GE HITRAP Q HP User's Manual
Contents
1. Fig 3 Recommended buffer substances for anion exchange chromatography The columns can be operated with a syringe peristaltic pump or a chromatography system 3 Operation Buffer preparation Water and chemicals used for buffer preparation should be of high purity It is recommended to filter the buffers by passing them through a 0 22 um filter immediately before use See Tables 2 and 3 Figs 2 and 3 for recommended buffers Sample preparation The sample should be adjusted to the composition of the start buffer This can be done by either diluting the sample with start buffer or by buffer exchange using HiTrap Desalting HiPrep 26 10 Desalting or PD 10 column The sample should be filtered through a 0 22 um or 0 45 um filter or centrifuged immediately before it is applied to the column See Table 4 uiue u sesbq OT ubui uns 01 f iAb46 J81DaJ5 sepnos nu fijuo sauinbay obijo pub YNG eppJ6 YNG lu gg IWS SZ dYN 10 2580 1 000S I sure1o4d S2 9 xepoydas Iu ST Iu OTt OT dVN TO 7S80 2T jo uonpoiyund 103 yum pexopdaJg lu OT J O S wdYN TO S80 T suonpJpdes JDjIuJIS pup sJaW OZT ubu Ja JOYs JOU sapljoajonu Palleqo Olpb1 Wot YNG payojsubJ 49 u pup 000S lt W Sulajoid 52 9 xappydas jo uonpJodes J04 yum pexopdaJd W t0 uU TO wiMDIN TO SS80 2 000S lt W uni 0 fiqin0s6 S 2012. 1 Equilibrate the column see Purification 2 Adjustthe sample to the chosen starting pH and ionic strength see Sample preparation 3 Determine the concentration of the specific proteins by UV SDS PAGE ELISA or other appropriate techniques p 15 4 IgG pH 8 2 HSA pH 8 2 Transferrin pH 8 2 IgG pH 9 0 mg protein eluted flow rate ml min Fig 4 Binding capacity of human IgG HSA and human transferrin at different pH s on HiTrap Q HP 1 ml 4 p 16 Apply the sample solution to the column with a pump or a syringe at a flow rate equal to the flow rate to be used in the purification method Collect fractions and continue sample application until the column is saturated Wash the column with 5 10 column volumes start buffer or until no material appears in the effluent Elute bound proteins with 3 5 column volumes of elution buffer start buffer with 1 M NaCl and collect eluate Analyse fractions and eluates from steps 4 and 6 for the specific protein and determine the breakthrough profile sample concentration as a function of the amount of sample applied The dynamic capacity is the amount that can be applied without any significant breakthrough The total capacity for the specific protein is determined from step 6 7 Scaling up For quick scale up of purifications back pressure will increase two or three HiTrap ion exchange columns of the same type can be connected in ser
3. Stepwise gradients are easy to produce and require minimal equipment Eluted peaks are very sharp and elution volumes minimal However care must be exercised in the design of the steps and the interpretation of results for substances eluted by a sharp change in pH or small differences in ionic strength Peaks tend to have sharp fronts and pronounced tailing since they frequently contain more than one component 2 Continuous salt gradients are the most frequently used type of elution Many types of gradient forming systems are available Two buffers of differing ionic strength the start and elution buffer start buffer 1M NaCl or higher buffer salt concentration are mixed together and if the volume ratio is changed linearly the ionic strength changes linearly Note Another but less common method to desorb bound material is to increase SP or decrease Q the pH of the eluent Continuous pH gradients are difficult to produce at constant ionic strength since simultaneous changes in ionic strength although small also occur buffering capacities are pH dependent Elution with stepwise ionic strength gradients Stepwise elution is the sequential use of the same buffer at different ionic strengths It is technically simple and fast and is suitable for syringe operation It is often used for sample concentration and sample clean up Stepwise elution gives small peak volumes and the resolution depends on the difference in elution powe
