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Amersham Biosciences offers you these highly renowned ionexchangers, based on cross-linked agarose, for preparativeprotein separations in both research and industrial applications.As part of the BioProcess™ Media range, Sepharose™ Fast Flowion exchangers carry full technical and regulatory support forproduction scale applications. The first choice in ready-to-useion exchange media for:

· High binding capacity

· High chemical and physical stabilities

· Fast flow characteristics

· Easy scale up

· Easy sanitization/CIP

Media characteristicsSepharose Fast Flow ion exchangers result from a continuousprogram to develop superior chromatography media with theexacting demands research and industry in mind. They can beused at very high flow rates and display remarkable chemicalstability. Sepharose Fast Flow is available with the weak exchangegroups DEAE and CM, and the strong exchange groups Q andSP attached to a highly cross-linked agarose matrix. The cross-linking has been optimized to give the matrix outstandingphysical and chemical stabilities together with excellent flowproperties. Typical linear flow rates are 300–400 cm/h througha 15 cm bed height at a pressure of 1 bar. The exceptionalflow characteristics make these ion exchangers the first choicefor separation of crude mixtures, for example early inpurification schemes, when fast, initial enrichment is required.The media are easy to handle and tolerate harsh workingconditions and effective cleaning can be carried out in situ.The media’s characteristics are given Table 1.

The functional groups of the four types of Sepharose ionexchangers are shown in Figure 2. Sepharose Fast Flow ionexchangers satisfy process chromatography requirements interms of quality, performance, stability, scalability and supplyin large quantities. Each of the media are supported withRegulatory Support Files containing information that can besubmitted to regulatory authorities.

Fig. 2. Ion exchange groups of Sepharose Fast Flow ion exchangers.

High binding capacityAvailable capacity depends on the nature and size of themolecules being separated. Dynamic binding capacities are inthe range of 50–100 mg/ml medium. Capacity data are givenin Table 1. Titration curves for all the ion exchangers areshown in Figure 3.

BioProcess media

Fig. 1. Sepharose Fast Flow ion exchangers for fast proteinseparations in both research and industrial applications.

Sepharose Fast Flow ion exchangers

High physical and chemical stabilityThe cross-linked structure of the Sepharose Fast Flow ionexchangers renders them chemically stable. Their high rigidityminimizes volume variations during change of pH or ionicstrength. Under acidic conditions, limited hydrolysis of thepolysaccharide chains may occur. The strong cation exchangerSP Sepharose is somewhat susceptible to hydrolysis. Sepharosemedia display a notable resistance to microbial attack due tothe presence of the unusual sugar 3,6- anhydro-L-galactosewithin the structure. Chemical stability data are given in Table 1.

OperationSepharose Fast Flow ion exchangers are supplied pre-swollen.Decant the 20% ethanol solution and replace with startingbuffer before use. After packing, the media should beequilibrated with approximately 5 bed volumes of the startingbuffer. Resolution is controlled by varying the pH, sampleload, flow rate, and the volume and shape of the pH or ionicstrength gradient.

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Fig. 3. Titration curves; Approx. 5 ml media of SP, Q, DEAE and CM Sepharose Fast Flow, in 50 ml 1 M KCl. (Work from AmershamBiosciences, Uppsala, Sweden).

Process hygieneRegenerationRegeneration of Sepharose Fast Flow ion exchangers is easilycarried out, directly in the column, without re-packing. Afterevery run, very tightly bound material is eluted using either highionic (e.g. 1 M NaCl) strength or a change in pH. The mediumis re-equilibrated with starting buffer before each run.

Easy sanitization/Cleaning-in-placeCleaning-in-place, CIP, is the on-line elimination and theprevention of the build up of very tightly bound, precipitatedor denaturated substances that would affect media capacity,flow properties and performance. In some applications,substances such as lipids or denaturated proteins may remainin the column bed and not be eluted by the regenerationprocedures. A specific CIP protocol has to be designed accordingto the type of contaminants known to be present in thefeedstream. The frequency of CIP cycles depends on the natureand the condition of the starting material, but a CIP protocolis typically recommended every 5 cycles. Examples of CIPprotocols are shown in Table 2.

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Table 1. Media characteristics.

