HiPrep Sephacryl High Resolution columns and Sephacryl High Resolution media

Transcription

1 data file Gel filtration HiPrep Sephacryl High Resolution columns and Sephacryl High Resolution media Introduction Sephacryl High Resolution (HR) gel filtration media give fast and reproducible separations at both laboratory and industrial scales. Five media are available in laboratory and larger pack sizes; Sephacryl S-100 HR, S-200 HR, S-300 HR,, and S-500 HR. Their main features are listed below: Excellent separations over a wide molecular weight range High reproducibility due to high stability Easy to pack, run, and maintain High flow rates and recoveries Well-suited to industrial- scale use Three of these media, Sephacryl S-100 HR, S-200 HR, and S-300 HR are also available in prepacked HiPrep columns. Each medium is available in two different prepacked column sizes, 16/60 (V T : 120 ml) and 26/60 (V T : 320 ml). HiPrep Sephacryl HR gel filtration columns provide the excellent separation properties of High Resolution media in a convenient, ready to use format. The many applications of HiPrep Sephacryl HR columns include preparative purifications and sample clean-up. Their main characteristics are: Convenient, easy-to-use, prepacked gel filtration columns in two different column sizes Choice of three selectivities covering a wide molecular weight range Reliable and reproducible preparative separations Run with simple equipment or chromatography systems Well-suited for work where columns must be discarded after use Fig 1. Sephacryl High Resolution media and HiPrep Sephacryl High Resolution prepacked columns offer the user a wide range of choice and reliable gel filtration separations. Media characteristics Sephacryl High Resolution is a cross-linked copolymer of allyl dextran and N,N-methylenebisacrylamide. This crosslinking gives good rigidity and chemical stability. The narrow particle size distribution, together with steep selectivity curves, results in good preparative characteristics with maintained high resolution. The hydrophilic nature of Sephacryl High Resolution media minimizes non-specific adsorption and maximizes recovery. Its excellent resolution and fast flow characteristics, longterm physical and chemical stability, and ease of handling make Sephacryl High Resolution the medium of choice for routine separations, particularly when handling large amounts of crude sample. The five media available have different porosity ranges. Figure 2 shows selectivity curves for each AB, p1

2 K av Globular proteins* K av Proteins** K av Dextran standards* S-500 HR S-200 HR S-300 HR S-100 HR Molecular weight, M r Molecular weight, M r Molecular weight, M r * In 0.05 M phosphate buffer 5 M NaCl, ph 7.0. ** In 6 M guanidine hydrochloride. Fig 2. Selectivity curves for Sephacryl High Resolution. For peptides and small proteins, Sephacryl S-100 HR is the best choice. When fractionating proteins in the molecular weight ranges of and , use Sephacryl S-200 HR and Sephacryl S-300 HR respectively. Note that these ranges include monoclonal antibodies and serum proteins. Sephacryl and Sephacryl S-500 HR are recommended for separating polysaccharides and other macromolecules with extended structures, and even small particles such as plasmids. Table 1 gives more details of media characteristics. Most types of column, including wide diameter production columns with bed heights of cm, can be packed and equilibrated successfully at high flow rates. This results in fast overall separation times. Table 1. Characteristics of Sephacryl High Resolution media. Sephacryl S-100 HR S-200 HR S-300 HR S-500 HR Useful fractionation range (M r ) Globular proteins Dextrans DNA exclusion limit (base pairs) Mean