4. x utajoJd jo fijuo seuinbay aBupyoxa 18jjnq S2 9 xeppudes bunjbpseq puo bunjosep 104 yum pexobdajd lu Sg ju sz OT dd 10 TS80 2 000S lt W uns 01 duund S 901 x uta101d D sauinbay auls bunjbpseg Jo ebBupuoxe Jayjng S2 9 xapoydas ju ST 01 92 puo bunjosep 104 yum pexopdaJg W O2 ST 01 dn deldlH 10 80S unio duund Jo eBuufis o 000S 4 S1901xe8 seunbesy eutyjedns ulajojd jo eDubu xe S2 9 wxepoydas bunjpseq 19jjnq pup Dunjpsep 104 yum pexopdaJg W 0 T lu S T TO doJLIH I0 8091 uonpoijddy sjueuluo euinjo uonn 3 euinjoA Buippo uun o9 ON apo aBupyoxa Jajyng pup bunjpsep 104 suuunjoo pexopdaudg amp ajqDL o a a Purification 1 C N wn 2 ov o Ko 10 Fill the syringe or pump tubing with start buffer low ionic strength Remove the stopper and connect the column to the syringe with the provided adaptor drop to drop to avoid introducting air into the column Remove the snap off end at the column outlet Wash out the preservatives with 5 column volumes of start buffer at 1 ml min for the 1 ml column and 5 ml min for the 5 ml column Wash with 5 column volumes of elution buffer start buffer with 1M Nacl Finally equilibrate with 5 10 column volumes of start buffer Apply the sample at 1 or 5 ml min for the 1 ml and 5 ml columns respectively using a syringe fitted to the luer adaptor or by pumping it onto the column Wash with at least 5 column volumes o
5. Figs 2 and 3 In the majority of cases a concentration of at least 10 mM is required to ensure adequate buffering capacity The ionic strength of the buffer should be kept low 5 mS cm so as not to interfere with sample binding Salts also play a role in stabilizing protein structures in solution and it is important the ionic strength should not be so low that protein denaturation or precipitation occurs p 6 The buffering ion should carry the same charge as the ion exchange group and should have a pKa within 0 5 pH units of the pH used in the separation Buffering ions of opposite charge may take part in the ion exchange process and cause local disturbances in pH Starting pH Cation exchangers SP At least 1 pH unit below the pl of substance to be bound Anion exchangers Q At least 1 pH unit above the pl of substance to be bound Table 2 Buffers for cation exchange chromatography pH interval Substance Conc mM Counter ion pKa 25 C 14 24 Maleic acid 20 a 192 2 6 3 6 Methyl malonic acid 20 Na or Li 3 07 2 6 3 6 Citric acid 20 a 3 13 3 3 4 3 Lactic acid 50 a 3 86 3 3 4 3 Formic acid 50 Na or Li 3 75 3 7 4 7 5 1 6 1 Succinic acid 50 a 4 21 5 64 4 3 5 3 Acetic acid 50 a or Li 4 75 5 2 6 2 Methyl malonicacid 50 Na or Li 5 76 5 6 6 6 MES 50 a orti 6 27 67 77 Phosphate 50 a 7 20 7 0 8 0 HEPES 50 Na or Li 7 56 7 8 8 8 BICINE 50 a 8 33 Ref Handbook of chemistry and physic
6. GE Healthcare Instructions 71 7149 00 AN HiTrap ion exchange columns HiTrap SP HP 1 ml and 5 ml HiTrap Q HP 1 ml and 5 ml HiTrap SP HP and HiTrap Q HP are prepacked ready to use cation and anion exchange columns for method scouting group separations sample concentration and sample clean up of charged biomolecules HiTrap SP HP and HiTrap Q HP provide fast reproducible and easy separations in a convenient format The columns can be operated with a syringe peristaltic pump or liquid chromatography system such as AKTAdesign or FPLC System HiTrap Code No Designation No supplied 7 1151 01 HiTrap SP HP 5x1ml 7 1152 01 HiTrap SP HP 5x5ml 7 1153 01 HiTrap Q HP 5x1ml 7 1154 01 HiTrap Q HP 5x5ml Connectorkit Connectors supplied Usage No supplied 16 male luer female Connection of syringe to top of HiTrap column Tubing connector Connection of tubing e g Peristaltic langeless M6 female Pump P1 to bottom of HiTrap column Tubing connector Connection of tubing e g Peristaltic langeless M6 male Pump P1 to top of HiTrap column Union 1 16 female Connection to original FPLC System M6 male through bottom of HiTrap column Union M6 female Connection to original FPLC System 1 16 male through top of HiTrap column Stop plug female 1 16 Sealing bottom of HiTrap column 2 50r7 Union 1 16 female M6 male is also needed Union M6 female 1 16 male is also needed Table of
