Related Manuals for Sartorius stedim Sartobind Phenyl nano 3 ml
Summary of Contents for Sartorius stedim Sartobind Phenyl nano 3 ml
- Page 1 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...
- Page 2 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.
- Page 3 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...
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Page 4: Table Of Contents
Table of Contents 1. Storage conditions ............... 6 2. Introduction .................. 7 3. Technical data ................11 4. Materials ..................12 5. Binding capacity ................. 13 6. Installation .................. 14 7. Operation ..................17 Venting ................17 Preconditioning ..............19 Recommended flow rates .......... - Page 5 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 Installation for test ............30 Operation of test ..............30 8.2.1 Pre-washing of capsule ...........
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Page 6: Storage Conditions
1. Storage conditions 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. -
Page 7: Introduction
2. Introduction 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. - Page 8 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.
- Page 9 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.
- Page 10 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.
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Page 11: Technical Data
3. Technical Data Bed height (all capsules) 8 mm Membrane volume | nano capsule 3 ml | 110 cm Membrane volume | area 150 ml capsule 150 ml | 0.55 m Membrane volume| area Jumbo 5 l capsule 5 l |18.2 m Typical dynamic binding capacity* 10% for IgG 0.4 mg/cm... -
Page 12: Materials
4. Materials Housing Polypropylene Membrane matrix Stabilized reinforced cellulose, nominal pore size >3 µm Membrane thickness 275 µm | 10.8 mils (average) Ligand Phenyl... -
Page 13: Binding Capacity
5. Binding capacity Data are based on dynamic binding capacity measurements 10% using 3 layers of 5 cm membrane discs (15 cm 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/cm with 0.9 M in 50 mM phosphate buffer pH 7.5... -
Page 14: Installation
6. Installation 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. - Page 15 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).
- Page 16 The direction of flow is imprinted on the banderole of the unit. External channel Membrane (8 mm) Central core Internal channel Fig. 3: Flow pattern inside the Jumbo 150 ml and 5 l. The feed flow is from outside to inside channel.
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Page 17: Operation
7. Operation 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. - Page 18 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. Outlet Inlet Fig. 4a: Filling the Sartobind nano with a Luer syringe...
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Page 19: Preconditioning
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. -
Page 20: Recommended Flow Rates
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. -
Page 21: Buffer Conditions
7.4 Buffer conditions Proteins are bound to the phenyl membrane at salt concentrations typically above 500 mM. Larger proteins tend to bind better than smaller ones. Differences in protein hydrophobicity have influence on the choice of salt concentration. The strength of the interaction depends mainly on salt concentrations but also on the sufficient number of exposed hydrophobic groups of the sample and on membrane ligand type and density. - Page 22 Binding buffer examples To bind IgG 0.8 M (NH in 50 mM potassium phosphate, pH 7.5 To bind Bovine serum 2 M (NH in 50 mM albumin or lysozyme potassium phosphate, pH 7.0 Choose salt concentrations as low as possible to bind the protein.
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Page 23: Selection Of Ph Conditions And Temperature
7.4.1 Selection of pH conditions and temperature The effect of pH on binding is much less than in ion exchange chro- matography. Higher temperature typically promotes stronger binding of the sample solute as known from entropy driven reactions. Thus temperature control is important to achieve reproducible results. 7.5 Contaminant removal from proteins in flow through mode The loading conditions can be chosen to selectively retain contami- nants with higher hydrophobicity and allow the target molecule with... -
Page 24: Washing
! Important Unfiltered feed will block the Membrane Adsorber and lead to capac- ity loss and increased back pressure. We recommend inline filtering during operation. With increase of pressure replace filter and restart. 7.7 Washing When using capsules in bind & elute mode, wash with 30 ml (nano), 1.5 l (150 ml device) or 25 l (5 l device) of equilibration buffer after sample loading. -
Page 25: Draining
7.9 Draining You may drain the capsule by application of air or nitrogen pressure (<1 bar |14.5 psi) to the inlet of the capsule. ! Important A dual air regulator system is recommended to prevent over-pressur- ization of the Sartobind capsule. The first regulator should reduce line air pressure to the Sartobind capsule to 2 bar. -
Page 26: Stability
7.11 Stability The capsules are stable against all commonly used buffers, 8 M urea or 8 M guanidine hydrochloride, ethanol and acetone. Do not use oxidizing agents, such as hypochlorite or H 7.12 Operation of the Sartobind nano with peristaltic pumps or LC systems After the unit is filled completely with equilibration buffer;... -
Page 27: Scaling Up
7.13 Scaling up Complete break through experiments for the target compound to be bound on the membrane matrix. After optimization of binding conditions of the contaminants, the purification step can be scaled up to a larger capsule. Recommendations: – Maintain –... - Page 28 Scale up calculations are done preferably by the membrane volume as the calculation is most simple. Other methods for scale up via resi- dence time will lead to similar result. Using the Sartobind nano 3 ml the scale up factor for flow rate and binding capacity is equal to a multiplication factor of membrane volumes of the listed scale up device: Device bed...
