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Toyopearl Instruction Manual

1. E ull N TL utl Mr y X OPERATION OF NANYO MANUFACTURING COMPLEX BEGINS SCIENTIFIC INSTRUMENTS DIVISION FORMED FIRST GPC COLUMN USING TSK GEL DEVELOPED BY TOSOH HIGH PERFORMANCE LIOUID CHROMATOGRAPHY COLUMN PLANT IS COMPLETED TOSOH DEVELOPS TOYOPEARL MEDIA TOSOH DEVELOPS HYDROPHOBIC INTERACTION MEDIA TOSOHAAS US OPERATIONS FORMED IN MONTGOMERYVILLE TOSOHAAS GMBH OPERATIONS FORMED IN STUTTGART TOSOH NANYO GEL FACILITY RECEIVES ISO 9001 IN NOVEMBER FORMER TOSOHAAS US OPERATIONS BECOMES TOSOH BIOSEP LLC A 100 SUBSIDIARY OF TOSOH CORPORATION IN JANUARY FORMER TOSOHAAS GMBH EUROPEAN OPERATIONS BECOMES TOSOH BIOSEP GMBH A 100 SUBSIDIARY OF TOSOH CORPORATION ll TOSOH CORPORATION ANNOUNCES THAT ALL TOSOH AFFILIATED SCIENTIFIC AND DIAGNOSTIC SYSTEM RELATED COMPANIES IN EUROPE WILL BE UNIFIED UNDER THE NEW NAME TOSOH BIOSCIENCE ECOSEC THE 7TH GENERATION GPC SYSTEM IS INTRODUCED GLOBALLY 20TH ANNIVERSARY OF TOSOH BIOSCIENCE GMBH STUTTGART m i T I
2. velocity Pressure Gauge needed needed not needed 2 1 Constant Velocity Semi Constant Pressure Methods a If used place the packing reservoir on the column The total volume of the column and the reservoir should be sufficient to allow the entire resin slurry to be poured in one operation b Ensure that the column is leveled prior to packing Wet the bottom frit or screen in the column with buffer Allow the buffer to drain a few seconds to remove any air bubbles Plug the outlet of the column and leave 1 2 cm of buffer in the bottom of the column FIGURE6 Bubble free liquid covered bottom frit I z 1 1 c Resuspend the resin slurry to assure homogeneity FIGURE7 Homogenise slurry 7 WWW TOSOHBIOSCIENCE COM d Carefully pour the resin slurry slowly down along the inside wall of the column Prevent air from being trapped in the resin slurry e After the resin slurry is transferred to the column rinse the inside walls of the column using a squirt bottle containing packing buffer FIGURE 8 Pouring the resin f Immediately place the flow adapter of the column onto the resin slurry There should be no trapped air between the flow adapter and the buffer g Open the column outlet and start the pump Start slowly to flow packing buffer through the column FIGURE 9 Adjusting column h Two different Packing Methods can be app
3. TOSOH TOYOPEARL INSTRUCTION MANUAL E TOSOH BIOSCIENCE 1 TOSOH BIOSCIENCE GMBH ZETTACHRING 6 70567 STUTTGART GERMANY 49 0 711 13257 0 49 0 711 13257 89 WWW TOSOHBIOSCIENCE COM AV 2 TOSOH BIOSCIENCE LLC 156 KEYSTONE DRIVE MONTGOMERYVILLE PA 18936 9637 USA 1 215 283 5000 1 215 283 5035 INFO TBL TOSOH COM WWW TOSOHBIOSCIENCE COM 1935 1936 1971 1974 1979 1983 1987 1989 1995 2000 2001 2002 2003 2008 2009 i TOSOH HISTORY FOUNDING OF TOYO SODA MANUFACTURING CO LTD 3 TOSOH CORPORATION 3 8 2 SHIBA MINATO KU TOKYO 105 8623 JAPAN 81 3 5427 5180 F 81 3 5427 5220 INFO TOSOH CO JP WWW TOSOHBIOSCIENCE COM y NS 4 TOSOH SHANGHAI CO LTD ROOM 2618A INTERN TRADE CENTER NO 2201 YAN AN WEST ROAD SHANGHAI 200336 CHINA T 86 21 6270 2810 86 21 6270 2820 PAN TOSOH COM CN WWW TOSOHBIOSCIENCE COM Dy
4. 3 000 4 000 60 c m h salt 3 500 3 900 300 cm h salt HIC 65 um IEC 65 um GE Healthcare Lifesciences AxiChrom BPG Chromaflow Index 60 x 20 20 30x 11 25 40 80 x 15 24 20 35 x 28 32 20 x 15 25 8 000 100 cm h salt 4 000 11 000 40cm h salt 3 000 5 000 100cm h salt 14 20 000 20c m h acetone 3 000 6 000 100c m h acetone HIC 65 um IEC HIC 65 um IEC 65 um IEC 20 um IEC HIC 35 65 um 20 30 x 15 30 20 x 30 2 500 3 500 100cm h acetone 7 000 250cm h acetone IEC 65 um IEC 20 um Millipore IsoPak Access QuikScale Moduline 44 x 25 44 x 13 30 100 160 x 15 25 140 x 25 160 x 13 15 200 x 30 20 30x 13 20 14 30 13 33 63 x 17 140 x 20 25 1 2 1 6 1 3 1 6 1 2 1 4 0 8 6 000 9 000 acetone 60 cm h 3 000 8 000 130 20 cm h 4 000 6 000 salt 60 cm h 5 000 7 000 salt 60cm h 600 900 acetone 100 cm h 4 000 5 500 100cm h salt 4 000 10 000 acetone 100 cm h 2 500 5 000 acetone 100 cm h 2 500 4 000 acetone 130 cm h 5 000 6 000 salt 30cm h IEC 35 um IEC HIC 65 um IEC HIC 65 um IEC 35 um IEC 100 um HIC 65 um HIC 35 um IEC 65 um IEC 65 um IEC 65 um Pall Euroflow 40 80x 12 32 40 80x 14 32 40 100 x 21 28 100 140 x 20 25 16 000 19 000 salt 60cm h 3 000 7 000 salt 30c m h 1 000 3 000 salt 100c m h 3 000 7 000 salt 80cm h HIC 35 um H
