HiLoad Superdex 30 prep grade HiLoad Superdex 75 prep grade HiLoad Superdex 200 prep grade

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1 GE Healthcare Life Sciences Data file AE Gel filtration HiLoad Superdex 3 prep grade HiLoad Superdex 75 prep grade HiLoad Superdex 2 prep grade HiLoad columns are XK laboratory columns prepacked with Superdex prep grade for gel filtration. Superdex prep grade is a matrix of dextran and highly cross-linked agarose. The steep selectivity of the dextran component and the high chemical and physical stability of the agarose give high resolution separations at flow rates up to 5 cm/h. HiLoad columns offer a number of significant advantages for high resolution work: Prepacked for convenience and reproducibility High-resolution separation of biomolecules High chemical stability and easy scale-up Easy connection to chromatography systems, such as ÄKTA design Three types of Superdex prep grade are available in prepacked HiLoad columns: Superdex 3 prep grade, Superdex 75 prep grade, and Superdex 2 prep grade. All are prepacked to 6 mm bed heights in 6 mm or 26 mm diameter columns. Each column is expertly packed and individually tested. This combination of prepacked convenience and reproducibility make HiLoad Superdex prep grade columns a confident choice for fast, high-resolution gel filtration at preparative scale. The columns run with a wide variety of equipment, including simple pump-based configurations and ÄKTA design systems. Superdex gel filtration media are produced by the covalent binding of dextran to highly cross-linked porous agarose beads. Fig. HiLoad Superdex 3, 75, and 2 prep grade columns bring convenience and high resolution to gel filtration. Each is available in two column sizes, HiLoad 6/6 and HiLoad 26/6. Steep selectivity curves give unmatched resolution for biomolecules in the molecular weight range up to for Superdex 3 prep grade (Fig ), 3 to 7 for Superdex 75 prep grade, and to 6 for Superdex 2 prep grade (Fig 2). Moreover, the mean particle size of 34 µm and narrow particle size distribution of Superdex prep grade media give good separation performance without creating high backpressure.

2 Superdex 3 pg PEG peptides HiLoad 26/6 Superdex 3 prep grade 5 µl mix of five synthetic peptides in % TFA : M r : M r : M r : M r 596 5: M r 827 Buffer: 2 mm Tris-HCl,.25 M NaCI, ph 8.5 ml/min (3 cm/h) A 28 nm TF A 3 M r pg 2 pg Superdex 75 pg Superdex 2 pg Dextrans Volume (ml) Fig 3. Separation of test substances on HiLoad 26/6 Superdex 3 prep grade. Superdex 3 prep grade is optimized for proteins/peptides below M r M r 75 pg Superdex 75 pg Superdex 2 pg 2 pg Globular proteins Columns: HiLoad 6/6 Superdex 75 prep grade HiLoad 6/6 Superdex 2 prep grade. Myoglobulin,.5 mg/ml, M r 7 2. Ovalbumin, 5 mg/ml, M r Albumin, human 5 mg/ml, M r IgG,.2 mg/ml, M r Ferritin,.24 mg/ml, M r 44 Sample volume: 5 µl Buffer:. M phosphate buffer.4 M NaCl,.27 M KCl, ph 7.4 (PBS) ml/min (3 cm/h) 5 25 HiLoad 6/6 3 2 Superdex 75 prep grade and M r 5 Fig 2. Selectivity curves from Superdex 3 pg, Superdex 75 pg, and Superdex 2 pg. Figures 3 and 4 show separations of different model proteins on HiLoad 6/6 Superdex 3 pg, 75 pg, and 2 pg. Table summarizes the characteristics of the media and columns HiLoad 6/6 Superdex 2 prep grade Fig 4. Comparison of the selectivity of Superdex 75 prep grade and Superdex 2 prep grade for model proteins. Superdex 75 prep grade ( gives excellent resolution of the three proteins in the M r range 7 to 67 while the two largest elute together in the void volume. Superdex 2 prep grade ( resolves these two largest proteins. The ferritin (5) contains aggregates and thus results in a double peak AE