- Page 29 Example: After breakthrough experiments with the nano, you realize that a 1500-fold binding capacity is needed for a large scale run. You choose the 5 liter Jumbo capsule. To determine the running conditions of the Jumbo and to keep consistent upscaling, increase flow rate by a factor of ~1670.
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Page 30: Diffusion Test Of 150 Ml And 5 L Capsules
8. Diffusion test of 150 ml and 5 l capsules The integrity of the capsule can be controlled by a diffusion test. The testing procedure describes the diffusion test for Sartobind capsules with Phenyl membranes. The test is intended to discrimi- nate between defective and intact capsules and to detect major bypasses, large holes and faulty assembly. -
Page 31: Diffusion Measurement With
! Important: The capsule needs to be pre-washed with the testing solvent, to remove any glycerol. The washing solution should be at room temperature. Keep the unit in an upright position for proper venting and open the vent screw on top of the unit until all air is replaced by testing solvent. -
Page 32: Result And Evaluation
Vent valve Membrane Adsorber Compressed Air 3–10 bar Open to atmosphere Fig. 5: Diffusion test with Sartocheck 4 plus, 4 or 3 plus 8.2.3 Results and evaluation – Diffusion < 15 ml/min: Test passed (diffusion value on the print out) –... -
Page 33: Troubleshooting
9. Troubleshooting Problem Possible cause Action Air bubbles Incomplete air Small air bubbles seen in the top of can be seen removal the unit do not interfere with the purification as long as they do not touch the membrane bed. If too much air is enclosed, repeat removal as described in chapter 7.1 Venting. - Page 34 Problem Possible cause Action High back Material has Prefilter with 0.2 µm filter before pressure not been processing through the unit (prefer- during sam- filtered entially inline). ple loading Material has Proteins can form aggregates within been filtered hours or during operation. Thus we but was stored recommend to prefilter inline by before purifica-...
- Page 35 Problem Possible cause Action Target mole- Conditions for Increase salt concentration, control cule is not binding are other process parameters as type of bound insufficient salt, pH and temperature Binding Process Use larger adsorber device, or: con- capacity is optimization nect two adsorbers (same size) in not sufficient series (i.e connect outlet of first...
- Page 36 Problem Possible cause Action Reuse is Laboratory The major application of Phenyl needed work is eased, capsules is the single use and they are economic constructed in plastic housing and or practical validated like this. Technically they reasons can be reused. The reuse validation has to be performed by the user.
- Page 37 Problem Possible cause Action Binding Improper Prefilter with 0.2 µm filter before capacity filtration processing (preferentially inline). decreases after several uses Some molecule Use capsule only once. species binds tightly and cannot be removed with 1 N NaOH 1 h Proteins or Perform a regeneration cycle.
- Page 38 Problem Possible cause Action Change of Wrong storage No action. A slight change of mem- membrane brane’s color is due to oxygen and color light exposure of the membrane and does not affect adsorptive properties of the membrane or performance of the device.
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Page 39: Quality Assurance
Capsules and membranes are manufactured in a controlled environment. The product meets all Sartorius Stedim Biotech standards for trace- ability, production and specifications as given here or exceeded them as certified in the quality assurance certificate enclosed (except nano... -
Page 40: Ordering Information
11. Ordering information 11.1. Capsules Order number Description Quantity 96HICP42EUC11--A Sartobind Phenyl nano 3 ml, Luer female conectors, manual 96HICP42E9BFF Sartobind Phenyl 150 ml, 3” sanitary clamp connectors, manual, certificate 96HICP42E3ESS Sartobind Phenyl Jumbo 5 l, 11” sanitary clamp, 2 end cap protectors manual, certificate... -
Page 41: Accessories
11.2 Accessories Order number Description Quantity 9ZGL--0102 Trolley for Jumbo 5 l, stainless steel 7ZAL-V0013 Reducing adapter 11”– 3”; 50.5 mm to 25 mm, sanitary 7ZAL-V0010 Reducing adapter 2"–11"; 64/50.5 mm sanitary... -
Page 42: Dimensions And Connections
12. Dimensions and Connections Capsule size 3 ml 150 ml Membrane [cm 5500 Membrane volume [ml] Dimensions 37 + 33 190 + 77 (height + diameter) Frontal surface area (cm Connectors Luer Sanitary 3” 25 mm outer, female 14 mm inner diameter Gaskets –... - Page 43 Capsule size Membrane [cm 182,000 Membrane volume [ml] 5000 Dimensions 850 + 302 (height + diameter) Frontal surface area (cm 6100 Connectors Sanitary 11” 50.5 mm outer, 36 mm inner diameter Gaskets 11”, internal diameter 35.8 mm Approximate weight 16 kg 20 kg (wet) 23 kg (filled) Void volume*...
- Page 44 Sartorius Stedim Biotech GmbH Copyright by Sartorius Stedim Biotech GmbH, Goettingen, Germany. August-Spindler-Strasse 11 37079 Goettingen, Germany All rights reserved. No part of this publication may be reprinted or Phone +49.551.308.0 translated in any form or by any means without the prior written Fax +49.551.308.32 89...
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