Thermo Fisher Scientific POROS ® Affinity Operating instructions

Brand: Thermo Fisher Scientific

Model: POROS ® Affinity

Type: Operating instructions

Operating Instructions

1 Product Description

POROS® Affinity Depletion Cartridges and columns are available in

two affinity chemistries:

•POROS Anti-HSA Cartridges – Packed with POROS

Perfusion Chromatography media covalently coupled with an

affinity-purified goat polyclonal antibody against human serum

albumin (HSA).

•POROS Protein G Cartridges – Packed with POROS

Perfusion Chromatography media covalently coupled with

recombinant Protein G.

POROS® Affinity Depletion Cartridges can be operated:

• Singly or in tandem with a cartridge of a different chemistry.

• Manually or on an automated MDLC (multi-dimensional liquid

chromatography) workstation (for example, Applied

Biosystems VISION™ or BioCAD workstations).

Table 1 Product Characteristics

Support Matrix Cross-linked poly(styrene-divinylbenzene)

Immobilized Ligand • POROS Anti-HSA – Affinity-purified

goat anti-HSA polyclonal antibody

•POROS G – Recombinant protein G

Saturation Capacity

@ 500 cm/hr •POROS Anti-HSA – 2 mg/mL. For

complete removal of HSA in serum

samples, do not exceed 50% of the

saturation capacity. For more

information, see the Sample Volume

ranges listed on page 3.

•POROS G – 15 mg/mL. For complete

removal of IgG, do not exceed 50% of

the saturation capacity. When used in

tandem with a POROS Anti-HSA

cartridge, do not exceed the Anti-HSA

cartridge capacity.

Shipping Solvent 0.1 M phosphate buffer pH 7.0,

0.05% sodium azide

Shrinkage/Swelling <1% from 1 to 100% solvent

Cartridge Dimensions 4 mmD × 15 mmL (0.2 mL)

6.2 mmD × 66.5 mmL (2.0 mL)

7.5mmD × 100 mmL (4.4 mL)

Table 2 Chemical Resistance

pH Range • POROS Anti-HSA – 2 to 10

•POROS G – 2 to 9

Ionic Strength Range 0 to 5 M, all common salts

Buffer Additives • POROS Anti-HSA – Agents that may

degrade the protein ligands are not

recommended.

•POROS G – All common agents,

including 4 M urea, 2 M guanidine

hydrochloride, ethylene glycol, and

detergents. Agents that may degrade

the protein ligands are not

recommended.

Solvents Water, 0 to 30% alcohols, acetonitrile,

other common organic solvents.

NOTE: Do not expose to strong oxidizers

(such as hypochlorite), oxidizing acids

(such as nitric acid), or strong reducing

agents (such as sulfite).

Operating

Temperature 5 to 40 °C

Contents Page

1 Product Description........................................................1

2 Connecting the Cartridge ...............................................2

3 Buffers............................................................................2

4 Preparing the Anti-HSA Cartridge..................................2

5 Cartridge Operating Conditions......................................3

6 Running a Cleanup Method ...........................................4

7 Storing the Cartridge......................................................5

8 Accessories, Spare Parts, and Ordering Information.....5

9 Technical Support...........................................................5

POROS® Affinity Depletion

Cartridges

2 Connecting the Cartridge

POROS Affinity Depletion Cartridges can be used singly or in tandem

with a second cartridge of different chemistry.

2.1 Single-Cartridge Connection

Figure 1 illustrates connections of a single cartridge for manual use.

For use with an automated MDLC workstation (for example, Applied

Biosystems VISION™ or BioCAD workstations), plumb the cartridge

inlet using standard 10/32 HPLC fittings (PEEK or stainless steel).

Figure 1 Cartridge Connection for Manual Use

2.2 Tandem-Cartridge Connection

For tandem-cartridge connection, connect a 5-cm piece of blue PEEK

tubing (1/16-inch O.D., 0.010-inch I.D.) between cartridges using

standard 10/32 HPLC fittings.

3Buffers

In general, when you prepare buffers:

• Use buffers of the highest purity practical.

• Degas and filter (0.22 or 0.45 µm) all buffers before use.

Prepare the following buffers.

