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BioProcess media

Sepharose Fast Flow ion exchangers

Amersham Biosciences offers you these highly renowned ion exchangers, based on cross-linked agarose, for preparative protein separations in both research and industrial applications. As part of the BioProcess Media range, Sepharose Fast Flow ion exchangers carry full technical and regulatory support for production scale applications. The first choice in ready-to-use ion exchange media for:
High binding capacity High chemical and physical stabilities Fast flow characteristics Easy scale up Easy sanitization/CIP

Media characteristics
Sepharose Fast Flow ion exchangers result from a continuous program to develop superior chromatography media with the exacting demands research and industry in mind. They can be used at very high flow rates and display remarkable chemical stability. Sepharose Fast Flow is available with the weak exchange groups DEAE and CM, and the strong exchange groups Q and SP attached to a highly cross-linked agarose matrix. The crosslinking has been optimized to give the matrix outstanding physical and chemical stabilities together with excellent flow properties. Typical linear flow rates are 300400 cm/h through a 15 cm bed height at a pressure of 1 bar. The exceptional flow characteristics make these ion exchangers the first choice for separation of crude mixtures, for example early in purification schemes, when fast, initial enrichment is required. The media are easy to handle and tolerate harsh working conditions and effective cleaning can be carried out in situ. The medias characteristics are given Table 1. The functional groups of the four types of Sepharose ion exchangers are shown in Figure 2. Sepharose Fast Flow ion exchangers satisfy process chromatography requirements in terms of quality, performance, stability, scalability and supply in large quantities. Each of the media are supported with Regulatory Support Files containing information that can be submitted to regulatory authorities.

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

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

High binding capacity

Available capacity depends on the nature and size of the molecules being separated. Dynamic binding capacities are in the range of 50100 mg/ml medium. Capacity data are given in Table 1. Titration curves for all the ion exchangers are shown in Figure 3.

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High physical and chemical stability

The cross-linked structure of the Sepharose Fast Flow ion exchangers renders them chemically stable. Their high rigidity minimizes volume variations during change of pH or ionic strength. Under acidic conditions, limited hydrolysis of the polysaccharide chains may occur. The strong cation exchanger SP Sepharose is somewhat susceptible to hydrolysis. Sepharose media display a notable resistance to microbial attack due to the presence of the unusual sugar 3,6- anhydro-L-galactose within the structure. Chemical stability data are given in Table 1.

Process hygiene
Regeneration
Regeneration of Sepharose Fast Flow ion exchangers is easily carried out, directly in the column, without re-packing. After every run, very tightly bound material is eluted using either high ionic (e.g. 1 M NaCl) strength or a change in pH. The medium is re-equilibrated with starting buffer before each run.

Easy sanitization/Cleaning-in-place
Cleaning-in-place, CIP, is the on-line elimination and the prevention of the build up of very tightly bound, precipitated or denaturated substances that would affect media capacity, flow properties and performance. In some applications, substances such as lipids or denaturated proteins may remain in the column bed and not be eluted by the regeneration procedures. A specific CIP protocol has to be designed according to the type of contaminants known to be present in the feedstream. The frequency of CIP cycles depends on the nature and the condition of the starting material, but a CIP protocol is typically recommended every 5 cycles. Examples of CIP protocols are shown in Table 2.

Operation
Sepharose Fast Flow ion exchangers are supplied pre-swollen. Decant the 20% ethanol solution and replace with starting buffer before use. After packing, the media should be equilibrated with approximately 5 bed volumes of the starting buffer. Resolution is controlled by varying the pH, sample load, flow rate, and the volume and shape of the pH or ionic strength gradient.

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 Amersham Biosciences, Uppsala, Sweden).

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Table 1. Media characteristics.

Q Sepharose Fast Flow Type of ion exchanger Total capacity (mmol/ml) Exclusion limit (globular proteins) Bead form Bead structure Chemical stability Working pH Operational pH stability Cleaning pH stability Physical stability Linear flow rate at 25C. 1 bar 15 cm bed height XK 50/30 column Autoclavable Stored in Dynamic binding capacity** Thyroglobulin HSA IgG -lactalbumin Bovine COHb Ribonuclease BSA 3 mg/ml medium 120 mg/ml medium 110 mg/ml medium N.D.* N.D.* 50 mg/ml medium 50 mg/ml medium 70 mg/ml medium 120 mg/ml medium 3.1 mg/ml medium 110 mg/ml medium 100 mg/ml medium N.D.* N.D.* 15 mg/ml medium 30 mg/ml medium 50 mg/ml medium strong anion 0.180.25 4 x 106 SP Sepharose Fast Flow strong cation 0.180.25 4 x 106 DEAE Sepharose Fast Flow weak anion 0.110.16 4 x 106 CM Sepharose Fast Flow weak cation 0.090.13 4 x 106

Spherical, diameter 45165 m. Cross-linked agarose, 6% Stable to all commonly used aqueous buffers: 1 M NaOH, 8 M urea, 8 M guanidine hydrochloride, 70% ethanol. 212 212 114 400700 cm/h 413 413 314 400700 cm/h 29 29 114 300600 cm/h 610 413 214 300600 cm/h

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

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. 20% ethanol 20% ethanol containing 0.2M sodium acetate 20% ethanol 20% ethanol

- lactoglobulin
* Not determined

95 mg/ml medium

** 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 sterilization

Sanitization is the use of chemical agents to inactivate microbial contaminants in the form of vegetative cells; it also helps to maintain a high level of both process hygiene and process economy. Like CIP, sanitization protocols are applied to chromatography systems and packed columns. Recommended sanitization and sterilization protocols for columns packed with Sepharose ion exchange media are given in Table 2.