3 Table. Characteristics of HiLoad Superdex 3 prep grade, HiLoad Superdex 75 prep grade and HiLoad Superdex 2 prep grade. Matrix Dextran covalently bound to highly cross-linked agarose Average particle size 34 µm Separation range (M r ) < (Superdex 3 pg) globular proteins (Superdex 75 pg) (Superdex 2 pg) dextrans (Superdex 75 pg) 3 5 (Superdex 2 pg) Column volume 2 ml (HiLoad 6/6) 32 ml (HiLoad 26/6) Sample volume Up to 5 ml (HiLoad 6/6) Up to 3 ml (HiLoad 26/6) Recommended flow rate 5 cm/h at room temperature (.3.6 ml/min for HiLoad 6/6, ml/min for HiLoad 26/6) Theoretical plates >3 m - Maximum pressure over the packed bed during operation, p.3 MPa, 3 bar, 42 psi HiLoad column hardware pressure limit.5 MPa, 5 bar, 73 psi Column fittings /6 (valco) ph stability long term and working range 3 to 2 short term to 4 Solutions in which the media is stable Storage Superdex 3 prep grade Superdex 75 prep grade All commonly used aqueous buffers, ph 3 2 M acetic acid M sodium hydroxide 8 M urea 6 M guanidine hydrochloride 3% isopropanol 3% acetonitrile 7% ethanol.2 M sodium acetate in 2% ethanol at 4 C to 3 C.2 M sodium acetate in 2% ethanol at 4 C to 3 C Superdex 2 prep grade 2% ethanol at 4 C to 3 C The media also withstand the rigorous conditions used in process hygiene procedures such as sanitization. All strong oxidizing agents should, however, be avoided. Extensive studies, including long-term exposure to harsh chemical treatment, have demonstrated that Superdex prep grade has extremely high chemical stability. Figure 5 illustrates the long-term stability of Superdex 2 prep grade to. M HCl and M NaOH, a feature that is important for CIP procedures. Nonspecific interaction Studies have demonstrated varying degrees of nonspecific interaction between the chromatography media and acidic, as well as basic proteins in the absence of salt. Such interactions are negligible in salt concentrations between.5.5 M NaCl Accumulated exposure to. M HCl 2 3 Time (h) Accumulated exposure to M NaOH Cytidine Lysozyme Cytochrome C Myoglobin α-chymotrypsin A β-lactoglobulin Ovalbumin BSA IgG Ferritin H 2 O at room temperature. 2 3 Time (h) Chromatography media characteristics Chemical stability Chromatography media based on highly cross-linked agarose are very stable. Low sample volumes give high resolution even at high flow rates. Superdex 3 prep grade, Superdex 75 prep grade, and Superdex 2 prep grade may be used in aqueous solutions over the range ph 3 to 2 for continuous operation and over the range ph to 4 for cleaning-in-place (CIP). Chaotropic agents such as 6 M guanidine hydrochloride or 8 M urea, detergents (ionic and non-ionic), and polar organic solvents such as 7% ethanol can also be used for CIP. Fig 5. Performance of Superdex 2 prep grade measured as values of a protein mixture after repeated treatment with (. M HCl and ( M NaOH. The chromatography medium was exposed for repeated 8 h periods at temperature of 22 C. After each exposure period the value was determined for a test mixture of proteins. Following an accumulated exposure time of 5 h, the exposure periods were increased to 6 h. Even after more than 3 h accumulated exposure, values did not change significantly AE 3

