IEX Sepharose Fast Flow labpacks and prepacked columns

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1 GE Healthcare Data file -77- Ion exchange chromatography IEX Sepharose Fast Flow labpacks and prepacked columns The popularity of Sepharose Fast Flow ion exchangers reflects the leading role they play in protein purification today. Their reliability and well-documented performance, and regular additions to the basic range, has ensured that they remain the first choice for capture and intermediate purification of proteins in both research and industry. Sepharose Fast Flow ion exchangers offer many practical advantages: High binding capacity and excellent flow properties High chemical and physical stabilities Reliable and reproducible Easy and effective CIP/sanitization Convenient HiPrep and HiTrap prepacked columns Predictable scale-up Ion exchange chromatography Ion exchange chromatography is probably the most frequently used and versatile method for fractionating proteins and peptides; even those with small differences in charge. Furthermore, binding and elution conditions are easy to optimize, resulting in fast separations that are reproducible and cost-effective to scale up. The technique is based on reversible interactions between charged molecules and immobilized ion exchange groups of opposite charge. The charged molecules bind to the separation medium at low ionic strength and are then eluted with a salt or gradient. Continuous gradient elution is most often used when good resolution is needed, while simple stepwise gradient elution is employed for sample preparation, group separation, or concentration. Fig. Sepharose Fast Flow ion exchangers, available in lab packs and prepacked columns, continue to play a leading role in preparative protein separations. Sepharose Fast Flow ion exchangers include media that are called weak (CM, DEE, and NX) or strong (SP and Q). The binding capacity of weak ion exchangers varies with considerably more than that of strong ion exchangers, which might affect selectivity. In contrast, the ligands of strong ion exchangers remain charged and consistently maintain high capacity over broad working ranges.

2 Media characteristics Sepharose Fast Flow ion exchange media comprise SP Sepharose Fast Flow, CM Sepharose Fast Flow, Q Sepharose Fast Flow, DEE Sepharose Fast Flow, and NX Sepharose Fast Flow (high sub). SP, CM, Q, and DEE are based on a robust, % highly crosslinked beaded agarose matrix with excellent flow properties and high loading capacity. Typical linear flow rates of 3 to cm/h through a 5-cm high bed at a pressure of bar, give fast separation cycles, which is especially important early in purification when rapid enrichment is required. For washing and equilibration, flow rates extend up to 75 cm/h. NX Sepharose Fast Flow (high sub) is based on a % highly cross-linked agarose matrix. This results in a medium with higher porosity, which is particularly useful for purifying high molecular weight proteins. Like the other Sepharose Fast Flow ion exchange media, NX has high flow rates for processing. Table. Characteristics of Sepharose Fast Flow ion exchangers Cation exchangers SP Sepharose Fast Flow CM Sepharose Fast Flow Matrix % highly cross-linked agarose % highly cross-linked agarose verage particle size 9 µm 9 µm Type of medium Strong cation Weak cation Charged group - -CH - CH -CH SO 3 -O-CH COO - Total ionic capacity..5 mmol H+/ml medium.9.3 mmol H+/ml medium Dynamic binding capacity 7 mg ribonuclease /ml medium 5 mg ribonuclease /ml medium Flow rate 7 cm/h 3 cm/h stability Short term 3 3 Long term 3 3 Storage temperature C to 3 C C to 3 C Storage buffer % ethanol,. M sodium acetate % ethanol Chemical stability ll commonly used buffers, M NaOH, M urea, M guanidine hydrochloride, and 7% ethanol void Oxidizing agents, cationic detergents, and buffers nion exchangers Q Sepharose DEE Sepharose NX Sepharose Fast Flow Fast Flow Fast Flow (high sub) Matrix % highly cross-linked agarose % highly cross-linked agarose % highly cross-linked agarose verage particle size 9 µm 9 µm 9 µm Type of medium Strong anion Weak anion Weak anion Charged group -N + (CH 3 ) 3 -N + (C H 5 ) H * -N + (C H 5 ) H * Total ionic capacity..5 mmol Cl - /ml medium.. mmol Cl - /ml medium.3. mmol Cl - /ml medium Dynamic binding capacity mg HS/ml medium mg HS/ml medium 5 mg thyroglobulin/ml medium Flow rate 7 cm/h 3 cm/h min cm/h stability Short term 3 Long term 3 3 Storage temperature C to 3 C C to 3 C C to 3 C Storage buffer % ethanol % ethanol % ethanol Chemical stability ll commonly used buffers, M NaOH, M urea, M guanidine hydrochloride, and 7% ethanol void Oxidizing agents, anionic detergents, and buffers Determination of dynamic binding capacity: DEE Sepharose Fast Flow, Q Sepharose Fast Flow, SP Sepharose Fast Flow, and CM Sepharose Fast Flow: Samples were applied at 75 cm/h until 5% breakthrough. Columns:.5 5 cm. uffers:.5 M Tris, M NaCl (in the elution buffer), 7.5 (Q and DEE) or. M acetate, M NaCl (in the elution buffer), 5. (SP and CM). NX Sepharose Fast Flow (high sub): Sample was applied at 3 cm/h until % breakthrough. Column:. 