7. amount of the sample at 1 or 5 ml min for the 1ml and 5 ml columns respectively Collect eluate 5 Wash with at least 5 column volumes start buffer or until no material appears in effluent Collect eluate 6 Elute bound material with elution buffer 3 5 column volumes is usually sufficient Other volumes may be required depending on the chosen operational conditions Collect eluate 7 Analyze all eluates for example by activity assay and SDS PAGE and determine the purity and the amount bound to the column 8 Perform steps 2 7 for the next buffer pH 9 Decide which pH should be used for the selected purification strategy 10 To decide on starting ionic strength conditions a similar screening is done but the buffer pH is held constant and the ionic strength is varied in the interval 0 0 5 M with intervals of 0 05 to 0 1 M salt between each buffer Further optimization The recommendations given above will give a sound basis for developing an efficient purification step Details of how flow rate sample loading particle size and elution scheme may be optimized to meet the special needs can be found in the handbook lon Exchange Chromatography amp Chromato foucsing Principles and Methods Code No 11 0004 21 GE Healthcare supplies a wide range of ion exchange chromatography media for purification of biomolecules at all scales See Ordering information and visit www gehealthcare com hitrap 5 Choice of gradient type 1
8. arks 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 representative for the most current information 2006 General Electric Company All rights reserved GE Healthcare AB a General Electric Company imagination at work 71 7149 00 AN 08 2006 K Elanders Ostervala 2006
9. contents 1 Description 3 2 Selection of ion exchanger and conditions 5 3 Operation 9 4 Optimization 12 5 Choice of gradient type 13 6 Determination of binding capacity 15 7 Scaling up 17 8 Storage 17 9 Ordering Information 17 p 2 1 Description Media properties SP Sepharose High Performance and Q Sepharose High Performance are strong cation and strong anion exchangers respectively Both remain charged and maintain high capacity over broad pH ranges The functional groups are coupled to the matrix via chemically stable ether linkages Characteristics of HiTrap SP HP and HiTrap Q HP 1 and 5 ml columns are isted in Table 1 Column HiTrap Q HP and HiTrap SP HP are 1 ml and 5 ml columns made of polypropylene which is biocompatible and non interactive with biomolecules The top and bottom frits are manufactured from porous polyethylene It is delivered with a stopper on the inlet and a snap off end on the outlet The separation can be easily achieved using a syringe together with the supplied luer adaptor a peristaltic pump or in a chromatography system such as AKTA or FPLC Note To prevent leakage it is essential to ensure that the adaptor is tight The column cannot be opened or refilled Table 1 HiTrap SP HP and HiTrap Q HP columns characteristics Column volumes Column dimensions Total ionic capacity Dynamic binding capacity Mean particle size Bead structure Maximum flow rates Reco