5. S M C SP 650 S M C SP 550C MegaCap II SP 550EC GigaCap S 650M 50 100 um GigaCap CM 650M 50 100 um S 35 um M 65 um C 100 um EC 200 um HIC Toyopearl Resin Ether 650 S M PPG 600M Phenyl 600M Phenyl 650 S M C Butyl 650 S M C Butyl 600M SuperButyl 550C Hexyl 650C S 35 um 65 um C 100 um Pore Size 400 500 1 000 1 000 750 1 000 1 000 500 500 1 000 1 000 Pore Size 1 000 750 750 1 000 1 000 750 500 1 000 Toyopearl Resin Toyopearl Reactive Resins AF Amino 650M AF Carboxy 650M AF Formyl 650M Toyopearl Activated Resins AF Epoxy 650M AF Tresyl 650M Toyopearl Ready to use Resins AF BlueHC 650M AF Chelate 650M AF HeparinHC 650M AF Red 650ML M 65 um ML 265 um z SEL Toyopearl Resin HW 40 S F C HW 50 S HW 55 5 HW 65 5 F C HW 75 S 30 um F 45 um C 75 um 7 WWW TOSOHBIOSCIENCE COM Pore Size 1 000 1 000 1 000 1 000 1 000 1 000 1 000 1 000 1 000 Pore Size 50 125 500 1 000 1 000 7 TOSOHBIOSCIENCE PROCESS TOSOH BIOSCIENCE PROCESS 15 PROCESS COLUMN INSTALLATIONS Column Manufacturer various bed dimensions ID x L in cm Column Performances M plate count N m TOYOPEARL Resin Type 20 45 x 15 25 130 x 24
6. 1 1 General Considerations for Packing Toyopearl It is best to pack Toyopearl resins by the application of pressure from 0 5 to 3 bar 7 to 45 psi across the bed length Although it is not recommended Toyopearl resins can be packed by simple gravitational settling The equipment components shown in Fig 1 required to successfully pack Toyopearl resins are a pump a pressure gauge a level glasses acrylic or PEEK or stainless steel column and a packing reservoir optional Equipment required for packing 7 WWW TOSOHBIOSCIENCE COM FIGURE 2 Toyopearl base particle t es 40 i T p P SN X SEES M OY FR 4 1 7 mu 1 2 Removal of Fines Tosoh Bioscience recommends that fines be removed Fines in the gel slurry may obstruct screens or sintered filters and may eventually increase the pressure drop across the column The following decantation process is required to remove fines from the resin slurry a The settled resin in the shipping containers should be suspended by vigorous agitation or stirring with a rod or paddle do not use a magnetic stirrer it will grind the resin generating fines Once suspended transfer the required amount of suspension approximately 4 volumes suspension 3 volumes resin into a container of sufficient volume to hold 4 times the volume of resin being prepared Add distilled water or buff
7. Decant d Add three times the resin volume of either distilled water or packing buffer to the decantation vessel and re suspend the resin by gentle overhead stirring Do not use a magnetic stir bar it will grind the resin generating fines e Repeat steps c and d at least two more times 1 3 Buffer Equilibration When choosing a packing buffer it is best to choose empirically since the optimal buffer will vary with your specific application In general the highest ionic strength mobile phase to be used in the separation including the cleaning and sanitization steps is a suitable starting point Some typical packing buffers are listed in Table 1 SEC HW 40 HW 50 HW 55 HW 65 and HW 75 IEC DEAE type QAE Q type CM type SP type MegaCap II SP 0 1 M Na S0 NaNO or NaCl in 50 mM phosphate or Tris buffer 1 M NaCl in 50 mM phosphate Tris or acetate buffer HIC Ether 650 Phenyl type Butyl type 2 M Na SO NH SO or NaCl in Hexyl 650 PPG 600 50 mM phosphate buffer AFC AF Tresyl and AF Epoxy 650 0 5 M NaCl in 0 1 M NaHCO or phosphate buffer AF Formyl 650 AF Amino 650 1 M NaCl in 100 mM phosphate AF Carboxy 650 Protein A orNaHCO buffer amp AF Chelate 650 AF Blue HC 650 0 5 M NaCl or 0 2 M glycine in and AF Red 650 20 mM phosphate or Tris buffer 1 4 Slurry Preparation After de fining the resin the slurry concentration can be adjusted for packing th
8. F 45 um S 30 um F 45 um F 45 um S 35 um M 65 um C 100 um C 100 um EC 100 300 um S 35 um M 65 um C 100 um M 65 um Please call our product specialists for your individual discussion Packing velocity flow rate cm hr 30 40 60 80 120 160 25 35 50 70 25 35 50 70 20 75 40 150 40 150 400 600 800 1000 800 1200 700 1000 800 1200 400 600 800 1000 800 1200 700 1000 800 1000 Not all resins are available in all particle sizes Operating velocity cm hr 10 25 25 50 50 100 10 20 25 35 10 20 25 35 10 15 15 30 15 30 45 65 80 130 80 600 80 240 80 500 45 65 80 130 80 500 80 240 30 130 ml min 0 6 1 6 1 6 3 2 3 2 6 4 0 6 1 3 1 6 2 2 0 6 1 3 1 6 2 2 0 6 1 0 1 0 1 9 1 0 1 9 2 0 8 0 5 TOYOPEARL o TOYOPEARL TOSOH BIOSCIENCE TOSOH PACKING The following descriptions are valid for packing under flow If you have other equipment or pack greater than 5 liters please call our Technical Specialists We have experience with many different column designs and brands qb M REM EE Features of packing methods Packing Constant Constant Assisted Method Pressure Velocity Gravity race 9 fast fast slow Velocity Flow Rate Range up to high up to high limited to low Pump constant constant
