BioPilot™ Column Mono Q™ 60/100
Contents
1. 2 Inserta new prefilter making sure the lower support screen is still in place 3 Replace the upper support screen and replace the top of the prefilter holder to finger tightness It is recommended to change the inlet prefilters regularly 6 12 Changing the adapter distribution unit Equilibrate the column in 2096 ethanol before removing the adapter Adapter removal 1 Disconnect the column inlet from the system 2 Note the height of the packed bed 3 Remove the column adapter unit by turning the head of the column counter clockwise until the unit can be lifted out 14 BioPilot Column Mono Q 60 100 User Manual 71 6087 00 AH 6 Maintenance 6 1 Column 6 1 2 Changing the adapter distribution unit Distribution unit replacement 1 2 Remove the adapter pre filter top piece and the black securing ring Lift the adapter pre filter housing free A length of preflanged tubing connects the pre filter housing to the distribution unit at the bottom of the adapter Use the supplied plastic wrench to disconnect the tubing from the pre filter housing Use the supplied filter tool to loosen the distribution filter unit by turning counter clockwise Remove the distribution filter unit from the adapter body and replace with the new unit Tighten the unit with the filter tool Reconnect the preflanged tubing of the new distribution unit to the pre filter housing Use the plastic wrench to tighten a halfturn Replace the pre filter2. 52 Ra 3 Description Description Media Mono Q is a strong anion exchanger based on MonoBeads a beaded hydrophilic resin The functional group on the matrix is the quarternary amino group identical to that used in Q Sepharose High Peformance and Q Sepharose Fast Flow Figure 3 1 The selectiv ity of Mono Q is analogous to both Q Sepharose High Performance and Q Sepharose Fast Flow The exclusion limit of Mono Q is approximately equivalent to M 107 for globular proteins allowing higher dynamic capacity for both large and small molecular weight proteins The ionic capacity of Mono O is 0 27 to 0 37 mmol Cl ml medium The particle size range is 10 um 0 6 um CH N CH3 Figure 5 1 Quaternary amine group Column BioPilot Column 60 100 has an internal diameter of 60 mm and a packed bed height of 100 mm The volume of the packed bed is 300 ml All wetted parts of BioPilot Column 60 100 are manufactured from fully biocompatible materials fluoroplastic borosilicate glass polypropylene polyethylene ETFE tubing and Titanium The pressure limit on BioPilot Column Mono Q 60 100 is 2 MPa 20 bar in the BioPilot system The maximum flow rate is 94 ml min flow velocity 200 cm h at room temperature in aqueous buffer This corresponds to a process capacity of 20 column volumes per hour Note Flow velocity cm min is calculated by dividing the volumetric flow rate ml min by the cross sectional area of the column
3. Chemical stability BioPilot Column Mono Q 60 100 may be used in aqueous media over the pH range 2 to 12 for continuous operation It is also designed to withstand the rigorous conditions used for cleaning in place CIP and process hygiene procedures For details see Sec tion 5 3 Cleaning in place CIP on page 11 Section 5 4 Column hygiene on page 12 and Section 6 2 Media on page 15 Chaotropic agents 6 M guanidinium hydrochloride 8 M urea and detergents cationic and non ionic can be used as well as polar organic solvents such as ethanol All strong oxidizing agents should be avoided BioPilot Column Mono Q 60 100 User Manual 71 6087 00 AH 7 3 Description 3 4 Column properties 3 4 Column properties Property Description Type of exchanger Strong anionic lonic capacity 0 27 to 0 37 mmol ml Column volume 300 ml Max flow rate 94 ml min 200 cm h Max column pressure 2 MPa 20 bar Bead size dp50 10 um Efficiency gt 25 000 N m Exclusion limit M 107 Chemical stability for CIP 1M NaOH 1 M acetic acid 8M urea 6 M guanidinium hydrochloride 3096 isopropanol 3096 acetonitrile 7096 ethanol pH stability working range 2to 12 pH stability CIP 2to 14 l inBioPilot System 8 BioPilot Column Mono Q 60 100 User Manual 71 6087 00 AH 4 Installation 4 Installation BioPilot Column Mono Q 60 100 is designed to connect to BioPilot System 1 Remove the stop plug from
