UNOsphere SUPrA™ Affinity Chromatography Media - Bio-Rad
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1. 4029300 Israel 03 963 6050 Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460 Mexico 52 555 488 7670 The Netherlands 0318 540666 New Zealand 0508 805 500 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 495 721 14 04 Singapore 65 6415 3188 South Africa 27 861 246 723 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 95 55 Taiwan 886 2 2578 7189 United Kingdom 020 8328 2000 Sig 0308 10014430 Rev C2. hr 25 2 mg ml 450 cm hr 20 2 mg ml 10 mM Hydrochloric acid 6 M Guanidine hydrochloride 0 1 M Arginine pH 2 8 0 1 M Citrate pH 2 8 0 1 M Glycine pH 2 8 3 11 6 M Guanidine hydrochloride 10 mM Hydrochloric acid 0 1 M Sodium hydroxide 1 M Acetic acid 20 Ethanol 100 600 cm hr 2 40 C 50 slurry in 20 Ethanol 2 8 C 1 Minimum 20 mg ml at 300 cm hr 10 breakthrough capacity determined with 1 0 mg ml polyclonal human IgG in 1 1 x 10 cm column 2 No significant change in chromatographic performance after 24 hr storage at room temperature 3 0 2 5 2 0 1 5 Pressure bar 1 0 0 5 0 0 100 200 300 400 500 600 700 Linear flow cm hr Fig 1 Flow performance of UNOsphere SUPrA media in Bio Rad InPlace column 20 cm x 20 cm packed to 13 axial compression Section 3 Preparation for Packing UNOsphere SUPrA affinity chromatography media are supplied fully hydrated in 20 ethanol as a 50 v v slurry For column packing removal of the shipping buffer is recommended Small volumes of UNOsphere media are easily washed in a B chner funnel with 4 5 volumes of packing buffer For large volumes cycling through 3 4 settling and decanting steps with packing buffer is recommended Complete removal of fine particles from UNOsphere SUPrA media is not required since the media are manufactured with a very narrow particle size range If fine particles fines have been generated during ha
3. plates meter is not required HETP L N N 5 54 Ve Wyn L Bed height cm N Number of theoretical plates Ve Peak elution volume or time Wyp Peak width at peak s half height in volume or time Ve and Wyp should always be in the same units Note Peaks should be symmetrical without significant leading or trailing shoulders A split peak may indicate a cracked bed which requires repacking Peak asymmetry factor calculation See Figure 2 Ag b a a Front end of peak width at 10 of peak height bisected by line denoting Ve b Back end of peak width at 10 of peak height bisected by line denoting Ve Bed height 20 cm Column volume 201 Flow rate 100 cm h Vo 10l Wip 11 HETP 0 036 cm Wy p width at half height 10 of peak A meres Fig 2 A simulated chromatography profile from which HETP and Ag values are calculated Section 6 Screening Conditions Because different antibodies will have differing levels of affinity for UNOsphere SUPrA media it is highly recommended to assess the behavior of the target antibody on the media To do so it is best to test for binding under conditions that will bind the widest range of antibodies followed by a linear elution protocol to assess optimal elution conditions before further refining the method It is important before undertaking this process to ensure that the target antibody is stable and soluble under the full range of conditions us
4. Evaluation After the completion of the packing operation it is highly recommended and often routine to verify the quality of the packing This verification can also be done at anytime throughout the lifetime of the packed bed to verify efficiency The verification consists of determining height equivalent to theoretical plate HETP as well as the asymmetry factor Ay To determine HETP equilibrate the column with 3 5 column volumes CV of starting buffer or until a baseline conductivity or UV trace is achieved To test the effectiveness of column packing inject a sample of a low molecular weight unretained compound e g acetone or 1 M NaCl If acetone is used as the test marker use an absorbance monitor set at 280 nm the starting buffer must have a salt concentration less than 100 mM If 1 M NaCl is the test marker use a conductivity monitor then the testing buffer salt concentration should be 100 200 mM The sample injection volume should be 1 3 of the total column volume The column testing should be performed at approximately 100 cm hr To obtain comparable HETP values between columns the same conditions must be applied The number of theoretical plates is often expressed in terms of plates per meter N m to normalize for column bed height Minimum theoretical plate values should be approximately 4 000 7 000 plates m However since protein A based separations are not plate based obtaining a particular number of theoretical