9. V p TOSOH BIOSCIENCE 7 PROCESS Toyopearl Instruction Manual Table of Contents l Packing 1 Preparation for Packing 1 1 General Consideration for Packing 1 2 Removal of Fines 1 3 Buffer Equilibration 1 4 Slurry Preparation 15 Alternative Slurry Preparation 2 Packing Procedure 2 1 Constant Velocity Semi constant Pressure Method 2 0 Alternative Packing Method Assisted Gravity 3 Equilibration and Efficiency Evaluation Il Column Operation 1 Chromatographic Separation 1 1 Size Exclusion SEC 1 2 lon Exchange IEC 13 Hydrophobic Interaction HIC 14 Affinity AFC 2 Cleaning 3 Storage 4 Sterilization Depyrogenation Preservative Removal Column Frits Ill Toyopearl Product Overview IV Process Column Installations 10 10 10 10 10 11 11 12 13 14 15 TOYOPEARL TOYOPEARL TOSOH BIOSCIENCE TOSOH PACKING Introduction Toyopearl chromatographic resins are macroporous polymeric packings for bioprocess chromatography They are applicable for the laboratory and process scale purifications of globular proteins peptides nucleic acids and other biologically derived materials These resins are a modified methacrylate polymer which gives the resin a hydrophilic surface due to the presence of ether and hydroxyl groups It also confers upon the resin excellent pressure flow characteristics and pH stability Packing 1 Preparation for Packing
10. in cases of extreme contamination 7 WWW TOSOHBIOSCIENCE COM 3 Storage SEC IEC and HIC Store the column or used bulk resin in distilled water containing a bacteriostatic agent such as 20 ethanol preferably at 4 C to 25 C AFC Store the column or used bulk resin in a neutral solution of 1 M sodium chloride or potassium chloride containing a bacteriostatic agent such as 20 ethanol preferably at 4 C to 10 C For AF Formyl 650M store the column or used bulk resin in a neutral solution of 1 M sodium chloride or potassium chloride in 196 gluteraldehyde preferably at 4 C to 10 C Please note that dye affinity chromatographic resins may release a small amount of dye during storage Be sure to wash the dye affinity resin before each use to remove any released dye TOSOHBIOSCIENCE PROCESS TOSOH BIOSCIENCE PROCESS COLUMN OPERATION 4 Sterilization Depyrogenation Preservative Removal Column Frits Sterilization Toyopearl resins can be sterilized by autoclaving at 121 C for 20 min without altering their properties Alternatively columns already packed may be exposed to 200 ppm sodium hypochlorite for periods up to 12 hours without loss of function Depyrogenation Toyopearl resins are recommended for use from pH 2 to 12 However short exposures lt 12 hours to higher pH 0 5 N NaOH are acceptable for depyrogenation Typically endotoxin levels are reduced by at least 4 logs fol
11. wash the column with 3 5 column volumes of starting buffer to remove unadsorbed impurities Two kinds of elution methods are commonly used in affinity chromatography nonspecific and specific Nonspecific elution generally is achieved by increasing the salt concentration in the eluent Most proteins are eluted with a solution containing 2 M sodium chloride or 3 M potassium chloride Proteins not eluted with these eluents can be eluted with solutions listed in Table 11 In specific elution an enzyme is eluted with a solution containing its substrate or coenzyme A substrate or coenzyme concentration below 10 mM usually is sufficient for elution 11 Eluents for exhaustive elution from AF Toyopearl resins 2 M KCI or 3 M NaCl 1 96 Triton X 100 1 M NaSCN 75 ethylene glycol 4 M urea or 0 1 M NaOH 4 2 M NH SO Choice Choice 2 Cleaning Toyopearl resins can be cleaned in the column or removed from the column and treated in bulk The cleaning method and duration of treatment depend on the extent of contamination At least three bed volumes of cleaning solution are typically employed in column washing procedures SEC Resins In most cases the resins can be cleaned simply by washing with distilled water to desorb remaining proteins For more tenaciously bound materials the following solutions may be required lonically bound materials For moderately bound materials 0 5 1 M aqueous salt solutions c