4 Column characteristics The XK columns prepacked with Superdex prep grade media are easy to use laboratory columns. Each has a precision bore borosilicate glass tube and a fitted thermostatic jacket. Dead volumes make up less than.% of the total column volume, keeping sample dilution and band broadening to a minimum. Valco fittings (/6 ) are standard and provide easy and direct connection to ÄKTA design systems. Every prepacked HiLoad column is tested for number of theoretical plates per meter (N/m), asymmetry factor (Af) and bed height (mm). These stringent control measures ensure that HiLoad columns give reproducible results time after time. Applications HiLoad Superdex 3 prep grade HiLoad Superdex 3 prep grade is optimized for proteins and peptides (Fig 7), but it can also be used with success to separate oligosaccharides (Fig 6). Sample volume: HiLoad 6/6 Superdex 3 prep grade (2 ml) ml Buffer:.5 M Tris-HCl, M NaCl,.% Triton X-, ph H (c.p.m.) -3 4 C (c.p.m.) 5 2 ml/min (3 cm/h) HiLoad 6/6 Superdex 3 prep grade (2 ml) ml tryptic digest of human inter-α-inhibitor Buffer: PBS, mm NaCl, ph 7.4 ml/min (3 cm/h) Detection: UV, absorbance 22 nm A 22 nm Fig 7. Purification of tryptic digest of human inter-α-inhibitor using HiLoad 6/6 Superdex 3 prep grade resulted in seven separated peaks (I VII). Pool I contains the large peptide fragment including the intact carbohydrate cross-link. The size of this fragment is mainly due to the large hydrodynamic volume of the GAG (glycosaminoglycan). Gel filtration is a good choice for isolating peptides containing large carbohydrate moieties. Reproduced by kind permission of Dr. J. Enghild, Dept. of Molecular and Structural Biology, University of Aarhus, Denmark. HiLoad 6/6 Superdex 75 prep grade (2 ml) IGF-, ZZ fusion protein and uncleaved material Buffer:.5 ammonium acetate, ph ml/min (22.5 cm/h) A 28 nm. Pool I II III IV V HiLoad Superdex 75 prep grade HiLoad Superdex 75 prep grade separates proteins and peptides in the molecular weight range, M r 3 7 and performs best between 8 and 5 (Figs 8 and 9). VI VII Volume (ml).5 V IGF- V t Fig 6. Separation of oligosaccharides on HiLoad 6/6 Superdex 3 prep grade. The numbers above each peak indicate the number of monosaccharide units per molecule/chain. The oligosaccharides are applied one at a time. Each chromatogram represents seven superimposed analyses. Reproduced by kind permission of Dr. K. Lidholt, University of Uppsala, Sweden. Fraction Time (h) Fig 8. Separation of recombinant IGF- (insulin-like growth factor, M r 76) from its fusion protein partner (ZZ, M r 4 5) and uncleaved material. V = Column void volume. V t = Total column volume AE

5 HiLoad 6/6 Superdex 75 prep grade (2 ml) 2 ml endo-β-,4-glucanase (cellulase) from blue mussel (Mytilus edilus). Concentrated, eluted endoglucanase active material from from an affinity step Buffer: 2 mm sodium phosphate buffer,.3 M NaCl, ph 7. A 28 nm ml/min (3 cm/h) 28 nm endoglucanase activity Relative endoglucanase activity Columns: HiLoad Superdex 2 prep grade Column volumes, V T : a) 2 ml (6/6) b) 32 ml (26/6) Mouse monoclonal cell supernatant, IgG 2b incl. % Fetal Calf Serum Sample pretreatment: Concentration 4 in concentration cell Sample volume:.2 ml 3.2 ml Buffer: 5 mm NaH 2 PO 4,.5 NaCI, ph 7. Flow rates: a).6 ml/min (5 cm/h) b) 4.4 ml/min (5 cm/h) (max recommended flow rates).6.4 A 28 nm IgG 2b HiLoad 6/6 Superdex 2 prep grade Volume (ml) Fig 9. Intermediate purification step in lab scale on HiLoad 6/6 Superdex 75 prep grade. Two ml concentrated sample of the endoglucanase active material eluted from an affinity step was applied on the column. Reproduced by kind