Thermo Fisher Scientific ID™ Self Pack ® Packing Owner's manual

Brand: Thermo Fisher Scientific

Model: ID™ Self Pack ® Packing

Type: Owner's manual

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

cartridge. If you are immobilizing ligand before packing, refer to the

Immobilizing Ligand for Perfusion Immunoassays document provided

before continuing.

Related documents

You receive two documents with the POROS® Self Pack® media:

•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 cartridge.

•Performing Unlabeled Perfusion Immunoassays—Refer to

this document after immobilizing ligand and packing the

cartridge.

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.

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

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.

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.

PB1000362

Chamber

O-ring Cap

Backpressure regulator

Outlet End Inlet End

Lock

nut Center

cover

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

ID™ Self Pack®

Packing Device

for

ImmunoDetection™

Cartridges

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:

•You can connect an empty 2.1 mm diameter PEEK cartridge.

•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.)

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

•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.

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

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 cartridge.

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.

PB100365

End fitting

Frit

Tube

End plug

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).

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

cartridge.

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

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 cartridge.

2. Position your empty beaker below the end of the cartridge.

3. Seat the packing chamber O-ring in the depression around the rim

of the chamber opening (shown in Figure 1 on page 1).

Table 1 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

4 Packing a Cartridge

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 (Vt) and the volume of

settled media (Vm). 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.

NOTE: Do not overtighten the cap. Overtightening can damage the

threads of the chamber and the cap.

Figure 3 shows a packing device setup on a BioCAD®/SPRINT™

System.

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

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 cartridge.

PB1000367

Packing chamber

Cap

PEEK cartridge

Backpressure

regulator

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

cartridge.

To pack a cartridge, do the following:

1. Use a total volume of at least 25 ml packing solvent to pack the

cartridge.

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 cartridge. 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 cartridge.

Packing a Cartridge Using Other Systems

NOTE: Monitor the highest sustained pressure that occurs as you pack

the cartridge. Do not exceed this pressure when you run the packed

cartridge.

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

cartridge.

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 cartridge.

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 cartridge.

4.4 Removing and Capping the Packed Cartridge

To remove the cartridge, do the following:

1. Remove the backpressure regulator from the cartridge.

2. Loosen the end fitting on the cap of the packing device to release

the pressure in the cartridge.

3. Unscrew the cartridge from the packing device. Grasp the bottom

end fitting of the cartridge. (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.

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 cartridge.

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

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.

Accessories, Cartridge

Frits, 2.1 mm PEEK Pkg of 5 1-9124-05

EZ Grip Fittings (SS) Pkg of 5 P5-1011-05

Table 2 Spare Parts and Accessories (Continued)

Description Quantity Part Number

500 Old Connecticut Path

Framingham, MA 01701

Tel: (508) 383-7700

Tel: (800) 899-5858

Center of Excellence

www.pbio.com

Headquarters

850 Lincoln Centre Drive

Foster City, CA 94404

Tel: (650) 638-5800

Tel: (800) 345-5224

www.pebiosystems.com

For international office locations, please call the headquarters or refer to our web site at www.pebiosystems.com.

PerSeptive Biosystems

PE Biosystems

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.