GE AxiChrom 50 User manual

GE Healthcare
Life Sciences
AxiChrom™ 50, 70 and 100
columns
Operating Instructions
Original instructions

Table of Contents
51 Introduction ..........................................................................................................
51.1 General introduction ...........................................................................................................................
51.2 Scope of this Operating Instructions ...........................................................................................
61.3 Important user information .............................................................................................................
81.4 Regulatory information ......................................................................................................................
102 Safety instructions ...............................................................................................
102.1 Safety precautions ...............................................................................................................................
142.2 Column pressure and over-pressure ...........................................................................................
152.3 Labels .........................................................................................................................................................
173 Specifications and characteristics ....................................................................
173.1 Principles of column function .........................................................................................................
223.2 Primary specifications ........................................................................................................................
233.3 Column lengths and bed support materials ............................................................................
233.4 Materials ...................................................................................................................................................
263.5 Chemical resistance ............................................................................................................................
293.6 Packing flow rates ................................................................................................................................
304 Getting started .....................................................................................................
304.1 Checking the packing list ..................................................................................................................
314.2 Identifying the main components of the column ..................................................................
324.3 Bed supports ...........................................................................................................................................
344.4 Wetting instructions for plastic bed supports .........................................................................
354.5 Other equipment ...................................................................................................................................
364.6 Routine procedures .............................................................................................................................
504.7 Over-pressure situation .....................................................................................................................
534.8 Preparing the column for a run ......................................................................................................
574.9 Installation in basic configuration ................................................................................................
60
4.10 Installing the UNICORN Intelligent Packing strategy on ÄKTAexplorer and ÄKTApi-
lot .................................................................................................................................................................
614.11 Selecting the Intelligent Packing strategy and component ..............................................
625 Operation ..............................................................................................................
635.1 Intelligent Packing of the column with ÄKTAexplorer and ÄKTApilot ...........................
795.2 Intelligent packing of the column with ÄKTA avant 150 .....................................................
805.2.1 Create a column type .......................................................................................................................
845.2.2 Creating an Intelligent Packing method using ÄKTA avant 150 ....................................
935.2.3 Preparing the slurry, priming and packing the column using ÄKTAavant ................
945.3 Manual packing of the column ......................................................................................................
1005.4 Performance evaluation of the column .....................................................................................
1025.5 Unpacking a packed column ..........................................................................................................
1035.6 Cleaning-in-place (CIP) ......................................................................................................................
1035.7 Sanitization ..............................................................................................................................................
AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG 3
Table of Contents

1055.8 Emptying the column ..........................................................................................................................
1065.9 Storage ......................................................................................................................................................
1086 Maintenance and service ...................................................................................
1086.1 About maintenance and service ...................................................................................................
1116.2 Performing maintenance and service ........................................................................................
1426.3 Performing a drainage test ..............................................................................................................
1436.4 Performing a column leakage test ...............................................................................................
1457 Troubleshooting ...................................................................................................
1457.1 Performance ...........................................................................................................................................
1497.2 Adapter ......................................................................................................................................................
1527.3 Top plate ...................................................................................................................................................
1527.4 Mechanical locking equipment ......................................................................................................
1527.5 Other ...........................................................................................................................................................
1538 Ordering information ..........................................................................................
1538.1 Useful accessories and spare parts .............................................................................................
1558.2 Contact information ............................................................................................................................
156A Parts list and diagrams AxiChrom 50 column ................................................
166B Parts list and diagrams AxiChrom 70 column ................................................
176C Parts lists and diagrams AxiChrom 100 column ............................................
186D Tubing kits and connections ..............................................................................
190Index .......................................................................................................................
4 AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG
Table of Contents

