber with a wrench if necessary, but hand tightening is usually sufficient. You purchase empty cartridges, ID Self Pack media, and reagents separately from the packing device. In These Instructions These instructions describe how to use the packing device and related hardware. They explain the following: How to assemble the packing device How to adapt the packing device to different LC instruments How to pack your own cartridges How to unpack a cartridge Section Page 1 Product Description... 1 2 Additional Items Needed to Pack Your Cartridge... 2 3 Preparing Your Equipment for Packing... 2 4 Packing a Cartridge... 4 5 Running the Packed Cartridge... 6 6 Unpacking a Cartridge... 6 7 Storing Packing Device and Cartridge... 6 8 Accessories, Spare Parts, and Ordering Information... 6 9 Technical Support... 7 1

The inlet end of the chamber connects to your LC instrument. It includes the following: A screw-on 2-piece cap with a 10-32 female fitting. You can adapt this fitting to M-6 metric systems (such as the Pharmacia FPLC system) or 1/4-28 systems by using the fitting adaptor kit supplied with the packing device. An O-ring seated in a depression around the rim of the inlet opening. The O-ring lets you hand-tighten the cap, even when you use the packing device with high-pressure instruments. The outlet end is the narrow end of the chamber, where: Two end plugs Two EZ Grip fittings and ferrules that are used to connect the packed cartridge to a system End fittings are assembled on the tube, and the end plugs and frits are installed. Figure 2 shows a PEEK cartridge without end plugs. End fitting You can connect an empty 2.1 mm diameter PEEK A white Teflon washer recessed in the outlet end of the packing chamber creates a proper seal when a cartridge is screwed in place. (This washer is not visible in Figure 1.) Tube Frit Fitting Adaptor Kit The fitting adaptor kit adapts the Self Pack packing device to M-6 metric or 1/4-28 LC systems. (For ordering information, see Table 2 on page 6.) The kit provides these fittings: 2 red Fingertight fittings 2 beige ferrules (barrel shaped) 2 black metric nuts 2 green nuts 2 blue ferrules (cone shaped) 2 pieces of orange 0.020-inch I.D. PEEK tubing To use the fitting adaptor kit, do the following: 1. Connect one end of the orange tubing to an M-6 or 1/4-28 system. For M-6 systems: Slip a black nut over the tubing followed by a blue ferrule, with the conical end pointing toward the nut. Connect to the LC system and finger tighten. For 1/4-28 systems: Slip a green nut over the end of the tubing, followed by a blue ferrule, with the conical end pointing away from the nut. Connect to the LC system and finger tighten. 2. Put a red Fingertight fitting on the other end of the tubing, followed by a beige ferrule. The fitting is now ready to be connected to the 10-32 fitting on the cap of the packing device. Because the cap is heavy, do not attach it now. Wait until you are ready to attach the cap to the chamber (see Section 4.2, Filling the Packing Device). 2 Additional Items Needed to Pack Your Cartridge In addition to the packing device, to pack a cartridge you need an empty PEEK cartridge, POROS Self Pack media (purchased separately), and your own ligand for immobilization. You can order this equipment from PerSeptive Biosystems. For ordering information, see Section 8, Accessories, Spare Parts, and Ordering Information. PEEK Cartridges Empty PEEK ID cartridges are sold in packages of 5, 10, or 20. Each empty PEEK cartridge comes with these parts: One hollow tube Two end fittings, which screw onto threads at either end of the cartridge tube Two frits that are placed in the end fittings to retain the media PB100365 Figure 2 PEEK Cartridge Parts 3 Preparing Your Equipment for Packing To pack a cartridge, do the following: Clean the cartridge and packing device Prepare the slurry solvent and packing solvent Prepare the packing device Prepare your LC system Assemble the packing device and empty cartridge Position the packing device Lab Equipment Needed In addition to the packing device and empty cartridge, you need this equipment to pack a cartridge: LC system Spatula Glass beaker of 50 ml or greater Squirt bottle Laboratory tissue 25 ml graduated cylinder or 20 ml pipette Deionized water 3.1 Cleaning the Cartridge and Packing Device NOTE: Observe good laboratory practice. Wear safety glasses during the following procedures. The cartridge and packing device are reusable. You clean them differently depending on whether you have used them before. Never Used End plug If the cartridge and packing device have never been used, do the following: Cartridge: Unscrew the end fittings and flush the tube and end fittings with deionized water. Do not reassemble the Set all the pieces aside. Packing device: Wash or sonicate the packing chamber and cap with 50% isopropanol, ethanol, or other alcohol to ensure cleanliness. Set all the pieces aside. 2