10. e g AKTAprime plus Atwo pump system e g FPLC or AKTA Choose starting conditions as outlined under Optimizing starting conditions 2 Equilibrate the column see Purification 3 Adjustthe sample to the chosen starting pH and ionic strength see Sample preparation 4 Applythe sample at 1 or 5 ml min for the HiTrap 1 or 5 ml column respectively Collect eluate 5 Wash with 5 10 column volumes of start buffer or until no material appears in effluent 6 Start the gradient elution A gradient volume of 10 20 column volumes and an increase in ionic strength to 0 5 M NaCl is usually sufficient 7 Regenerate the column by washing with 5 column volumes of start buffer with 1 M NaCl followed by 5 10 column volumes of start buffer The column is now ready for a new sample 6 Determination of binding capacity The amount of sample which can be applied to a column depends on the capacity of the column and the degree of resolution required The capacity is dependent on the sample composition choosen starting conditions of pH and ionic strength and the flow rate at which the separation is done The influence of flow rate and pH on the capacity for some model proteins are shown in Figure 4 Samples were applied until 596 of the start material appeared in the eluent The column was then washed with 10 ml start buffer 20 mM Tris HCl pH 8 2 or 9 0 before elution with elution buffer 20 mM Tris HCl 1 0 M NaCl pH 8 2 or 9 0
11. f start buffer or until no material appears in the effluent Elute with 5 10 column volumes of elution buffer see section Choice of gradient type The purified fractions can be desalted using a HiTrap Desalting HiPrep 26 10 Desalting or a PD 10 columns if necessary After the completed elution regenerate the column by washing with 5 column volumes of regeneration buffer start buffer with 1 M NaCl followed by 5 10 column volumes of start buffer The column is now ready for a new sample For a first experiment the following conditions are recommended Flow rate 1 ml min using HiTrap 1 ml column 5 ml min using HiTrap 5 ml column Start buffer See Tables 2 and 3 Elution buffer Start buffer 1 M NaCl Gradient volume 20 ml Note Ifa P1 pump is used a max flow rate of 1 3 ml min can be run on aHiTrap 1 ml column packed with Sepharose High Performance media 4 Optimization If sample composition is unknown a simple screening test with the aid of a syringe or pump can be performed to optimize starting pH and ionic strength 1 Setupa series of buffers with different pH s in the range 4 8 SP or 5 9 Q with 0 5 1 pH unit intervals between each buffer Make one series with 1 M NaCl included in the buffers regeneration buffer and the other without NaCl start buffer 2 Equilibrate the column see Purification 3 Adjustthe sample to the chosen start buffer see Sample preparation 4 Apply a known constant
12. ies For further scale up SP Sepharose High Performance and Q Sepharose High Performance are available in prepacked HiLoad columns or bulk media packs See Ordering Information 8 Storage HiTrap SP HP Rinse with water and then wash with 5 column volumes of 2096 ethanol 0 2 M sodium acetate HiTrap Q HP Rinse with water and then with 5 column volumes of 2096 ethanol Seal the column with the supplied stoppers The recommended storage temperature is 4 to 30 C 9 Ordering Information Product No Supplied Code No HiTrap SP HP 5x1ml 17 1151 01 HiTrap SP HP 5x5ml 17 1152 01 HiTrap Q HP 5x1ml 17 1153 01 HiTrap Q HP 5x5ml 17 1154 01 Related products No Supplied Code No HiTrap IEX Selection Kit 7x1ml 7 6002 33 HiTrap Desalting 5x5ml 7 1408 0 HiTrap Desalting 100 x 5 ml 1 0003 29 HiPrep 26 10 Desalting 1x53ml 7 5087 0 HiPrep 26 10 Desalting 4x53ml 7 5087 02 PD 10 Desalting column 30 7 0851 0 HiLoad 16 10 SP Sepharose High Performance 1x20ml 7 1137 0 HiLoad 26 10 SP Sepharose High Performance 1x53ml 7 1138 0 HiLoad 16 10 Q Sepharose High Performance 1x20ml 7 1064 0 HiLoad 26 10 Q Sepharose High Performance 1x53ml 7 1066 0 SP Sepharose High Performance 75 ml 7 1087 0 Q Sepharose High Performance 75 ml 7 1014 0 Special pack size delivered on specific order Accessories No Supplied Code No 1 16 male luer female 2 8 1112 5 Tubing connector flangeless M6 female 2 8 1003 68 Tubing connector flangele