5. UNOsphere SUPrA Affinity Chromatography Media Instruction Manual Catalog s 156 0218 156 0219 156 0220 156 0221 156 0222 156 0250 Please read these instructions prior to using UNOsphere media If you have any questions or comments regarding these instructions please contact your Bio Rad Laboratories representative Table of Contents Section 1 Section 2 Section 3 Section 4 Section 5 Section 6 Section 7 Section 8 Section 9 Section 10 Section 11 Section 12 Introduction uuu2200000000n0nn ann ann nun nun mann nun nnnnunn ann nun nunnannannnn 1 Technical Description u zu220022000000000n0nn0nn00nnunnannannn0n 2 Preparation for Packing uuauunuu2000000n0nnannannannnnnnannnnnnn 3 3 1 Determining Slurry Percentage or Media Volume 4 Column Packing uuuaunnunnunnaunnunnunnunnannnunnunnannnnnnunnannnnnnn 4 4 1 Packing in Open Column Systems 4 4 2 Packing in Closed Column Systems 5 4 3 Stall packing Closed Column Systems 6 Column Packing Evaluation uu uu22u0000000n0nn00n0nnnannannannnn 6 Screening Conditions u 2u 2u0nu0000n00nnunnannannnnnnunnunnannnnn 8 Operation and Maintenance uuunuunaunnunnunnannnunnunnannannnnn 9 Cleaning In Place CIP uuau022002000000000n0nn00nnnnnannannnnnnn 9 Sanitization uunuunnannnunnunnunnannnunnunnunnannnunnunnunnannnunnunnnnnn 10 Storage uunuunnunnunnunnannnunnnnnunn ann nun nun nann ann nun
6. clear supernatant packing buffer is observed above the bed Replace the top flow adapter piston assembly and lower into supernatant layer Engage the seal Lower the top piston in order to remove any trapped air and purge the top piston assembly of air Once all of the air is removed begin flow packing at approximately 300 cm hr This value may change depending on your process requirements o Columns equipped with speed controlled dynamic axial compression capabilities such as Bio Rad EasyPack columns can be packed using a combination of flow packing and axial compression or only using axial compression o Columns without axial compression capabilities may be flow packed The optimal compression percentage for UNOsphere SUPrA media is 12 4 2 Packing in Closed Column Systems Using Bio Rad InPlace Columns Bio Rad InPlace columns are typically packed using a Bio Rad media transfer device and a slurry tank The slurry tank should be filled with slurry at 30 50 concentration and should be equipped with a low shear impeller for gentle mixing The column should be cleaned and levelled with the piston lifted to the uppermost position Ensure that there is no air trapped in the bottom process valve or frit by reverse flowing with packing buffer for a short period or by introducing packing buffer into the column and allowing it to drain Verify that about 2 cm of packing buffer remains at the bottom of the column Close the b
7. ed for the screening Using a BioScale Mini column 1 or 5 ml packed with UNOsphere SUPrA media A 0 02 M sodium phosphate 0 02 M sodium citrate pH 7 5 B 0 02 M sodium citrate 0 1 M sodium chloride pH 3 Equilibrate column the with 10 CV buffer A see note below Inject a small sample of antibody either as is or at 1 10 dilution in buffer A see note below Wash the loaded columns with buffer A until effluent absorbance returns to baseline Elute with a 10 CV linear gradient to buffer B collect fractions Wash the column with 5 CV buffer B Note Boric acid may be added to the binding equilibration buffer to obtain a higher pH range Up to 1M sodium sulfate may be added to enhance binding Section 7 Operation and Maintenance UNOsphere SUPrA M media are designed to achieve high productivity usability and scalability to allow users to process at high linear velocities A linear flow rate of up to 600 cm hr is well within the operational capabilities of the media UNOsphere SUPrA media can be used with all buffer systems common in monoclonal antibody purification The media are also designed to withstand multiple compression and decompression cycles at the recommended compression rate of 12 Section 8 Cleaning in Place CIP During operation it is recommended that the column bed be periodically cleaned to remove bound substances that can adversely impact the separation performance of the column The accumulated sub