Previously Used If the cartridge or packing device has been used before, clean it before each reuse to avoid contamination with packing material left over from a previous use. Cartridge: Sonicate the tube, end fittings, and frits in 50% isopropanol, ethanol, or other alcohol for 10 minutes. Rinse all pieces thoroughly with deionized water. Set all the pieces aside. Packing device: Flush the packing device cap with deionized water and thoroughly wash the threads at the outlet end of the chamber. Residual media gets trapped in the threads during normal use. Wash the O-ring. Flush with organic solvent if you see any media residue on surfaces after the water wash. Rinse again with water to remove the organic solvent. Do not reassemble the packing device. Set all the pieces aside. 3.2 Preparing the Slurry Solvent and Packing Solvent To prepare the slurry solvent and packing solvent, do the following: 1. Prepare your slurry solvent and packing solvent (Table 1). Table 1 2. Fill the squirt bottle with the slurry solvent. 3.3 Preparing the Packing Device Make sure the white Teflon washer is installed in the outlet end of the chamber. The washer is required for a proper seal to pack the 3.4 Preparing Your LC System To prepare your LC system, do the following: Prepare the tubing Set the pressure limit Prepare the pumps Preparing the Tubing Slurry Solvents and Packing Solvents for Self Pack Media POROS Self Pack Media Slurry Solvent Packing Solvent AD 100 % ethanol 5 % ethanol BA, EP, AL, XL, PG, PA Water Water Make sure that the fitting on the end of your LC system pump outlet tubing is compatible with the fitting on the packing device cap. If it is not, use the fitting adaptor kit. See Fitting Adaptor Kit on page 2. Do not connect the free end of the pump outlet tubing to the cap of the packing device yet. For example, on an FPLC system, do the following: 1. Connect one end of the orange tubing of the fitting adaptor kit to the outlet of the FPLC mixing chamber with the M-6 black nut. See Fitting Adaptor Kit on page 2. 2. Reserve the free end of the orange tubing for the red Fingertight fitting that will connect into the cap of the packing device. Setting the Pressure Limit For Perfusion Chromatography or conventional HPLC systems: Set the pressure limit of your system at 3,000 psi. For medium-pressure systems: Set the pressure limit at the maximum allowable on the system. For example, on an FPLC system, set the pressure limit at 4.0 MPa. Preparing the Pumps Flush the pumps and the outlet tubing with the packing solvent listed in Table 1. NOTE: On an FPLC system, you must flush both pumps with packing solvent because both pumps are used during packing. 3.5 Assembling the Packing Device and Empty Cartridge To assemble the packing device and cartridge for packing, do the following: 1. Locate the equipment needed for packing. See Lab Equipment Needed on page 2. 2. Write the name of your POROS Self Pack media on one of the blank labels provided with the media. These labels identify the cartridges and also identify the top of the cartridge (the end connected to the packing chamber). Use the top as the inlet end. Attach the label to an end fitting. Reserve this fitting to be the top end fitting. 3. Set the cartridge end fittings on the countertop so that you can look down into them. 4. Drop a frit into each end fitting. 5. Pick up one end fitting containing a frit. 6. Screw the cartridge tube into the end fitting until finger-tight. 7. Tighten the end fitting a little more: grip the cartridge tube with one hand and the end fitting in the other hand and tighten securely. 8. Screw the free end of the cartridge tube into the outlet end of the packing chamber. Tighten securely with two hands. 9. Set the unused end fitting aside. 10. Screw the backpressure regulator into the cartridge (see Figure 3 on page 4). 3.6 Positioning the Packing Device 1. Clamp the open packing device/cartridge assembly on the front of your LC instrument. If clamps are not available on the instrument, use a ring stand. Make sure that the following conditions are met: The packing device is positioned vertically. The pump outlet tubing can reach the top of the packing device. You can fit a 50 ml beaker under the end of the 2. Position your empty beaker below the end of the 3. Seat the packing chamber O-ring in the depression around the rim of the chamber opening (shown in Figure 1 on page 1). 3