particle size 47 µm 47 µm 47 µm 47 µm 47 µm Bead structure Spherical, allyl dextran and N,N -methylene bisacrylamide Chemical stability Stable in all commonly used buffers: M NaOH, M HCl, 1 M acetic acid, 8 M urea, 6 M guanidine hydrochloride, 1% SDS, 2 M NaCl, 24% ethanol, 30% propanol, 30% acetonitrile (tested at +40 ºC for 7 days), 0.5 M NaOH (only for cleaning in place) ph stability long term short term Physical stability Negligible volume variation due to changes in ph or ionic strength Autoclavability At +121 C, ph 7 for 30 min (media only) Storage 20% ethanol 20% ethanol 20% ethanol 20% ethanol 20% ethanol AB, p2

3 Table 2. Bed volumes of Sephacryl High Resolution media in common organic solvents starting from 100 ml sedimented aqueous gel. Bed volume (ml) Bed volume (ml) Bed volume (ml) Bed volume (ml) Bed volume (ml) Solvent S-100 HR S-200 HR S-300 HR S-500 HR Formamide Dimethyl sulfoxide Methanol Ethanol Acetone Column diameter 1.6 cm Column diameter 2.6 cm MPa* MPa* (a) (b) S-200 HR S-300 HR S-100 HR S-500 HR S-200 HR S-100 HR S-300 HR S-500 HR Linear flow rate cm/h Linear flow rate cm/h * Pressure drop (MPa) determined with acetone at a flow rate of cm/h. Fig 3. Pressure drop as a function of flow rate for Sephacryl High Resolution media. Bed height approximately 60 cm in distilled water at +25 C. To calculate volumetric flow rate multiply linear flow rate by the cross-sectional area of column. (2 cm 2 for i.d. 16, 5.3 cm 2 for i.d. 26.) Stability Sephacryl High Resolution is extremely stable, both chemically and physically. It is compatible with all aqueous buffers within the ph range 2 11 commonly used in biochemistry, and withstands strong bases (e.g. M NaOH) and strong acids (e.g. M HCl and 1.0 M acetic acid). Furthermore, the presence of detergents (e.g. 1% SDS), chaotropic salts or dissociating agents (e.g. 8 M urea and 6 M guanidine hydrochloride) will not affect the separation properties of the media. The data on chemical stability given in Table 1 refer to the results of tests where the gel was exposed to different chemical agents for one week at +40 C. In no case was any significant change found in the chromatographic properties of the gel. Sephacryl High Resolution also demonstrates notable thermal stability. It may be autoclaved repeatedly at +121 C, ph 7 for 30 min without affecting its chromatographic performance. Note: Chromatographic media should never be exposed to chemical of physical extremes for longer than absolutely necessary. The gel will withstand occasional short contact with 0.5 M NaOH provided it is washed with buffer or water immediately afterwards. A suitable cleaning procedure is described in the Cleaning section. Sephacryl High Resolution is normally used with aqueous eluents. However, due to the high stability of the matrix, it can also be used in organic solvents. Table 2 lists the gel volumes in various organic solvents for an original 100 ml of gel sedimented in water. The chemical structure of Sephacryl High Resolution renders the medium more rigid than standard gel filtration media. This rigidity is illustrated by the pressure/flow diagram in Figure 3. Operation Sephacryl High Resolution is supplied ready to use in prepacked columns or as a suspension in 20% ethanol. With its high rigidity and narrow particle size distribution, Sephacryl High Resolution is easy to pack. Simply take the required amount of gel slurry, dilute with eluent buffer, and pour it into the column. Complete packing and operating instructions are provided with each pack of gel AB, p3

