Waters Protein-Pak Hi Res IEX Columns User Manual

[ CARE AND USE MANUAL ]

columns

Protein-Pak Hi Res IEX Columns and Standards

CONTENTS

I. INTRODUCTION

II. CONFIGURING AN ACQUITY SYSTEM FOR
USE IN IEX PROTEIN SEPARATIONS

III. GETTING STARTED
a. eCord Installation

b. Column Connectors
c. Column Installation
d. Column Equilibration
e. Useful Functional Tests for
Benchmarking a New Column

IV. COLUMN SPECIFICATIONS AND USE
a. Specifications

b. Sample Preparation
c. Operating pH
d. Solvents
e. Pressure
f. Flow Rate
g. Temperature

I. INTRODUCTION

Thank you for choosing a Protein-Pak™ ion-exchange column.

Waters Protein-Pak Hi Res IEX family of columns were developed

to assist in the UPLC® characterization of recombinant proteins

and monoclonal antibodies found in many of today’s novel

biopharmaceutical therapeutics. T he non-porous, high-ligand

density particles overcome some of the high molecular weight

limitations present using traditional porous, IEX particles. In

addition, use of a multi-layered network of ion-exchange groups

(SP, CM, or Q) on non-porous particles assist the effective diffusion

and binding of charged proteins or other biomolecules onto the

particles to deliver relatively high sample loading capacities and

component resolution while minimizing column fouling.

The available ion-exchange chemistries include a strong-anion

and weak- and strong-cation exchangers (Table 1), all of which are

stable, over a wide pH range (3–10), high salt concentrations and

standard pressures (see Column Specifications and Use section) via

use of polymer-based particles. Only when the Protein-Pak Hi Res

IEX column series is combined with the ACQUITY UPLC® system

will the full performance benefits of this column be realized.

V. TROUBLESHOOTING

VI. COLUMN CLEANING, REGENERATION,
AND STORAGE
a. Cleaning and Regeneration

b. Storage

VI. INT RODUCING ECORD INTELLIGENT
CHIP TECHNOLOGY
a. Introduction

b. Installation
c. Manufacturing Information
d. Column Use Information

VIII. ORDERING INFORMATION

Table 1

Abbreviation Ion-Exchange Ligand

SP Sulfopropyl

CM Carboxymethyl

Q Quaternary ammonium

[ CARE AND USE MANUAL ]

2 mm

II. CONFIGURING AN ACQUITY SYSTEM FOR
USE IN IEX PROTEIN SEPARATIONS

Although Waters Protein-Pak Hi Res IEX columns do not require

the high pressure capability of an ACQUITY UPLC system, the full

performance benefits of this column will only be realized with the

low system volume and low detector dispersion of the optimized

ACQUITY UPLC system. However, ion-exchange chromatography

may require modifications to an existing ACQUITY UPLC system.

Please refer to “Size Exclusion and Ion-Exchange Chromatography

of Proteins using the ACQUITY UPLC System”, (715002147A) for

specific recommendations that can be obtained at www.waters.

com/chemcu.

III. GETTING STARTED

An Analysis Report and an Inspection Data Sheet are generated

and available for each shipped Protein-Pak Hi Res IEX column.

The Analysis Report is specific to each batch of packing material

and includes the batch number and chromatographic separation

obtained using defined protein standards. This document is

available upon request http://www.waters.com/waters/form.

htm?id=10048430&ev=10048691&locale=en_US. The

Inspection Data Sheet is included with each shipment and is

specific to each individual column and contains the following

information: batch number, column serial number, plate count,

tailing factor, retention factor, and chromatographic separation

conditions. These data should be stored for future reference.

b1. The ACQUITY UPLC system utilizes tubing and gold plated

compression screws that have been designed to meet

stringent tolerance levels and to minimize extra column

volumes. The columns should be attached to the ACQUITY

UPLC injector with a column stabilizer.

b2. Waters Protein-Pak Hi Res columns require end fittings that

have a 2 mm depth between the ferrule and the end of the

extending stainless steel tubing (Figure A) that must perfectly

seat to the end of the Protein-Pak Hi Res column. Connecting

a different style connector to a Waters Protein-Pak Hi Res

IEX column may leave a gap between the end of the stainless

steel tubing and the column that will result in undesired peak

broadening (Figure B).

To correct this problem, disassemble the finger-tight fitting of

the stabilizer and remake the connection (i.e., 2 mm depth) to the

Waters Protein-Pak Hi Res IEX column.