permission of Dr. B. Xu, University of Uppsala, Sweden A 28 nm HiLoad Superdex 2 prep grade Figure shows the purification of mouse monoclonal IgG 2b.6.4 IgG 2b HiLoad 26/6 Superdex 2 prep grade directly from cell supernatant. The reproducibility when scaling up from a HiLoad 6/6 to a HiLoad 26/6 column is also shown. HiLoad Superdex 2 prep grade has a separation range of M r 6 and separates with highest selectivity between 3 and 25. Superdex 2 separates monoclonal antibodies from critical contaminants and aggregates (e.g., Fig ) Fig. Purification of mouse monoclonal IgG 2b from cell supernatant using HiLoad 6/6 Superdex 2 prep grade, column volume 2 ml and HiLoad 26/6 Superdex 2 prep grade, column volume 32 ml. Almost identical separations are the result, even using prepacked columns of different sizes. Sample volume: Buffer: HiLoad 6/6 Superdex 2 prep grade Monoclonal antibodies purified on HiScreen MabSelect SuRe LX ml. M phosphate buffer,.4 M NaCl,.27 M KCl, ph 7.4 (PBS) ml/min (3 cm/h) Sample volume: Buffer: HiLoad 26/6 Superdex 2 prep grade Monoclonal antibodies purified on HiScreen MabSelect SuRe LX 3 ml. M phosphate buffer,.4 M NaCl,.27 M KCl, ph 7.4 (PBS) 2.5 ml/min (28 cm/h) Monomers Monomers 2 Aggregates/dimers 5 Aggregates/dimers Fig. Separation of monoclonal antibody monomers from aggregates/dimers on HiLoad 6/6 Superdex 2 prep grade and HiLoad 26/6 Superdex 2 prep grade. 85% of IgG 4 was monomers (9.5 mg) AE 5

6 Ordering Information Product Quantity Code no. HiLoad 6/6 Superdex 3 prep grade 2 ml HiLoad 26/6 Superdex 3 prep grade 32 ml HiLoad 6/6 Superdex 75 prep grade 2 ml HiLoad 26/6 Superdex 75 prep grade 32 ml HiLoad 6/6 Superdex 2 prep grade 2 ml HiLoad 26/6 Superdex 2 prep grade 32 ml Accessories No. supplied Code no. Accessory kit XK 6* Accessory kit XK 26* Support screen XK Support screen XK Net ring ( μm) XK Net ring ( μm) XK O-ring XK O-ring XK Stop plug female, / HiTrap/HiPrep /6 male connector for ÄKTA design Transport device Related products Quantity Code no. Superdex Peptide PC 3.2/3 2.4 ml Superdex Peptide /3 GL 24 ml Superdex 3 prep grade 5 ml Superdex 75 PC 3.2/3 2.4 ml Superdex 75 5/5 GL 3 ml Superdex 75 /3 GL 24 ml Superdex 75 prep grade 5 ml Superdex 2 PC 3.2/3 2.4 ml Superdex 2 5/5 GL 3 ml Superdex 2 /3 GL 24 ml Superdex 2 prep grade 5 ml Related literature Code no. Gel Filtration, Principles and Methods Gel Filtration Columns and Media, Selection Guide Prepacked chromatography columns for ÄKTA design systems, Selection Guide * Accessory kits XK 6 and XK 26 are suitable for repacking purposes and contain: 2 support screens, 5 net rings, 2 O-rings, 2 stop plugs, HiTrap/HiPrep /6 male connectors for ÄKTA design, and tool for dismantling. For local office contact information, visit GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden imagination at work GE, imagination at work, and GE monogram are trademarks of General Electric Company. ÄKTA, HiLoad, HiScreen, MabSelect SuRe, and Superdex are trademarks of GE Healthcare companies. All third party trademarks are the property of their respective owners General Electric Company All rights reserved. First published 26. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare UK Limited Amersham Place Little Chalfont, Buckinghamshire, HP7 9NA UK GE Healthcare Europe, GmbH Munzinger Strasse 5, D-79 Freiburg Germany GE Healthcare Bio-Sciences Corp. 8 Centennial Avenue, P.O. Box 327, Piscataway, NJ USA GE Healthcare Japan Corporation Sanken Bldg., 3-25-, Hyakunincho, Shinjuku-ku, Tokyo Japan AE 5/2