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.
2

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.
3

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
4
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
5

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.
6
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.
7
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.
8

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+
9
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.
10

Bed height (all capsules) 8 mm
Membrane volume | nano capsule 3 ml | 110 cm2
Membrane volume | area 150 ml capsule 150 ml | 0.55 m2
Membrane volume| area Jumbo 5 l capsule 5 l |18.2 m2
Typical dynamic binding
capacity* 10% for IgG 0.4 mg/cm2
Ligand capacity 3 µeq/cm2
Maximum pressure at 20°C
nano 3 ml and 150 ml 4 bar, 0.4 MPa, 58 psi
Jumbo 5 l 3 bar, 0.3 MPa, 44 psi
Maximum pressure during venting 0.5 bar, 0.05 MPa, 7 psi
Short term** pH stability 2-14
Long term*** pH stability 3-13
* see also section 5. Binding capacity
** Short term refers to cleaning in place and regeneration procedures during operation
of units, e.g. with 1 N NaOH or 0.01/0.1 N HCl for 1 hour
*** Long term refers to storage e.g. overnight and longer. Preferably store unit s in
20% ethanol | buffer within pH limits
11
3. Technical Data

Housing Polypropylene
Membrane matrix Stabilized reinforced cellulose, nominal
pore size >3 µm
Membrane thickness 275 µm | 10.8 mils
(average)
Ligand Phenyl
12
4. Materials

Data are based on dynamic binding capacity measurements 10%
using 3 layers of 5 cm2 membrane discs (15 cm2 total area) arranged
in a holder and run at 10 ml/min.
Sample protein for Sartobind Phenyl:
polyclonal IgG at a concentration of 1 mg/ml
Typical dynamic binding capacity 10%: 0.4 mg/cm2 with 0.9 M
(NH4)2SO4 in 50 mM phosphate buffer pH 7.5
Unit Area Dynamic binding capacity
10%
Sartobind Phenyl
nano 3 ml 110 cm2 44 mg
Sartobind Phenyl
150 ml 5500 cm2 2.2 g
Sartobind Phenyl
5 l 18.2 m2 72.8 g
13
5. Binding capacity

The contents of the package are described in chapter 11.1. When
unpacking capsule, protect inlet and outlet connec tors from damage.
The capsule should be installed in an upright position in the process
flow. In this position the inlet is up. The flow is guided to an
external channel passing through the 8 mm membrane layer to an
internal channel and to the outlet of the capsule.
Install the capsule in-line with a prefilter (0.2 µm) in front of the
device.
! Important note
Capsules should be visually inspected before use. In case of visible
damage, the capsule should be replaced.
Close vent valves before use by screwing the valve in clockwise.
For unpacking of Jumbo 5 l capsule, take the capsule including the
styrene foam end protectors, out of the box and place it upright on
the end protectors.
14
6. Installation

Move the Jumbo trolley (accessory) in place. Then remove upper
foam protection and transparent bag. Lift the Jumbo directly onto
the trolley (inlet is up and the arrow imprinted on the banderole is
pointing down). We recommend to connect the Jumbo with the
trolley by the three screws delivered with the trolley (accessory).
To ensure safe unpacking, the protective caps on inlet and outlet
should stay until you use the unit.
The Jumbo 5 l carries protective caps on vent valves.
Remove before venting.
15

Fig. 3: Flow pattern inside the Jumbo 150 ml and 5 l.
The feed flow is from outside to inside channel.
16
External channel
Membrane (8 mm)
Central core
Internal channel
The direction of
flow is imprinted
on the banderole of
the unit.

7.1 Venting
It is important to remove air from the unit completely.
The 150 ml and 5 l capsules carries vent valves. The vent valves are
equipped with hose barb connectors for the fluid spilled out during
venting. Before opening the vent valve, please connect the valves
with flexible tubing (inner diameter 6 mm) to waste. During venting
please do not exceed 0.05 MPa (0.5 bar | 7.3 psi) pressure, as the
vent valve O-ring could change its position which will result in
insufficient closing of the valve. For appropriate venting, open the
vent valve screw 1/3 turn to left until all air is replaced by fluid.
For the 3 ml nano capsule fill a 10–20 ml Luer syringe with equili-
bration buffer and connect to the nano capsule, then hold capsule
upright (outlet is up) and expel air as shown in Fig. 4a. If you still
detect any air in the filled unit, close it at the outlet, flip the syringe
and the nano capsule so that the syringe is on top and the nano 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.
17
7. Operation

Very small air bubbles observed directly below the inlet do not disturb
separations. The capsules will function normally as long as the small
air bubbles remain outside of the membrane bed.
Fig. 4a: Filling the Sartobind nano with a Luer syringe
18
Outlet
Inlet

Fig. 4b: Fig. 4c: Sartobind 150 ml
Sartobind nano 3 ml with vent valves (arrows)
7.2 Preconditioning
Prior to sample loading, a sanitization and flushing procedure
is recommended.
1. For sanitization use 30 membrane volumes (MV)
of 1 N NaOH solution at a flow rate of 1 MV/min.
2. Flush with 35 MV of water at 5 MV/min.
19
L
L

7.3 Recommended flow rates
Membrane adsorbers can be run at much higher flow rate per
volume than columns. The recommended flow rates for membrane
adsorbers with 8 mm bed height are at 5 membrane volumes per
minute. This recommendation is only a guideline since buffers and
samples have different compositions and viscosities. Please test your
planned flow rates using a small scale device to ensure that they
fit into your pump capacities and pressure limits. Membranes can
be operated also at lower flow rates without any loss of performance
if the pump capacity is not sufficient to reach high flow rates. Please
consider that lowering the flow rate will not drastically improve
binding capacity.
20