Phenyl Sepharose® 6 Fast Flow (low sub)
Contents
1. he separation It is therefore very important to pack and test the column according to the following recommendations Begin the packing procedure by determining the optimal packing flow rate Guidelines are given below for determining the optimal packing flow rates or columns with adaptors and fixed bed heights Determining optimal packing flow rates The optimal packing flow rate is dependent on column size and type medium volume packing solution and temperature The optimal packing low rate must therefore be determined empirically for each individual system To determine the optimal packing flow rate proceed as follows Calculate the amount of medium needed for the slurry this is especially important for columns with fixed bed heights The quantity of medium required per litre packed volume is approximately 1 15 litres sedimented medium 2 Prepare the column exactly as for column packing 3 Begin packing the medium at a low flow rate 30 cm h 4 Increase the pressure in increments and record the flow rate when the pressure has stabilised Do not exceed the maximum pressure of the column or the maximum flow rate for the medium 5 The maximum flow rate is reached when the pressure flow curve levels off or the maximum pressure of the column is reached Stop the packing and do not exceed this flow rate The optimal packing flow rate pressure is 70 100 of the maximum flow rate pressure 6 Plot the pressure flow rate cur2. and start packing the bed by suction through the bed at the predetermined flow rate see Fig 2 Keep the flow rate constant during packing 5 When the bed has stabilised the top of the bed surface should be just below the junction between the column and the packing device If when stabilised the level of the bed is incorrect add or remove slurry Always stir the slurry thoroughly before packing 6 Just before the last of the solution has entered the packed bed before the surface starts to dry close the valve on the column outlet stop the pump quickly remove the packing device and replace it with the final lid This final operation should be completed as quickly as possible because the bed will start to expand as soon as flow through it stops 7 Start pumping buffer with upward flow through the column to remove any air bubbles trapped under the lid p 9 Hydraulic packing INdEX columns INdEX columns are supplied with a hydraulic function which allows an extremely simple rapid and reproducible packing procedure to be used The medium is packed at the same time as the adaptor is lowered into position at the correct pressure The adaptor is pushed down by a constant hydraulic pressure forcing water through the slurry and compressing it so that a packed bed is gradually built up The hydraulic pressure can be generated using a pump and a pressure relief valve When the adaptor reaches the surface of the settled medium it
3. continues downwards under hydraulic pressure compressing the medium The extent to which the medium is compressed depends upon the pressure from the adaptor and the elasticity of the medium The quantity of medium required when packing Sepharose 6 Fast Flow based media by hydraulic pressure is approximately 1 2 litres sedimented medium per litre packed bed 1 Pour some water or packing solution into the column Make sure that there is no air trapped under the bottom net Leave about 2 cm of liquid in the column 2 Pour the 75 slurry carefully into the column Fill the column with buffer solution up to the edge of the glass tube Mix the slurry and buffer solution Allow the medium to settle to below the bevel G on the glass tube see Fig 2 3 Rest the adaptor against the bevel G on the glass tube Tilt the lid slightly to avoid trapping air bubbles under the net when mounting it on the column Lower and secure it in place 4 Connect a pump to the inlet of the hydraulic chamber A in line with a manometer and a pressure relief valve between the pump and the hydraulic chamber The manometer should be placed after the valve in the direction of the flow 5 Open the hydraulic inlet A and the hydraulic outlet C Start the pump and flush the hydraulic chamber E free of air and any residual medium 6 Close C and open the elution inlet outlet B to expel trapped air in the adaptor net Hydra
4. includes other HIC media information is available upon request as well as media for ion exchange and affinity chromatography Further Columns For information about process scale columns please ask for the following Data Files Data File Code No BPG 100 140 200 300 450 18 1115 23 INdEX 18 1115 61 BioProcess Stainless Steel 18 1121 08 CHROMAFLOW columns 18 1138 92 p 19 www gehealthcare com GE Healthcare Bio Sciences AB Bjorkgatan 30 751 84 Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Ltd Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan IndEX Sepharose BioProcess BPG CHROMAFLOW HiPrep and HiTrap are trademarks of GE Healthcare companies GE imagination at work and GE monogram are trademarks of General Electric Company All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them GE Healthcare reserves the right subject to any regulatory and contractual approval if required to make changes in specifications and features shown herein or discontinue the product described at any time without notice or obligation Contact your local GE Healthcare represen
