Sartorius stedim Sartobind Phenyl nano 3 ml User manual

Operating Instructions
Sartobind® Phenyl
nano 3 ml | 150 ml |
Jumbo 5 l
A Separation Technology
Based on Macroporous Membranes,
8 mm Bed Height
85032-540-05
85032-540-05

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.
Capsules should be visually inspected before use.
The capsules are supplied as non-sterile.
The membrane is dried from glycerol.
Intended use
The products are intended and validated for single use to avoid
carryover as well as tedious and costly cleaning validation procedures.
However, it is technically possible to reuse after cleaning in place
depending on application, character of sample and process.
Additional validation steps will be needed to assure constant
binding capacity and flow rate after each cycle.
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Sartobind nano 3 ml capsules have been developed for working
with small sample volumes while retaining the cylindrical design
of large scale Membrane Adsorber.
They are perfect for screening of buffers, binding and flow properties
in flow through and bind and elute purifications.
Sartobind 150 ml capsules have been developed for intermediate
and pilot scale for the downstream processing of therapeutic
proteins for the removal of hydrophobic contaminants or bind and
elute purifications.
Sartobind Jumbo 5 l capsules have been developed for production
purposes in the biopharmaceutical industry.
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1. Storage conditions ....................................................................... 6
2. Introduction .................................................................................. 7
3. Technical data ............................................................................. 11
4. Materials ..................................................................................... 12
5. Binding capacity ......................................................................... 13
6. Installation .................................................................................. 14
7. Operation .................................................................................... 17
7.1 Venting ............................................................................... 17
7.2 Preconditioning ................................................................. 19
7.3 Recommended flow rates .................................................. 20
7.4 Buffer conditions ............................................................... 21
7.4.1 Selection of pH conditions and temperature ........ 23
7.5 Contaminant removal from proteins in
flow through mode ............................................................ 23
7.6 Sample preparation ............................................................ 23
7.7 Washing .............................................................................. 24
7.8 Elution ................................................................................ 24
7.9 Draining .............................................................................. 25
7.10 Regeneration and storage ................................................. 25
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Table of Contents

7.11 Stability............................................................................... 25
7.12 Operation of the Sartobind nano with
peristaltic pumps or LC systems........................................ 26
7.13 Scaling up ........................................................................... 27
8. Diffusion test of 150 ml and 5 l capsules ............................... 30
8.1 Installation for test ............................................................ 30
8.2 Operation of test ................................................................ 30
8.2.1 Pre-washing of capsule ........................................... 30
8.2.2 Diffusion measurement with
Sartocheck® 4 ........................................................... 31
8.2.3 Result and evaluation ............................................. 32
9. Troubleshooting .......................................................................... 33
10. Quality assurance ....................................................................... 39
11. Ordering information ................................................................. 40
11.1 Capsules .............................................................................. 40
11.2 Accessories.......................................................................... 41
12. Dimensions and connections ..................................................... 42
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Keep the Sartobind capsules in the transport box until use at +4 to
+8°C in a clean, dry and dark place. When not in use, the end caps
of the nano should be attached to the units to avoid oxygen all the
times. The sealed bag of the 150 ml and 5 l capsule should not be
opened unless you use it. After use store the cleaned capsules in 20%
ethanol in the dark at +4 to +8°C.
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1. Storage conditions

The Sartobind capsules are ready-to-use devices and can be used out
of the box. They can be operated with a peristaltic pump or liquid
chromatography system. For the Sartobind nano, a pair of adapters
(Luer male to UNF10-32 female) are enclosed in the shipment.
Sartobind membranes are made from regenerated, stabilized cellu-
lose. The stabilization | cross-linking generates high chemical stabili-
ty. Ligands are covalently attached to a macroporous membrane
support. The membranes are rolled up to form a cylindrical chro-
matographic bed and incorporated into a housing. The capsules are
constructed with optimized fluid channels. The 150 ml and 5 l units
carry a central core to minimize void volume. Fluid models have
been developed to reduce the channel width to 3 mm. These prod-
ucts are mainly for single use to avoid carryover as well as tedious
and costly cleaning validation procedures. However, it is technically
possible to reuse after cleaning in place. They are validated for con-
taminant removal from proteins in flow-through mode (negative
chromatography) in single use mode, to bind e.g. aggregates or
other hydrophobic contaminants. The capsules can also be used to
capture proteins.
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2. Introduction

Fig. 1: Jumbo 5 l capsule Fig. 2: Jumbo 5 l on stainless steel
trolley for Jumbo (accessory)
Hydrophobic interaction chromatography (HIC) separates and puri-
fies biomolecules based on differences in their hydrophobicity. The
phenyl membrane adsorber follows the same rules known from the
conventional hydrophobic interaction chromatography. Due to the
large pore size, Membrane Adsorbers show excellent flow properties.
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There is almost no diffusion limitation of mass transport detectable
which is the issue with conventional beads. On average 50% of a
protein or peptide surface is accessible for hydrophobic interaction.
Buffers with high concentrations of salt promote the adsorption of
proteins on the hydrophobic membrane matrix. The effect of anions
and cations on protein precipitation is described in the Hofmeister
series:
Anions: PO43-, SO42-, CH3·COO-, Cl-, Br-, NO3-, ClO4-, I-, SCN-
Cations: NH4+, K+, Na+ Cs+, Li+, Mg2+, Ca2+
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Increasing precipitation
Salting out effect
Stronger binding
Increasing chaotropic
Salting in effect
Weaker binding

Typically ammonium sulfate containing salting-out buffers are used
to promote ligand-protein interaction. With increased concentration
more protein is bound until the protein precipitates. Preferably, the
protein binding is performed in the region where the amount of
bound protein increases linearly with the salt concentration. Proteins
are eluted by decreasing the salt concentration in the elution buffer.
Using step or linear gradient elution proteins are eluted in the order
of their hydrophobicity. Regeneration and cleaning of the phenyl
membrane adsorber can be achieved with water, up to 1 N NaOH,
70% ethanol or 30% isopropanol. In contrast to HIC, reverse phase
chromatography uses ligands in much higher density. This results in
higher hydrophobicity: HIC matrices allows moderate separation
conditions in which the molecular structure is preserved.
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