4 Cleaning When necessary, Sephacryl High Resolution can be cleaned in situ with 1 2 bed volumes of M NaOH or a non-ionic detergent. After cleaning, ensure that the column is fully reequilibrated with 2 3 bed volumes of buffer before re-using. We also recommend that 20% ethanol is used as a bacteriostatic storage solution when the column is not in use. Characteristics of HiPrep columns Sephacryl S-100 HR, Sephacryl S-200 HR, and Sephacryl S-300 HR are also available in two diameters of prepacked HiPrep columns. HiPrep 16/60 columns have an internal diameter of 16 mm and a bed height of 60 cm. The media bed volume is approximately 120 ml. HiPrep 26/60 columns have an internal diameter of 26 mm and a bed height of 60 cm. The media bed volume is approximately 320 ml. HiPrep colums are made of polypropylene, which are noninteractive with biomolecules. The column is not designed to be opened or repacked. The good separation characteristics of the packed column are ensured by carefully testing bulk gel selectivity, nonspecific interaction, and particle size distribution. The columns are then packed using validated packing procedures that include testing the column efficiency (the number of theoretical plates per meter [N/m] for each production batch). Table 3 summarizes the main characteristics of HiPrep Sephacryl High Resolution gel filtration columns. Separation of standard proteins Figure 4 compares the separation of a mixture of standard proteins on HiPrep 16/60 Sephacryl S-100 HR, S-200 HR, and S-300 HR columns. Operation HiPrep columns are simple to run with a single pump, e.g. Peristaltic Pump P-1 or with a chromatographic system, such as ÄKTA design or FPLC System. Flanged inlet and outlet tubing with fitted M6 connectors (metric threads, 6 mm) is supplied for connection to the column top and bottom end pieces. Connectors are also included for connection to ÄKTA design. Detailed instructions for setting up and running the columns are provided. Cleaning Regular cleaning will prolong the long-term separation performance of HiPrep columns. Wash the column with onehalf column volume of M NaOH at 15 cm/h Table 3. Characteristics of HiPrep Sephacryl High Resolution gel filtration columns. Property Matrix: Mean particle size Description Cross-linked copolymer of allyl dextran and N,Nmethylenebisacrylamide 47 µm (25 75 µm) Separation range, globular proteins: Sephacryl S-100 HR M r : Sephacryl S-200 HR M r : Sephacryl S-300 HR M r : HiPrep 16/60 HiPrep 26/60 Column volume 120 ml 320 ml Sample volume* up to 5 ml up to 13 ml Recommended flow rate ** 0.5 ml/min 1.3 ml/min (3 15 cm/h) (1 15 cm/h) Maximum flow rate** 1.0 ml/min 2.7 ml/min (30 cm/h) (30 cm/h) Maximum pressure over the packed bed during operation 5 MPa, 5 MPa, 1.5 bar 1.5 bar HiPrep column hardware pressure limit 0.5 MPa, 5 bar 0,5 MPa, 5 bar Theoretical plates >5 000 m -1 >5 000 m -1 ph stability long term and working range Short term Chemical stability All commonly used aqueous buffers ph 3 11, 1M acetic acid, 8 M urea, 6 M guanidine hydrochloride, 30% isopropanol, 30% acetonitrile, 24% ethanol, 0.5 M NaOH Storage 20% ethanol 20% ethanol * Optimal sample volume depends on the complexity of the sample and the flow rate. If the sample contains substances with small differences in size, either decrease the sample volume, or decrease the flow rate. (In very difficult cases, it may be necessary to decrease both.) ** +25 ºC in distilled water (0.5 ml/min HiPrep 16/60 or 1.3 ml/min HiPrep 26/60) to remove most proteins that are non-specifically adsorbed to the gel. Cleaning in place (CIP) procedures for removing protein and lipids bound more strongly to the gel are also included in instructions supplied with each column AB, p4

