Sartobind IEX nano 3 ml

Transcription

1 Operating Instructions Sartobind IEX nano 3 ml A Separation Technology Based on Macroporous Membranes, 8 mm Bed Height

2 Information Please read the operation instruction carefully before using Sartobind capsules. For more information about other Membrane Adsorber types please contact your local Sartorius Stedim Biotech representative, visit our homepage or send mail to 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. Follow safety regulations and wear gloves, safety glasses and a lab coat during operation. 2

3 Contents 1. Storage conditions 4 2. Introduction 4 3. Technical data 5 4. Installation 6 5. Operation Venting Recommended buffer volumes flow rates Buffer conditions Selection of ph conditions Contaminant removal from proteins Sample preparation Washing Elution Stability Operation with peristaltic pumps or LC systems Scaling up Quality assurance Ordering information 14 3

4 1. Storage conditions Sartobind nano capsules should be stored clean and dry and away from direct sunlight in the box at room temperature. 2. Introduction Sartobind nano capsules have been developed for working with a small sample volume but keeping the cylindrical design of large scale Membrane Adsorbers. The Sartobind nano is a ready-to-use device and can be used out of the box. It can be operated with a syringe, pristaltic pump or liquid chromatography system. 4

5 3. Technical data Membrane base matrix Stabilized and reinforced cellulose Ion exchange ligand Q Strong basic anion exchanger: quaternary ammonium (R-CH 2-N + (CH 3) 3) Ion exchange ligand S Strong acidic cation exchanger: sulfonic acid (R-CH 2 SO - ) 3 Number of layers 30 8 mm bed height Membrane volume/area 3 ml/110 cm 2 Binding capacity per cm 2 >0.8 mg bovine membrane area serum albumin on Q >0.8 mg lysozyme on S Binding capacity per unit 87 mg Ion capacity per cm μ equiv of membranes Recommended flow rate ml/min Maximum pressure 4 bar (0.4 MPa, 58 psi) Short term ph stability Q 2 14 S 3 14 Long term ph stability Q 2 12 S

6 Connectors Housing Female Luer Polypropylene, FDA approved 4. Installation Important note: Capsules should be visually inspected before use. In case of damage, the capsule has to be replaced. The Sartobind nano capsule should be installed in the process flow as indicated by the arrow on the capsule. The flow pattern inside the capsule is from outside of the cylinder through the membrane bed to the inside and to outlet. 6

7 5. Operation 5.1 Venting It is important to remove any air from the unit completely. Fill a ml Luer syringe with equilibration buffer and connect to the nano capsule. Then hold capsule upright (outlet is up) and expel air as shown in Fig. 1. Fig. 1: Filling the Sartobind nano with a Luer syringe If you still detect any air in the filled unit, close it at the outlet, hold the syringe up and move the plunger slightly 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 capsule will function normally as long as the small air bubbles remain outside of the membrane bed. 7

8 5.2 Recommended buffer volumes flow rates New membranes are dried from glycerol to avoid shrinking. For prewashing 30 ml equilibration buffer is needed to remove any glycerol. Use the same volume for equilibration. You may use the nano at highest flow rate possible up to 4 bar maximum pressure. The recommended flow rate is ml/min. Lower flow rates do not affect the performance. 5.3 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 filter before use and the quality of water and chemicals should be of high purity. Do not apply pure water as it leads 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. 5.4 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 buffer eluting the molecule. 8

9 The ph value at which a biomolecule has no net charge is the isoelectric point: pi. Below the isoelectric point (rule of the thumb: at least 1 ph unit) a protein for example carries a positive net charge and will bind to a cation exchanger (Sartobind S). Above its isoelectric point (at least 1 ph unit] it will bind to an anion exchanger (Sartobind Q). 5.5 Contaminant removal from proteins For contaminant removal from products 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, some host cell proteins and viruses. The product of interest, the monoclonal antibody with pis of for example, will not be bound and pass through Sartobind Q. To remove contaminating proteins with Sartobind S in flow through mode, process impurities have to be charged positively to bind to S while the target protein stays negative as the ph of the buffer is above the pi of the protein product and flows through without binding. 5.6 Sample preparation The sample should be adjusted to the starting buffer and be prefiltered through a 0.2 μm membrane filter. For small volumes in the ml range use a Minisart with Luer outlet (order no K), for a larger volume e.g. Sartobran P capsule ( H ). 9

10 5.7 Washing After loading wash with 30 ml of equilibration buffer. 5.8 Elution To elute the target protein use equilibration buffer with e.g. 1 M NaCl. 5.9 Stability The capsules are stable against all commonly used buffers, 8 M urea, 8 M guanidine hydrochloride. They are not compatible with organic solvents, except lower alcohols. Do not use oxidizing agents, such as hypochlorite or H 2O Operation with peristaltic pumps or LC systems For the operation of Membrane Adsorbers with peristaltic pump or a liquid chromatography system, Luer adapters are needed and enclosed in the nano pack. 10

11 When the unit is filled completely with equilibration buffer; close the outlet of the Sartobind nano and remove the syringe. Start your LC system or peristaltic pump at a low flow rate. When fluid emerges, stop the pump, connect the tubing via Luer adapter to the inlet of the Sartobind nano. 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 manual to remove any 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. 6. Scaling up Complete breakthrough experiments for the target compound to be bound on the membrane matrix. After optimisation of binding conditions of the contaminants, the purification step can be scaled up to a larger capsule. 11

12 Recommendations Maintain Bed height is automatically kept constant when using capsules with 8 mm bed height Linear flow is automatically kept constant when scaling within the capsules with 8 mm bed hight and using scaling factors as shown below Sample concentration Increase Sample loading Volumetric flow rate Membrane volume Scale up calculations are done preferably by the membrane volume as the calculation is most simple. Other methods for scale up via residence 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 the membrane volumes of the target scale up device. Important Keep sample concentration constant in lab and production scale. Watch out for piping and flow in whole system. 12

13 7. Quality assurance The Sartobind nano capsule is tested for total protein binding capacity and flow rate. Sartobind membranes have been tested for protein binding capacity and flow rate. Capsules and membranes are manufactured in a controlled environment. The product meets all Sartorius Stedim Biotech standards for traceability, production and specifications as given here or exceeded them. The capsules are supplied as non-sterile. The membrane is dried from glycerol. The product is for in vitro use only. 13

14 8. Ordering information Order number Description and Quantity type of connectors Nano capsules 92IEXQ42EUC11--A Sartobind Q nano 3 ml, 110 cm mm bed height, Luer female 92IEXS42EUC11--A Sartobind S nano 3 ml, 110 cm mm bed height, Luer female 14

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16 Sartorius Stedim Biotech GmbH August-Spindler-Strasse Goettingen, Germany Phone Fax USA Toll-Free UK France Italy Spain Japan Sartobind is a trademark of Sartorius Stedim Biotech GmbH, Goettingen Technical data are subject to change without notice. Printed and copyrighted by Sartorius Stedim Biotech Status: September 2008 W4A000 G Publication No.: SL-6146-e08091