Cellufine Chromatography media

Transcription

1 hromatography media

2 is the liquid chromatography media for the purification of proteins, enzymes and other bio-active substances. Since it is made from spherical cellulose particles having high chemical stability, high mechanical strength and bio-compatibility, it is suitable for the production in pharmaceutical and food industry. Leaking from this matrix is much less than that from the synthetic polymer media. product range covers all liquid chromatography modes (Gel Filtration, Ion Exchanger, Affinity and Hydrophobic Interaction chromatography) in order to purify target molecules to requested specifications. In addition, we can provide custom made grades corresponding to user requests, such as the ion exchanger made from big beads and the affinity with a designated ligands. The production of is guaranteed by ISO 9001 and HOMATOGAPHY MEDIA PODUT AGE ADSOPTIO PATITIO IO AFFIITY HYDOPHOBI GEL EXHAGE ITEATIO FILTATIO For initial separation and For high specificity For purification For purification of biofinal polishing of proteins, separations of a wide of molecules and proteins peptides and enzymes range of molecules proteins and enzymes by molecular size DEAE Weak Anion Virus & Heparin Binding Butyl MW 50-3,000 kd A-200 Proteins,oagulation Factors Phenyl GL-2000 A-500 Sulfate A-800 For salt and solvent ucleic acid bunding protein Mini-column removal, and buffer QA Strong Anion & uclease,histon Butyl 1ml exchange Q-500 Phosphate Phenyl 1ml GH-25 M Weak ation Glycoprotein,glycated proteins -500 PB (Phenyl Boronate) Mini-column A500 1ml Endotoxin removal Q500 1ml ET-clean L 500 1ml ET-clean S IMA,His-Tag protein helate Activated Supports Amino Formyl Mini-column Sulfate Phosphate PB helate ET-clean L&S 1ml 1ml 1ml 1ml 1ml 2

3 Affinity Group specific affinity gel Sulfate For concentration, purification and depyrogenation of virus, viral/microbial antigens and heparin-binding proteins. Especially, for purification of Influenza virus, manufacture of influenza vaccine.. haracteristics A B ligand ligand conc μm sulfate ester approx. 8μM/ml lysozyme >3mg/ml HBsAg 6-8mg/ml For concentration, Purification of glycoprotein,glycated-protein and diol-compound. Phenyl borate affinity ligand. haracteristics Ligand boron conc. PB (Phenyl Borate) Phenyl borate > 600μmol/g-dry Picture A:The surface of Sulfate(before influenza adsorption) B:The surface of Sulfate after influenza adsorption. The data representing from professor Kida.(Hokkaido Univ. Graduate school of veterinary medicine. B free B ph6.5 HO HO ' ' bound B O O - ph7.5 ' + O conalbumin >7mg/mL Adsorption and desorption mechanism of PB Phosphate conventionalce lulose phosphate e lufine Phosphate alconc. For nucleic acid relate protein,dehydrogenase,phosphate relate protein, cation exchange chromatography. Abs. 280nm ATP Ovalbumin Lysozyme yt. al haracteristics ligand ligand conc. phosphate ester 2-4meq/g-dry lysozyme 20mg/mL time (min.) The example of separation of the mixed protein by Phosphate 3

4 helate O O 2+ i OO- O 2+ i 2 IMA:Immobilized Metal Affinity hromatography. For His-tag protein, histizine,cysteine,tryptophan contain protein. haracteristics Protein-His Imidazole helate OO- O i 2+ OO- 2 OO- OO- 2+ i OO- + ligand μm iminodiacetic acid Zn μmol/ml u μmol/ml Adsorption and desorption mechanism of helate IMA. Affinity Endotoxin removal gel ET clean L/S The (r) ETclean is poly(ε-lysine) immobilized (cellulose spherical beads). The beads bind and remove endotoxin from your sample solution. The poly(ε-lysine) is a microbial poly(amino acid) that consist of lysine residues produced by Streptomyces albulus. The poly(ε-lysine) as ligand and the cellulose beads act as matrix ands are products of hisso orporation. haracteristics products μm μg/ml exclusion limit ET clean-s <10 3 ET clean-l