4 Preparing the Anti-HSA Cartridge

Before injecting human serum for the first time on the Anti-HSA

cartridge, perform one blank run (no sample) that includes the

following steps:

• 5 cartridge-volume equilibration step

• 5 cartridge-volume elution step

• 5 cartridge-volume clean step

• 5 cartridge-volume equilibration step

The Equilibration step followed by the Elution step removes any weakly

bound antibody from the cartridge and increases the run-to-run

reproducibility.

Syringe

Needle-port

adapter

Cartridge holder

with cartridge

Compression screw

1/16-inch O.D. PEEK

outlet tubing

Syringe needle

Buffer Composition

Equilibration, Load,

and Wash Phosphate buffered saline (PBS), pH 7.2

Elution 12 mM HCl

CHEMICAL HAZARD.

Hydrochloric acid (HCl) causes severe

eye, skin, and respiratory tract burns.

Read the MSDS, and follow the handling

instructions. Wear appropriate protective

eyewear, clothing, and gloves.

Cleaning • Anti-HSA – 1 M NaCl

•Protein G – 2 M urea in 1 M NaCl or

10% acetic acid in 1 M NaCl

CHEMICAL HAZARD.

Read the MSDS, and follow the handling

instructions. Wear appropriate protective

eyewear, clothing, and gloves.

Urea may cause eye, skin and respiratory

tract irritation.

10% acetic acid in 1 M sodium chloride

is a flammable liquid and vapor. Exposure

causes eye, skin, and respiratory tract

burns. It may be harmful if inhaled,

swallowed, or absorbed through the skin.

Keep away from sparks, heat, and flame.

DANGER

CAUTION

5 Cartridge Operating Conditions

BIOHAZARD. Biological samples such as tissues, body fluids, and blood of humans and other animals have the potential to

transmit infectious diseases. Read and follow the guidelines published in Biosafety in Microbiological and Biomedical Laboratories

(http://bmbl.od.nih.gov) and in the Occupational Safety and Health Standards, Bloodborne pathogens (http://www.access.gpo.gov/nara/cfr/

waisidx_01/ 29cfr1910a_01.htm). Follow all applicable local, state/provincial, and/or national regulations. Wear appropriate protective eyewear,

clothing, and gloves.

Step/Buffer Condition Settings

0.2-mL Cartridge 2.0-mL Cartridge 4.4 mL-Cartridge

Equilibration

PBS, pH 7.2 Flow Rate

(5 to 10 cartridge volumes) 1 mL/min 2.4 mL/min 3.5 mL/min

Load – Anti-HSA

PBS, pH 7.2 Note: The concentration of albumin in diluted human serum is 3 to 7 mg/mL. Adjust sample volume to accommodate the

albumin concentration in your sample. For example, if the albumin concentration in your 10% diluted human serum sample

is ~3 mg/mL, you can load up to the maximum of 70 µL. If the albumin concentration in your sample is ~7 mg/mL, load a

lower sample volume of 30 µL.

Sample Volume

(10% diluted human serum with PBS) 30 to 70 µL 300 to 700 µL 600 to 1,600 µL

Flow Rate 0.5 mL/min 1.2 mL/min 1.8 mL/min

Comment Capture the Load step effluent in up to five 0.5-CV fractions. Determine which

fractions contain protein (measure by UV absorbance at 280 nm), then pool the

protein-containing fractions for further proteomics analysis.

If breakthrough of albumin occurs, it may be because the concentration of

albumin in the sample exceeds the cartridge capacity. Repeat the run and use a

smaller injection volume.

Load – Protein G

PBS, pH 7.2 Note: The concentration of IgG in diluted human serum is 0.8 to 1.8 mg/mL. Adjust sample volume to accommodate the

IgG concentration in your sample. For example, if the IgG concentration in your 10% diluted human serum sample is

~0.8 mg/mL, you can load up to the maximum of 2 mL. If the IgG concentration in your sample is ~1.8 mg/mL, load a lower

sample volume of 1 mL.

Sample Volume

(10% diluted human serum with PBS) 1 to 2 mL 20 to 10 mL N/A

Flow Rate 0.5 mL/min 1.2 mL/min N/A

Comment Capture the Load step effluent in up to five 0.5-CV fractions. Determine which

fractions contain protein (measure by UV absorbance at 280 nm), then pool the

protein-containing fractions for further proteomics analysis.