1 Introduction
1.1 General introduction
AxiChrom column family has been designed to deliver reproducible results from process
development to production scales. This is facilitated by the innovative Intelligent Packing
where UNICORN™ software, ÄKTA™ systems and AxiChrom columns work together to
facilitate a convenient operation for packing of the bed via axial compression.
AxiChrom 50, 70 and 100 are designed to meet process development needs, such as
scale-up trials, and also be used in pilot scale production of biomolecules. The ÄKTA
systems ÄKTA avant, ÄKTAexplorer and ÄKTApilot can be used for the AxiChrom 50 col-
umn, the 70 column can be used with ÄKTA avant and ÄKTApilot, while the 100 column
is compatible with ÄKTApilot only. These columns are packed using internal hydraulic
axial compression. The 70 and 100 columns are mounted on a pivot stand, which allows
easy handling and unpacking. AxiChrom 50, 70 and 100 come in two column tube lengths
to allow bed heights between 10-30 cm and 30-50 cm. The columns are validatable and
use materials classified according to USP Class VI and FDA CFR 177. The material is free
from material of animal origin or has been produced under manufacturing conditions
complying with EMEA/410/01.
1.2 Scope of this Operating Instructions
This Operating Instructions is designed as a general introduction and guide to using
AxiChrom 50, 70 and 100. It is recommendedthat the user reads the complete Operating
Instructions before using the column. Information concerning safety precautions is
presented in this manual in conjunction with general information about the features
and operation of the column.
Specifically, the instructions cover:
General introduction, intended use of the columns, and labels.Chapter 1
Safety instructions.Chapter 2
AxiChrom 50, 70 and 100 specifications, including principles of
column function and column specifications.
Chapter 3
Preparation of a column for use, including unpacking, installa-
tion, column and system setup, use of AxiChrom Master and
instructions for cleaning the column before use.
Chapter 4
Preparations required before packing the column, including
slurry preparation and priming the column.
Chapter 5
AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG 5
1 Introduction

Maintenance and service of AxiChrom 50, 70 and 100.Chapter 6
Troubleshooting guide.Chapter 7
Ordering information.Chapter 8
Spare parts information and exploded diagrams.
Tubing kits and connections. Packing buffers and recommended
packing factors. Column and crate weights and dimensions.
Appendices
Referencing column components
Many of the procedures in this Operating Instructions make reference to specific com-
ponents on the AxiChrom column. To facilitate easy identification of a specified compo-
nent, numbers or letters are given within square brackets, [ ]. These correspond to a
commonly assigned item in the parts list and diagrams for each of the column sizes in
the Appendices.
In some cases bold style characters, either a number or letter may be contained in { }
brackets. These correspond to parts on the 100 column only that deviate from the
commonly assigned items identified in square brackets (see above).
1.3 Important user information
Read this before using the
AxiChrom system
All users must read the entire Operating Instructions before installing, using, or
maintaining the system. Always keep the Operating Instructions at hand when using
AxiChrom 50, 70 and 100 columns.
Do not operate AxiChrom 50, 70 and 100 columns in any other way than described in
the user documentation. If you do, you may be exposed to hazards that can lead to
personal injury, and you may cause damage to the equipment.
6 AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG
1 Introduction
1.2 Scope of this Operating Instructions

Intended use
AxiChrom 50, 70 and 100 are designed for chromatographic separations in process de-
velopment, scale-up trials, and small scale production.
The columns shall be used in accordance with the safety instructions and technical
specifications given in this Operating Instructions so as to not cause injury or to not
damage the column or other equipment.
AxiChrom columns should not be used in any clinical or in vitro procedures for diagnostic
purposes.
Safety notices
This user documentation contains WARNINGS, CAUTIONS and NOTICES concerning the
safe use of the product. See definitions below.
Warnings
WARNING
WARNING indicates a hazardous situation which, if not avoided,
could result in death or serious injury. It is important not to proceed
until all stated conditions are met and clearly understood.
Cautions
CAUTION
CAUTION indicates a hazardous situation which, if not avoided,
could result in minor or moderate injury. It is important not to pro-
ceed until all stated conditions are met and clearly understood.
Notices
NOTICE
NOTICE indicates instructions that must be followed to avoid
damage to the product or other equipment.
AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG 7
1 Introduction
1.3 Important user information

