Toyopearl HW Media for Scale-up Size Exclusion Chromatography of Nucleic Acids and Proteins

Transcription

1 Toyopearl HW Media for Scale-up Size Exclusion Chromatography of Nucleic Acids and Proteins CHEMISTRY OF TOYOPEARL RESINS Toyopearl resins are hydrophilic macroporous packings for bioprocessing chromatography. These resins are semi-rigid, spherical beads synthesized by a copolymerization of ethylene glycol and methacrylate-type polymers. They are highly resistant to chemical attack and are not degraded by microbes. Toyopearl packings are available in the size ranges preferred for production chromatography and can be packed easily into columns up to the largest industrial size. PROPERTIES OF TOYOPEARL HW MEDIA Toyopearl HW media were developed for size exclusion (gel filtration) chromatography. There are five different products having increasing pore sizes and exclusion limits for proteins and water soluble polymers, as shown in Table I. Table II lists the features and benefits of Toyopearl HW resins. Table I - Properties and Molecular Weight Separation Ranges for Toyopearl HW Resins Toyopearl Resin Particle Size (µm) Pore Size (Å) Molecular Weight of Sample Polyethylene Globular Proteins Dextrans Glycols & Oxides HW-0S HW-0F HW-0C , , ,000 HW-50S HW-50F , , ,000 HW-55S HW-55F , ,000,000-00, ,000 HW-65S HW-65F ,000-5,000,000 0,000 -,000, ,000,000 HW-75F 0-60 > ,000-50,000,000 00,000-0,000,000,000-5,000,000 Table II - Features and Benefits of TSK-GEL HW Resins Constant packing volume over a wide range of salt Bed stability concentrations Low cost chromatography High capacity for water soluble proteins Little loss or fouling High yield recovery using various aqueous eluents Can be regenerated with base or acid Stable from ph to for routine operation Stable from ph to for cleaning Excellent flow rates, high linear velocities, can be scaled to very large columns Stable up to bars of pressure Stable in eluents containing surfactants and organic solvents Can be autoclaved repeatedly in clean water at 0 C Can be used for very hydrophobic proteins Easy to sanitize

2 FRACTIONATION RANGE Figure shows the fractionation range of the Toyopearl HW media. It can be seen that the product line covers the full range of normally encountered water soluble proteins. Also given is an indication of how Toyopearl HW resins compare with Sephadex and Sephacryl gels sold by Pharmacia. Figure I Fractionation Range for Globular Proteins: Toyopearl HW Resins Compared with Pharmacia Gels 0 8 SIZE SORTING OF PROTEINS The molecular weight of proteins can be estimated using size exclusion chromatography. Figure shows an example of this process using Toyopearl HW 55F in a chaotropic buffer. Figure Size Exclusion Chromatography of Proteins in 6M Guanidine Hydrochloride 0 5 Molecular Weight (Daltons) G-5 G-50 G-75 Toyopearl HW-50 G-00 G-50 Toyopearl HW-55 G-00 S-00 S-00 Toyopearl HW-65 6B B Toyopearl HW-75 B Molecular Weight (Daltons) 0 Bovine serum albumin Ovalbumin Chymotrypsinogen A Cytochrome C Parathyroid hormone 0 G-0 G-5 Insulin 0 BED STABILITY IN VARIOUS SOLVENTS Toyopearl Resins, due to their high degree of crosslinking, are quite resistant to changes in eluents as can be seen in Figure. Gel Volume with Various Solvents (gel in water) Figure Swelling Properties of Toyopearl HW-0S in Various Eluents H 0 MeOH Acetone ChCl ChCl /MeOH Elution Volume (ml) Packing: Toyopearl HW-55F.cm ID x 00cm 6M guanidine HCl ml/min Temperature: 0 C Detector: 80nm page