12. High HETP Low HETP Probe molecule retained on column due to interaction with functional group or backbone Injection sample or detector too far from column Injection volume too high Column not packed efficiently HETP Height Equivalence of a Theoretical Plate TOYOPEARL TOSOH BIOSCIENCE TOSOH COLUMN OPERATION Il Column Operation 1 Chromatographic Separation 1 1 Size Exclusion Chromatography SEC Equilibrate the resin with 5 10 column volumes of an appropriate buffer solution see Table 1 Size exclusion separations on Toyopearl HW columns are performed under isocratic conditions using buffered salt solutions of moderate ionic strength Sample volumes are usually 1 3 of the column packed bed volume If retention times are shorter or longer than expected changes in the mobile phase may be necessary Please refer to Table 7 for suggested mobile phase changes ele Non ideal SEC behavior Observation Cause Solution Retention time Is shorter than expected Sample can be partially or totally excluded from column confirm MW of sample and use a resin with higher exclusion limit if necessary Anionic molecules can be repulsed by ionic exclusion increase the ionic strength of the mobile phase Retention time Cationic molecules can be Is longer than expected retarded by ionic attraction increase the ionic strength of the mobile phase Hydrophobic molecules
13. IC IEC 65 um IEC 100 200 um HIC 65 um Peak Biotech DAN Process 30 x 19 21 30 x 20 30 x 20 13 000 17 000 salt 100cm h 6 000 8 000 salt 100cm h 4 000 salt 80cm h HIC IEC 20 um HIC IEC 35 um IEC 65 um Proxcys CRIO radial flow 5 20 liter BV 6 116 cm L 1 0 1 2 3 000 7 000 salt 100cm h IEC 65 pm These examples show real values for any packing condition given It need not to be the achievable optimum We have more than 10 years of experience in packing production columns of various manufacturers Please call our specialist for your individual discussion In addition we assist you on site TOYOPEARL TOSOH BIOSCIENCE TOSOH YOUR NOTES 7 WWW TOSOHBIOSCIENCE COM 22 TOSOHBIOSCIENCE 7 PROCESS TOSOH BIOSCIENCE PROCESS To get an overview about the whole range of our bulk media for biopurifi columns and small bulk cation please request our media please request our process catalog chromatography catalog TOSOH Chromatography Catalog YOUR WEL iS iil TOSOH BIOSCIENCE For a deeper insight into applications and all questions related to the practical use TSK GEL and Toyopearl check our website www tosohbioscience com and the related catalogues or inst ruction manuals Our technical experts are happy to discuss your specific separa tion needs via hotline 49 0 1805 012299 or mail te
14. IC Resins In most cases the resins can be cleaned simply by washing with distilled water to desorb remaining proteins For more tenaciously bound materials the following solutions may be required lonically bound materials For moderately bound materials 0 5 1 M aqueous salt solutions can be used to clean the resin For more strongly bound materials 0 1 0 5 M sodium hydroxide Or an appropriate acid such as hydrochloric or sulfuric is appropriate Under no circumstances should nitric acid be used to clean Toyopearl resins Because acids sometimes cause protein aggregation first use an alkaline solution for removing proteins Hydrophobically bound materials 10 40 of an alcohol such as ethanol methanol or isopropanol can be used to remove hydrophobic materials Solvents such as acetonitrile and acetone can also be used It is Important to remember that solvents can sometimes cause protein aggregation Non ionic detergents may also be used for cleaning After using any base acid or organic solvent use distilled water as a final rinse AFC Resins High concentrations of neutral salts chaotropes or detergents such as those listed in Table 9 should be used as eluents prior to extensive cleaning efforts Remaining protein contaminants adsorbed on the resin can be removed by washing with two column volumes of 0 5 M sodium hydroxide followed by distilled water Sodium hydroxide should be used with AF Heparin and AF Protein only