Q Sepharose SP Sepharose DEAE Sepharose CM SepharoseFast Flow Fast Flow Fast Flow Fast Flow

Type of ion exchanger strong anion strong cation weak anion weak cation

Total capacity (mmol/ml) 0.18–0.25 0.18–0.25 0.11–0.16 0.09–0.13

Exclusion limit 4 x 106 4 x 106 4 x 106 4 x 106

(globular proteins)

Bead form Spherical, diameter 45–165 µm.

Bead structure Cross-linked agarose, 6%

Chemical stability Stable to all commonly used aqueous buffers:1 M NaOH, 8 M urea, 8 M guanidine hydrochloride, 70% ethanol.

Working pH 2–12 4–13 2–9 6–10

Operational pH stability 2–12 4–13 2–9 4–13

Cleaning pH stability 1–14 3–14 1–14 2–14

Physical stability Negligible volume variation due to changes in pH or ionic strength.

Linear flow rate at 25°C. 400–700 cm/h 400–700 cm/h 300–600 cm/h 300–600 cm/h1 bar 15 cm bed heightXK 50/30 column

Autoclavable With Cl- and Na+ respectively as counter Ions, at 121 °C, pH 7, for 30 min.For SP Sepharose Fast Flow we recommend 0.2 M sodium acetate for autoclaving.

Stored in 20% ethanol 20% ethanol containing 20% ethanol 20% ethanol0.2M sodium acetate

Dynamic binding capacity**

Thyroglobulin 3 mg/ml medium N.D.* 3.1 mg/ml medium N.D.*

HSA 120 mg/ml medium N.D.* 110 mg/ml medium N.D.*

IgG – 50 mg/ml medium – 15 mg/ml medium

α-lactalbumin 110 mg/ml medium – 100 mg/ml medium –

Bovine COHb – – 50 mg/ml medium - 30 mg/ml medium

Ribonuclease – 70 mg/ml medium 50 mg/ml medium

BSA – 120 mg/ml medium – –ß- lactoglobulin – 95 mg/ml medium – –

* Not determined

** Capacity determined at a flow velocity of 75 cm/h. For anion exchangers (DEAE and Q) the starting buffer was 0.05 M Tris, pH 8.3, and for cation exchangers

(CM and SP) 0.1 M acetate buffer, pH 5.0. Limit buffers were the respective start buffers containing 2.0 M NaCl.

Working pH: pH interval in which the medium binds protein as intended. This may differ from operational pH due to uncharged weak ion exchanger.

Operational pH stability: pH interval in which the medium can be operated without significant change in function.

Cleaning pH stability: pH interval to which the medium can be subjected for cleaning- or sanitization-in-place, without significant change in function.

Sanitization and sterilizationSanitization is the use of chemical agents to inactivate microbialcontaminants in the form of vegetative cells; it also helps tomaintain a high level of both process hygiene and processeconomy. Like CIP, sanitization protocols are applied tochromatography systems and packed columns. Recommendedsanitization and sterilization protocols for columns packedwith Sepharose ion exchange media are given in Table 2.

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Fig. 4. Pressure/flow rate curves for Q Sepharose Fast Flow. BP 113column, cross section 100 cm2. Bed height 15 cm. BP 252 column,cross section 500 cm2. Bed height 15 cm. Pressure/flow rate curvesfor SP Sepharose Fast Flow. BPG™ 300 column, cross section 706 cm2.Bed height 15 cm. XK 50/30 column, cross section 19.6 cm2. Bedheight 15 cm. (Work from Amersham Biosciences, Uppsala, Sweden.)

0

0 0.4 0.8 1.61.2 2.0 3.23.02.4

100

200

300

400

500

700

600

Flow ratecm/h

Operating pressure (bar)

XK 50/30

BP 133

BPG 300

BP 252

Q Sepharose Fast Flow

SP Sepharose Fast Flow

Fig. 5. This illutrates the excellent scale up reproducibility withSepharose Fast Flow ion exchangers. The analysis of the resultsshowed no significant difference in pattern or in purity of the individualpeaks. (Work from Amersham Biosciences, Uppsala, Sweden.)

0

0 10 20 30

300

500

A 280 nm

StartingBuffer

ElutionBuffer

Time (min)

Table 2. CIP protocols, sanitization and sterilization procedures.

CIP protocolIonically bound Wash with 0.5 column volumes of filteredproteins 2 M NaCI. Contact time 10–15 min

reversed flow direction

Precipitated proteins Wash with 1 M NaOH at 40 cm/h.and hydrophobically Contact time 1–2 h.bound proteins orlipoproteins

Lipids and very Wash with 70% ethanol, reversed flow athydrophobic proteins 40 cm/h for 1-2 hours. Alternatively wash

with saw-tooth gradients of 0–30%–0isopropanol, contact time 1–2 hours.