3 EFFECT OF SALT ON PROTEIN RETENTION Protein retention on HW resins can change depending on the pi of the protein and the ph of the mobile phase. These effects can be minimized by the addition of a salt to the mobile phase as shown in Figure. Figure Effect Of Salt on Protein Retention 5.5 Urease (.8-5.) Catalase (5.5) γ-globulin (6.6) Yeast ADH (5.) [NaCl] = 0.5M ph 7.5 in the absence of NaCl in the presence of 0.5M NaCl Log MW 5.0 Apo Transferrin (5.5) Hemoglobin ( ).5 Porcine Pepsin (.0) Subtilisin Carlsberg (9.) Subtilisin BPN' (9.) Thermolysin (5.) A (9.5).0 T Lysozyme (.0) RNase A (9.5) (90ml) Cytochrome C (0.6) Elution Volume (ml) Chromatography was done on a T TrisHCl buffer fer). HW-55F column (.5cm x 50cm) with 5mM PRESSURE/FLOW CHARACTERISTICS The superior rigidity of the Toyopearl Resin allows much higher flow rates than conventional gel products as illustrated in Figure 5. 0 Figure 5 Flow Rate as a Function of Pressure Drop On Toyopearl HW-55F Bed Height Flow Rate (ml/hr - cm ) Sephadex G-00 Sephadex G-00: 50cm Sephacryl S-00: 80cm Toyopearl HW-55F: 90cm Sephacryl S-00 Toyopearl HW-55F Pressure Drop (kg/cm ) Packing pressure: cm I.D. x 70cm.5 to.5 kg/cm, using a peristaltic pump to.5ml/min page

4 EFFECT OF PARTICLE SIZE ON RESOLUTION In Figure 6, two different grades of Toyopearl HW-55 are compared --- an exercise often done in scale-up research and development. This figure shows that resolution is best with the S-grade (0 to 50µm), while the F-grade provides adequate performance. With these options, the most economical solution for scale-up chromatography can be chosen. Figure 7 TSK-GEL G000SW XL, TSK-GEL G000PW XL (5µ A: TSK-GEL G000SWXL n 5 Figure 6 Comparison of Resolution on Different Particle Sizes of Toyopearl HW-55 Fine grade 5 0 B: TSK-GEL G000PWXL 5 Superfine grade Elution Time (hr) To Sample:. hyroglobulin (0.%); ( ;. l 06ml/hr (0cm/hr) Te C Detector: UV (80nm) C: Toyopearl HW-55F 5 0 SCALING UP PROTEIN SEPARATIONS FOR HPLC Due to surface and pore characteristics which are almost identical, Toyopearl HW resins behave very much as TSK-GEL HPLC columns as shown in Figure Elution Ti ) Columns: A. TSK-GEL XL B. XL C. T Sample:. Th γ-globulin;. Ovalbumin d 50mM phosphate buffer (ph 7.0) containing l ml/min Temperature: 5 C Detector: UV (80nm) page

5 ADSORPTION CHROMATOGRAPHY EXAMPLES SEPARATION OF NUCLEIC ACID-RELATED COMPOUNDS Figure 8 shows a chromatogram of various nucleic acid related compounds obtained with Toyopearl HW-0S. This product is widely used for this kind of adsorption purification as well as for the separation of oligosaccharides. Figure 8 Separation of Nucleic Acid Related Compounds by Toyopearl HW-0S Elution Time (hr).6cm ID x 9cm Sample:. Blue dextran;. ATP;. ADP;. '-AMP; 5. '-AMP; 6. ', 5'-cAMP; 7. Adenosine; 8. Adenine 80ml/hr Te C Detector: UV (80nm) CHROMATOGRAPHY IN ORGANIC SOLVENTS Figure 9 shows an example of purification of a plantorigin polyphenol in 50% methanol. Toyopearl HW-0 is being used in ways similar to ODS reverse phase packings or Sephadex LH-0 for the separation of polyphenolic substances. ENZYME PURIFICATION EXAMPLES PURIFICATION OF GLYCOPROTEINS 5 6 Membrane-bound enzymes, most of which are glycoproteins, tend to lose activity during conventional slow gel filtration. This process is due to excessive elution times as well as non-specific interaction between the glycoproteins and polysaccharide gel. High-speed elution with Toyopearl HW-55F solved this problem in the example shown in Figure 0. Recovery of enzymatic activity was up to 80%. Specific activity was increased as well, while chromatography with dextran gels destroyed 90% of the activity. 7 8 Absorbance at 80nm ( ) Figure 9 Elution Pattern of Polyphenolic Substances Obtained from Chestnut Galls.0.0 Absorbance at 80nm CH OH CH OH O O OH Chestanin OH OH OC OH O O OH CH OC OH O Fraction Number Toyopearl HW-0F, Sample: Detector: Fraction size: 50% (v/v) methanol.5ml/hr 0.ml 80nm g/tube Figure 0 Chromatogram of Post-Proline Dipeptidyl Aminopeptidase from Pig Kidney Te Sample load: Fraction Number, 8ml/tube 0 0 Toyopearl HW-55F,.5cm ID x cm fer containing 0ml/hr 5ml Enzyme aactivity units/ml ( ) page 5

