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ANX Sepharose 4 Fast Flow (high sub)

Fig. 1. ANX Sepharose 4 Fast Flow (high sub).

Introduction

ANX Sepharose ? 4 Fast Flow (high sub) is a weak anion exchanger, designed to support the increasing need for ion exchange media with different selectivity and higher chemical stability at high pH. In addition, the pore size distribution of ANX Sepharose 4 Fast Flow (high sub) has been optimized for the separation of large proteins. ANX Sepharose 4 Fast Flow is a BioProcess ? Medium and is supported with a Regulatory Support File.

?Different selectivity compared with established weak anion exchangers

?Applicable for separation of high molecular mass proteins ?

Developed in co-operation with leading large-scale pharmaceutical manufacturers

Characteristics

ANX Sepharose 4 Fast Flow (high sub) is a weak anion exchanger medium with tertiary amine groups attached to the base matrix Sepharose 4 Fast Flow via ether linkage and a hydrophilic spacer arm. The diethylaminopropylgroup is coupled in a mode that precludes formation of quaternary groups, which are typically seen on traditional DEAE media,resulting in a truly weak anion exchanger. The base matrix,Sepharose 4 Fast Flow, is a highly cross-linked, 4% agarose derivative with high chemical and physical stability and broad separation range.

Fig. 2. The ion exchance group of ANX Sepharose 4 Fast Flow (high sub).

–CH 2CHOHCHH 2NH +

(CH 2CH 3)2

Type of ion exchanger Weak anion

-Matrix structure Cross-linked 4% agarose Exclusion limit 3 ′ 107 (globular proteins)Particle form

Spherical, 45-165 μm Mean particle size 90 μm

Chemical stability 1

Stable in all commonly used aqueous buffers – 1.0 M NaOH

– 20 and 70% ethanol – 8.0 M guanidine HCl

– 8.0 M urea

– 1.0 M acetic acid

Physical stability

Negligible volume variation due to changes in pH or ionic strength Recommended pH working range 2

3–10 Cleaning-in-place 2–14

Recommended working flow velocity

300-500 cm/h Temperature stability +4 – +40 °C Storage

20% ethanol

1No significant change in ionic binding capacity after one year storage at room temperature 2

The group is charged over this pH range

Table 1. Characteristics of ANX Sepharose 4 Fast Flow (high sub).

BioProcess Media

Different selectivity

ANX Sepharose 4 Fast Flow (high sub) displays different selectivity in comparison with the already established Fast Flow ion-exchangers and therefore offers an additional choice when selecting anion-exchange media (see Figure 3).

Separation of high molecular mass proteins

ANX Sepharose 4 Fast Flow (high sub) has larger pores than DEAE Sepharose Fast Flow which improves the dynamic binding capacity when separating larger molecules e.g thyroglobulin (M

r

= 6.5′105). See Table 2. Excellent chemical stability

Leakage of carbon from the base matrix is minimized, even at extreme pH, due to the improved coupling chemistry (see Figure 4). This feature allows the use of harsher cleaning-in-place (CIP) and sanitization protocols and also increases the lifetime of the medium.Developed in co-operation with leading large-scale drug manufacturers

ANX Sepharose 4 Fast Flow (high sub) has been developed together with leading large-scale drug manufacturers. This has resulted in an adsorbent which is suitable for industrial processes, where parameters such as packing, scalability, low leakage, consistent performance and long lifetime are crucial.

Operation

ANX Sepharose 4 Fast Flow (high sub) ion exchanger is supplied as a suspension in 20% ethanol. Decant the 20% ethanol solution and replace with starting buffer before use. After packing, the medium should be equilibrated with approximately 5 bed volumes of starting buffer before use. Varying the pH, sample load, flow rate and the volume and shape of the pH or ionic strength gradient will affect resolution.

Regeneration

Regeneration of ANX Sepharose 4 Fast Flow (high sub) is easily carried out in the column, without the need for re-packing. After every run very tightly bound material is eluted using either high ionic strength (e.g. 1 M NaCl) or change in pH. The medium is re-equilibrated with starting buffer before each run.

Easy sanitization/cleaning-in-place

CIP is the on-column elimination and prevention of the build-up of very tightly bound, precipitated or denaturated substances that affect media capacity, flow properties and performance. In some applications, substances such as lipids

Fig. 4. The influence of pH, temperature, and time on the release of carbon from ANX Sepharose 4 Fast Flow (high sub).

pH

BioProcess Media

Anion exchange Q

b BSA (mg/ml Q

b

Thyroglobulin

medium drained medium)(mg/ml drained

medium)

ANX Sepharose 4 Fast

Flow (high sub)435

DEAE Sepharose Fast

Flow471

1No significant change in ionic binding capacity after one year storage at room temperature Table 2. Breakthrough capacity (Q

b

) at 10% breakthrough of BSA and thyroglobulin for ANX Sepharose 4 Fast Flow (high sub) and DEAE Sepharose Fast Flow at 300 cm/h.

or denaturated proteins may remain in the column bed and not be eluted by the regeneration procedures. Therefore, a specific 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. Examples of CIP protocols are shown in Table 3.