15. M 2 2 5 000 3 500 3 000 5 5 5 000 3 900 10 8 5 000 2 500 im 21 0 4 000 2 200 1 500 31 0 2 000 1 200 40 0 1 800 1 000 2 2 6 000 4 000 2 000 5 5 6 000 4 000 IEC 10 8 6 000 4 000 21 0 4 000 2 600 2 000 31 0 2 000 1 000 40 0 1 500 750 2 2 6 000 4 000 2 000 5 5 6 000 4 000 HIC 10 8 6 000 4 000 21 0 4 000 2 600 2 000 31 0 2 000 1 000 40 0 1 500 150 2 2 4 000 55 4 000 10 8 4 000 AEG 21 0 2 600 31 0 2 000 40 0 1 500 If there is a large deviation from expected plate height number and assymetry factors please repeat the packing procedure If column diameters 40 cm are utilized the number of plates M can slightly decrease For further details call our Technical Specialists TOYOPEARL How to calculate efficiency amp asymmetry Factor Efficiency Plates per column N 5 54 VW y 2 V elution volume at the peak maximum Wy width of peak at half height h 2 jection Asymmetry Asymmetry A b a A is measured at 1076 of the peak height h jection Troubleshooting performance evaluation Column not packed tight enough Clogged screens or frits at top or bottom of the column Small void at top of column Air pockets in column hardware void Overpacking the column Packing at too high pressure Column bed cracking spaces Poor injection technique
16. S COLUMN OPERATION TABLE 10 SST Mobile phase additives for HIC Organic Additives Detergents Chaotropic Agents ethanol Triton X 100 guanidine hydrochloride methanol octyl glucoside tetraethylammonium chloride Isopropanol Tween 20 urea n butanol SDS potassium thiocyanate acetonitrile CHAPS ethylene glycol Emulgen 911 CTAB Lubrol PX 1 4 Affinity Chromatography AFC Included among the Toyopearl affinity resins are both group specific ligand resins Chelate Red and Blue HC and resins with surface chemistries that allow attachment of custom ligands by the end user Contact Tosoh Bioscience Technical Service for information concerning coupling chemistries for the attachment of ligands to Formyl Carboxy Amino Epoxy and Tresyl Toyopearl Equilibration AF Red AF Blue HC and Chelate resins should be equilibrated with 3 5 column volumes of the appropriate starting buffer such as phosphate or Tris with little or no salt The dye affinity chromatographic resins may release a small amount of conjugated dye during storage Be sure to wash the dye affinity columns before each use to remove the released dye Wash a column containing new resin with 1 M sodium chloride or 1 M potassium chloride Use 2 M potassium chloride or 4 M urea for washing used resin Equilibrate a column containing old or new resin with an appropriate starting buffer such as 20 mM phosphate at pH 7 5 Loading and Elution After applying the sample
17. an be used to clean the resin For more strongly bound materials 0 1 0 5 M sodium hydroxide or 0 1 0 5 M hydrochloric or sulfuric acid is appropriate Under no circumstances should nitric acid be used to clean Toyopearl resins Nitric acid can react violently with Toyopearl resins Because acids sometimes cause protein aggregation first use an alkaline solution for removing proteins Hydrophobically bound materials About 10 20 of an alcohol such as ethanol methanol or isopropanol can be used to remove hydrophobic materials Solvents such as acetonitrile and acetone can also be used It is important to remember that solvents can sometimes cause protein aggregation After using any base acid or organic solvent use distilled water as a final rinse IEC Resins For moderate contamination wash with 0 5 1 M sodium chloride then equilibrate with the starting buffer For severe contamination wash with 0 1 0 5 M sodium hydroxide then with 0 1 0 5 M sodium chloride then equilibrate with the starting buffer For extremely severe contamination of DEAE and OAE resins wash with 0 1 0 5 M sodium hydroxide then distilled water then 0 1 0 5 M hydrochloric acid and then 0 1 0 5 M sodium chloride Equilibrate with the starting buffer A high salt mobile phase can be used as a final rinse to assure the correct counter ion is present TOYOPEARL TOYOPEARL TOSOH BIOSCIENCE TOSOH COLUMN OPERATION H
