Sartorius Sartobind IEX MA 15, Sartobind IEX MA 75, Sartobind IEX MA 100, Sartobind IEX MA Series Operating Instructions Manual
Operating Instructions
Sartobind
®
IEX MA 15 | 75 |100
A Separation Technology
Based on Macroporous Membranes
85032-539-13
Read operational instructions carefully before using Sartobind capsules.
!
Important
Use of the product in applications not specified or not described in
this manual, may result in improper function, personal injury, or
damage of the product or material.
Membrane Adsorber (MA) units should be visually inspected before
use.
The units are supplied as non-sterile.
The membrane is dried from glycerol and should be prewashed with
equilibration buffer before use.
If you plan to scale up later, preferentially use Sartobind capsules
with 4 or 8 mm bed height: e.g. Sartobind Q nano with 1 or 3 ml as
downscale.
2
Intended use
The products are intended for ion exchange (IEX) chromatography
work in a laboratory for research purposes only.
Sartobind MA 15 units with 3 membrane layers can be used for
screening of buffer conditions and quantitative purifications at
exceptional high flow rate.
Sartobind MA 75 units contain 15 membrane layers and have been
developed for bind and elute and flowthrough applications at high
flow rate.
Sartobind MA 100 units with 5 membrane layers can be used for
screening of buffer conditions and quantitative work at exceptional
high flow rate.
3
4
1. Storage conditions 5
2. Introduction 5
3. Technical data 7
4. Materials and pH
stability 8
5. Binding capacity 9
6. Installation 10
7. Operation 11
7.1 Venting 11
7.2 Recommended buffer
volumes and flow rates 11
7.3 Buffer conditons 13
7.4
Selection of
pH conditions
14
7.5 Contaminant removal
from proteins in flow
through operation 14
Table of Contents
7.6 Sample preparation
and equilibration 15
7.7 Washing 16
7.8 Elution 16
7.9 Regeneration,
cleaning and storage 17
7.10 Stability 18
7.11 Operation with
peristaltic pump or
LC system 19
8. Troubleshooting 20
9. Quality assurance 24
10. Ordering information 25
10.1 MA units 25
10.2 Accessories 26
11. Dimensions and
connections 27
5
Sartobind MA units should be stored clean, dry and away from direct
sunlight in the box at room temperature.
2. Introduction
Traditional chromatography uses porous particles packed into
columns. As liquid flows through the column and around the beads,
bio-molecules in the liquid diffuse into the pores of the beads to
binding sites on the inner surface of the pores. The limiting factor in
low pressure column chromatography is the time required for the
molecules to diffuse into and out of the pores. The various steps of
equilibration, loading, washing, elution and regeneration can take
hours.
Sartobind membranes are made from regenerated and stabilized cellulose. The stabilization and cross-linking generate high chemical
stability. Conventional ion exchange ligands are covalently attached
to the macroporous membrane support. The membrane layers form
chromatographic bed between 3 and 15 layers which is incorporated
into housings.
1. Storage conditions
Pressure forces the liquid through the pores of the membrane,
bringing target substances to direct contact with the binding sites.
This direct convection to the binding sites minimizes diffusion
limitation of mass transfer and increases the speed of operation to
10 – 30 fold compared to traditional column chromatography.
The Luer-Lok connectors on top and bottom allow you to run the
units with a syringe and a prefilter or to connect several units to
each other to get a larger bed volume or to run units with different
chemistries.
6
7
MA 15 MA 75 MA 100
Membrane Area [cm2] 15 75 100
Number of layers 3 15 5
Bed height [mm] 0.8 4.0 1.4
Membrane volume [ml] 0.41 2.1 2.8
Membrane diameter [mm] 25 25 50
Typical dynamic binding 12 60 80
capacity 10% for Q [mg/unit]*
Typical dynamic binding 10.5 52.5 70
capacity 10% for S [mg/unit]*
Typical dynamic binding capacity – 60 –
10% for D [mg/unit]**
Ligand density [μeq/cm2] 2 – 5 (Q, S), 2 – 4 (D)**
Maximum pressure at 20°C 6 bar | 0.6 MPa | 87 psi
* See also section 5. Binding capacity
** D type is available as MA 75 only.
3. Technical data
Housing and basic membrane
Housing MA 15, 75, 100 Polysulfone
Membrane matrix Stabilized reinforced cellulose,
nominal pore size >3 μm
Membrane ligands Quaternary ammonium (Q)
Strong basic anion exchanger R-CH2–N+(CH3)
3
Strong acidic cation exchanger Sulfonic acid (S)
R-CH2–SO
3
–
Weak basic anion exchanger Diethylamine (D)
R-CH
2
–N(C2H5)
2
8
4. Materials
9
Data are based on dynamic binding capacity measurements 10%
using MA15 run at 10 ml/min.