5 Comparing the separation of standard proteins Column: a) HiPrep 16/60 Sephacryl S-100 HR b) HiPrep 16/60 Sephacryl S-200 HR c) HiPrep 16/60 Sephacryl S-300 HR Sample: 500 µl of a mixture containing IgG (M r ), BSA (M r ), β-lactoglobulin (M r ), cytochrome C (M r ) and cytidine (M r 240) Buffer: 0.05 M sodium phosphate, 5 M NaCl, ph 7.0 Flow rate: 0.8 ml/min (24 cm/h) Separation of insulin chains Column: HiPrep 26/60 Sephacryl S-100 HR Sample: 1 ml of a mixture containing bovine insulin chain A (M r 2532) and chain B (M r 3496), 0.5 mg/ml of each Buffer: 0.05 M sodium phosphate, 5 M NaCl, ph 7.0 Flow rate: 2.0 ml/min (22 cm/h) a) HiPrep 16/60 Sephacryl S-100 HR BSA IgG Cytochrome C Cytidine β-lactoglobulin Vol. (ml) b) HiPrep 16/60 Sephacryl S-200 HR BSA Cytochrome C IgG Cytidine β-lactoglobulin Vol. (ml) Fig 5. Separation of insulin chains on HiPrep Sephacryl 26/60 S-100 HR. Purification of E. coli PPR1-fragment a) Initial separation by ion exchange Column: HiLoad 16/10 S Sepharose FF Sample: 35 ml E. coli extract Start buffer: 10 mm triethylamine acetate, 1 mm ZnCl 2, 100 mm NaCl, ph 7.0 Elution buffer: 10 mm triethylamine acetate, 1 mm ZnCl 2, 1 M NaCl, ph 7.0 Flow rate: 5 ml/min (150 cm/h) a) 40 Chain A Chain B Vol. (ml) 1.0 M NaCl c) HiPrep 16/60 Sephacryl S-300 HR 0.8 BSA IgG Cytochrome C Cytidine β-lactoglobulin Pool Vol (ml) Fig 4. Comparison of the selectivity of HiPrep Sephacryl 16/60 S-100 HR, HiPrep Sephacryl 16/60 S-200 HR, and HiPrep Sephacryl 16/60 S-300 HR columns. Applications Sephacryl High Resolution media are well-proven and used for many applications by life scientists and industrial manufacturers all over the world. Figures 5 to 11 show a number of laboratory scale separations on the five Sephacryl media, including purifications run on HiPrep columns. The industrial use of Sephacryl High Resolution is discussed later. Insulin consists of two chains (A and B) held together by -S-S- bonds. Figure 5 shows that when these links have been broken, the two chains can, in spite of the small difference in molecular weight, be separated on HiPrep 26/60 Sephacryl S-100 HR. b) Vol. (ml) b) Separation on HiPrep 16/60 Sephacryl S-100 HR Column: HiPrep 16/60 Sephacryl S-100 HR Sample: 0.5 ml pooled fraction from (a) Buffer: 10 mm triethylamine acetate, 1 mm ZnCl 2, 100 mm NaCl, ph 7.0 Flow rate: 1 ml/min (30 cm/h) Pool PPR1-fragment Vol. (ml) Fig 6. Purification of PPR1-fragment from E. coli using HiPrep 16/60 Sephacryl S-100 HR. Courtesy of C. Fuller, Department of Biochemistry, University of Cambridge, UK. Figure 6 shows a partially purified E. coli PPR1-fragment run on a HiPrep 16/60 Sephacryl S-100 HR column AB, p5

6 Separation of hgh dimers and monomers Gel: Sephacryl S-100 HR Sample: 14 ml hgh solution, containing 6% dimers Column: K26/100, bed size 2.6 x 90 cm Buffer: Glycine-phosphate, ph 7.0 Flow rate: ml/min (4 cm/h) hgh dimers 2. hgh monomers Separation of membrane proteins Gel: Sephacryl S-300 HR Sample: 500 µl of a mixture containing integral membrane proteins from human erythrocytes Column: K26/70, bed size 2.6 x 64 cm Buffer: PBS, ph 7.0 Flow rate: 0.8 ml/min (9.4 cm/h) Anion transporter, dimer 2. Anion transporter, monomer 3. Glycophorine A, dimer 4. Glucose transporter 5. Possibly lipids and SDS Vol. (ml) Fig 7. Human Growth Hormone (hgh) dimers and monomers are efficiently separated on Sephacryl S-100 HR. Figure 7 shows the highly efficient separation of hgh dimers and monomers. Phytohemagglutinin M is degraded in an acidic environment into an active protein and inactive polysaccharide. Figure 8 shows the separation of PHA-M and purified PHA-P on HiPrep 26/60 Sephacryl S-200 HR. Separation of phytohemagglutinin Column: HiPrep 26/60 Sephacryl S-200 HR Sample: a) 1 ml PHA-M, 2 mg/ml in acetic acid b) 1 ml PHA-P, 2 mg/ml in acetic acid Buffer: 0.05 M sodium phosphate, 5 M NaCl, ph 7.0 Flow rate: 2.0 ml/min (22 cm/h) a) Fig 