Notes and tips
A Note is used to indicate information that is important for trouble-free and
optimal use of the product.
Note:
A tip contains useful information that can improve or optimize your procedures.
Tip:
Typographical conventions
Software items are identified in the text by bold italic text. A colon separates menu levels,
thus File:Open refers to the Open command in the File menu. Hardware items are
identified in the text by bold text (e.g., Power switch).
1.4 Regulatory information
This section describes the directives and standards that are fulfilled by AxiChrom 50, 70
and 100.
Manufacturing information
Address of manufacturerName of manufacturer
GE Healthcare Bio-Sciences ABGE Healthcare Bio-Sciences AB
Björkgatan 30, SE-751 84 Uppsala, Sweden
Other regulatory information may be found in the Declaration of Conformity supplied
with the product.
CE Conformity
This product complies with the European directives listed in the table, by fulfilling the
corresponding harmonized standards. A copy of the Declaration of Conformity is supplied
with the product.
TitleDirective
Machinery Directive (MD)2006/42/EC
8 AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG
1 Introduction
1.3 Important user information

International standards
This product is designed in accordance with the requirements of the following standards
and regulations:
NotesDescriptionStandard
EN ISO standard is
harmonized with
EU directive
2006/42/EC
Safety of machinery, General principles
for desgn, Risk assessment and risk reduc-
tion.
EN ISO 12100
CE Marking
The CE marking and the corresponding Declaration of Conformity is valid for the instru-
ment when it is:
•used as a stand-alone unit, or
•connected to other CE-marked products recommended or described in the user
documentation, and
•used in the same state as it was delivered from GE Healthcare, except for alterations
described in the user documentation.
The Declaration of conformity is valid only for systems that are marked with the
CE-marking.
AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG 9
1 Introduction
1.4 Regulatory information

2 Safety instructions
2.1 Safety precautions
This Operating Instructions does not guarantee user safety. It is the responsibility of the
user’s employer to ensure safe operation and training. Training courses are available
and can be arranged by contacting your local GE Healthcare representative. Information
given in this Operating Instructions is suggested best working practice and shall in no
way take precedence over individual responsibilities or local regulations.
Great effort has been placed on the design and manufacture of the various parts of the
equipment so that it will comply with all applicable safety aspects for this type of
equipment. The level of risk is highly dependent on the application and environment in
which the column is operated. In order to secure the safe operation of the equipment it
is the user’s responsibility to perform a risk assessment. This assessment results in
safety instructions for installation, operation and maintenance, use of proper personal
protective equipment, and/or other arrangements needed to operate the process safely.
For personal safety it is important that the user has a proper knowledge of the entire
system of which the column is a part of, and that this Operating Instructions and the
rest of the System documentation have been studied. Additionally, the user shall study
any complementary safety instructions and use appropriate personal equipment for the
specific application and operating environment.
During the operation and during other work with a column, it is always each individual's
responsibility to consider:
•Their own and others' personal safety.
•The safety of the equipment through correct use in accordance with the descriptions
and instructions given in this Operating Instructions.
General warnings
WARNING
Only use qualified personnel with a thorough knowledge of the
entire system and processes to perform the installation, operation
and maintenance of the column.
10 AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG
2 Safety instructions

WARNING
AxiChrom columns are not designed to handle flammable liquids.
The columns are not approved for work in a potentially explosive
atmosphere.
WARNING
AxiChrom columns must NOT be used for other purposes than
protein separations, such as:
•Storage tank for chemicals etc.
•General pressure vessel
•Fermentation vessel
•Gas storage, including compressed air
WARNING
When using hazardous chemicals take all suitable protective
measures, such as wearing appropriate personal protective
equipment resistant to the chemicals used. Follow local regulations
and instructions for safe operation and maintenance of the column.
General cautions
CAUTION
Ensure that there is no pressure in the column before disconnecting
any tubing.
CAUTION
Ensure that all tubing and hoses are placed so that the risk for
tripping accidents is minimized.
AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG 11
2 Safety instructions
2.1 Safety precautions

CAUTION
Do not use the stand to step on when reaching for the top mobile
phase inlet/outlet. Use proper equipment to climb/stand on when
necessary to reach the top mobile phase inlet/outlet.
CAUTION
Remove any spillage on the floor immediately to minimize the risk
for slipping accidents.
CAUTION
Never exceed the maximum rated pressure for the column. Be
aware that the column has a ‘packing pressure’ and an ‘operating
pressure’. For details of packing and operating pressures, see
specifications.
CAUTION
Use a pressure gauge, pressure relief valve or other pressure
safety equipment to ensure that the maximum operating pressure
of the column is not exceeded.
General notices
NOTICE
Ensure that the liquids and samples used in the process are com-
patible with the chemical resistance properties of the materials of
construction.
NOTICE
Ensure that the pressure not exceed the specified operating pres-
sure of the medium packed in the column.
12 AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG
2 Safety instructions
2.1 Safety precautions

