GE Healthcare

Instruction 18-1060-44 AC

SOURCE 15RPC
Important user information
Please read these instructions carefully before using SOURCE 15RPC. Should you have any comments on this instruction manual, we will be pleased to receive them at: GE Healthcare Bio-Sciences AB Bjrkgatan 30 SE-751 84 Uppsala Sweden

Warranty and liability

GE Healthcare warrants that the Product(s) has been tested according to the established test procedures of GE Healthcare and that it conforms with the specifications published by GE Healthcare. This warranty shall be void if the Product(s) has not been used in accordance with the operating instructions supplied by GE Healthcare, or if the Product(s) has been altered, misused, or damaged.

GE Healthcare disclaims any warranty for merchantability and any responsibility for the suitability of the Product(s) for any particular purpose. Nor shall GE Healthcare be held liable for any incidental or consequential damages, including without limitation, lost profits or loss of use caused by any defect or malfunction of the Product(s). The delivery of the Product(s) may be subject to and conditional to the obtaining of an export license or other requisite governmental authorizations. GE Healthcare shall not be responsible for any delay or non delivery caused by denial, withdrawal, or other obstacles in obtaining such licenses or authorizations.

1. Introduction
SOURCE 15RPC is a polymer-based matrix for high performance reversed phase chromatography (RPC). It meets the demands of today's industrial bioprocessing for reproducibility, scalability, chemical and physical stability, and reliability of supply. These instructions contain information about medium characteris tics, operation (including column packing), method optimization and scale up, maintenance, and equipment. To ensure best performance and trouble-free operation, please read these instructions before using SOURCE 15RPC.

2. Characteristics
SOURCE 15RPC is based on rigid, monodisperse, 15 m diameter polystyrene/ divinyl benzene beads. The matrix is underivatized and has a selectivity unique for RPC. These characteristics, together with the controlled pore size distribution, give reproducible and scalable results and make SOURCE 15RPC even suitable for difficult separations such as those encountered in the final stages of an industrial purification process. Table 1 summarizes the characteristics of SOURCE 15RPC. Figure 1 shows typical pressure flow-rate characteristics.

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Table 1. Characteristics of SOURCE 15RPC. Matrix Bead form Particle size Pore volume Dynamic capacity* Polystyrene/divinyl benzene Rigid, spherical, porous, monodisperse, 15 m 1.9 ml/g 10 mg BSA /ml medium at 300 cm/h 30 mg bacitracin/ml medium at 300 cm/h 50 mg insulin/ml medium at 300 cm/h pH stability working range Max. linear flow rate Typical flow rate range Operating temp. Delivery conditions Autoclavable 112 1800 cm/h 200900 cm/h 440 C 20% ethanol 20 min at 121 C cleaning range (CIP/SIP) 114

* To measure the dynamic capacity, 5 mg bacitracin (MW 1 400), 2 mg BSA (MW 67 000) or 5 mg insulin (MW 5 700) per ml 0.1% TFA were applied at 300 cm/h. RESOURCE RPC 1 ml and FPLC system were used.
Pressure (MPa) 3.0 Isopropanol Ethanol Water Acetonitrile

2.0

1.0

3.0 0 180 360 540 720 900 1080 1260 1440 Linear flow rate (cm/h)

Fig 1. Pressure flow-rate characteristics of SOURCE 15RPC in a 6.4 100 mm pre-packed column (RESOURCE RPC, 3 ml).

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2.1 Stability
SOURCE 15RPC can be used with all aqueous and organic solvents commonly used in reversed phase chromatography. The chemical stability is excellent, with a pH working range of 112 and a pH cleaning range of 114. Swelling/shrinking in water/organic solvents is less than 3%.

3. Packing columns
SOURCE 15RPC is supplied in 20% ethanol. Decant the ethanol solution and replace with packing solvent before use.

3.1 Recommended columns

SOURCE 15RPC can be used with columns and equipment commonly used for preparative RPC.

3.2 Packing recommendations

Columns can be packed in different ways depending on the type of column and equipment used. Always read and follow the relevant column instruction manual carefully. Recommended packing solvent is 100% methanol. The best slurry concentration is between 2% and 10% gel but up to 35% is acceptable. Higher slurry concentrations often lead to more assymetric peaks. Do not exceed the maximum operating pressure of the column and other equipment in use.

3.3 Evaluation of packing

Column performance can be evaluated using conventional efficiency and asymmetry factor testing.

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Suggested protocol:
Eluent: Sample: 100% methanol 3% acetone

Sample volume: 1% of bed volume Flow rate, linear: 2 cm/min UV detection: 280 nm

Typical efficiency values are greater than 15 000 plates/m when measuring the peak width at 50% of the peak height. Typical values for the asymmetry factor are 0.81.8 when measuring half-peak widths at 10% of the peak height. Note that system dead volumes should be reduced as much as possible to achieve the required efficiency.

4. Maintenance
All samples should be filtered and free of particulate matter. Use a filter with a porosity less than 2 m. Fouling can be prevented and medium lifetime lengthened if regeneration procedures are developed for each application. The high chemical stability of SOURCE 15RPC allows harsh cleaning and sanitization procedures to be used. The following cleaning agents, alone or in combination, are generally efficient for regeneration. Up to 90% acetonitrile or iso-propanol 0.51 M NaOH 90% acetic acid 1 M HCl or 3% TFA

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4.1 Storage
Store SOURCE 15RPC in 20100% alcohol to prevent microbial growth.