Wash

PBS, pH 7.2 Flow Rate

(15 to 30 cartridge volumes) 1 mL/min 2.4 mL/min 3.5 mL/min

Elution

12 mM HCl Flow Rate

(5 to 10 cartridge volumes) 1 mL/min 2.4 mL/min 3.5 mL/min

Comment For optimum performance, run an Elution step during each cycle to remove the

bound serum proteins and prepare the cartridge for the next cycle.

Cleaning

•Anti-HSA –

1 M NaCl

•Protein G –

2 M urea in 1 M

NaCl or 10%

acetic acid in

1 M NaCl

Flow Rate

(5 to 10 cartridge volumes) 1 mL/min 2.4 mL/min 3.5 mL/min

Comment Perform a Cleaning step after 1 to 2 cycles to reduce fouling that may decrease

cartridge performance.

WARNING!

6 Running a Cleanup Method

In some applications, or after many cycles, sample molecules may not

fully elute or may precipitate on the cartridge and cause any of the

following symptoms:

• Increased bandspreading

• Loss of binding capacity

• Loss of recovery

• Increased pressure drop

• Trace or “ghost” peaks different from the usual RI (refractive

index) shift peak during blank runs

Clean the cartridge if any of the symptoms listed above appear.

NOTE: In any cleanup method, reversing the flow direction is

recommended to help flush out particulates and to prevent

contamination of the lower part of the bed. Also, to expose the

cartridge to the regeneration solution for several minutes at each step

of the cleaning protocol, slowing the flow rate is recommended.

Recommended Cleaning Solutions

CHEMICAL HAZARD. Urea may cause eye, skin

and respiratory tract irritation. Read the MSDS, and follow the handling

instructions. Wear appropriate protective eyewear, clothing, and

gloves.

CHEMICAL HAZARD. 10% acetic acid in 1 M

sodium chloride is a flammable liquid and vapor. Exposure causes

eye, skin, and respiratory tract burns. It may be harmful if inhaled,

swallowed, or absorbed through the skin. Keep away from sparks,

heat, and flame. Read the MSDS, and follow the handling instructions.

Wear appropriate protective eyewear, clothing, and gloves.

Cleaning by Pumping

To clean the cartridge, follow these guidelines:

1. Wash with 15 to 30 column volumes of the solution recommended

for your cartridge to remove contaminants.

2. For Protein G cartridges only, you can remove precipitated protein,

bound lipids, and so on, by other means, for example:

• Brief washes with alcohols, acetonitrile, or mixtures of these

solvents with 20% acetic acid

• 2 M guanidine hydrochloride

CHEMICAL HAZARD. Acetonitrile (ACN) is a

flammable liquid and vapor. Exposure may cause eye, skin, and

respiratory tract irritation, central nervous system depression, and

damage to the heart, blood system, liver, and kidneys. Read the

MSDS, and follow the handling instructions. Wear appropriate

protective eyewear, clothing, and gloves.

Cartridge Cleaning Solution

Anti-HSA 1 M NaCl

Protein G 2 M urea in 1 M NaCl

10% acetic acid in 1 M NaCl

CAUTION

DANGER

WARNING!

CHEMICAL HAZARD. 20% Acetic acid is a

flammable liquid and vapor. Exposure causes eye, skin, and

respiratory tract burns. It may be harmful if inhaled, swallowed, or

absorbed through the skin. Keep away from sparks, heat, and

flame. Read the MSDS, and follow the handling instructions. Wear

appropriate protective eyewear, clothing, and gloves.

CHEMICAL HAZARD. Guanidine

hydrochloride may be harmful if swallowed or absorbed through

the skin. Exposure may cause eye, skin, and respiratory tract

irritation and adverse effects on the central nervous system and

bone marrow. Read the MSDS, and follow the handling

instructions. Wear appropriate protective eyewear, clothing, and

gloves.

Cleaning by Making Multiple Injections

For very aggressive or very viscous solvents, use multiple injections of

cleaning solutions instead of pumping cleaning solutions directly.