NOTICE
If leaving the column unattended always rest it horizontally on the
planar surfaces of the flanges, or hanging on the pivot stand.
NOTICE
Always wear gloves when handling the bed support to prevent fat
from unprotected fingers coming into contact with the bed support.
NOTICE
Disconnect all tubing before moving the column.
NOTICE
Ensure that any water used with the column is particle-free (down
to 1 µm), as particles can block and damage the bed supports.
NOTICE
Handle stainless steel parts, especially the bed supports, with care.
Damage to surfaces may lead to corrosion.
NOTICE
Chlorides and low pH can cause corrosion on stainless steel. Rinse
thoroughly with clean water after use.
Inspect the column regularly for signs of corrosion, which may
cause column damage if untreated.
NOTICE
Keep a visual check on the adapter when it is moving, to monitor
smooth movement.
AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG 13
2 Safety instructions
2.1 Safety precautions

NOTICE
Perform a leakage test whenever any of the wetted parts have
been re-fitted or changed.
2.2 Column pressure and over-pressure
AxiChrom columns have been stringently tested for their pressure tolerances and the
effects of over-pressure. At high over-pressure levels, the borosilicate glass will tend to
snap rather than shatter and splinter when filled with liquid.
Section 3.2 Primary specifications, on page22 presents the maximum pressure that may
be used for packing and operation procedures using AxiChrom 50, 70 and 100. For safe
use of the columns, these pressure limits shall be observed. Particular attention shall be
taken when connecting manual pumps to the column, or deviation in normal operating
procedures using ÄKTAexplorer or ÄKTApilot that can lead to an over-pressure. AxiChrom
70 and 100 columns are not compatible with ÄKTAexplorer.
GE Healthcare recommends that the pressure inside the column is continually measured
to detect and deal with any potential over-pressure situations. It is also recommended
that over-pressure safety equipments are fitted. For instructions on how to handle
overpressures, see Handling over-pressure in the column, on page 51.
14 AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG
2 Safety instructions
2.1 Safety precautions

2.3 Labels
This section describes the identification and safety labels that are attached to AxiChrom
50, 70 and 100.
Identification label
The label for serial number, code number, and manufacturing year of the specific column
is located on the outer edge of the top bayonet.
Figure 2.1: The AxiChrom 50, 70 and 100 label for serial number, code number, and
manufacturing year.
Label for operating conditions
The label for operating conditions is located on the column tube near the bottom flange.
Figure 2.2: The AxiChrom 50, 70 and 100 label for operating conditions.
AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG 15
2 Safety instructions
2.3 Labels

Text in safety label
MeaningLabel
Maximal operating pressure (bar g)Max operating pressure
Maximal packing pressure (bar g)Max packing pressure
Operating temperature range (°C)Operating temperature
Symbols used in safety labels
MeaningLabel
Warning! Read the user documentation before using the system. Do not
open any covers or replace parts unless specifically stated in the user
documentation.
The system complies with applicable European directives.
16 AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG
2 Safety instructions
2.3 Labels

3 Specifications and characteristics
3.1 Principles of column function
Axial compression
Packing of AxiChrom 50, 70 and 100 is achieved by internal hydraulic axial compression
(Figure 3.1).
ABC D
Packing liquid
forced out
Packing liquid
forced out
Packing liquid
forced out
Adapter reaches
consolidated bed
Compression of bed according to Packing Factor
Check valve
prevents
backflow
Hydraulic chamber inlet
Hydraulic liquid drives adapter down
Consolidated
bed
Slurry
Figure 3.1: Principle of internal hydraulic axial compression. (A) Hydraulic liquid pushes
the adapter down. Packing liquid is forced out of the slurry. (B) The slurry forms a consoli-
dated bed. (C) The adapter reaches the surface of the consolidated bed. (D) The bed is
compressed to a predetermined packing factor.
Axial compression is achieved by driving the adapter downwards in the column tube by
pumping hydraulic liquid, typically 20% v/v ethanol or 0.01 M NaOH, into the space be-
tween the top plate and the top of the adapter. This space is referred to as the hydraulic
chamber. The space between the bottom bed support and the top bed support is the
process chamber into which a slurry of medium has been poured. As the adapter moves
downwards in the column, packing liquid is forced from the slurry through an outlet in
AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG 17
3 Specifications and characteristics