Figure A

Figure B

a. eCord Installation

The eCord™ button should be attached to the side of the Waters

ACQUITY UPLC column heater module. The eCord button is

magnetized and does not require specific orientation.

b. Column Connectors

There are two options for connecting the Protein-Pak Hi Res

column to an ACQUITY system. The first option is to use the

reusable fitting on the 150 mm and 100 mm column stabilizers

(sect b1). This fitting/tubing is supplied with ACQUITY UPLC

column heaters. A second alternative is to remove this fitting and

replace it with a non removable fitting (i.e. steel). If the second

option is employed, specific directions must be followed (sec b2).

In a proper tubing/column connection, the tubing touches the

bottom of the column end fitting with no void between them.

(Figure C).

Figure C

Protein-Pak Hi Res IEX Columns and Standards

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[ CARE AND USE MANUAL ]

c. Column Installation

Note: The flow rates given in the procedure below are for a Protein-

Pak Hi Res CM or SP, 7 µm, 4.6 mm i.d. column.

1. Purge the solvent delivery system of any organic or water-

immiscible mobile phases and connect the inlet end of the column

to the injector outlet. An arrow on the column identification label

indicates the correct direction of solvent flow.

2. Flush column with 100% aqueous buffer (line A1) by setting

the pump flow to 0.2 mL/min.

3. When the buffer is flowing freely from the column outlet attach

the column outlet to the detector. Monitor the system pressure

to ensure the column is within its pressure limitations.

4. Gradually increase the flow rate, by not more than 0.5mL/min

at a time (as described in step 2).

5. Once a stable backpressure has been achieved, proceed to the

next section.

d. Column Equilibration

Protein-Pak Hi Res columns are shipped in 20 mM sodium sulfate

with 0.05% sodium azide. It is important to ensure mobile-phase

compatibility before changing to a different mobile-phase system.

Equilibrate the column with a minimum of 10 column volumes of

the buffer to be used (refer to Table 2 for column volumes).

Table 2. Empty Column Volumes in mL
(multiply by 10 for flush solvent volumes).

Column Dimension Approximate Volume

4.6 x 100 mm 1.66 mL

e. Useful Functional Tests for Benchmarking a New Column

It may be useful to benchmark the column performance with

a sample that is representative of the intended application. A

separation of common proteins with an appropriate method is

suggested for your new column. Two standards were specially

designed for this purpose. The IEX Cation Test Standard is

specifically designed with proteins that resolve well on cation-

exchange columns. The IEX Anion Test Standard is specifically

designed with proteins that resolve well on anion-exchange

columns. This test can also be used to monitor the condition of

your column after extended use.

The following sets of conditions and chromatograms show

examples of using the IEX Standards for benchmarking and

monitoring column and system health. T he exact results observed

in any laboratory will depend on the instrument in use. Similar

results can only be expected with a Waters ACQUITY UPLC system.

These tests are valuable for monitoring the life of the column

and for troubleshooting separation difficulties that may arise.

Protein-Pak Hi Res Cation-Exchange Columns and Standards

Sample Diluent: 20 mM Sodium Phosphate Buffer, pH 6–7

The IEX Cation Test Standard is packaged in a Waters TruView

Max Recovery Vial (p/n 186005668cv) and is lyophilized for

prolonged storage and stability. The standard is shipped at room

temperature but it upon arrival, it is recommended to store this

standard at -20 ˚C.

Protein Sigma P/N Conc. mg/mL

Bovine Ribonuclease A R5500 4

Cytochrome C, bovine heart C2037 4

Chicken Lysozyme L6876 4

Column: Protein-Pak Hi Res CM, 4.6 x 100 mm,

or Protein-Pak Hi Res SP, 4.6 x 100 mm

Injection Volume: 10.0 μL

Flow Rate: 1 mL/min

Mobile Phase A: 20 mM sodium phosphate buffer, pH 6.6

Mobile Phase B: 20 mM sodium phosphate buffer,

0.5 M NaCl, pH 6.6

Weak Needle Wash: Mobile phase A

Strong Needle Wash: Mobile phase B

Seal Wash: 90/10 H20/MeOH

Temperature: 30 ˚C

Detection: 280 nm

Gradient:

Time %A %B Curve

1 100 0 6

34 40 60 6

36 40 60 6

38 100 0 6

55 100 0 6

Protein-Pak Hi Res IEX Columns and Standards

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