4. housing and secure with the black securing ring Adapter replacement 1 Fill the adapter with 20 ethanol by connecting to the system and starting the pump at a flow rate of 10 ml min Disconnect the adapter Replace the adapter unit in the column body making sure the groove in the adapter is in line with the Allen screw in the column body Turn the head of the column clockwise until the adapter rests on the packed bed Continue to turn the column head until the bed is compressed as much as possible Reconnect the column to the system and pump 20 ethanol through the column at 2 MPa 20 bar back pressure until the level of the bed has dropped 1 mm Stop the pump and readjust the adapter position Repeat this procedure until the bed has been compressed to approximately its original height Finally turn the adapter head 1 turn Equilibrate the column as described in Section 4 1 Startup and equilibration on page 9 If more extensive washing of the frit is required remove the o ring from the adapter distribution unit or bottom piece and place the unit or bottom piece complete with mounted frit only in the cleaning solution 6 2 Media If fouling of the media is suspected specific cleaning procedures should be employed When the cause of fouling i e the problem causing material present in the raw material is known one or more of the following procedures should prove useful BioPilot Column Mono Q 60 100 User Manual 71 6087
5. of General Electric Company BioPilot Mono Q MonoBeads Sephadex and Sepharose are trademarks of GE Healthcare companies 2010 2012 General Electric Company All rights reserved First published Sep 2010 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 repre sentative for the most current information GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Japan Corporation Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan 71 6087 00 AH 07 2012
6. 00 AH 15 6 Maintenance 6 2 Media 6 2 1 Precipitated proteins 6 2 1 Precipitated proteins Use the procedure employed for the extraction of the protein Wash with 1 M NaOH at a flow velocity of 20 cm h 10 ml min for 2 hours rinse with 1 5 column volumes of water then elute the solubilized protein with 1 column volume of a strong salt solution e g 2 M NaCl If the protein is soluble in acetic acid wash with 1 M acetic acid at a flow velocity of 20 cm h for 2 hours Fill the column with 0 1 pepsin in 0 05 M HCl and leave overnight at room temperature 6 2 2 Hydrophobic peptides These are usually soluble in organic solvents e g ethanol If the percentage organic solvent which best dissolves the contaminant is known run this overnight Use low flow velocities 20 cm h to conserve solvents as the contact time is most important If the optimal percentage is not known wash with saw tooth gradients 0 100 0 of organic solvent and water 6 2 Nucleic acids charged sugars DNA and RNA are very soluble in solutions of low ionic strength e g 10 mM Tris HCl 1 mM EDTA pH 8 0 Prolonged washing 24 hours with this solution can in most cases resolubilize nucleic acids which have precipitated on the column 6 2 4 DNA RNA chemical fragmentation A 2 M NaOH wash for 1 hour will hydrolyze both DNA and RNA Rinse with 1 5 column volume of water and desorb the small DNA RNA fragments with 1 column volume of 3 M KCI 6 25 Lipid