Membrane Typical dynamic binding Reference protein
capacity 10% (mg/cm2) and buffer
Q 0.8 BSA (bovine serum
albumin) in 20 mM
Tris/HCl, pH 7.5
S 0.7 Lysozyme in 10 mM
potassium phosphate,
pH 7.0
D 0.8 BSA (bovine serum
albumin) in 20 mM
Bis Tris, pH 6.0
5. Binding capacityy
The contents of the packages are described in chapter 10.1.
The Sartobind Membrane Adsorber (MA) units are ready-to-use
devices and can be used out of the box. They can be operated with a
syringe (chapter 7.1 to 7.8), peristaltic pump or liquid chromatography (LC) system (follow chapter 7.11 first).
The unit should be installed in an upright position in the process
flow. The female Luer connector is the inlet and the male Luer connection is the outlet of the unit. The flow is guided from top
through the 3 to 15 membrane layer to the outlet. The units can
also be connected in series to increase the binding capacity.
Fig. 1 Flow direction of buffer
10
6. Installation
11
7.1 Venting
It is important to remove air from the unit completely.
Fill a 10 – 20 ml Luer syringe with equilibration buffer and connect
to Luer female inlet, then hold unit upright (outlet is up) and expel
air until the first fluid is seen at the outlet. If you still detect any air
in the filled unit, close it at the outlet with enclosed cap, flip the
syringe and the MA so that the syringe is on top and the unit is on
the bottom, hold the syringe up and move the plunger a few strokes
up and down that air bubbles can ascend into the syringe. Very small
air bubbles observed directly below the inlet do not disturb separations. The unit will function normally as long as the small air bubbles remain outside of the membrane bed.
7.2 Recommended buffer volumes and flow rates
Membranes are dried from glycerol to avoid shrinking. For pre-washing with e.g. equilibration buffer or 1 M NaCl the following flushing
volumes are recommended. Lower flow rates than the recommended
ones can also be used.
7. Operation
Tab. 1: Buffer consumption of Sartobind MA units [ml]
MA 15 MA 75 MA 100
Equilibration 10 15 20
Wash 10 – 20 10 – 20 10 – 20
Elution 5 – 20 5 – 20 5 – 20
Regeneration 5 – 10 10 – 20 10 – 20
Tab. 2: Flow rate recommendation
MA 15 MA 75 MA 100
Recommended flow rate [ml/min] 10 – 30 10 – 30 20 – 50
12
13
7.3 Buffer conditions
In the majority of applications an equilibration buffer concentration
of 10 mM provides sufficient buffering capacity and prevents the
protein of interest from precipitation. The ionic strength should
be kept as low as possible to avoid reduction of binding capacity.
The buffer should have a pKa within 0.5 pH units of the pH used.
It should be filtered with 0.2 μm filters before use and the quality
of water and chemicals should be of high purity.
!
Important
Do not apply pure water through the units as it may lead to a reversible
swelling and decrease of the flow rate of the membrane. The buffering
ion should carry the same charge as the ion exchange ligand.
Binding | equilibration buffer examples
To bind BSA on Sartobind Q 20 mM Tris/HCl pH 7.5
To bind BSA on Sartobind D 20 mM Bis Tris pH 6.0
To bind lysozyme on Sartobind S 10 mM Potassium phosphate
buffer pH 7.0
7.4 Selection of pH conditions
In ion exchange chromatography a charged molecule is bound to
oppositely charged groups attached to the insoluble matrix. This
binding is reversible by application of salt ions to the elution buffer.
The pH value at which a biomolecule has no net charge is the
isoelectric point (pI). In buffers below the pI (at least 1 pH unit),
a protein, for example, carries a positive net charge and will bind
to a cation exchanger (Sartobind S). In buffers above its pI (at least
1 pH unit), it will bind to an anion exchanger (Sartobind Q or D).
7.5 Contaminant removal from proteins in flow through operation
For contaminant removal from proteins such as monoclonal antibodies, pH conditions in the range of pH 6 to 8 are used in order
to bind highly negatively charged DNA, endotoxins, contaminating
proteins, some host cell proteins and viruses. The product of interest,
the monoclonal antibody with pI of 8 – 9.5 for example, will not bind
and pass through Sartobind Q or D.