13. le binding of charged sample molecules to oppositely charged groups attached to an insoluble matrix The pH value at which a biomolecule carries no net charge is called the isoelectric point pl When exposed to a pH below its pl the biomolecule will carry a positive charge and will bind to a cation exchanger SP At pH s above its pl the protein will carry a negative charge and will bind to an anion exchanger Q If the sample components are most stable below their pl s a cation exchanger should be used If they are most stable above their pl s an anion exchanger is used If stability is high over a wide pH range on both sides of pl either type of ion exchanger can be used Figure 1 Selection of buffer pH and ionic strength Buffer pH and ionic strength are critical for the binding and elution of material both target substances and contaminants in ion exchange chromatography Selection of appropriate pH and ionic strength for the start and elution buffers allows the use of three possible separation strategies g amp 2 9 2 i S 3 2 a Attached to 9 i anion exchangers o T T T T T 1 S 8 1C Attached o pH wm tocation 2 exchangers Fig 1 The net charge or a protein as a function of pH Strategy 1 Binding and elution of all sample components Binding is achieved by choosing a start buffer with a low pH for HiTrap SP HP or a high pH for HiTrap Q HP The ionic strenght should be kept as low as
14. mmended flow rates Maximum backpressure Chemical stability Charged group pH stability Short term Working Long term Storage temperature Storage buffer Avoid 1mlor5 ml 0 7 x 2 5 cm 1 ml and 1 6 x 2 5 cm 5 ml 0 14 0 20 mmol CI ml medium QJ 0 15 0 20 mmol H ml medium SP SP approx 55 mg ribonuclease ml medium 0 1 M sodium acetate pH 6 0 at 1 ml min Q approx 50 mg HSA ml medium 20 mM Tris HCI pH 8 2 at 1 ml min 34 um 696 highly cross linked spherical agarose HiTrap 1 ml 4 ml min HiTrap 5 ml 20 ml min HiTrap 1 ml 1 ml min HiTrap 5 ml 5 ml min 0 3 MPa 3 bar 42 psi All commonly used buffers SP CH CH CH SO Q CHN ICHy P 3 14 Q 1 14 P 4 13 Q 2 12 P 4 13 Q 2 12 4 to 30 C P 20 ethanol 0 2 M sodium acetate Q 20 ethanol SP Oxidizing agents cationic detergents and buffers Q Oxidizing agents anionic detergents and buffers to t Co CO CO Theranges given are estimates based on our knowledge and experience Please note the following pH stability long term refers to the pH interval where the medium is stable over along period of time without adverse effects on its subsequent chromatographic performance pH stability short term refers to the pH interval for regeneration cleaning in place and sanitization procedures p 4 2 Selection of ion exchanger and conditions lon exchange chromatography is based on adsorption and reversib
15. possible to allow all components to bind to the ionic exchange 5 mS cm This results in a concentration of the target substance and a complete picture of the whole sample The drawback of this strategy is that the binding capacity of the ion exchanger for the target substance is dependent on the amount of contaminant in the sample Strongly binding contaminants can also displace bound target protein if a large volume of sample is loaded Note Start conditions are subject to the stability of the sample components Strategy 2 Enrichment of target protein This is achieved by choosing a start buffer with a pH optimized to allow maximal binding of target protein and as high as possible ionic strength to suppress binding of sample contaminants This strategy results in a concentration of the target substances Strategy 3 Binding of sample contaminants This is achieved by choosing a start buffer with a pH and ionic strength that promotes the binding of some or all contaminating substances but allows the substance of interest to pass through the column The drawback ofthis approach is that the target substance is not concentrated and the sample volume applied to the ion exchanger is dependent on the amount of contaminants in the sample Start buffer The concentration of buffer required to give effective pH control varies with the buffer system A list of suitable buffers and suggested starting concentrations is shown in Tables 2 and 3