9. Elution profile of integral membrane proteins from human erythrocytes on Sephacryl S-300 HR. The proteins were resolved into four fractions (1 4), which contained mainly: dimer and monomer of anion transporter, dimer of glycophorin A, and glucose transporter. The last peak (5) is probably a mixture of lipids and SDS. (Data provided by E. Greijer and P. Lundahl, Dept. of Biochemistry, Biomedical Centre, University of Uppsala, Sweden.) Separation of phospholipid vesicles Gel: Sephacryl Sample: Integral membrane proteins prepared from human erythrocytes solubilized in M phosphate, 100 mm SDS, 1 mm EDTA, 1 mm DTE, ph 7.4 Sample volume: 2 ml (2 mg/ml) Column: K26/70, bed size 2.6 x 61 cm Buffer: M phosphate, 50 mm SDS, 1 mm EDTA, 1 mm DTE, ph 7.4 Flow rate: 1 ml/min (11 cm/h) Elution volume (ml) 1. Large phospoholipid vesicles (LPLV) 2. Small phospholipid vesicles (SPLV) b) Vol. (ml) Elution volume (ml) Vol. (ml) Fig 8. Separation of phytohemagglutinin on HiPrep 26/60 Sephacryl S-200 HR. Fig 10. Gel filtration on Sephacryl can quickly separate phospholipid vesicles (liposomes) into large (LPLV) and small (SPLV) phospholipid vesicles. (Data provided by E. Greijer and P. Lundahl, Dept. of Biochemistry, Biomedical Centre, University of Uppsala, Sweden.) AB, p6

7 Rapid separation of plasmids Gel: Sephacryl S-500 HR Sample: 1.0 ml DNA-solution from E. coli HB 101 containing plasmid prit-18 Column: HR 16/10, bed size 1.6 x 10 cm Buffer: M Tris-HCl, M NaCl, 1mM EDTA, ph 8.0 Flow rate: 3 ml/min (90 cm/h) 1. Plasmid 2. Linear DNA and RNA Preparation Method Culture E. coli HB 101 containing plasmid prit-18 at +37 C overnight in the presence of amplicin (70 mg/l and Tryptic Soy Broth (30 g/l) Centrifuge the cells (4 000 x g, 5 min) and suspend in solution I* Add solution II* and incubate in a waterbath 10 min, +50 C, stirring every other minute. Add solution III* and cool on ice for 5 min. Centrifuge ( x g, 5 min) and filter the supernatant through 5 µm or 2 µm filter. * Solution I: M Tris, M EDTA, 1% glucose, ph 8.0 Solution II: M NaOH, 1.5% SDS Solution III: 3.0 M Na acetate, 2.0 M acetic acid The volumes of solutions I, II, and III should be one fifth of the culture volume. A 254 nm Fig 11. Rapid separation gives high-purity plasmids using Sephacryl S-500 HR. (Data provided By T. Moks, Department of Biochemistry, Royal Institute of Technology, Stockholm, Sweden.) Industrial use of Sephacryl High Resolution With its excellent resolution and high chemical stability, Sephacryl High Resolution is ideally suited to industrial use. Sephacryl S-200 HR in particular is well-established for use in a number of large-scale applications, especially in the field of serum protein purification. For example, the high resolution of Sephacryl S-200 HR has been used to optimize a polymer removal step in a major albumin production process. Here, no less than three production batches are now processed in a single column volume by successive sample additions (see Fig 12). The productivity of the process increased 3-fold. A second significant industrial application is in the final step of the purification of monoclonal antibodies (MAb) from cell culture supernatants. The challenge for gel filtration in this case is to remove contaminants such as transferrin and albumin that have been added earlier to the culture to increase MAb production yield. Removing these contaminants is critical, especially in clinical and other applications that demand extreme purity. Sephacryl S-200 HR has proven extremely valuable in this demanding role (Fig 13) Vol. (ml) Repeated cycles of albumin fractionation Sample: Albumin fraction from previous ion exchange step (sample volume equivalent to 4% of V t. (V t =column volume) Sample