7. 1 6087 00 AH 11 5 Operation 5 3 Cleaning in place CIP Purpose Procedure Protocol 3 Removal of lipid and very hydropho Wash the column with 5 column volumes of bic proteins 70 ethanol at 40 cm h All flow velocities are defined at room temperature 5 4 Column hygiene 5 4 1 Sterilization 12 NaOH is an effective cleaning agent for chromatographic media and it has also been shown to be very effective for the destruction of bacteria and yeast cells which could contaminate the media Tables 5 1 and 5 2 show the relationship between the concen tration of NaOH and the inactivation time of Gram negative Pseudomonas aeruginosa bacteria and Gram positive Bacillus subtilis spores Table 5 1 Time required for inactivation of Pseudomonas aeruginosa at room tem perature 0 15 16 0 25 16 0 5 5 1 0 4 Data from Dr F Hasko Institute of Haematology Budapest Hungary Table 5 2 Time required for 99 inactivation of Bacillus subtilis spores at different temperatures NaOH conc M 4 C 20 C 0 5 76h 8h 1 0 22h 2h Data from Whitehouse R L Clegg L F L Journal of Dairy Research 30 315 322 1963 Note The efficiency of NaOH is both time and concentration dependent For efficient sanitation 0 5 M NaOH should be used and the sanitation should preferably be carried out at room temperature to minimize the contact time required Mono Q is well suited to these sani
8. 2 MPa 20 bar The column is now ready for use 4 2 Solvent and raw material preparation To ensure long column life and trouble free operation of the BioPilot columns it is essential to take great care in preparing both the buffers and the raw material for chromatography Itis very important to filter all liquids entering the column All solvents should be degassed by sonication or vacuum filtration and filtered through 0 45 um filters When running under sterile conditions 0 22 um filtration should be employed Water should be of highest possible purity Solvents salts and buffers should be of analytical or pharmaceu tical quality to ensure maximum column life length BioPilot Column Mono Q 60 100 User Manual 71 6087 00 AH 9 4 Installation 4 2 Solvent and raw material preparation Recommended buffers are listed in Chapter 8 The quality and composition of the raw material will directly determine the life time of the column e For ion exchange the lipid content should be kept as low as practically possible e Turbid solutions should not be applied e Endogenous protein should be fully solubilized e Particulate matter should be removed by filtration or centrifugation An efficient way to get maximum protein loading capacity on the BioPilot column is to condition the raw material by transferring the protein solution to the column starting buffer by buffer exchange using Sephadex G 25 Medium or Coarse Low molecular weight charged
9. GE Healthcare Life Sciences BioPilot Column Mono Q 60 100 User Manual Table of Contents Table of Contents RR YR HNN eae serein erent RHWYF HWY 5 E IMPAR 6 3 DCSE TON aria 7 3 1 Media 7 3 2 Column ee 7 SA Ghemical stabilibu een ee 7 34 gt COMA Properties huequito 8 G A O FFR TPN ee ee 9 41 StdrbupandedguiliDratiOP e ciertos artis 9 4 2 Solvent and raw material preparation uuuuuuuuu LLU IIIUUHHIIUHH 9 43 LOGGING CODGOCIby etarra 10 OPEN Ran 11 5 1 Regeneration sala 11 5 2 dEquilibtatior ia 11 5 3 Cledning in place Pinal 11 54 OO 12 544 Stenliza Mtra bi rd dida Heinen 12 5 47 SA 13 6 Maintendnc aeu eie Y Y dui GYF GT Y FF iu GT FFF GYLL 14 GU CO TE Y oda NFYFC ANY HN AN NWFN FF HN 14 613 Changing the Preferred mesitas mico 14 6 1 2 Changing the adapter distribution unit 14 6 2 Medigi etm dicite i ea FFF FN 15 6 21 Precipitated proteins sse ii 16 6 2 2 Hydrophobic Deptides issues nacimos 16 6 23 Nucleic acids charged sugdrs i eter dci 16 6 2 4 DNA RNA chemical fragmentation m 16 625 idone titius GU Dae AEE edidi ease 16 6 3 Testing the CONTI iuueni ctr A 17 AN AAA 17 6 35 2 BdCK DIeSsul amp enu tete 17 7 Spore parts and accessories ae 18 8 Choice Of DUMMET nee iaa 19 o SEODUEG re ran cites nr enter adatra tsaa UE MEER 20 seriei control lee NE meneame 21 BioPilot Column Mono Q 60 100 User Manual 71 6087 00 AH 3 1 Introdu