3 cm. uffer:.5 M Tris, M NaCl (in the elution buffer), 7.5. bar ( kpa), XK 5/3 column, bed height 5 cm (SP, CM, Q, and DEE) or bar ( kpa), XK 5/ column, bed height 5 cm (NX). 3 Refers to the interval for regeneration and cleaning. Refers to the interval where the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. * Note: The active end of the charged group is the same for DEE Sepharose Fast Flow and NX Sepharose Fast Flow (high sub). The difference is the length of the carbon chain of the charged group attached to the medium. DEE Sepharose Fast Flow has a diethylaminoethyl-group bound to the agarose while NX Sepharose Fast Flow has a diethylaminopropyl-group attached. Data file -77-

3 M NaOH (ml) Q Sepharose Fast Flow M NaOH (ml) NX Sepharose Fast Flow (high sub) DEE Sepharose Fast Flow M NaOH (ml) SP Sepharose Fast Flow M NaOH (ml) CM Sepharose Fast Flow M NaOH (ml) ll media are chemically very robust and withstand rigorous cleaning-in-place (CIP) and sanitization without problem (see Chemical stability below). Their functional ion exchange groups are attached to the matrices via chemically stable ether linkages. The main media characteristics are listed in Table. Figure shows the working ranges of the ion exchangers as titration curves, that is, the ranges over which the functional groups are charged. Packing in laboratory columns Sepharose Fast Flow media are supplied in laboratory packs for users who prefer the flexibility of packing columns of their choice. Straightforward and well-proven recommendations for packing, operation, and maintenance are included in the instructions supplied with each pack. Columns and accessories from the XK range are recommended. Prepacked Sepharose Fast Flow ion exchange columns y providing extra speed, convenience, and reproducibility, prepacked columns extend the usefulness of Sepharose Fast Flow ion exchangers. In addition, ÄKTdesign TM chromatography systems include preset method templates for these prepacked columns, which further simplifies operation, especially reproducibility, and saves time. ll five media are supplied in two types of column: HiPrep / ( ml) and HiTrap, the latter available in -ml and 5-ml sizes. The five HiTrap -ml columns are also included in HiTrap IEX Selection Kit together with SP Sepharose XL and Q Sepharose XL. HiPrep / columns HiPrep / SP FF, HiPrep / CM FF, HiPrep / Q FF, HiPrep / DEE FF, and HiPrep / NX FF (high sub) are prepacked, ready to use IEX columns. They are simple to operate and compatible with single pump configurations as well as ÄKTdesign systems. Rapid enrichment during the initial capture of proteins from the start material is an ideal application for any of the columns named above. HiPrep / columns are made of polypropylene, which is biocompatible with biomolecules. Its bed height of cm gives a volume of ml. The supplied set of connectors makes it easy to connect the columns to different equipment. See Table for the main characteristics of the HiPrep / column. Note that HiPrep columns cannot be opened or repacked. Fig. Titration curves for CM, SP, DEE, and Q Sepharose Fast Flow and NX Sepharose Fast Flow (high sub) showing the ranges over which the functional groups are charged. Data file -77-3