Sanitization Wash with 0.5–1 M NaOH for 30–60 min.

Sterilization Equilibrate the ion exchanger with Cl¯ forthe anion exchangers (DEAE and Q) or Na+

for the cation exchangers (CM and SP) atpH 7. Autoclave the media at 121 °C for30 min. For SP Sepharose Fast Flow werecommend 0.1 M sodium acetate.

0

0 10 20 30

5.0

10.0

A 280 nm

StartingBuffer

ElutionBuffer

Time (min)

Medium: SP Sepharose Fast FlowColumn: BPG 300/500 15 cm bed height, 10.5 I HETP 0.013 cmSample: Diafiltered bovine plasma in 0.1 sodium acetate pH 5.2 Loading: 6.5 I sample (approx. 25 g/l)Starting buffer: 0.1 M sodium acetate, pH 5.2Elution buffer: 0.4 M sodium acetate, pH 8.0Flow velocity: 70 I/h, 100 cm/hSystem: BioProcess System I

Medium: SP Sepharose Fast FlowColumn: XK 26/30 15 cm bed height, 80 ml HETP 0.012 cmSample: Diafiltered bovine plasma in 0.1 M sodium acetate pH 5.2Loading: 50 ml sample (approx. 25 g/l)Starting buffer: 0.1 M sodium acetate, pH 5.2Elution buffer: 0.4 M sodium acetate, pH 8.0Flow velocity: 530 ml/h, 100 cm/hSystem: BioPilot™

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StorageStore the medium in the salt form in a buffer containing asuitable anti-microbial agent e.g. 20% ethanol. Recommendedstorage at +4 to +30 °C. Packed columns should be equilibratedin starting buffer with the addition of 20% ethanol. SP SepharoseFast Flow should be stored in 0.2 M sodium acetate buffercontaining 20% ethanol.

Further informationThe strong ion exchangers are also available as prepackedcolumns with convenient and reproducible performance. Forfurther information about Sepharose Fast Flow ionexchangers and ion exchange chromatography, please contactyour local Amersham Biosciences office.

Ordering information

Product Pack size Code No.

DEAE Sepharose Fast Flow 500 ml 17-0709-0110 l 17-0709-05

Q Sepharose Fast Flow 300 ml 17-0510-015 l 17-0510-04

CM Sepharose Fast Flow 500 ml 17-0719-0110 l 17-0719-05

SP Sepharose Fast Flow 300 ml 17-0729-015 l 17-0729-04

ReferencesHandbook of Process Chromatography: A Guide to optimization, Scale up and validation.Sofer, G. K., Nyström, L-E. Academic Press Limited, London, U.K. 1997ISBN 0-12-654266-X. Amersham Biosciences code number 18-1121-56.

Ion Exchange Chromatography. Principles and Methods.Amersham Biosciences, code number 18-1114-21.

ApplicationsSepharose Fast Flow for production andinitial purificationSepharose Fast Flow ion exchangers are the natural choice forthe initial purification of protein extracts, culture supernatantsand other samples. The usual way of working with SepharoseFast Flow ion exchangers is to choose conditions so that thecomponents of interest bind to the ion exchanger while mostof the contaminants pass through. The components of interestcan then be eluted in a small volume for further purification.Excellent flow properties and high capacities make SepharoseFast Flow media especially suited to industrial use, wherelarge volumes of raw material must be processed withmaximum overall economy in mind. Processing time and costcan be reduced without compromise in end-product quality.

Fast flow characteristicsThe cross-linking of the matrix has been optimized to giveprocess flow characteristics, with typical linear flow rates of300-400 cm/h through 15 cm bed height at a pressure of1 bar. Figure 4 shows the pressure/flow rate relationship.

Easy scale upThe exceptional performance of the media from research -scale through pilot-scale and into production, makesSepharose Fast Flow ion exchangers the media of choice forthe biotechnology industry. The purification of bovine plasmaillustrates the performance of SP Sepharose Fast Flow with ascale up factor of 130. (See Fig. 5.)

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BioPilot, BioProcess, BPG and Sepharose are trademarks of Amersham Biosciences Limited. Amersham and Amersham Biosciences are trademarks of Amersham plc.

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