10. btained before the raw material is applied to the column 5 3 Cleaning in place CIP After repeated separation cycles at high protein loads a progressive buildup of contam inating material can occur on the column This material may not be removed by the re generation step described above The nature and degree of contamination will depend on the raw material and the chromatographic conditions employed and these should be considered when designing a cleaning protocol The following are CIP protocols which can be used as guidelines to formulate a cleaning protocol specific for the raw material to be applied The frequency of use will depend on the raw material but it is recommended to use a CIP protocol at least every 10 cycles during normal use If the nature of the contamination is unknown then protocols 1 3 can be used in sequence separated by a wash of one column volume of water After cleaning in place CIP procedures using acidic or basic solutions wash the column with two column volumes of neutral buffer before final re equilibration or storage in 20 ethanol Purpose Procedure Protocol 1 Removal of strongly ionically bound Wash the column with a 1 2 column volume protein of filtered 2 M NaCl solution at 50 cm h Protocol 2 Removal of strongly bound hydropho Wash the column with 4 column volumes of bic proteins or lipoproteins 1 M NaOH solution at 40 cm h BioPilot Column Mono Q 60 100 User Manual 7
11. ction 1 Introduction BioPilot Column Mono Q 60 100 are prepacked ion exchange columns for high perfor mance separation of proteins and peptides at pilot and small volume production scale They are intended for operation with BioPilot System This column contains a titanium frit in the adapter distribution unit and bottom piece After cleaning in place CIP procedures using acidic or basic solutions wash the column with two column volumes of neutral buffer before final re equilibration or storage in 20 ethanol If more extensive washing of the frit is required remove the o ring from the adapter distribution unit or bottom piece and place the unit or bottom piece complete with mounted frit only in the cleaning solution The following instructions should be used as guidelines when handling BioPilot Columns to ensure trouble free use and maximum column life length BioPilot Column Mono Q 60 100 User Manual 71 6087 00 AH 5 2 Unpacking 2 Unpacking When unpacking your BioPilot Column carefully check the contents of the box against the following list Code no BioPilot Column Mono Q 60 100 17 1002 01 Contents BioPilot Column Mono Q 60 100 Quality control test sheet Instruction manual Pressure expansion syringe Accessory package containing Filter tool 60 mm Filter kit Prefilter 6000 Tubing connector Filter tool Stop plug Wrench plastic 6 BioPilot Column Mono Q 60 100 User Manual 71 6087 00 AH 31
12. exposure to Tween 80 and ether Prince A M Horowitz B Brotman B et al Vox Sang 46 361 43 1984 Disinfection and inactivation of the human T lymphotropic virus type lll lymphoadenopathy associated virus Martin L S McDougal J S Loskowski S L J Infect Diseases 152 400 403 1984 3 Parenteral Drug Association Technical Report No 7 1985 PDA Inc Philadelphia USA BioPilot Column Mono Q 60 100 User Manual 71 6087 00 AH 13 6 Maintenance 6 Maintenance An increase in pressure drop over the column discoloration of the top of the packed bed or non reproducible function indicates contamination and or blockage in the column Thefollowing sections describe recommended procedures for dealing with contamination 61 Column Blockage of the column can be identified by isolating the column programme column by pass in BioPilot System and checking the back pressure in the system If an increased back pressure is observed locate the blockage point in the system by systematically isolating the different components in the line of flow Ifthe back pressure increase is due to a blockage in the column the following procedures should be employed 6 11 Changing the prefilter The prefilters are located at the column inlet and outlet Each prefilter is held in place by an upper and lower support screen 1 Unscrew the top of the prefilter holder and remove the upper support screen and prefilter using the filter tool