18. brate the column in the initial mobile phase Elute adsorbed proteins by decreasing the concentration of salt in the eluent Proteins with lower hydrophobicity are eluted earlier and at higher salt concentrations than more hydrophobic proteins If the desired protein is not eluted by this method add a small percentage of organic solvent or nonionic detergent change the eluent pH or lower the temperature See Table 10 for suggestions on what organic solvents detergents or chaotropes to use If sample profiles are inconsistent first increase the column equilibration step by using an additional 3 to 10 column volumes of starting eluent If the desired protein is not adsorbed on the column increase the concentration of salt in the starting buffer or adjust the pH of the buffer closer to the isoelectric point of the protein TABLEY Neutral salts used in HIC Salt listed in decreasing order Comments of strength Sodium Citrate May exhibit high UV absorbency prone to microbial growth Not stable above pH 8 low UV interference resists microbial growth most commonly used salt for HIC Solubility is low 1 5 M at 25 C Halide salt can be corrosive to stainless steel inexpensive Ammonium Sulfate Sodium Sulfate Sodium Chloride Halide salt can be corrosive to stainless steel Potassium Chloride based on the Hofmeister series of lyotropic salts 7 TOSOHBIOSCIENCE PROCESS TOSOH BIOSCIENCE PROCES
19. can be retarded by hydrophobic attraction decrease the ionic strength of the mobile phase or add a small percentage 10 20 96 of an organic solvent such as methanol ethanol or acetonitrile 1 2 lon Exchange Chromatography IEC Equilibrate the column with 5 to 10 column volumes of an appropriate starting buffer solution Table 8 The elution is performed by increasing the salt concentration or changing the pH of the eluent If the ion exchanger fails to adsorb the desired protein change the pH of the equilibration buffer to enhance the electrostatic interaction between the protein and the or decrease the salt concentration in the equilibration buffer 7 WWW TOSOHBIOSCIENCE COM TABLE8 TTT Examples for buffers used in IEC Resin Type Buffer Buffering Range Cation Exchangers Acetic acid 4 8 5 2 Citric acid 4 2 52 MES 5 5 6 7 Phosphate 6 7 7 6 HEPES 7 6 8 2 Anion Exchangers L Histidine 5 5 6 0 Imidazole 6 6 7 1 Triethanolamine 1 3 1 1 Tris HCI 7 5 8 0 Diethanolamine 8 4 8 8 1 3 Hydrophobic Interaction Chromatography HIC Equilibrate the column with an appropriate buffer solution containing a concentrated generally 1 M to 3 M neutral salt such as one listed in Table 9 High ionic strength enhances the hydrophobic interaction between proteins and the resin and thus facilitates adsorption Before introducing a sample onto the column make at least one blank analysis and equili
20. chsupport sep tosoh com TOSOH TOSOH BIOSCIENCE Zettachring 6 70567 Stuttgart Germany Tel 49 0 711 132570 Fax 49 0 711 13257 89 info sep eu tosoh com www tosohbioscience com 100 from well managed forests 3 9 18 www fsc org Zert Nr IMO COC 028664 FSC 1996 Forest Stewardship Council
21. e column The slurry concentration is calculated as the volume of settled gel divided by the total volume of the slurry and the slurry concentration is adjusted as follows a Resuspend the resin slurry in the de fining vessel and transfer the homogeneous slurry to a graduated cylinder b Allow the slurry to settle overnight gt 12 hours for best results c Determine the settled resin volume and adjust the slurry concentration to 30 50 by adding or removing packing buffer d For packing a column of a given volume use the following amounts of settled resin HW 40 HW 50 HW 55 use approximately 1 1 x the column HW 65 and HW 75F sss Ether 650 Phenyl type Butyl type Hexyl 650 PPG 600 DEAE type Q type CM 650 SP type Giga Cap type and all affinity QAE 550C and SP 550C Toyopearl MegaCap II SP 550EC volume use approximately 1 2 x the column volume 3 TOYOPEARL W I TOYOPEARL TOSOH BIOSCIENCE TOSOH PACKING 1 5 Alternative Slurry Preparation a Re suspend the resin slurry in the de fining vessel and transfer the homogeneous slurry to a Buchner funnel or equivalent b Filter the slurry under suction until the slurry becomes a wetcake all excess liquid has been removed c Weigh out the appropriate amount of resin wetcake 1 g of wetcake 1 ml of gravity settled gel using the above table d Transfer the wetcake to a beaker and add enough pac