5 emoval of LPS from a Protein Solution by ET-lean Sample solution ET-lean S (μ=0.05, ph7.0) ET-lean L (μ=0.40, ph7.0) protein pi LPS con. in protein solution pg ml -1 emain LPS pg ml -1 ecovery of Protein % emain LPS pg ml -1 ecovery of Protein % BSA <10 97 γ-globuline <10 97 ytochrome <10 98 The removal of LPS was determined by a batchwise methods with 0.3 ml of wet adsorbent and 2mL of a protein solution containing natural LPS(protein:1mg ml -1,pH=7.0,m=0.05 or 0.40). Affinity Ligand coupling gel Activated Supports for Immobilization of Antibodies, Antigens, Affinity Ligands and Enzymes Amino haracteristics active group active group conc. (-H2) 15-20μmol/ml μm The scheme of the structural formula of Amino. oupling with carboxyl group and aldehyde group. 5

6 Formyl haracteristics active group active group conc. formyl(-ho) 10-15μmol/ml μm The aldehyde active group on Formyl packings reacts with primary amine groups on the ligand to form a Schiff s base complex (see Figure ). A mild reducing agent is used to convert the Schiff s base to a highly stable linkage. Preincubation reductive amination O H + H H O O educing agent H H H H -Formyl Ligand Schiff base Ligand-immobilized Ion exchange chromatography Ion Exchangers are based on spherical particles manufactured from crosslinked cellulose. Each offers excellent flow properties, mechanical stability and chemical resistance. These ion exchangers are ideally suited for both laboratory and process scale chromatography of proteins, peptides and other biomolecules. Applications include the purification of antibodies, growth factors, albumin, enzymes, nucleic acids, etc. A-200/A-500/A-800 DEAE H 3 H M O O Q-500 Adsorb 500 Adsorb A500,Q500 positive charge negative charge QA H 3 H 3 H 3 + > Low ph + + < High ph 6 isoelectric point

7 haracteristics type (μm) ion exchange (meq/g) BSA (mg/ml) lysozyme (mg/ml) A A A Q Hydrophobic Interaction hromatography Butyl O H 3 Phenyl O Hydrophobic Interaction hromatography (HI) is a method which separates proteins on the basis of their differential interactions with a mildly hydrophobic surface. HI media are porous chromatography particles, manufactured from crosslinked cellulose to which either a butyl, phenyl functionality has been covalently bonded via a short spacer. adsorb high desorption Salt conc. low The Hofmeister (lyotropic) series PO 3-4 >SO 2-4 >H 3 OO - >l->br - >lo 4- >I - >S - H 4+ >K + >a + >s + >Li + >Mg 2+ >a 2+ >Ba + 2 increase Adsorption force decrease 7

8 Gel Filtration hromatography GL-2000 This media offer an extraordinarily broad selection of fractionation ranges: from the unique GL-2000 for very high molecular weight protein complexes. GH-25 GH-25 desalting media is based on porous, spherical, cellulose particles. The sharp 3kD exclusion limit allows proteins to pass through the column in the void volume while retarding smaller molecular weight solutes in the internal pores. Mini-olumn Mini-olumns offer convenience and expedience for isolating, purifying, and concentrating biomolecules from aqueous samples. HA-Titer HA-titer OD OD280 kd Fraction o Lane 1:Marker 2:HA vaccine 3:Allantoic fluid 4:Pass 5:Fr.44 6:Fr.45 7:Fr.46 8:Fr.47 9:Fr.48 10:Fr.49 11:Fr.50 12:Fr.52 Purification of influenza virus on Mini-column Sulfate. ondition Mini-column Sulfate,1mL (ID 8 20mm) Flow ate: 1mL/min Equilibration buffer : 0.01M Phosphate buffer, ph 7.4 Washing buffer: 0.01M Phosphate buffer, 0.19M al, ph 7.2 Elution buffer: 0.01M Phosphate buffer, 3M al, ph 7.0 Virus strain: Vac-2/04(H77) β-propiolactone --- Allantoic Fluid was filtered after centrifuged 8

9 Product code list (Part 1 / Affinity media) Product ame Pack Size atalogue # Product ame Pack Size atalogue # Sulfate helate ETclean L Amino Formyl ETclean S Phosphate PB M = Mini-olumn 9

10 Product code list (Part 2 / Ion Exchange media, Hydrophobic Interaction media, and Gel Filtration media) Product ame Pack Size atalogue # Product ame Pack Size atalogue # A Butyl A Phenyl A GH Q GL ml x 5 (M)