the bottom plate. Compression of the bed occurs when the bed support comes into
contact with the surface of the consolidated bed and continues moving downwards.
The adapter is stopped when the compression corresponds to a predetermined Packing
Factor, and thereby attains the target bed height. The adapter remains in place by the
pressure of the hydraulic liquid above it.
Definitions associated with the
Packing Factor
Bed settled through exposure to an external force,
e.g. flow.
Consolidated bed:
Bed in its final condition for use.Packed bed:
Bed settled only by gravity.Gravity settled bed:
PF= consolidated bed height
packed bed height
Packing Factor (PF):
where the consolidated bed height is read when
the adapter reaches the consolidated bed,
where the packed bed height is read when the bed
has been mechanically compressed to its final
condition for use.
CF= gravity settled bed height
packed bed height
Compression Factor (CF):
where the gravity settled bed height is measured
after settling the medium by gravity, read when
the medium surface no longer continues to move
downwards (usually a few hours up to 48 hours
depending on the type of medium),
where the packed bed height is read when the bed
has been mechanically compressed to its final
condition for use.
18 AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG
3 Specifications and characteristics
3.1 Principles of column function

Inlet and outlet system
The inlet and outlet system of the column comprises (see figure):
1
3
4
5
2
Figure 3.2: Main components of the column. The 50 column is delivered with AxiChrom
foot (shown in picture), and the 70 and 100 columns with the AxiChrom pivot stand (not
shown here).
FunctionPart
Top mobile phase inlet/outlet1
Vent valve, Hydraulic chamber (hidden behind adaptor rod)2
Unpacking (Hydraulic chamber outlet)3
Hydraulic chamber inlet4
Bottom mobile phase inlet/outlet5
AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG 19
3 Specifications and characteristics
3.1 Principles of column function

Inlet and outlet functions
•Top mobile phase inlet/outlet on the adapter - note that the column may be run
upflow or downflow.
•Hydraulic chamber vent valve on the top plate is used to get rid of air trapped in the
hydraulic chamber. Viewed from above, this valve has a small hole and is fitted with
a round nipple.
•Hydraulic chamber outlet on the top plate is used to channel the hydraulic liquid out
of the column during unpacking. Viewed from above, this outlet has a larger hole
than the hydraulic chamber vent valve, and is fitted with a hexagonal stop plug.
•Hydraulic chamber inlet on the top bayonet is used to fill the hydraulic chamber. The
hydraulic chamber inlet has an integrated check valve.
•Bottom mobile phase inlet/outlet on the bottom plate - note that the column may
be run upflow or downflow.
Port inner diameter (mm) of inlets and outlets
Column diameter (mm)
Inlet/outlet connection thread 1007050
2.9 mm2.9 mm1.7 mmBottom mobile phase inlet/outlet, 5/16”
2.9 mm2.9 mm1.7 mmTop mobile phase inlet/ outlet, 5/16”
2.9 mm2.9 mm1.7 mmInternal adapter tubing, 5/16”
2.9 mm1.7, 2.91mm1.7 mmAdapter
2.0 mm1.5 mm1.5 mmHydraulic chamber inlet, 5/16”
4.0 mm3.5 mm3.0 mmHydraulic chamber outlet, 5/16”
0.9 mm0.9 mm0.9 mmHydraulic chamber vent valve, M8
1Dependant upon media e.g.: 1.7 mm for Sepharose HP, 2.9mm for Capto media.
20 AxiChrom 50, 70 and 100 columns Operating instructions 28-9331-08 AG
3 Specifications and characteristics
3.1 Principles of column function