13. material such as amino acids and even phospholipids can be removed by this operation 4 5 Loading capacity 10 The practical working range of BioPilot Column Mono Q 60 100 is up to 20 mg protein per ml of medium depending on the starting material for continuous gradient elution The feed volume is not generally regarded as a limiting factor in ion exchange chromatog raphy since feed volumes of up to 20 times the column volume may be applied for con tinuous gradient elution The time taken to process large volumes however can be limiting Therefore when handling large volumes of starting material such as hybridoma cell culture supernatants rapid concentration of the starting material should be consid ered to reduce total cycle time This can be performed using Sepharose Fast Flow ion exchange media BioPilot Column Mono Q 60 100 User Manual 71 6087 00 AH 5 Operation 5 Operation 5 1 Regeneration After each separation cycle the column is regenerated by passing two column volumes of a 1M solution of the elution buffer salt through the column This step will remove any proteins that bind strongly to the column by ionic interaction under the conditions em ployed during separation 5 2 Equilibration After regeneration the column is equilibrated for the next separation step by passing at least two column volumes of the start buffer through the column at a flow velocity of 200 cm h 94 ml min A steady UV baseline should always be o
14. nd applying the appropriate flow rate BioPilot Column Mono Q 60 100 User Manual 71 6087 00 AH 17 7 Spare parts and accessories 7 Spare parts and accessories Table 7 1 Spare parts and accessories Item Code no Distribution unit 60 mm 18 4610 60 Filter tool 60 mm 18 4284 01 Filter kit Prefilter 6000 18 4584 01 Tubing connector 18 4274 01 Filter tool 18 1153 20 Stop plug 18 1102 21 Wrench plastic 19 7481 01 Spare keys for the BioPilot Column box can be obtained from your local GE Healthcare representative 18 BioPilot Column Mono Q 60 100 User Manual 71 6087 00 AH 8 Choice of buffer 8 Choice of buffer Table gives recommended buffers for various pH intervals based on runs performed in our laboratories All values were determined at room temperature Table 8 1 Buffers recommended for Mono Q on the basis of experiments performed in our laboratories pHinterval Substance Concentration pKa Counterion 5 5 to 6 0 L histidine 20 mM 5 96 Cl 5 8 to 6 4 bis Tris 20 mM 6 46 Cl 7 3 to 7 7 triethanolamine 20 mM 7 76 Cl 7 6 to 8 0 Tris 20 mM 8 06 CI 8 4 to 8 8 diethanolamine 20 mM 8 88 Cl 9 0to 9 5 ethanolamine 20 mM 9 5 Cr BioPilot Column Mono Q 60 100 User Manual 71 6087 00 AH 19 9 Column storage 9 Column storage BioPilot Column 60 100 should be stored in a solution containing an effective bacterio static agent 2096 etha
15. nol at neutral pH is recommended After cleaning in place CIP procedures using acidic or basic solutions wash the column with two column volumes of neutral buffer before storage in 2096 ethanol 20 BioPilot Column Mono Q 60 100 User Manual 71 6087 00 AH 10 Quality control test 10 Quality control test To maintain high quality standards the efficiency of each BioPilot Column is tested using the experimental conditions listed in Table 10 1 Table 10 1 Conditions for column efficiency test Sample 1 0 ml acetone 20 mg ml Eluent 50 ethanol Flow velocity Flow rate 60 cm h 30 ml min Detection 280 nm 2 mm cell 0 1 AUFS Chart speed 3 cm min The efficiency of the column is calculated using the equation N m 5 54 Vg wp 1000 L L bed height mm Vp peak retention elution volume Wh peak width at half peak height The parameters Vp and w can be measured as shown below A VR Wh Figure 10 1 Peak retention and peak width parameters The efficiency of BioPilot Column Mono Q 60 100 exceeds 25 000 theoretical plates per meter N m BioPilot Column Mono Q 60 100 User Manual 71 6087 00 AH 21 For local office contact information visit www gelifesciences com contact GE Healthcare Bio Sciences AB Bj rkgatan 30 75184 Uppsala Sweden www gelifesciences com protein purification imagination at work GE imagination at work and GE monogram are trademarks