5. of fines and allows efficient maintenance procedures for increased media life time Typical pressure flow rate curves are shown in Fig 1 Flow rate cm h 700 600 J 500 400 300 high sub low sub 200 100 T T T T T T T T T T T 1 0 0 0 2 0 4 0 6 0 8 1 0 1 2 Pressure bar Fig 1 Typical pressure flow rate curves for Phenyl Sepharose 6 Fast Flow low sub and Phenyl Sepharose 6 Fast Flow high sub in an XK 50 30 column bed height 15 cm mobile phase 0 1 M NaCl p 3 Method design and optimisation The main purpose of optimising a chromatographic step is to reach the pre defined purity level with highest possible product recovery by choosing the most suitable combination of the critical c hromatographic parameters In process chromatography in contrast to analytical or small scale preparative chromatography this has to be accomplished as quickly and economically as possible i e finding the conditions tha productivity and process economy give the highest possible Recommendations for optimising the critical operational parameters which affect the maximum utilisation of a HIC step can be found in our handbook Hydrophobic Interaction Chromatography Principles and Methods Code number 18 1020 90 available from your local GE Healthcare office Media screening To help with screening and selection of media a HiTrap HIC Test Kit is available It consists of 6 x 1 ml HiTrap columns packed wi
6. GE Healthcare Instructions 71 5002 39 AC Hydrophobic Interaction Chromatography Phenyl Sepharose 6 Fast Flow low sub Phenyl Sepharose 6 Fast Flow high sub Phenyl Sepharose 6 Fast Flow low sub and Phenyl Sepharose 6 Fast Flow high sub are part of the GE Healthcare media range for hydrophobic interaction chromatography HIC They are also part of the Fast Flow media range developed for preparative separations They are based on rigid 90 um 45 165 um diameter highly cross linked agarose beads designed to meet industrial demands on reliability and scalability These instructions contain information about media characteristics column packing and maintenance To ensure best performance and trouble free operation please read these instructions before using Phenyl Sepharose 6 Fast Flow low sub or Phenyl Sepharose 6 Fast Flow high sub Table of contents 1 Characteristics 2 Column packing guidelines 3 Evaluation of column packing 4 Media and column maintenance 5 Further information 6 Ordering information 14 17 18 19 1 Characteristics The base matrix Sepharose 6 Fast Flow is a highly cross linked 6 agarose derivative with excellent kinetics making them ideal for process scale applications particularly during initial capture and intermediate stages of a separation process when high flow rates are required The high physical and chemical stability of the matrix prevents bed compression and formation
7. ccumulate on the column they may affect the chromatographic properties of the column If the fouling is severe it may also block the column increasing back pressure and reducing flow rate The following are suggested methods to remove strongly bound hydrophobic proteins lipoproteins and lipids e Wash the column with 4 10 bed volumes of up to 70 ethanol or 30 isopropanol followed by 3 4 bed volumes of water Apply gradients to avoid air bubble formation when using high concentrations of organic solvents e Alternatively wash the column with 1 2 bed volumes of 0 5 non ionic detergent e g in 1 M acetic acid followed by 5 bed volumes of 70 ethanol to remove the detergent and 3 4 bed volumes of water Caution Specific regulations may apply when using 70 ethanol since it can require the use of explosion proof areas and equipment Consult your local safety regulations for more information To remove other contaminants the following method is suggested e Wash the column with 4 bed volumes of 0 5 1 0 M NaOH at 40 cm h followed by 2 3 bed volumes of water The CIP protocols given above should be used as guidelines when ormulating a cleaning protocol specific for the raw material used The requency of CIP will depend on the raw material applied to the column but it is recommended to use a CIP procedure at least every 5 cycles during normal use Depending on the nature of the contaminants different protocols may have to be use