22. er to 4 times the resin volume and stir thoroughly Example for Fine Removal 5 liter resin ordered 7 8 liter of suspension in total 65 70 slurry concentration Fill in a 20 liter vessel and fill up with 12 liter water b Allow the resin to settle Settling time is dependent on the vessel height the slurry concentration the solvent and the resin particle size The average settling times for Toyopearl resins in water in a typical measurement cylinder are Toyopearl Pore Size Minutes Coarse C Grade Oum 000 15 30 Medium M Grade 65 um 30 45 Fine Grade 45 um 45 60 Superfine S Grade 35 um 60 90 TOSOHBIOSCIENCE PROCESS TOSOH BIOSCIENCE PROCESS PACKING In larger tanks sedimentation of particles takes longer 50 slurry 25 slurry 65 um particles need 65 um particles need 3 4hours per meter SD 1 5 2 5 hours per meter SD 35 um particles need 35 um particles need 5 7 hours per meter SD 2 3 5 hours per meter 65 um particles need 65 um particles need 3 5 hours per meter SD 2 3 hours per meter SD 35 um particles need 35 um particles need 12 16 hours per meter SD 3 7 hours per meter SD in 1 8 M 65 um particles need 65 um particles need NHj SO 6 9hourspermeterSD 4 8hourspermeterSD in 20 65 um particles need ethanol 6 hours per meter SD SD Sedimentation Distance c Once the resin has settled carefully decant the supernatant Re suspend
23. esin slurry Column filled to half height with Packing Buffer Peristaltic Pump To Waste I After bed consolidation is complete stop the pump and shut the bottom outlet m Loosen the seal on the flow adapter and gently place the flow adapter onto the resin bed Be careful not to allow resin past the column seal n Repeat steps I through m until there is no further bed compression from the flow adapter 0 5 cm o In the final step lower the adapter 1 5 mm into the bed p The column is now ready for an efficiency evaluation see page 9 TOSOH BIOSCIENCE 7 PROCESS PACKING 3 Equilibration and Efficiency Evaluation Once the packing operation is completed equilibrate the column with 5 10 column volumes of low ionic strength buffer Test the effectiveness of the packing procedure by injecting a sample 0 25 196 of the column volume of a low molecular weight unretained compound i e acetone Vitamin B12 sodium chloride and determine the column plate count and asymmetry as shown in Figure 15 Columns packed according to the above procedures and operated at linear velocities of 50 250 cm h depending on the particle size should have the minimum plate counts listed in Table 5 and asymmetries between 0 8 1 5 when tested JABLE5 _ Typical packing buffer Mode Column S Grade FGrade M Grade C Grade ID plates M plates M plates M plates
24. igure 14 attach a peristaltic pump to the bottom outlet of the column c Ensure that the column is leveled prior to packing d With the pump running in the upflow direction backflow packing buffer into the column until it is about 50 full Stop the pump e With the pump running at the desired flow rate in the downflow direction slowly add the homogeneous resin slurry to the column Pour the slurry down along the inner wall of the column to prevent the formation of air bubbles f When the bed is almost entirely formed and with approximately 2 3 cm of buffer above the bed shut off the pump and column outlet valve g Gently rinse down the inside walls of the column with a squirt bottle containing packing buffer h Carefully place the flow adapter into the column with the adapter just touching the packing buffer i With the adapter firmly in place place the pump in front of the column Eliminate air in tubing j Start the pump at a low flow rate open the bottom valve k Slowly ramp up to the target flow rate This prevents hydraulic shock to the forming bed and therefore prevents uneven packing of the column The flow rate can be ramped up in several ml minute increments over the initial phase of the packing The size and duration of these increments will be determined by the size of the column which is being packed see Table 4 7 WWW TOSOHBIOSCIENCE COM Assisted cravity packing method Beaker with 50 r
25. king buffer to make a slurry concentration of 30 50 96 TSKgel perQ 5PW 20 D amp a cc __ Wa aa HBUHEB Settled resin in water 57 7 WWW TOSOHBIOSCIENCE COM 3 come f c ll Ml Mm E 100 Soe 2 TSKgee 22 uperQ 5PW 20 z rer dad Mati S J 25mL 43383 7 TOSOHBIOSCIENCE 9 PROCESS TOSOH BIOSCIENCE PROCESS PACKING 2 Packing Procedures Do not pack Toyopearl like traditional soft gels For best results Toyopearl should be packed at a higher flow rate and pressure Packing and operating velocities for Toyopearl resins LABORATORY SCALE Resin Type SEC HW 40 HW 50 HW 55 HW 65 HW 75 IEC DEAE 650 SuperQ 650 CM 650 SP 650 Giga Cap S CM Q SP 550 QAE 550 Toyopearl MegaCap II SP 550 HIC Ether 650 Hexyl 650 Butyl 600 Phenyl 650 PPG 600 Butyl 650 SuperButyl 550 PPG 600 Phenyl 600 AFC AF Amino 650 AF Tresyl 650 AF Carboxy 650 AF Blue 650 AF Formyl 650 AF Chelate 650 AF Epoxy 650 AF Blue 650 PROCESS SCALE The packing velocity in process scale columns should be at least 1 5 x the operating velocity Column Size cm ID x cm L 2 2x 60 2 2 x 60 2 2 X 60 2 2 x 60 2 2 X 60 2 2 x 20 2 2 x 20 2 2 x 20 22 10 Grade S 30 um F 45 um C 75 um S 30 um F 45 um S 30 um