5. Method design and optimization

The main purpose of optimizing a preparative chromatographic step is to reach the pre-defined purity level with the highest possible recovery and productivity by choosing the most suitable combination of the critical chromatographic parameters. We recommend you optimize the procedure at laboratory scale; this will save both time and material.

5.1 Selectivity, loading capacity and recovery

The following parameters are important for selectivity, loading capacity and recovery: Type and concentration of organic solvent Acetonitrile is usually considered to give the best resolution. It also has low viscosity and good UV transparency. However, because of the toxicity of acetonitrile, low alcohols are often preferred for process scale applications. They are cheaper but more viscous, giving much higher back pressures than with acetonitrile (see pressure/flow-rate data in Fig. 1). Shallow solvent gradients are often necessary to achieve the required resolution; a 5% increase/hour is not unusual. We recommend you begin gradients with at least 5% of organic solvent and do not exceed 95%. Use of 0% and 100% organic solvent will result in very long equilibration times. The risk of precipitating sample substances and salts at high solvent concentrations should also be considered. Type and concentration of ion-pairing agents and buffer components Trifluoroacetic acid (TFA) is widely used as ion-paring agent. It gives high resolution of peptide and protein separations and it is volatile and relatively easy to remove. Useful alternatives that can offer different selectivities are p7

triethylammonium phosphate or acetate (TEAP, TEAA). Other buffer components used are acids e.g. phosphoric or acetic acid and neutral salts like ammonium sulphate. pH The excellent pH stability of SOURCE 15RPC make it possible to use a wide pH range to improve selectivity. This is illustrated in Figure 2 where the separation of two Angiotensin peptides is improved significantly by going from pH 2 to pH 12.
Column: Sample: Sample load: Eluent A: Eluent B: RESOURCE RPC 3 ml Mixture of Angiotensin II and Angiotensin III 25 g in 150 l 0.1% TFA (a), 10 mM NaOH (b) 0.1% TFA, 60% acetonitrile (a), 10 mM NaOH, 60% acetonitrile (b) 2 ml/min 10% B in 2 min, 1065% B in 10 min

Flow rate: Gradient:

A214nm 0.14 pH 2

A214nm

pH 12

0.12

Angiotensin II Angiotensin III

0.40

0.10
0.30

0.08

Angiotensin II

Angiotensin III

0.06

0.20

0.04
0.10

0.02

0.00

5.00

10.00

15.00 Time (min)

5.00

10.00

15.00 Time (min)

Fig 2. Separation of two Angiotensin peptides at pH 2 (a), and pH 12 (b).

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 Temperature Temperature can affect the performance of an RPC method. However, in process scale applications, the choice of temperature is often restricted by practical reasons. Develop your method within the same temperature ranges that will be used when running the final separation.

5.2 Resolution vs productivity

Apart from working on the factors affecting the selectivity, you can often increase resolution further by decreasing the sample load, gradient slope, flow-rate and/or increasing the bed height. However, these changes will have a negative effect on the through-put. For process scale methods, it is thus important to optimize sample load, gradient slope, flow rate and bed height to get the required resolution at the best possible productivity.

6. Scaling up
After the RPC step has been optimized at laboratory scale, the method can be scaled up. Scale up is carried out by increasing the diameter of the column. Parameters that remain constant include bed height, linear flow, sample concentration and volume (in relation to bed volume), and the ratio gradient volume/bed volume. The column diameter and volumetric flow rate will increase. The larger equipment needed when scaling up may cause some deviations from the method optimized at small scale. In such cases check the solvent delivery system and monitoring system and try and minimize the effects of liquid delays and volume changes in the flow path. The increased lengths and diameters of outlet pipes can also cause zone spreading with larger systems.

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7. Ordering information
Product SOURCE 15RPC SOURCE 15RPC SOURCE 15RPC SOURCE 15RPC SOURCE 15RPC Pack size 10 ml 200 ml 500 ml 1 litre 5 litre Code No. 17-0717-20 17-0727-02 17-0727-03 17-0727-04 17-0727-05

SOURCE 15RPC is also available in pre-packed 1 ml and 3 ml RESOURCE columns for fast and convenient separations on FPLC and HPLC systems.
Product RESOURCE RPC, 1 ml RESOURCE RPC, 3 ml Code No. 17-1181-01 17-1182-01

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www.gehealthcare.com/protein-purication www.gehealthcare.com GE Healthcare Biosciences AB Bjrkgatan 30 751 84 Uppsala Sweden

GE Healthcare Europe GmbH Munzinger Strasse 5 D-79111 Freiburg Germany GE Healthcare UK Ltd Amersham Place Little Chalfont Buckinghamshire, HP7 9NA 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
Produced by Wikstrms, Sweden 1060187, 03.2006 Printed matter. Licence 341 051

SOURCE, RESOURCE, SuperPac and FPLC are trademarks of GE Healthcare companies. GE imagination at work and GE monogram are trademarks 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 specications 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. GE Healthcare Bio-Sciences AB, a General Electric company. 2006 General Electric Company All rights reserved.

18-1060-44 AC 02/2006