8. d in combination If fouling is severe the protocols may have to be further optimised During CIP flow direction hrough the column should be reversed Sanitisation For inactivation of microbial contaminants equilibrate the column with 0 5 1 0 M NaOH at a flow rate of approximately 40 cm h contact time 30 60 minutes Wash the column thoroughly with running buffer after sanitization Sterilisation To sterilise Phenyl Sepharose 6 Fast Flow high sub or low sub dismantle the column and autoclave the medium for 20 minutes at 120 C Storage Store Phenyl Sepharose 6 Fast Flow high sub or low sub in 20 ethanol at 4 30 C to avoid microbiological growth 5 Further information Please read these instructions carefully before using Phenyl Sepharose 6 Fast Flow media For further information visit www gehealthcare com or contact your local GE Healthcare representative 6 Ordering information Product Pack size Code No Phenyl Sepharose 6 Fast Flow low sub 25 ml 7 0965 10 200 ml 7 0965 05 1litre 7 0965 03 5 litres 7 0965 04 Phenyl Sepharose 6 Fast Flow high sub 25 ml 7 0973 10 200 ml 7 0973 05 1 litre 7 0973 03 5 litres 7 0973 04 10 litres 7 0973 06 HiTrap HIC Test Kit 6x1ml 1 0034 53 Handbook Hydrophobic Interaction Chromatography Principles and Methods 8 1020 90 HiPrep 16 10 Phenyl low sub 20 ml 7 5094 01 HiPrep 16 10 Phenyl high sub 20 ml 7 5095 01 The complete range of Sepharose Fast Flow media
9. etention for the molecules to be separated can be substantial at any specific ionic strength Therefore relatively short columns can be used if the selectivity of the adsorbent is exploited in an optimal way Recommended bed heights range from 3 to 15 cm which will minimise back pressure and allow high throughput Recommended process scale columns e BPG variable bed glass columns inner diameters from 100 450 mm bed volumes up to 130 litres bed heights max 58 cm e BioProcess Stainless Steel BPSS fixed bed columns inner diameters from 400 1400 mm fixed bed volumes from 19 230 litres fixed bed height 15 cm e INdEX variable bed columns inner diameters from 70 200 mm bed volumes up to 25 litres bed heights max 61 cm e CHROMAFLOW variable and fixed bed columns Inner diameters from 280 2000 mm Packing process scale columns General packing procedures Columns can be packed in different ways depending on the type of column and equipment used Always read and follow the relevant column instruction manual carefully Sepharose 6 Fast Flow based media are easy to pack since their rigidity allows the use of high flow rates see Fig 1 Four types of packing methods are described e Pressure packing for columns with adaptors e Suction packing for large columns with fixed bed heights e Hydraulic pressure packing e CHROMAFLOW packing p 6 How well the column is packed will have a major effect on the result of
10. her than expected from the hydraulic pressure applied during packing It is therefore important to carefully optimize the hydraulic packing pressure in order to achieve the same flow properties as for columns packed with conventional techniques using a pump Packing CHROMAFLOW columns Procedure Prepare the column for packing as described in the User Manual Packing from the top 1 Set the top nozzle to the pack position mid position Fully retract the bottom nozzle run position Ensure that the top mobile phase is closed Open the bottom mobile phase non FW nN Open Inlet C and start the packing pump Adjust the flow to achieve the required packing conditions for the selected medium Monitor column pressure and the outlet flow rate in order to record column packing parameters Remember to stir the medium slurry during packing to prevent it from settling 6 Continue pumping until the column is fully packed and the pump stalls due to build up of medium in its pipelines Turn off the packing pump 7 Fully retract the top nozzle to its run position Close Outlet C Open Inlet B from the water buffer tank and open Outlet D The pump should now be restarted to rinse the top slurry lines If the nozzle is full of liquid when in the packing position make sure that the waste slurry outlet is open before retracting the nozzle 8 To clean in place exchange the buffer tank for wash buffer tank containing cleaning so
11. iple of operation CHROMAFLOW columns p 13 Note Itis also possible to use a slightly different p the amount of medium is predetermined In alount of medium is packed into the column acking method where his case the complete causing compression of the bed When all medium has entered the column the pump is stopped the top nozzle is retracted the bot closed and the medium is allowed to decom 3 Evaluation of column pa To check the quality of the packing and to monitor i deteriorate sample such as 1 acetone solution to the column be used as a test substance Use a concentration o 0 5 M NaCl in water as eluent Sometimes a concen 10 fold is preferred vary depending on the test conditions and it should reference value only It is also important that condi ept constant so that results are comparable Chan eluent sample volume flow rate liquid pathway te influence the results om mobile phase valve press within the column cking during the working life of the column column efficiency should be tested directly after packing at regular intervals afterwards and when separation performance is seen to The recommended method of expressing the efficiency of a packed column is in terms of the height equivalent to a theoretical plate HETP and the asymmetry factor As These values are easily determined by applying a Sodium chloride can also 2 0 M NaCl in water with rated buffer solution e g ti