8. m hr with the top flow adapter at least 10 cm above the packed bed Stop the flow and open the top of the column Allow the bed to rebound for at least 15 min to determine the bed volume prior to compression Section 4 Column Packing General column packing procedures are outlined below for the two main process scale column types open and closed column systems Please consult the user guide of the specific column you are using for complete instructions See Figure 1 for the flow properties of UNOsphere SUPrA media Caution Some column systems require recirculation of slurry for extended periods through the packing pump Avoid this operation with UNOsphere SUPrA media as vigorous recirculation can damage the media and create an excessive amount of fine particles Consult Bio Rad Technical Services for alternate methods 4 1 Packing in Open Column Systems e Completely remove top piston assembly Make sure the column is level e Ensure that there is no air trapped in the bottom process valve or frit by performing a brief up flow with packing buffer or by pouring packing buffer into the column and allowing it to drain e Leave about 2 cm of packing buffer at the bottom of the column Close the bottom process valve Carefully transfer the slurry to the column using the pre calculated volume Slurry can be transferred using a diaphragm pump or other gentle transfer method Allow the slurry to settle until a 2 5 cm layer of
9. ndling resuspend reslurry the media let settle then decant the supernatant containing the fines Repeat this process several times until a clear supernatant is obtained When preparing a homogenous slurry from settled material take special care not to crush the settled media with a mixing paddle this can create fines Use a side to side motion or J stroke with a PTFE mixing paddle or other plastic paddle to disturb the top layers of the settled bed until the slurry becomes homogenous Alternatively gently roll the sealed container back and forth in a rocking motion to resuspend the media 3 1 Determining Slurry Percentage or Media Volume The recommended slurry percentage for column packing is 30 50 There are several methods that can be used to determine the slurry percentage of a solution Using disposable open columns e g Bio Rad Econo Pac columns Method 1 Fill the column with slurry and use a marker to indicate the filled height Open the bottom of the column and drain the buffer Mark the bed height of the remaining media The height of the remaining media divided by the height of the total slurry volume will yield the approximate slurry percentage Method 2 Pour slurry into a graduated cylinder and allow to settle overnight gt 12 hrs The height of the settled bed divided by the height of the total volume will yield the approximate slurry percentage Method 3 Ifthe media are packed into a column flow at 300 c
10. nun mann ann nunnunnannn 10 Regulatory Support uusaunnunnunnnunnunnunnunnnunnunnnnnannnunnunnnnn 10 Ordering Information u u 2u0200000n000000n0nnnnnannnnnnunnnnnn 10 Section 1 Introduction UNOsphere SUPrA media are affinity chromatography support based on recombinant protein A The media are designed for process scale purification of monoclonal antibodies The protein A ligand is produced in E coli without the use of material from animal origin The UNOsphere base bead is a macroporous polymeric bead that is designed for robust and scaleable process applications See Table 1 in the next section for the technical description of the product The outstanding flow pressure performance of UNOsphere chromatography media allows its use in large scale process applications The flow characteristics of UNOsphere SUPrA can be found in Figure 1 in section 2 of this manual UNOsphere SUPrA affinity chromatography media come with full regulatory support and are backed by the support of Bio Rad s global application and development team Section 2 Technical Description Composition Particle Size Range Ligand Coupling Chemistry Dynamic Binding Capacity Chemical Stability2 Working pH Range Cleaning In Place CIP Recommended Mobile Phase Velocity Range Working Temperature Range Delivery Conditions Storage Conditions Highly Crosslinked Polymer 53 61 um Recombinant Protein A Epoxy 150 cm hr 30 3 mg ml 300 cm