To clean by injections:

• Make the injection volume as large as possible.

• Use a low flow rate to expose the cartridge to the cleaning

solution for several minutes.

DANGER

WARNING

7 Storing the Cartridge

When you store your cartridge, always be sure to:

• Store cartridges in the refrigerator. DO NOT FREEZE

CARTRIDGES!

• Store the cartridge with the endcaps in place, carefully sealed

to prevent drying. Drying results in decreased chromatographic

efficiency.

Short-Term Storage (less than 1 day)

1. Equilibrate the cartridge in PBS.

2. Remove the cartridge from the cartridge holder and insert end caps

to prevent the cartridge from drying out.

3. Store cartridges at 4 °C.

Long-Term Storage (more than 1 day)

1. Equilibrate the cartridge in PBS with 0.05% sodium azide.

Sodium azide may react with lead and copper

plumbing to form highly explosive metal azides. If disposing of

solutions that contain sodium azide down laboratory sinks, flush

the drains with large volumes of water to prevent azide buildup.

2. Remove the cartridge from the cartridge holder and insert end caps

to prevent the cartridge from drying out.

3. Store at 4 °C.

8 Accessories, Spare Parts, and Ordering

Information

Available accessories for your POROS® Affinity Depletion cartridges

are listed below:

9 Technical Support

Applied Biosystems is dedicated to helping you use POROS Affinity

Depletion Cartridges to the fullest extent possible.

Applied Biosystems publishes a continuing series of Proteomics

Application and Technical Notes. Please contact Applied Biosystems

directly for a publication list.

For further details or for answers to questions on POROS Affinity

Depletion cartridges, Perfusion Chromatography, or other products,

please contact Applied Biosystems. Refer to the back page of this

document for contact information.

WARNING

Table 3 Affinity Cartridge Accessories

Description Quantity Part

Number

POROS Affinity Depletion

Anti-HSA Cartridge ___ ___

4 mmD × 15 mmL (0.2 mL) 1 4337325

6.2 mmD × 66.5 mmL (2.0 mL) 1 4337344

7.5mmD × 100 mmL (4.4 mL) 1 4337345

POROS Affinity Depletion

Protein G Cartridge ___ ___

4 mmD × 15 mmL (0.2 mL) 1 4337331

6.2 mmD × 66.5 mmL (2.0 mL) 1 4337347

Cartridge Holder ___ ___

0.2 mL 1 4326688

2.0 mL 1 4337843

Needle-Port Adapter 1 4326689

Outlet Connector 1 4326690

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Phone: +1 650.638.5800

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Fax: +1 650.638.5884

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Applied Biosystems vast distribution and service

network, composed of highly trained support and

applications personnel, reaches 150 countries on six

continents. For international office locations, please

call our headquarters or see our Web site at

www.appliedbiosystems.com.

Technical Support

In North America, call +1 800.899.5858.

Outside North America, see our Web site at

www.appliedbiosystems.com, or call our local sales

office.

www.appliedbiosystems.com

Applied Biosystems is committed to providing

the world’s leading technology and information

for life scientists.

Printed in the USA, 04/2011

Part Number 4333109 Rev. B

For Research Use Only. Not for use in diagnostic procedures.

Information in this document is subject to change without notice.

Applied Biosystems assumes no responsibility for any errors that

may appear in this document. This document is believed to be

complete and accurate at the time of publication. In no event shall

Applied Biosystems be liable for incidental, special, multiple, or

consequential damages in connection with or arising from the use of

this document.

Applied Biosystems, BioCAD, Perfusion Chromatography, and

POROS, are registered trademarks of Applied Biosystems or its

subsidiaries in the U.S. and certain other countries.

AB (Design), Applera, and VISION are trademarks of Applied

Biosystems or its subsidiaries in the U.S. and certain other coun-

tries.

PEEK is a trademark of Victrex, PLC.

All other trademarks are the sole property of their respective owners.

Thermo Fisher Scientific POROS ® Affinity Operating instructions

Brand: Thermo Fisher Scientific

Model: POROS ® Affinity

Type: Operating instructions

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Thermo Fisher Scientific POROS ® Affinity Operating instructions

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