concentration 15 mg/ml. Column: BP 113/120 containing Sephacryl S-200 HR, bed height 100 cm Buffer: 0.05 M Tris-HCl, 5 M NaCl, ph 7.5 Flow rate: 7.5 cm/h Fig 12. Repeated cycles of gel filtration using Sephacryl S-200 HR of albumin obtained from a previous ion exchange chromatography step. The sample peaks are spaced so that three separations can be performed on the same column system simultaneously, within an elution volume of V t. The time equivalent to the passage of V t is shown. Purification of monoclonal antibodies Sample: IgG fraction from previous ion exchange step (sample volume equivalent to 1% of V t. (V t =column volume) Column: BP 113/120 containing Sephacryl S-200 HR, bed height 100 cm Buffer: 0.05 M Tris-HCl, 5 NaCl, ph 7.5 Flow rate: 7.5 cm/h 1 V t Immunoglobulin 1 2 Time (h) Elution volume Fig 13. Purification of monoclonal antibodies on Sephacryl S-200 HR. Inset shows analysis by gradient SDS-PAGE of the immunoglobulin pool. Lane 1, native sample; lane 2, sample reduced with 2-mercaptoethanol. Sephacryl S-200 HR is also very efficient in removing aggregated MAb, again increasing the quality and value of the final product AB, p7

8 Further information The Amersham Biosciences handbook Gel filtration: Principles and methods describes in full the methodology and applications of gel filtration with Sephacryl High Resolution and other media. For further information on scale-up and the operation of large scale chromatography systems, please contact your local Amersham Biosciences representative directly. Sephacryl High Resolution gel filtration media fulfill process chromatography requirements in terms of stability, scaleability, and bulk availability. As members of the BioProcess media family, they carry full technical and regulatory support for production-scale operations. Ordering information Column Bed vol. Code No. HiPrep 16/60 Sephacryl S-100 HR 120 ml HiPrep 26/60 Sephacryl S-100 HR 320 ml HiPrep 16/60 Sephacryl S-200 HR 120 ml HiPrep 26/60 Sephacryl S-200 HR 320 ml HiPrep 16/60 Sephacryl S-300 HR 120 ml HiPrep 26/60 Sephacryl S-300 HR 320 ml Medium Quantity Code No. Sephacryl S-100 HR 750 ml Sephacryl S-100 HR 10 liters Sephacryl S-200 HR 750 ml Sephacryl S-200 HR 10 liters Sephacryl S-300 HR 750 ml Sephacryl S-300 HR 10 liters Sephacryl 750 ml Sephacryl 10 liters Sephacryl S-500 HR 750 ml Sephacryl S-500 HR 10 liters Accessories Domed nut Union M6 female/1/16 male * Transport syringe * For connection to ÄKTA design systems Technical information Gel Filtration handbook Principles and Methods Gel Filtration Column and Media Guide to order: Asia Pacific Tel: Fax: Australasia Tel: Fax: Austria Tel: Fax: Belgium Tel: Fax: Canada Tel: Fax: Central, East, South East Europe Tel: Fax: Denmark Tel: Fax: Finland & Baltics Tel: +358 (0) Fax: +358 (0) France Tel: Fax: Germany Tel: Fax: Italy Tel: Fax: Japan Tel: Fax: Latin America Tel: Fax: Middle East and Africa Tel: +30 (1) Fax: +30 (1) Netherlands Tel: Fax: Norway Tel: Fax: Portugal Tel: Fax: Russian & other C.I.S. & N.I.S. Tel: +7 (095) , Fax: +7 (095) South East Asia Tel: Fax: Spain Tel: Fax: Sweden Tel: Fax: Switzerland Tel: Fax: UK Tel: Fax: USA Tel: Fax: Sephacryl, HiPrep, HiLoad, ÄKTA, FPLC, and BioProcess are trademarks of Amersham Biosciences or its subsidiaries. Amersham is a trademark of Nycomed Amersham plc. Amersham Biosciences Björkgatan 30, SE 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 USA. Amersham Biosciences Europe GmbH Munzinger Strasse 9, D Freiburg, Germany. Amersham Biosciences K.K. Sanken Building, , Hyakunincho, Shinjuku-ku, Tokyo , Japan. 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 2001 All rights reserved. Produced by Wikströms, Sweden , Printed matter. Licence AB, p8