11. on and store at 2 8 C Section 11 Regulatory Support A regulatory support file is available for UNOsphere SUPrA M affinity chromatography media Section 12 Ordering Information UNOsphere SUPrA affinity chromatography media are available in the following formats Bottled Media Catalog Description 156 0218 UNOsphere SUPrA affinity chromatography media 25 ml 156 0219 UNOsphere SUPrA affinity chromatography media 100 ml 156 0220 UNOsphere SUPrA affinity chromatography media 500 ml 156 0221 UNOsphere SUPrA affinity chromatography media 5 liters 156 0222 UNOsphere SUPrA affinity chromatography media 10 liters Prepacked Cartridges Catalog Description 732 4200 Bio Scale Mini cartridges prepacked with UNOsphere SUPrA media 1x1ml 732 4201 Bio Scale Mini cartridges prepacked with UNOsphere SUPrA media 5Bx1ml 732 4202 Bio Scale Mini cartridges prepacked with UNOsphere SUPrA media 1x5ml For larger volume quantities please contact your local Bio Rad representative to discuss your requirements 10 Life Science Group Bio Rad Laboratories Inc Web site www bio rad com USA 800 4BIORAD Australia 61 02 9914 2800 Austria 01 8778901 Belgium 09 385 55 11 Brazil 55 21 3237 9400 Canada 905 364 3435 China 86 21 6426 0808 Czech Republic 420 241 430 532 Denmark 44 52 10 00 Finland 09 804 22 00 France 01 47 95 6965 Germany 089 318840 Greece 30 210 777 4396 Hong Kong 852 2789 3300 Hungary 36 1 455 8800 India 91 124
12. ottom process valve The seal may be deflated prior to slurry transfer Using the media transfer device open the slurry valves Open the bottom valve on the slurry tank to allow the transfer of slurry into the InPlace column Turn on the diaphragm pump to increase the speed of transfer After completing the slurry transfer rinse the slurry tank and transfer lines with reserved buffer Close the slurry valves e Allow the slurry to settle until a 2 5 cm layer of clear supernatant packing buffer is observed above the settled bed e Lower the top piston into supernatant layer and engage the seal Continue to lower the top piston to remove any trapped air and to purge the top piston assembly of air e After ensuring that all of the air has been removed begin flow packing at approximately 300 cm hr This value may change depending on your process requirements o Columns equipped with speed controlled dynamic axial compression capabilities such as the Bio Rad EasyPack column may be packed using a combination of flow packing and axial compression or simply by using axial compression o Columns without axial compression capabilities may be flow packed without any precaution e The optimal compression percentage for UNOsphere SUPrA media is 12 4 3 Stall Packing Closed Column Systems When using process scale closed column systems that require stall packing consult the user manual of your specific column model Section 5 Column Packing
13. stances fall into two general categories a difficult to remove precipitated or denatured substances and b substances that are hydrophobically bound to the column bed To ensure that all bound substances are released and washed out of the column the following CIP cleaning protocols are recommended CIP Protocols The following protocols are suggested to remove precipitated or denatured substances from the bed Wash the bed with 2 5 column volumes in reverse flow with one of the following solutions e 6 M guanidine hydrochloride e 10 mM hydrochloric acid e 0 1 M sodium hydroxide e 1 M acetic acid 20 ethanol Followed by a reverse flow wash with at least 5 column volumes of binding buffer at neutral pH 7 8 To remove any hydrophobically bound substances from the bed wash the column with 2 5 column volumes in reverse flow of a non ionic surfactant detergent followed by a reverse flow wash with at least 5 column volumes of binding buffer at neutral pH Suggested contact time per cycle is 15 min at room temperature Section 9 Sanitization If microbial contamination of the packed bed is suspected the column can be periodically washed with a solution consisting of 0 1 M sodium hydroxide Allow to stand for 1 hour then wash with buffer until a neutral pH is reached Section 10 Storage To store UNOsphere SUPrA M media for extended periods or between purification campaigns equilibrate the media with a 20 ethanol soluti
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