12. lution Packing from below To pack from the bottom carry out the same procedure for the connections and flow path via the bottom nozzle The column is now ready to equilibrate and test p 12 SLURRY INLET MOBILE PHASE IMOBILE PHASE MOBILE PHASE BUFFER WASTE SLURRY WASTE SLURRY BUFFER Packing position The top nozzle is extended part of the way mid position into the column The bottom nozzle is fully retracted Slurry enters the column via the top nozzle and excess liquid exits via the bottom mobile phase outlet After packing the slurry lines are isolated from the mobile phase and can be cleaned independently from the rest of the column Running position The bottom and top nozzles are retracted Mobile phase enters the column directly into an annulus immediately behind the bed support The annulus is cut through at an angle to ensure that linear flow rate is kept constant during distribution of the mobile phase across the bed Unpacking position In this position both bottom and top nozzles are fully extended into the column thereby exposing a third passage through which medium leaves the column Cleaning solution can be pumped through the nozzles and sprayed into the column In this way the column is easily and effectively cleaned without exposing the interior or the medium to the environment or without dismantling the column Fig 3 Princ
13. n the bed has stabilised mark the bed height on the column tube close the bottom valve and stop the pump The bed starts rising in the column Loosen the O ring and lower the adaptor to 0 5 1 cm from the bed surface 6 Seal the O ring start the pump and continue packing Repeat steps 5 and 6 until there is a maximum of 1 cm between bed surface and adaptor when the bed has stabilised 7 Close the bottom valve stop the pump disconnect the column inlet and push the adaptor down to approximately 3 mm below the mark on the column tube without loosening the adaptor O ring The packing solution will flush the adaptor inlet Remove any trapped air by pumping liquid from the bottom after the inlet tubing and the bottom valve have been properly filled p 8 Suction packing BioProcess Stainless Steel BPSS columns BioProcess Stainless Steel Columns are supplied with fixed end pieces They are packed by sucking packing solution through the chromatographic bed at a constant flow rate 1 Fit a packing device on top of the column tube 2 Pour some water or packing solution into the column Make sure that there is no air trapped under the bottom net Leave 2 3 cm of liquid in the column 3 Mix the packing solution with the medium to form a 50 slurry sedimented bed volume slurry 0 5 Pour the slurry into the column Stir gently to give a homogeneous slurry 4 Connect the column outlet valve to the suction side of a pump
14. o 1 values between 0 8 1 5 are usually acceptable A change in the shape of the peak is usually the first indication of bed deterioration due to use p 15 Peak asymmetry factor calculation A b a where a 1st half peak width at 10 of peak height b 2nd half peak width at 10 of peak height Figure 4 shows a UV trace for acetone in a typical test chromatogram in which the HETP and As values are calculated UV absorption 4 Column BPG 300 EAI Media Sepharose 6 Fast Flow i Bed height 57 5 cm i Bed volume 40 6 litres in Eluent Distilled water pi Sample 1 05 litres 1 acetone i Flow rate 19 cm h Wh re V 18 7 W 0 9 HEPT 0 024 cm a 0 90 b 0 85 A 0 94 Volume Fig 4 UV trace for acetone in a typical test chromatogram showing the HETP and A value calculations p 16 4 Media and column maintenance Regeneration For best performance from the media bound substances must be washed from the column after each chromatographic cycle Wash with 2 bed volumes of water followed by 2 3 bed volumes of starting buffer To prevent a slow build up of contaminants on the column over time more rigorous cleaning protocols may have to be applied on a regular basis Cleaning in place CIP Cleaning in place CIP is the removal of very tightly bound precipitated or denatured substances from the purification system generated in previous purification cycles If such contaminants a
15. s of utmost importance to realise that the calculated plate number will therefore be used as a ions and equipment are ges in solute solvent mperature etc will For optimal results the sample volume should be a column volume and the flow rate between 15 and 3 maximum 2 5 of the 0 cm h f an acceptance limit is defined in relation to column performance the column plate number can be used as part of the acceptance criteria for column use Method for measuring HETP and A To avoid dilution of the sample apply it as close tot possible p 14 he column inlet as Conditions Sample volume 1 0 of bed volume Sample conc 1 0 v v acetone in water 2 0 M NaCl or 10x buffer Eluent 0 5 M NaCl in water or dilute buffer Flow rate 30 cm h Detection Acetone UV 280 nm NaCl buffer Conductivity Calculate HETP and the number of theoretical plates as follows HETP L N and N 5 54 Ve W where Bed height cm Number of theoretical plates ie Il Peak elution distance e W Peak width at half peak height v and W are in the same units To facilitate comparison of column performance the concept of reduced plate height is often used The reduced plate height is calculated as HETP d where d is the mean diameter of the beads As a guide line a value of lt 3 is normally acceptable The peak should be symmetrical and the asymmetry factor as close as possible t