14
15
To remove contaminating proteins and aggregates with Sartobind S
in flow through mode, process impurities have to be charged positively to bind while the target protein stays negative. At the pH of
the buffer above the pI, the protein product flows through without
binding.
7.6 Sample preparation and equilibration
The sample should be adjusted to the starting buffer and be prefiltered through a 0.2 μm membrane filter (not included). For small
volumes in the ml range use a Minisart
®
with Luer outlet (order no.
16534-K).
Fig. 2: Inline filtration with a Minisart
coupled to the inlet of Sartobind MA unit
Sartobind
Minisart
L
L
The Minisart shall be coupled directly to inlet of the MA unit during
use as shown in Fig. 2 to avoid clogging of the adsorber. This is
recommended also for usage of MA units with LC systems.
!
Important
Unfiltered feed will block the Membrane Adsorber and lead to capacity
loss and increased back pressure. We recommend inline filtering during
operation. With increase of pressure replace filter and restart.
7.7 Washing
When using MA units in bind and elute mode, wash with equilibration buffer (see Tab. 1 and 2) after the sample has been loaded.
7.8 Elution
Elute the target protein with elution buffer (Tab. 1 and 2), with
increased salt concentration or shifted pH.
Elution buffer examples
To elute BSA from Sartobind Q or D 1 M NaCl in binding buffer
To elute lysozyme from Sartobind S 1 M NaCl in binding buffer
16
17
7.9 Regeneration, cleaning and storage
After elution, wash the unit with equilibration buffer. If necessary,
use 1 N NaOH, 1 N HCl or 70% ethanol for 1 hour and store in
20% ethanol in equilibration buffer. Do not store units in water.
Do not freeze the units.
The MA 15, 75 or 100 can be cleaned in place directly after unpacking with 1 N NaOH, 1 hour at 20°C.
Example for cleaning of a Sartobind MA 75, bed volume 2.1 ml
1 1 N NaOH at the flow rate of 1 bed volume (BV)/min at 20°C
for1 h (in total ~130 ml)
2 10 BV (~20 ml) 1 M NaCl
3 10 BV (~20 ml) equilibration buffer
Do not autoclave.
7.10 Stability
The MA 15, 75 and 100 units are stable against all commonly used
buffers in chromatography, e.g. 8 M urea, 8 M guanidine hydrochloride, ethanol and acetone. They can be sanitized after prewashing
with 1 N NaOH, 1 hour at room temperature (20°C). Do not use
oxidizing agents such as hypochlorite or H
2O2
.
pH Stability
QS D
Short term* 2 – 14 3 – 14 2 – 14
Long term** 2 – 12 4 – 13 4 – 13
* Short term refers to cleaning in place and regeneration procedures during
operation of units
** Long term: Storage of units e.g. overnight and longer. Preferably store units
in 20% ethanol in buffer within pH limits
18
19
7.11 Operation with peristaltic pump or LC system
After the unit is filled completely with equilibration buffer, close the
outlet of the MA 15, 75 or 100 and remove the syringe. Start your
LC system or peristaltic pump at a low flow rate. When fluid
emerges, stop the pump and connect the tubing to the inlet of the
unit. Make sure that no air is introduced. Remove the cap from
outlet. Run the pump until fluid emerges from the outlet of the unit
and stop it. Then connect the outlet of the unit via Luer adapter to
the LC detector and proceed with loading. If your system pressure is
too high, refer to your LC system manual to remove any flow restrictor after the UV cell, as the system may generate a pressure above
the allowed maximum pressure. As Membrane Adsorbers run typically at much higher flow rates than columns, there is no risk of bubble
formation in the UV cell when removing the restrictor.
Problem Possible cause Action
High back Material has Prefilter with 0.2 μm filter
pressure not been before processing through the
during sample filtered unit.
loading
Material has Proteins can form aggregates
been filtered within hours or during
but was stored operation. Thus we recommend
before to prefilter inline by attaching a
purification 0.2 μm filter in front of the adsor-
ber. When you observe again
pressure built up, replace the filter.