4 Table. Characteristics of HiPrep / column Column volume Column dimensions Recommended flow rate Maximum flow rate Maximum pressure over the packed bed during operation Column hardware pressure limit ml. cm ml/min ( 3 cm/h) ml/min (3 cm/h).5 bar ( psi,.5 MPa) 5 bar (73 psi,.5 MPa) Water at room temperature. Flow rate is determined by v and = viscosity. < ml/min where v = flow rate HiTrap -ml and 5-ml columns HiTrap SP FF, HiTrap CM FF, HiTrap Q FF, HiTrap DEE FF, and HiTrap NX FF (high sub) are small, affordable, and easy-touse prepacked -ml or 5-ml IEX columns. The -ml column is often used for method screening to quickly establish optimal binding and elution conditions. Its fast and simple operation is ideally suited to this role, as well as to small-scale purifications. The larger 5-ml column is an excellent choice for group separations and sample concentration, and when the purification method has been established and larger amounts of protein need to be purified. For quick scale-up of purification, two or three HiTrap columns can easily be connected in series. Further scale-up can be done on HiPrep / columns, see Figure. HiTrap columns are made of polypropylene, which is biocompatible with biomolecules. The column is delivered with a stopper on the inlet and a snap-off end on the outlet. ll necessary connectors are included for connection to different systems, as well as to a laboratory pump and a simple syringe. Note that HiTrap columns cannot be opened or repacked. Table 3. Characteristics of HiTrap -ml and 5-ml columns Column volumes Column dimensions Recommended flow rate Maximum flow rate Maximum back-pressure Room temperature, aqueous buffers. Operating HiTrap columns ml and 5 ml.7.5 cm ( ml)..5 cm (5 ml) ml/min ( ml) 5 ml/min (5 ml) ml/min ( ml) ml/min (5 ml) 3 bar (3 psi,.3 MPa) Using a HiTrap column packed with a Sepharose Fast Flow ion exchanger is simple. Complete, easy-to-follow instructions are included for fast start-up and method optimization. Operation is straightforward whether you use a syringe and the provided Luer adapter (Fig 3), a peristaltic pump, or a chromatography system such as ÄKTdesign. C Fig 3. Using a HiTrap -ml column with a syringe. () Prepare buffers and sample. Remove the column s top cap and snap off the end. Wash and equilibrate. () Load the sample and begin collecting fractions. (C) Wash, elute, and continue collecting fractions. HiTrap IEX Selection Kit The -ml version of all five HiTrap columns named above is also included in HiTrap IEX Selection Kit, which also contains SP Sepharose XL and Q Sepharose XL. In offering seven well-known media in an easy-to-compare format, this kit offers a fast, simple, and convenient way to decide which ion exchange medium and ligand is best for a given application. In other words, it is a useful tool in speeding the development of an optimized ion exchange separation. For more information about the kit, request Data file HiTrap IEX Selection Kit, code no. --. Choice of buffer s noted earlier, ion exchange chromatography is based on reversible interactions between charged molecules and immobilized ion exchange groups of opposite charge. Charged molecules bind to the separation medium at low ionic strength and are then eluted with a salt or gradient. To avoid local disturbances in caused by buffering ions participating in the ion exchange process, select an eluent with buffering ions of the same charge as the substituent groups on the ion exchanger. See Figures and 5 for a selection of standard aqueous buffers and Table for a selection of volatile buffers. Chemical stability Good chemical stability allows the use of effective cleaningin-place (CIP) schemes that result in high recoveries over many purification cycles. Likewise, it allows regular sanitization to hinder microbial growth and maintain a high level of hygiene. oth CIP and sanitization thus promote good economy and are therefore key factors to consider when selecting ion exchange media and prepacked columns for preparative applications. Data file -77-

5 For CIP, regular washing with.5 to. M sodium hydroxide should be sufficient to remove most contaminating material, although very hydrophobic molecules might bind so tightly that they must be eluted with organic solvents like 7% ethanol or 3% isopropanol, or with strong detergents. General CIP and sanitization protocols for SP Sepharose Fast Flow, CM Sepharose Fast Flow, Q Sepharose Fast Flow, DEE Sepharose Fast Flow, and NX Sepharose Fast Flow (high sub) are supplied with the media. Note that specific protocols should be developed according to the nature and condition of the start material N-methyl piperazine Piperazine bis-tris bis-tris propane Triethanolamine Tris pka (5 C) N-methyldiethanolamine.5.3-diaminopropane. Ethanolamine 9.5 Piperazine diaminopropane.7 Piperidine Fig. Recommended buffers for anion exchange chromatography.. The excellent availability of suitable equipment also contributes to their effective use; especially as systems in the ÄKTdesign platform have method templates for both HiPrep and