6 OTHER EXAMPLES OF ENZYME PURIFICATION Figures and give two further examples of the use of Toyopearl HW resins for the recovery and purification of enzymes, one from a culture supernatant and the other from animal tissue. Figure Chromatogram of Extracellular Protease from Thermus Caldophilus GK Toyopearl HW-50S resin has been used to help isolate the ubiquitin-histone conjugate uha from the unicellular ciliated protozoan Tertrahymena pyriformis. Figure shows the separation of uha from the histone, HA. The sole difference between these two components is a small polypeptide, ubiquitin (ca. 8,500Da). Figure Isolation of a Complex Protein Conjugate on Toyopearl HW-50S A A /hr) HA Fraction Number Toyopearl HW-55F,.8cm ID x 9cm 00mM Tris-HCl buf containing mm CaCl ) Temperature: C Fraction size: Sample volume: 7ml Detector: 80nm ( ) Enzyme activity ( ) Figure Purification of mrna on Toyopearl HW-65F 0.0 Sample: Elution: Detection: uha 8 Fraction Number mm x 8cm fraction from crude Tetrahymena HA containing the ubiquitin-histone conjugate uha 0mM HCl 0.ml/min 0nm Gel filtration chromatography on a Toyopearl HW-75F column is also a sensitive and useful method for determining the purity of amylose specimens, and for demonstrating heterogeneity in molecular weight and branched structure. Figure shows the elution profiles of amylose subfractions isolated from potato. Such substances as wheat and tapioca have also been purified by gel filtration. Figure Isolation of Potato Amylose Subfractions on Toyopearl HW-75F page 6 Sample: Elution: Detection: Fraction Number (5.0ml/fraction) Toyopearl HW-65F, 5mm x 90cm 0mg of poly (A) RNA/5ml of formamide, sample from silkworm 0mM sodium citrate, 5mM EDTA, 0.5% SDS, 6M urea, ph.5 ml/hr 5, total mrna activity (- - -), mrna activity for major plasma proteins ( ) carbohydrate (µmol of glucose/ml) 0.8 F F Sample: Elution: Temperature: Detection: V o V o F F Fraction Number (5ml/fraction) β-amylolysis limits (%) Toyopearl HW-75F, 6mm x 00cm potato amylose, 5mg/ml 0.05M NaCl 0.67ml/min 5 C total carbohydrate: anthrone-sulfuric acid assay β-amylolysis limits: hydrolysis w. β-amylase

7 TOYOPEARL SEC RESINS: Part # Container Product Particle Exclusion size (ml) description size (µm) limit (Da) HW-0S 0-0 x HW-0F 0-60 x HW-0C x HW-50S x HW-50F x HW-55S x HW-55F x HW-65S 0-0 x HW-65F 0-60 x HW-75F x Conditions:Exclusion limits are +/- 0% and are determined using polyethylene glycol, polyethylene oxide, or dextran standards, as appropriate. TOYOPEARL LABPAKS: Part # Container Product description Particle size (ml) size (µm) 98 x 50 SECPAK LMW (HW-0F, HW-50F, HW-55F) x 50 SECPAK HMW (HW-55F, HW-65F, HW-75F) x 50 SECPAK HP (HW-0S, HW-50S, HW-55S, HW-65S) 0-0 page 7

8 TOSOH BIOSCIENCE TOSOH BIOSCIENCE LLC 56 Keystone Drive Montgomeryville, PA Orders & Service: (800) Fax: (5) Orders & Service: 8A07.0/0 Sephacryl and Sephadex are registered trademarks of Pharmacia Biotech AB.