4 Packing a Cartridge Figure 3 shows a packing device setup on a BioCAD /SPRINT System. The packing operation consists of five steps: Slurrying the media Filling the packing device Packing the cartridge Removing and capping the packed cartridge Cleaning up 4.1 Slurrying the Media 1. Determine the weight or volume of media to add to the packing device (100 mg of dry media or 0.2 ml of wet settled media per cartridge). NOTE: With wet media of unknown volume, allow it to settle in a graduated test tube. Note the total volume (V t ) and the volume of settled media (V m). Calculate the volume in ml of resuspended media needed using the following equation: V = 0.2 x (Vt/Vm) 2. Add 5 ml of the appropriate slurry solvent (see Table 1 on page 3) to the media and mix. 4.2 Filling the Packing Device Caution: Do not proceed without setting your system pressure as described in Section 3.4, Preparing Your LC System. To fill the packing device, do the following: 1. Add the media slurry into the open packing device. 2. Rinse the pipette or cylinder with a small amount of slurry solvent to ensure that all of the slurry is added to the packing device. 3. Add slurry solvent to the packing device until it is completely full. NOTE: Do not stir up the slurry at the bottom of the packing device. Direct the slurry solvent so that it runs down the inner wall of the packing device, instead of pouring directly on the slurry. 4. Quickly screw the 2-piece cap onto the packing device: place the center cover on the chamber, thread the lock nut down over the cap and hand tighten until snug. Use a lab tissue to wipe any liquid that comes out through the top as you tighten the cap. PB1000367 Figure 3 Typical Packing Device Setup 5. Attach the pump outlet tubing from the LC system to the cap of the packing device. Finger tighten the fitting. 4.3 Packing the Cartridge This section discusses these topics: Principles of Packing Packing a Cartridge Using the INTEGRAL 100Q System, VISION or BioCAD Workstation, BioCAD/SPRINT System, BioCAD/RPM System, or FPLC system Packing a Cartridge Using Other Systems Principles of Packing Cap Packing chamber PEEK cartridge Backpressure regulator As a cartridge packs, the backpressure on the cartridge rises. The optimal packing flow rate is the highest flow rate possible that allows the following: Packing to proceed within the pressure limit you set earlier A sufficient volume of packing solvent to pass through the cartridge to ensure an efficient and stable bed The actual packing flow rate is dependent on the system used, the particle size of the media, and the length of the NOTE: Do not overtighten the cap. Overtightening can damage the threads of the chamber and the cap. 4

Packing a Cartridge Using the INTEGRAL 100Q System, VISION or BioCAD Workstation, BioCAD/SPRINT System, BioCAD RPM Workstation, or FPLC System NOTE: Monitor the highest sustained pressure that occurs during packing. Do not exceed this pressure when you run the packed To pack a cartridge, do the following: 1. Use a total volume of at least 25 ml packing solvent to pack the 2. Set the flow rate for packing the cartridge at 9 ml/min. 3. Turn on the flow and monitor the volume of packing solvent in the waste beaker below the If the pressure is below the system limit increase the flow rate until a pressure of 2,000 psi is attained, or the maximum system flow rate is reached, whichever comes first. 4. Once at least 25 ml of packing solvent has passed through the cartridge and a constant pressure has been reached, stop the flow. 5. Let the cartridge sit for 3 minutes to allow the pressure to dissipate evenly across the packed bed. NOTE: The packing flow rate has been determined to allow you to pack your cartridge within the pressure limit of your system. However, variations in frit permeability or system backpressure may require you to adjust the flow rate slightly. 6. If your system is well below the pressure limit during packing, increase the flow rate in 0.5 ml/min increments until you approach the pressure limit. Pack cartridges at the highest flow rate possible without exceeding the system pressure limit. 7. If you reach the pressure limit before you finish packing the cartridge, stop the flow, restart the flow at 0.5 ml/min lower than the original setting, and continue packing. Repeat if necessary until you are able to pack the cartridge within the pressure limit. NOTE: Occasionally a defective frit generates excessive backpressure. If so, replace both frits and start again. 