HiTrap columns. Ease of scale-up is a key practical benefit of working with any Sepharose Fast Flow ion exchanger. Figure demonstrates this. laboratory protein separation was scaled up first five-fold and then twenty-fold on prepacked HiTrap Q FF and HiPrep / Q FF columns respectively with excellent reproducibility. Scaling up five-fold and twenty-fold using different prepacked Q Sepharose Fast Flow columns Sample: Sample volume:. Conalbumin, mg/ml. α-lactalbumin, mg/ml 3. Soy trypsin inhibitor, mg/ml Start buffer: 5 mm Tris-HCl, 7.3 CV (column volume) () ml, () 5 ml, (C) ml Elution buffer: 5 mm Tris-HCl,.5 M NaCl, 7.3 Gradient: % to % elution buffer in CV () ml, () ml, (C) ml Flow rate: () ml/min (5 cm/h), () and (C) 5 ml/min (5 cm/h) System: ÄKTexplorer Citric acid Lactic acid utanedioic acid cetic acid Malonic acid MES Phosphate HEPES ICINE pka (5 C) nm HiTrap Q FF, ml ml Fig 5. Recommended buffers for cation exchange chromatography. Table. Recommended volatile buffers for ion exchange chromatography Substances Pyridine/formic acid Trimethylamine/formic acid.. Trimethylamine/acetic acid.. Trimethylamine/HCl 7..5 mmonia/formic acid.5. mmonia/acetic acid 7.. Trimethylamine/CO. 9.5 mmonium carbonate/ammonia.5.5 Ethanolamine/HCl pplications Sepharose Fast Flow ion exchangers have many applications. Their speed, reliability, and documented success make them a natural choice for preparative protein separations in general. Separating protein mixtures early in purification schemes puts their high flow rates and capacities to very effective use. C nm nm HiTrap Q FF, 5 ml ml HiPrep / Q FF, ml 3 5 ml Fig. Five-fold and twenty-fold scale-up on prepacked Q Sepharose Fast Flow columns. () HiTrap Q FF, ml (.7.5 cm), () HiTrap Q FF, 5 ml (..5 cm), and (C) HiPrep / Q FF, ml (. cm). 3 3 Data file -77-5

6 Figure 7 shows a protein separation scaled up from laboratory scale (5-ml sample) to industrial production level (.5-l sample) on SP Sepharose Fast Flow, again with first-class reproducibility. nalysis of the separated proteins showed no significant differences in the pattern or purity of the individual peaks at either scale. Note that the HETP (Height Equivalent to the Theoretical Plate) values are essentially the same for both packed columns, despite their widely differing sizes. Separation of bovine plasma components on SP Sepharose Fast Flow at laboratory and industrial scales. nm Column: XK /, 5 cm bed height, ml bed volume Sample: Filtered bovine plasma in. M sodium acetate, 5. Sample volume: 5 ml sample (5 g/l) Start buffer:. M sodium acetate, 5. Elution buffer:. M sodium acetate,. Flow rate: 53 ml/h ( cm/h) System: iopilot Figure also compares separations, this time the effect of on the separation of standard proteins on a prepacked HiPrep / CM FF column. s can be seen, 7.5 gives the most distinct separation. Effect of on the separation of standard proteins on a HiPrep / CM FF column Column: Sample: uffer: Gradient: Flow rate: System: nm HiPrep / CM FF, ml mg apotransferrin, ribonuclease, and cytochrome C in ml CIEX ufferprep recipe in ÄKTexplorer % to 5% in 3 ml (5 CV) where 5% =.5 M NaCl ml/min (3 cm/h) ÄKTexplorer 5. % 5 5. Start buffer Elution buffer ml nm.5 %. 3 Time (min) 5. nm Column: PG 3/5, 5 cm bed height,.5 l bed volume Sample: Filtered bovine plasma in. M sodium acetate, 5. Sample volume:.5 l sample (5 g/l) Start buffer:. M sodium acetate, 5. Elution buffer:. M sodium acetate,. Flow rate: 7 l/h ( cm/h) System: ioprocess System C ml 7.5 nm % 5 5. Start buffer Elution buffer ml Fig. Separations of standard proteins on HiPrep / CM FF at () 5., ().5, and (C) Time (min) Fig 7. Scale-up from () a laboratory column (bed volume ml) packed with SP Sepharose Fast Flow to () a column suitable for industrial production (bed volume.5 l) with retained packing and separation characteristics. Not only the elution affects the separation profile. s Figure 9 illustrates, different ligands, in this case anion groups, can give small but significant differences in retention times. ll purifications were done on ÄKTexplorer. fter sample application, the columns were washed and eluted with the same linear gradient. Data file -77-