26. lied Constant Velocity Method Slowly increase to the final flow rate This prevents hydraulic shock to the forming bed and prevents uneven packing of the column bed The flow rate can be ramped up in several incremental changes These increments will be determined by the size of the column and target flow rate Some examples are listed in Table 4 TOSOHBIOSCIENCE 9 PROCESS TOSOH BIOSCIENCE PROCESS 7 PACKING Pressure Method Slowly ramp up to the target pressure This prevents hydraulic shock to the forming bed and therefore prevents uneven packing of the column The pressure can be maintained by manually decreasing the flow rate to keep a constant pressure on the forming bed The optimal packing pressure for Toyopearl resins is around 3 bar 44 psi across the bed length 4 Typical packing buffer Column Size MediaType TargetFlow Increment Hold Time ID x L Rate ml min ml min min 22cmx80cm HW 5S 0 05 05 9 30 QAESSC 300 0 2 E 25cmx30cm DEAE 650M 200 400 3 i After the bed has fully formed shut off the pump and close the column outlet z FIGURE 10 Clear supernatant of sedimenting resin j The entire bed should reside in the lower column section if using a packing reservoir Using a pipette or pump siphon the supernatant from the upper reservoir Remove the upper reservoir and the coupling ring k Carefully place the flow adapter into the column app
27. lowing a 4 hour treatment with 0 5 N NaOH followed by a wash with 3 column volumes of endotoxin free equilibration buffer TOYOPEARL PPG 600M i y it punit Part No 07477 lot Na 5 FON iN VITRO LSE OMF i ji Hi ca i 9 7 Preservative Removal Shipping solvents for Toyopearl resins contain 2096 ethanol with exception of some affinity products The resin preparation procedures outlined in this document will reduce the ethanol level in the packed column effluent Column Frits Pressure related problems are often caused by clogged column frits Remove the frits and clean thoroughly as recommended by the column manufacturer If the problem persists replace the frits TOYOPEARL i Las ag tv Led Chromatography ond Both Type Po Net Vol 1000mL poems d Part No 21948 SSE lot No 65BUMBO5H M sas TOSOH omm 156 Keystre Dra itm lm LIN en im B miam ril 1 n FOR IN VITRO USE ONLY TOSOH Mig gua sea mn E wie copes aa W TOYOPEARL mel A TOYOPEARL TOSOH BIOSCIENCE TOSOH TOYOPEARL PRODUCT OVERVIEW EL Toyopearl Resin Anion Exchangers SuperO 650 S M C OAE 550C DEAE 650 S M C GigaCap 650 M 75 um 600 AR S 35 um M 65 um C 100 um Cation Exchangers CM 650
28. roximately 2 3 cm away from the consolidated bed Avoid introduction of air into the column Secure the flow adapter in place begin the pump as described in Step h Pressure Method and open the column outlet TOYOPEARL FIGURE 11 Flow adapter place m The bed will compress further When compression is complete and pressure is stable stop the pump and close the column outlet n Carefully loosen the flow adapter seal and lower the adapter near to the resin bed Take care not to disturb the resin bed when moving the flow adapter 2 1 0 7 Bubble free recommendable Air disturbs homogenious settling procedure o Repeat Steps 1 n until there is no further compression of the resin bed from the flow adapter 0 5 cm It will usually take 2 3 iterations until the bed is stable p In the final step lower the adapter 1 5 mm into the bed q The column is now ready for efficiency evaluation see page 8 TOYOPEARL TOSOH BIOSCIENCE TOSOH PACKING 2 2 Alternative Packing Method Assisted Gravity Due to hardware constraints it may not be possible to use a reservoir when packing Toyopearl resin The following method was developed to pack the resin without a packing reservoir a Adjust the resin slurry concentration to 50 and gently resuspend the resin with overhead stirring Do not use a magnetic stirrer b As shown in F

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