8. Record the highest sustained pressure that occurred during packing. Do not exceed this pressure when you run the packed Packing a Cartridge Using Other Systems NOTE: Monitor the highest sustained pressure that occurs as you pack the Do not exceed this pressure when you run the packed If you are using systems other than the INTEGRAL Micro Analytical Workstation, VISION or BioCAD Workstation, BioCAD/SPRINT System, BioCAD RPM System, or FPLC system, you need to experiment to find the correct flow rate. Start by setting the flow at either 9 ml/min or the maximum allowable on the system, whichever is lower. When using other systems to pack the cartridge, note the following: 1. Use a total volume of at least 25 ml packing solvent to pack the 2. Turn on the flow and monitor the volume of packing solvent coming through the cartridge by observing the level in the waste beaker below the 3. If you reach the pressure limit before the required volume of packing solvent passes through the cartridge, stop the flow, restart the flow rate at 0.5 ml/min lower than the original setting, and continue packing. 4. If the pressure limit is again exceeded, lower the flow rate another 0.5 ml/min. 5. Continue lowering the flow rate in 0.5 ml/min increments until at least 25 ml of packing solvent passes through the cartridge and you reach a constant pressure; then, stop the flow. NOTE: Occasionally a defective frit generates excessive backpressure. If so, replace the frits and start again. 6. Let the cartridge sit for 3 minutes to allow the pressure to dissipate evenly across the packed bed. 7. Record the final flow rate. Use this flow rate the next time you pack a cartridge with your system. 8. Record the highest sustained pressure that occurred during packing. Do not exceed this pressure when you run the packed 4.4 Removing and Capping the Packed Cartridge To remove the cartridge, do the following: 1. Remove the backpressure regulator from the 2. Loosen the end fitting on the cap of the packing device to release the pressure in the 3. Unscrew the cartridge from the packing device. Grasp the bottom end fitting of the (Turn slowly at first to make sure the end fitting does not unscrew from the cartridge body.) Turn the cartridge to the left to unscrew it. A small bubble of excess packing material may cover the top end of the cartridge tube. If the material is wet, lightly touch the bubble with a lab tissue or paper towel to draw off the moisture. 4. Draw the flat side of the spatula across the top of the cartridge to remove the excess material and to level off the top of the packed bed. 5. With a damp tissue, carefully wipe any remaining packing material from around the rim at the top of the cartridge tube. Do not touch the top of the packed bed itself. 6. Holding the unused end fitting upright to ensure that the frit remains seated, turn the packed cartridge upside down and screw it into the end fitting. Keep track of the cartridge inlet end. 7. Tighten the end fitting with both hands. 8. Insert the end plugs into the cartridge end fittings. 9. Set the cartridge aside while you clean up from the packing procedure. 5

4.5 Cleaning Up To clean up, do the following: 1. Make sure an empty waste beaker is below the cartridge packing device. 2. Pump several milliliters of packing solvent through the packing device to push out excess packing material accumulated at the outlet end of the chamber. 3. Remove the connection at the top of the packing device. The pressure inside the packing device is released and the entire contents flush into the beaker. 4. Remove the packing device from the clamp, take off the cap, and clean all pieces thoroughly with water. 5. Rinse the threads at the outlet end to remove residual media. NOTE: The O-ring may stick to the cap of the packing device by surface tension. Use the spatula to pull it out. 5 Running the Packed Cartridge For information about running the packed cartridge, see the Performing Unlabeled Perfusion Immunoassays document provided. 6 Unpacking a Cartridge To unpack a cartridge, do the following: 1. Unscrew one end fitting and remove the frit. 2. Connect the other end of the cartridge to the pump outlet tubing of your LC system. 3. Position a beaker under the 4. Start the flow rate at 5 ml/min. 5. As soon as the media is pushed out of the cartridge into the beaker, stop the flow. 6. Remove the remaining end fitting. 7. Sonicate the tube, end fittings, and frits in 50% isopropanol, ethanol, or other alcohol for 10 minutes. Rinse all pieces thoroughly with deionized water. 