7 Influence of different anion functional groups on retention times for alkaline phosphatase Column: Sample: Sample application: nm ms/cm nm ms/cm ml ml nm ms/cm ml () HiTrap DEE FF, ml () HiTrap Q FF, ml (C) HiTrap NX FF (high sub), ml ml E. coli lysate clarified by centrifugation ml Start buffer: mm Tris-HCl, 7. Elution buffer: mm Tris-HCl,.5 M NaCl, 7. Equilibration: Wash: Elution: Flow rate: C ml start buffer ml start buffer ml, linear gradient, % to % elution buffer ml/min (5 cm/h) Fig 9. Clarified E. coli lysate separated on () HiTrap DEE FF, ml, () HiTrap Q FF, ml, and (C) HiTrap NX FF (high sub), ml. lkaline phosphatase activity measured at 5. Process development and scale-up to production The excellent performance of Sepharose Fast Flow ion exchangers for laboratory scale preparative applications naturally lends itself to process development and scale-up. The media are well supported for this task. s members of the ioprocess family, all ion exchangers are supported with special services and documentation to facilitate the development, scale-up, and routine operation of production applications. Validated manufacture, secure supply, and regulatory support comprise just part of this package. For more information, please contact GE Healthcare or visit Ordering information Products Quantity Code no. Q Sepharose Fast Flow 5 ml ml l 7-5- l l 7-5- SP Sepharose Fast Flow 5 ml ml l l l DEE Sepharose Fast Flow 5 ml ml l l CM Sepharose Fast Flow 5 ml ml l l NX Sepharose Fast Flow (high sub) 5 ml ml l 7-7- l l 7-7- Prepacked laboratory columns HiPrep / Q FF ml HiPrep / SP FF ml HiPrep / DEE FF ml HiPrep / CM FF ml HiPrep / NX FF (high sub) ml HiTrap Q FF 5 ml ml HiTrap SP FF 5 ml ml HiTrap DEE FF 5 ml ml HiTrap CM FF 5 ml ml HiTrap NX FF (high sub) 5 ml ml HiTrap IEX Selection Kit * 7 ml * May require a license; see legal information on the next page Literature Ion Exchange Chromatography & Chromatofocusing: Principles and Methods -- Ion Exchange Columns and Media, Selection Guide -7-3 HiTrap Column Guide -9- Column Packing CD, The Movie Data file -77-7

8 ccessories, HiPrep Quantity Code no. HiTrap/HiPrep, / male connector for ÄKTdesign -- To connect columns with / connections to FPLC System: Union M female// male * * Two units (in red polypropylene) are included in each HiPrep package ccessories, HiTrap / male/luer female * --5 Tubing connector flangeless/m female * -3- Tubing connector flangeless/m male * -7-9 Union / female/m male * --57 Union M female// male * Union Luerlock female/m female -7- HiTrap/HiPrep, / male connector for ÄKTdesign -- Stop plug female, / 5 -- Fingertight stop plug, / * One connector 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 on delivery GE Healthcare io-sciences jörkgatan 3 75 Uppsala Sweden GE, imagination at work, and GE monogram are trademarks of General Electric Company. ÄKTdesign, ÄKTexplorer, iopilot, ioprocess, PG, Drop design, FPLC, HiPrep, HiTrap, and Sepharose are trademarks of GE Healthcare companies. Separating viral particles with Q Sepharose XL products may require a license under US patent,537,793 and equivalent patents and patent applications in other countries owned by Centelion SS. Such a license is not included with the purchase of Q Sepharose XL but is included with the purchase of Q Sepharose XL virus licensed products. With the purchase of Q Sepharose XL virus licensed the customer is granted a free limited license under US patent,537,793 and equivalent patents and patent applications in other countries owned by Centelion SS to separate viral particles solely through use of the product purchased. ll third party trademarks are the property of their respective owners. 3-7 General Electric Company ll rights reserved. First published Oct. 3. ll goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare Europe GmbH Munzinger Strasse 5, D-79 Freiburg, Germany GE Healthcare UK Limited mersham Place, Little Chalfont, uckinghamshire, HP7 9N, UK GE Healthcare io-sciences Corp. Centennial venue, P.O. ox 37, Piscataway, NJ 55-37, US GE Healthcare io-sciences KK Sanken ldg. 3-5-, Hyakunincho Shinjuku-ku Tokyo 9-73, Japan sia Pacific Tel: Fax: ustralasia Tel: + 99 Fax: + ustria Tel: /57 3 Fax: /57 elgium Tel: 73 9 Fax: Canada Tel: 3 5 Fax: 57 Central, East, & South East Europe Tel: Fax: Denmark Tel: Fax: +5 5 Eire Tel: 7999 Fax Finland & altics Tel: Fax: France Tel: Fax: Germany Tel: 9 7 Fax: 9 7 Greater China Tel: +5 3 Fax: Italy Tel: 3 Fax: 399 Japan Tel: Fax: Korea Tel: 37 Fax: 33 Latin merica Tel: Fax: Middle East & frica Tel: Fax: Netherlands Tel: - Fax: - Norway Tel: Fax: Portugal Tel: Fax: 7 3 Russia & other C.I.S. & N.I.S Tel: Fax: Spain Tel: Fax: Sweden Tel: 9 Fax: 9 Switzerland Tel: Fax: UK Tel: Fax: 97 US Tel: Fax: imagination at work -77- /7 Elanders Östervåla 7