LC system Remove restrictor after the
generates high UV cell.
pressure
The MA 15, 75 Perform a regeneration cycle.
or 100 adsorber
is clogged
8. Troubleshooting
20
21
Problem Possible cause Action
Target mole- Conditions for Decrease conductivity, control other
cule is not binding are process parameters as type of buffer
bound insufficient and pH
Proteins or Perform a regeneration cycle
contaminants
are still bound
from last cycle
Binding capa- Improper Prefilter with 0.2 μm filter before
city decreases filtration processing through the unit.
after several
uses
Some molecule The binding capacity of the unit
species binds depends on the nature of sample
tightly (e.g. DNA and sample preparation,
on Sartobind Q) prefiltration as well as proper
and cannot be regeneration and application.
removed with Tightly bound molecules may be
1 N NaOH, 1 h removable by specific enzymes,
or use unit only once.
Problem Possible cause Action
Proteins or Run a 1 M NaCl buffer step to
contaminants elute tightly bound proteins
are still bound quantitatively. Then regenerate
from last cycle adsorber by loading with 1 N NaOH
and keep it for 1 hour at room
temperature (20°C).
Wrong storage Do not store in sodium hydroxide
containing buffers. Store long term*
in 20% ethanol-buffer (e.g. equilibration buffer) solution and do not
use oxidative chemicals in buffers.
22
23
Problem Possible cause Action
Early Binding Use larger adsorber device, or
break- capacity is connect two adsorbers (same size)
through of not sufficient in series (i.e. connect outlet of first
protein adsorber to inlet of second) to
achieve higher binding capacity.
As a rule of thumb the pressure
doubles when the flow rate is
kept constant and the number
of membrane layers is doubled.
We do not recommend to run
two adsorbers in parallel.
* Long term storage is defined as storage of units when not in operation,
e.g. storage overnight and longer
Sartobind membranes have been tested for protein binding capacity
and flow rate.
Sartobind MA units are manufactured in a controlled environment
and have been tested for protein binding capacity and flow rate.
The product meets all Sartorius Stedim Biotech standards for
traceability, production and specifications as given here or exceeded
them.
9. Quality assurance
24
25
10.1 MA units
Order number Description Quantity
Sartobind MA units (reusable)
93IEXQ42GB-12--A Sartobind Q 15, Luer female and 4
male connectors
93IEXS42GB-12--A Sartobind S 15 Luer female and 4
male connectors
93IEXQ42DB-12--V Sartobind Q 75, Luer female and 2
male connectors
93IEXS42DB-12--V Sartobind S 75, Luer female and 2
male connectors
93IEXD42DB-12--V Sartobind D 75, Luer female and 2
male connectors
93IEXQ42BC-12 Sartobind Q 100, Luer female and 1
male connectors
93IEXS42BC-12 Sartobind S 100, Luer female and 1
male connectors
10. Ordering information
10.2 Accessories
Order number Description Quantity
1ZA- - -0004 Luer male adapter to 1
UNF 10-32 female, PEEK
1ZA- - -0005 Luer female adapter to 1
UNF 10-32 female, PEEK
17002---140 2 + M6 female to female Luer and 1
2 + M6 female to male Luer Lok Adapter,
black Tefzel
26
27
MA 15 MA 75 MA 100
Membrane Area [cm2]1575100
Membrane volume [ml] 0.41 2.1 2.8
Dimensions 25 + 36 28 + 36 31 + 66
(height + diameter) [mm]
Frontal surface area [cm2]5510
Connector inlet Luer female
Connector outlet Luer male
Approximate weight [g] 9 10 35
11. Dimensions and Connections
Sartorius Stedim Biotech GmbH
August-Spindler-Strasse 11
37079 Goettingen, Germany
Phone +49.551.308.0
Fax +49.551.308.3289
www.sartorius-stedim.com
Published
28. February, 2011
Sartorius Stedim Biotech GmbH,
Goettingen, Germany
Copyright by Sartorius Stedim Biotech
GmbH, Goettingen, Germany.
All rights reserved. No part of this
publication may be reprinted or
translated in any form or by any
means without the prior written permission of Sartorius Stedim Biotech
GmbH. The status of the information,
specifications and illustrations in this
manual is indicated by the date given
below. All products are sold subject to
the terms and conditions ruling at the
time of delivery. Sartorius Stedim
Biotech GmbH reserves the right to
make changes to the technology,
features, specifications and design of
the equipment or discontinue products
described at any time without notice
or obligation.
Sartobind and Minisart are trademarks
of Sartorius Stedim Biotech GmbH,
Luer-Lok is a trademark of Becton,
Dickinson and Company.
Printed in Germany on paper that
has been bleached without any use
of chlorine
W4A000 Sartobind IEX MA 15|75|100
Publication No.: SL-6171-e11011
Ver. 02 | 2011
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