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 columns. Recommended sanitization and sterilization protocols for columns packed with ANX Sepharose 4 Fast Flow (high sub) are given in Table 3.

CIP protocol

Ionically bound proteins Wash with 0.5 column volumes of

filtered 2 M NaCl. Contact time

10–15 min, reversed flow direction. Precipitated proteins and Wash with 1 M NaOH at 40 cm/h hydrophobically bound Contact time 1–2 h.

proteins or lipoproteins

Lipids and very Wash with 70% ethanol, reversed hydrophobic proteins flow at 40 cm/h for 1–2 h. Sanitization Wash with 0.5–1 M NaOH

for 30–60 min.

Sterilization Equilibrate the ion exchanger with

Cl- at pH 7. Autoclave the media at

121 °C for 30 min in buffer at pH 7. 1No significant change in ionic binding capacity after one year storage at room temperature Table 3. CIP protocols, sanitization and sterilization procedures.Easy scale-up

The performance of Sepharose Fast Flow media at all scales is well-documented: During the development of ANX Sepharose 4 Fast Flow (high sub) with large-scale pharmaceutical manufacturers it was shown that going from research-scale through pilot-scale and into production was a straightforward operation with maintained performance.

Applications

ANX Sepharose Fast Flow (high sub) for intermediate purification of large protein molecules

ANX Sepharose 4 Fast Flow (high sub) is the natural choice for the intermediate purification of large protein extracts, culture supernatants and other samples. The usual way of working with ANX Sepharose 4 Fast Flow (high sub) is to choose conditions so that the compounds of interest binds 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.

Absorbance, 280 nm Conductivity

mS/cm mAU

600

10.0

40.0

500

400

300

200

100

30.0

20.0

10.0

20.030.040.050.0

0.0ml

A405

60.0

Column:HiTrap? ANX Sepharose 4 Fast Flow (high sub) 1 ml Sample: 2 ml E. coli lysate clarified by centrifugation

Start buffer:20 mM Tris?-HCl pH 7.4

Elution buffer:20 mM Tris HCl, 0.5 M NaCl pH 7.4

Flow velocity150 cm/h

Running parameters:Sample application: 2 ml

Wash: 10 ml start buffer

Elution: 40 ml, linear gradient, 0-100% elution buffer System:?KTA?explorer 100 controlled by UNICORN? 3.0

Fig. 5. Purification of alkaline phosphatase at 405 nm from E. coli lysate on HiTrap ANX Sepharose 4 Fast Flow (high sub).

BioProcess Media

P r o d u c e d b y W i k s t r ?m s , S w e d e n 1000185, 02. 00P r i n t e d m a t t e r . L i c e n c e 341 051

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?KTA, BioProcess, BPG, CHROMAFLOW, HiTrap, INdEX, Sepharose, UNICORN are trademarks of Amersham Biosciences Limited or its subsidiaries. Amersham and Amersham Biosciences are trademarks of Amersham plc. Tris is a trademark of Union Carbide Chemicals and Plastics Co. Amersham Biosciences AB Bj?rkgatan 30, SE-751 84 Uppsala, Sweden.Amersham Biosciences UK Limited Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England. Amersham Biosciences Inc 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855

USA. Amersham Biosciences

Europe GmbH Munzinger Strasse 9, D-79111 Freiburg, Germany. All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them. A copy of these terms and conditions is available on request. ? Amersham Biosciences AB 2000 – All rights reserved.

Ordering information

Product Pack size Code No.ANX Sepharose 4 Fast Flow (high sub)25 ml 17-1287-10

500 ml 17-1287-015 l 17-1287-0410 l 17-1287-0560 l 17-1287-60

All bulk media products are supplied in suspension in 20% ethanol.

Column

Inner diam.Bed volume Bed height (mm)(cm)Lab Scale:

HR 5/55up to 1 ml max. 5HR 10/1010up to 8 ml max. 10HR 16/2016up to 30 ml max. 15HR 26/2026up to 80 ml max. 15XK 16/2016up to 20 ml max. 10XK 26/20

26up to 53 ml max. 10Production scale:BPG? 100/500100up to 2.4 l max.30BPG 200/500200up to 9.4 l max.30BPG 300/500300up to 21 l max.30BPG 450/500

450up to 43 l max.27INdEX? 100/500100up to 2.4 l max. 30INdEX 200/500200up to 9.4 l max. 30CHROMAFLOW?400

18 l

15

400*

*For large-scale separation, CHROMAFLOW columns with diameters greater than 400 mm are available as Custom Designed Columns. Please contact your local Amersham Bio sciences representative for information.

Table 4. Recommended columns for ANX Sepharose 4 Fast Flow (high sub).

Storage

Store the medium in the salt form in a buffer containing a suitable anti-microbial agent e.g. 20% ethanol.Recommended storage is at +4 – +30 °C.

Equipment

Recommended columns are given the table below.