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Table 2. CIP protocols, sanitization and sterilization procedures.

CIP protocol
Ionically bound proteins Precipitated proteins and hydrophobically bound proteins or lipoproteins Lipids and very hydrophobic proteins Wash with 0.5 column volumes of filtered 2 M NaCI. Contact time 1015 min reversed flow direction Wash with 1 M NaOH at 40 cm/h. Contact time 12 h.

Medium: Column: Sample: Loading: Starting buffer: Elution buffer: Flow velocity: System:

SP Sepharose Fast Flow XK 26/30 15 cm bed height, 80 ml HETP 0.012 cm Diafiltered bovine plasma in 0.1 M sodium acetate pH 5.2 50 ml sample (approx. 25 g/l) 0.1 M sodium acetate, pH 5.2 0.4 M sodium acetate, pH 8.0 530 ml/h, 100 cm/h BioPilot

280 nm

Wash with 70% ethanol, reversed flow at 40 cm/h for 1-2 hours. Alternatively wash with saw-tooth gradients of 030%0 isopropanol, contact time 12 hours. Wash with 0.51 M NaOH for 3060 min. Equilibrate the ion exchanger with Cl for the anion exchangers (DEAE and Q) or Na+ for the cation exchangers (CM and SP) at pH 7. Autoclave the media at 121 C for 30 min. For SP Sepharose Fast Flow we recommend 0.1 M sodium acetate.

500

Elution Buffer

Sanitization Sterilization

300
Starting Buffer

0 0 10 20 30
Time (min)

Flow rate cm/h 700 600 500 400 300 XK 50/30

BP 133

BPG 300 BP 252

Medium: Column: Sample: Loading: Starting buffer: Elution buffer: Flow velocity: System:

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

280 nm

10.0

200 100 0 0 0.4 0.8 1.2

Q Sepharose Fast Flow SP Sepharose Fast Flow

Elution Buffer

5.0

1.6

2.0

2.4

3.0

3.2

Operating pressure (bar)

Starting Buffer

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

0 0 10 20 30
Time (min)

Fig. 5. This illutrates the excellent scale up reproducibility with Sepharose Fast Flow ion exchangers. The analysis of the results showed no significant difference in pattern or in purity of the individual peaks. (Work from Amersham Biosciences, Uppsala, Sweden.)

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Applications
Sepharose Fast Flow for production and initial purification
Sepharose Fast Flow ion exchangers are the natural choice for the initial purification of protein extracts, culture supernatants and other samples. The usual way of working with Sepharose Fast Flow ion exchangers is to choose conditions so that the components of interest bind to the ion exchanger while most of the contaminants pass through. The components of interest can then be eluted in a small volume for further purification. Excellent flow properties and high capacities make Sepharose Fast Flow media especially suited to industrial use, where large volumes of raw material must be processed with maximum overall economy in mind. Processing time and cost can be reduced without compromise in end-product quality.

Storage
Store the medium in the salt form in a buffer containing a suitable anti-microbial agent e.g. 20% ethanol. Recommended storage at +4 to +30 C. Packed columns should be equilibrated in starting buffer with the addition of 20% ethanol. SP Sepharose Fast Flow should be stored in 0.2 M sodium acetate buffer containing 20% ethanol.

Further information
The strong ion exchangers are also available as prepacked columns with convenient and reproducible performance. For further information about Sepharose Fast Flow ion exchangers and ion exchange chromatography, please contact your local Amersham Biosciences office.

Fast flow characteristics

The cross-linking of the matrix has been optimized to give process flow characteristics, with typical linear flow rates of 300-400 cm/h through 15 cm bed height at a pressure of 1 bar. Figure 4 shows the pressure/flow rate relationship.

Ordering information
Product
DEAE Sepharose Fast Flow Q Sepharose Fast Flow

Pack size
500 ml 10 l 300 ml 5l 500 ml 10 l 300 ml

Code No.
17-0709-01 17-0709-05 17-0510-01 17-0510-04 17-0719-01 17-0719-05 17-0729-01

Easy scale up
The exceptional performance of the media from research scale through pilot-scale and into production, makes Sepharose Fast Flow ion exchangers the media of choice for the biotechnology industry. The purification of bovine plasma illustrates the performance of SP Sepharose Fast Flow with a scale up factor of 130. (See Fig. 5.)

CM Sepharose Fast Flow SP Sepharose Fast Flow

5l

17-0729-04

References
Handbook of Process Chromatography: A Guide to optimization, Scale up and validation. Sofer, G. K., Nystrm, L-E. Academic Press Limited, London, U.K. 1997 ISBN 0-12-654266-X. Amersham Biosciences code number 18-1121-56. Ion Exchange Chromatography. Principles and Methods. Amersham Biosciences, code number 18-1114-21.

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