16. tative for the most current information 2005 General Electric Company All rights reserved GE Healthcare AB a General Electric Company imagination at work 71 5002 39 AC E Elanders Tofters 2005
17. th Butyl Sepharose 4 Fast Flow Butyl S Sepharose 6 Fast Flow Octyl Sepharose 4 Fast Flow Phenyl Sepharose 6 Fast Flow high and low substitution and Phenyl Sepharose High Performance cod p 4 e number 11 0034 53 Table 1 Characteristics of Phenyl Sepharose 6 Fast Flow low sub and Phenyl Sepharose 6 Fast Flow high sub Matrix Type of ligand Bead form Mean particle size Particle size distribution Degree of substitution PH stability working range cleaning range Chemical stability Autoclavable Linear flow rate at 25 C Operating temperature Delivery conditions Highly cross linked agarose 6 Phenyl R O CH CH OH CH O C H Rigid spherical macro porous 90 um diameter 45 165 um Low sub approx 20 umol phenyl ml medium High sub approx 40 pmol pheny ml medium 3 13 2 14 Stable in commonly used aqueous buffers 1 M NaOH 3 M Ammonium sulphate 30 isopropanol 70 ethanol 10 ethylene glycol 0 5 SDS 6 M guanidine hydrochloride 8 M Urea 20 min at 121 C 2 400 cm h at 100 kPa 1 bar 14 5 psi XK 50 30 15 cm bed height 4 40 C 20 ethanol The actual loading capacity in a real working situation will depend on the nature and concentration of contaminants in the sample and the degree of resolution required p 5 2 Column packing guidelines General Purifying biological macromolecules by HIC is a typical high selectivity technique where the difference in r
18. ulic inlet G Elution inlet outlet Hydraulic outlet E Elution inlet outlet Hydraulic chamber Slurry Packed bed a Bevel on glass tube a onmonpop Fig 2 Schematic representation of INdEX column with a 4 port 2 way valve mounted at the bottom outlet N Close B and open the elution inlet outlet D to start packing Apply a pre defined constant hydraulic packing pressure When packing Sepharose 6 Fast flow based media in an INdEX column to a bed height of 15 cm the recommended hydraulic packing pressure is 1 5 bar for INdEX 100 and 0 8 bar for INdEX 200 8 When the bed has finally settled no flow at the column outlet stop the packing procedure by closing A and D The adaptor stops moving when the hydraulic force acting downwards is equal to the mechanical force of the bed expressed upwards 9 To unpack the column connect the outlet from the pump to B and open C while keeping D closed This will cause the adaptor to rise from the bed surface Note The hydraulic pressure used for packing is not comparable to the back pressure generated when packing with a pump or pressure vessel When using hydraulic pressure packing the bed is mechanically compressed during the last part of the procedure As a result the flow properties of the packed bed will be limited by this mechanical compression At any flow rate the pressure drop over the bed under running conditions is hig
19. ve as in Fig 1 and determine the optimal packing flow rate The operational flow rate pressure should be lt 70 of the packing flow rate pressure Note For BPSS columns first pack the column by suction packing at a low flow rate then determine the flow pressure characteristics as above by pumping with downward flow through the column Packing methods Pressure packing BPG columns BPG glass columns are supplied with a movable adaptor They are packed by conventional pressure packing by pumping the packing solution through the chromatographic bed at a constant flow rate or back pressure 1 Pour some water or packing solution into the column Make sure that there is no air trapped under the bottom net Leave about 2 cm of liquid in the column 2 Mix the packing buffer with the medium to form a 50 70 slurry Sedimented bed volume slurry volume 0 5 0 7 Pour the slurry into the column Insert the adaptor and lower it to the surface of the slurry making sure no air is trapped under the adaptor Secure the adaptor in place 3 Seal the adaptor O ring and lower the adaptor a little further into the slurry to fill the adaptor inlet with packing solution 4 Connect a pump and a pressure gauge then start packing at the predetermined packing flow rate or pressure Keep the flow rate or pressure constant during packing and check the pressure at the column inlet Never exceed the pressure limit for column or medium 5 Whe
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