16. r between each step 1 Choose starting conditions as outlined under Optimizing starting conditions d Equilibrate ad the column see Purification Sample preparation 4 Apply the sample at 1 or 5 ml min for the HiTrap respective 5 Wash with appears in 6 Elute with stepwise e column vo column vo ute with next ionic s y Collect eluate at least 5 column volumes of start buf effluent Collect eluate Adjust the sample to the choosen starting pH and ionic strength see ml or 5 ml column er or until no material he first step ionic strength buffer The volumes required for ution depend on the operating conditions However 3 5 umes is usually sufficient Collect elua E 8 After completed elution regenerate the column b rength buffer Collect eluate e y washing with 5 umes of regeneration buffer start buf Elution with continuous ionic strength gradients er with 1 M NaCl ollowed by 5 10 column volumes of start buffer The column is now ready for a new sample Continuous salt gradient elution is the most frequently used type of elution in ion exchange chromatography It is very reproducible and leads to improved resolution since zone sharpening occurs during elution Continuous gradients can be prepared in different ways depending on available equipment p 14 Aperistaltic pump and a gradient mixer e g pump P 1 gradient mixer GM 1 Aone pump system
17. s 83 edition CRC 2002 2003 p 7 Table 3 Buffers or anion exchange chromatography pH interval Substance Conc mM Counter ion pKa 25 C 4 3 5 3 Methylpiperazine 20 g 4 75 4 8 5 8 Piperazine 20 Cl or HCOO 5 33 5 5 6 5 L Histidine 20 Cr 6 04 6 0 7 0 bis Tris 20 C 6 48 6 2 7 2 8 6 9 6 bis Tris propane 20 cr 6 65 9 10 7 3 8 3 riethanolamine 20 Cl or CH COO 7 76 7 6 8 6 ris 20 Cr 8 07 8 0 9 0 Methyldiethanolamine 20 SO 8 52 8 0 9 0 Methyldiethanolamine 50 Cl or CH COO 8 52 8 4 9 4 Diethanolamine 20atpH8 4 Cl 8 88 50 at pH 8 8 8 4 9 4 Propane 1 3 Diamino 20 Cr 8 88 9 0 10 0 Ethanolamine 20 Cr 9 50 9 2 10 2 Piperazine 20 Cr 9 73 10 0 11 0 Propane 1 3 Diamino 20 Cr 10 55 10 6 11 6 Piperidine 20 Cr 11312 Ref Handbook of chemistry and physics 83 edition CRC 2002 2003 pKa pH 2 5 3 4 5 6 7 8 9 25 C Citric 3 13 Lactic 3 86 Butanedioic 4 21 Acetic 4 75 Methyl Malonic 5 76 MES 6 27 Phosphate 7 20 HEPES 7 56 BICINE 8 33 Fig 2 Recommended buffer substances for cation exchange chromatography p 8 pKa 4 5 6 7 8 9 10 11 25 C N methyl piperazine 4 75 Piperazine 5 33 bis Tris 6 48 bis Trispropane 6 65 9 10 Triethhanolamine 7 76 Tris 8 07 N methyldiethanolamine 8 52 Propane 1 3 diamino 8 88 Ethanolamine 9 50 Piperazine 9 73 Propane 1 3 diamino 10 55 Piperidine 1112
18. ss M6 male 2 8 1017 98 Union 1 16 female M6 male 6 8 1112 57 Union M6 female 1 16 male 5 8 3858 01 Union luerlock female M6 female 2 8 1027 12 HiTrap HiPrep 1 16 male connector for AKTAdesign 8 28 4010 81 Stop plug female 1 6 5 1 0004 64 Fingertight stop plug 1 16 5 1 0003 55 Oneconnector included in each HiTrap package Two five or seven stop plugs female included in HiTrap packages depending on the product One fingertight stop plug is connected to the top of each HiTrap column at delivery p 18 Realated literature lon Exchange Chromatography amp Chromatofocusing Handbook Principles and Methods 1 11 0004 21 lon Exchange Columns and Media Selection Guide 1 18 1127 31 Convenient Protein Purification HiTrap Column Guide 1 18 1129 81 p 19 www gehealthcare com hitrap ce media ide GmbH unzinger Strasse www gehealthcare com D 79111 Freiburg Germany GE Healthcare Bio Sciences AB GEWeoitheare URLS Bjorkgatan 30 Amersham Place 751 84 Uppsala Little Chalfont Buckinghamshire HP7 9NA Sweden 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 HiTrap Sepharose Sephadex FPLC AKTA AKTAprime AKTAdesign Drop Design NICK NAP HiPrep and HiLoad are trademarks of GE Healthcare companies GE imagination at work and GE monogram are tradem
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