16. s 16 For lipoproteins or hydrophobic peptides wash the column with detergent in basic or acidic solution Use 0 596 non ionic detergent in 1 M acetic acid Wash at a flow velocity of 20 cm h Residual detergent can be removed by washing with 5 column volumes of 7096 ethanol Very hydrophobic lipids can be dissolved in non polar solvents Rinse with 3096 iso propanol Then run a gradient from 3096 isopropanol to 10096 water in 10 column volumes at a flow velocity of 20 cm h All flow velocities are defined at room temperature BioPilot Column Mono Q 60 100 User Manual 71 6087 00 AH 6 Maintenance 6 3 Testing the column 6 3 Testing the column 6 3 1 Efficiency After media and or column maintenance procedures the efficiency ofthe column should be checked The efficiency of the column is an indication of column packing and perfor mance Column efficiency N can be estimated using the following equation N m 5 54 Vg wp 1000 L L bed height mm Vn peak retention elution volume Wh peak width at half peak height The experimental conditions are described in the Quality Control Test Sheet supplied with the column 6 3 2 Back pressure Estimate the back pressure in the column at a given flow rate by subtracting the pressure generated in the system up to the column inlet A from the pressure generated by the total system plus column B A can be measured by disconnecting the pre flanged tubing from the column inlet a
17. the column inlet and the pressure expansion syringe and tubing from the column outlet 2 Place the column on the P 6000 pump housing 3 Connect the column inlet to port 2 of motor valve IMV 8 No 2 using 1 2 mm i d tubing as provided in the Start Up Kit with BioPilot System and the column outlet to port 2 of IMV 8 No 3 Up to 7 different columns can be connected to BioPilot System simultaneously Note When connecting pre flanged tubing turn the tubing connector until finger tight and then use the plastic wrench for a final half turn 4 1 Startup and equilibration The columnis delivered in 2096 ethanol as an antimicrobial agent This has to be washed out before the equilibration procedure can be started 1 Wash away the 2096 ethanol using low ionic strength buffer 1 5 column volumes are needed and the flow velocity should be limited to 50 cm h 25 ml min for 60 100 columns After 1 5 column volumes of buffer double the flow rate and wash with another 3 column volumes of buffer 2 Introduce the desired counter ion by washing with high ionic strength elution buffer for 5 column volumes at a flow velocity of 100 cm h 50 ml min 3 Eguilibrate the column to starting conditions by passing 5 column volumes of start buffer through the column at a flow velocity of 150 cm h 70 ml min Note the back pressure in the system as registered by the pumps BioPilot Column 60 100 should not be exposed to a system back pressure higher than
18. tizing conditions BioPilot Column Mono Q 60 100 User Manual 71 6087 00 AH 5 Operation 5 4 Column hygiene 5 4 2 Decontamination 5 4 2 Decontamination Inactivation of biologically active material can be divided into two categories 1 Inactivation of viruses 2 Inactivation of contaminating microbial products Certain detergents organic solvents and NaOH have been shown to be effective inacti vating agents for different viruses that may originate from mammalian cell culture tissue extracts and blood plasma Martin et al have shown that 0 1 M NaOH at room temperature inactivates the HTLV III LAV virus in 10 minutes Inactivation of biologically active bacterial products typically endotoxins in the form of lipopolysaccharides can prove to be a problem E coli endotoxin has been shown to be heat resistant Investigations at GE Healthcare have shown that NaOH is effective for inactivating E coli endotoxin Figure The inactivation efficiency is highly dependent on aOH concentration 100 19 O 0 1MNaOH T 05 M NaOH E 10M NaOH Endotoxin ng ml 40 y 03 T T T 1 4 50 Time h Figure 5 1 Inactivation of E coli endotoxin in alkaline environment at room temperature Further information concerning endotoxin inactivation can be obtained from chapters 1 and 8 in reference no 3 the PDA Technical Report No 7 1 Inactivation of Hepatitis B and Hutchinson Strain NonA NonB Hepatitis viruses by
Download Pdf Manuals
Related Search