7 Storing Packing Device and Cartridge Cartridge: To store a packed cartridge, see the cartridge Operating Instructions sheet included with your ID Self Pack media. To store an unpacked cartridge, keep the tube, end fittings, frits, and end plugs together. Packing Device: Store the packing device with the cap on to keep the threads from knocking against other equipment. If the threads are damaged, the device may not seal properly during packing. Also, damaged threads may cause the cap to lock in place irreversibly. Ideally, store the capped device in its original box. 8 Accessories, Spare Parts, and Ordering Information Table 2 Spare Parts and Accessories Description Quantity Part Number ID Self Pack Packing Device 1 ea 1-9544-00 Chemistry Trial Kit AD, AL, BA, EP, and XL Self Pack Media, with reagents and 5 empty cartridges 1 kit 2-3011-00 ID Sensor Cartridge Kits (cartridges with reagents) AD ID Sensor Kit 1 kit 2-3006-00 AL ID Sensor Kit 1 kit 2-3010-00 BA ID Sensor Kit 1 kit 2-3005-00 EP ID Sensor Kit 1 kit 2-3003-00 PA ID Sensor Kit 1 kit 2-3001-00 PG ID Sensor Kit 1 kit 2-3002-00 XL ID Sensor Kit 1 kit 2-3004-00 ID Sensor Cartridges (cartridges only) AD ID Sensor Cartridge 1 cartridge 2-1007-00 AL ID Sensor Cartridge 1 cartridge 2-1010-00 BA ID Sensor Cartridge 1 cartridge 2-1005-00 EP ID Sensor Cartridge 1 cartridge 2-1003-00 PA ID Sensor Cartridge 1 cartridge 2-1001-00 PG ID Sensor Cartridge 1 cartridge 2-1002-00 XL ID Sensor Cartridge 1 cartridge 2-1004-00 Self Pack Media AD (Adsorbed) 500 mg 2-3114-00 AL (Aldehyde) 500 mg 2-3115-00 BA (Streptavidin) 1 ml 2-3111-00 EP (Epoxide) 500 mg 2-3116-00 PA (Protein A) 1 ml 2-3117-00 PG (Protein G) 1 ml 2-3118-00 XL (Cross-linked Protein G) 1 ml 2-3119-00 Reagents Loading Buffer 1 pack 2-2101-00 Elution Buffer 1 pack 2-2102-00 AD ID Reagent Pack 1 set 2-3120-00 AL ID Reagent Pack 1 set 2-3121-00 EP ID Reagent Pack 1 set 2-3122-00 XL ID Reagent Pack 1 set 2-3124-00 Empty Cartridges 2.1 mmd/30 mml PEEK Pkg of 5 2-3008-05 2.1 mmd/30 mml PEEK Pkg of 10 2-3008-10 2.1 mmd/30 mml PEEK Pkg of 20 2-3008-20 Accessories, Packing Device Fitting Adaptor Kit 1 ea 1-9532-00 O-Ring 1 ea 1-9108-00 6

Table 2 Spare Parts and Accessories (Continued) Description Quantity Part Number Accessories, Cartridge Frits, 2.1 mm PEEK Pkg of 5 1-9124-05 EZ Grip Fittings (SS) Pkg of 5 P5-1011-05 9 Technical Support PerSeptive Biosystems is dedicated to helping you use Perfusion Chromatography and ID media to the fullest extent possible. Our biochromatographers, bioprocess engineers, and applications development laboratories are available for support ranging from telephone consultation to full method development. PerSeptive Biosystems offers a full line of ID media for Perfusion Chromatography in the reversed-phase, ion exchange, affinity, and other chromatographic modes. Please contact your PerSeptive Biosystems representative for technical and ordering information. PerSeptive Biosystems publishes a continuing series of Application and Technical Notes, highlighting specific purification solutions and technical aspects of Perfusion Chromatography. Please contact PerSeptive Biosystems directly for a publication list. For further details or for answers to questions on ID Self Pack, Perfusion Chromatography, or other products, please contact PerSeptive Biosystems. Refer to the back cover of this document for the number of the appropriate office. 7

PerSeptive Biosystems, POROS, Perfusion Chromatography, BioCAD, INTEGRAL, RPM, Self Pack, and the fractal icon are registered trademarks of PerSeptive Biosystems, Inc. ID, ImmunoDetection, SPRINT, and VISION are trademarks of PerSeptive Biosystems, Inc. Luer-Lok is a registered trademark of Becton-Dickinson and Company. Tris is a registered trademark of Rohm and Haas Company. FPLC is a registered trademark of Pharmacia Biotech AB. Teflon is a registered trademark of E. I. Du Pont de Nemours. EZ is a trademark of Scientific Systems, Inc. POROS products and perfusive chromatography are covered by US patents 5,019,270; 5,030,352; 5,228,989; 5,384,042; 5,552,041; 5,605,623; and 5,833,861; issued to PerSeptive Biosystems, Inc.; foreign patents pending. Chromatography materials of certain pore geometries, including wide-pore supports, can permit perfusive chromatography, that is, separations where the rate of intraparticle convection exceeds the rate of diffusion. Use of any such chromatography media that permit performance at high linear velocity, without license, constitutes infringement of US patent 5,019,270 and US patent 5,228,989. A limited license to use the patented perfusive chromatography process is granted with the purchase of POROS products from PerSeptive Biosystems. The license may not be transferred except with the transfer of POROS columns or media, and terminates upon expiration of the useful life of the product. Part Number 8-0122-40-0795, Rev. 2, 3/00 2000 Printed In USA Printed on recycled paper. PE Biosystems Headquarters 850 Lincoln Centre Drive Foster City, CA 94404 Tel: (650) 638-5800 Tel: (800) 345-5224 www.pebiosystems.com PerSeptive Biosystems Center of Excellence 500 Old Connecticut Path Framingham, MA 01701 Tel: (508) 383-7700 Tel: (800) 899-5858 www.pbio.com For international office locations, please call the headquarters or refer to our web site at www.pebiosystems.com.