Recombinant Adenoviral Vector Purification

Transcription

1 Recombinant Adenoviral Vector Purification 1. Purpose and Backgrounds 1.1. To provide a standardized method of producing a research grade purified adenoviral vector from a cell factory of mi293-hek cells. The procedure has been modified from the method of Gerard and Meidell in DNA Cloning: a Practical Approach, BD Hanes and D Glover, eds., Oxford University Press, Oxford, 1995, pp Materials 2.1. Equipment Eppendorf 5810R tabletop refrigerated centrifuge Beckman-Coulter J2-21 Superspeed Centrifuge with JA-14 fixed angle rotor Beckman-Coulter Ultracentrifuge with SW41 or SW28 rotor Forma Scientific Biological Safety Cabinet Model # Forma Scientific CO 2 incubator Model # 3110 equilibrated to 5% CO Barnstead NanoPureUltrapure water purification system Pipet-Aid with filter unit BioRad SmartSpec 3000 and quartz cuvette with 1 cm lightpath 2.2. Supplies mm Tissue Culture Dish (Falcon /Fisher Catalog # ) Cell factory with 10 levels (Nunc /Fisher Catalog # ) Sterilized 1 liter aspirator bottle (Fisher Catalog # K) with about 90 cm of Nalgene 50 silicone tubing (Nalgene /Fisher catalog # H) and cell factory port connector (Nunc /Fisher Catalog # ) The top opening of the aspirator bottle should be covered with aluminum foil. One end of the hose should be connected to the bottle's hose connector and the other end to the cell factory port connector (to the end without an O-ring). The port connector should be wrapped in aluminum foil or covered by a 50mL conical with a hole drilled through the blue lid. The entire assembly should be steam sterilized Sterilized hose (About 90 cm of Nalgene 50 silicone tubing) and port connector. These should be connected and placed into an autoclave sleeve, sealed, and steam sterilized Sterile 0.22 micron filter unit (Millipore SCGPU05RE) Sterile 0.45 micron filter unit (Nalgene ) Ten sterile 250 ml Nalgene centrifuge bottles (Nalgene catalog # ) Two SW41 (Beckman-Coulter catalog # ) or SW28 (Beckman-Coulter catalog # ) Ultra-Clear ultracentrifuge tubes Falcon 50 ml sterile conical tubes (Falcon ) Sepharose CL-4B, about 25 ml (Fluka or Sigma CL-4B-200) ml sterile polystyrene pipets (Falcon ) ml sterile polystyrene pipets (Falcon ) inch glass Pasteur pipets sterilized (Fisher Catalog # C) inch glass pasteur pipets with cotton plugs sterilized (Fisher Catalog # B)

2 Biohazard bags (Fisher C) Autoclave sleeve (4 x 100, Allegiance Healthcare Corporation Catalog # T90004) µl filtered pipet tips (CLP Cat# Bt1000) Corning 2.0 ml cryovials (Corning /Fisher ) Toughtag cryolabels (Diversified Biotech Cat # TTLW-2016) 2.3. Reagents Household chlorine bleach (5.25% sodium hypochlorite) % Chlorine bleach Common household chlorine bleach is typically 5.25% sodium hypochlorite. Dilute 1 part bleach with 4 parts water to make a solution suitable for wiping down contaminated surfaces % NP ml IGEPAL CA-630 (NP-40) (Fluka catalog # 56741) ml de-ionized water Mix thoroughly. Store at room temperature % PEG, 2.75 M NaCl g Polyethylene Glycol 8000 (Fisher Scientific catalog # BP233-1) g NaCl (Sigma Cat# S9625) Add de-ionized water to 1 liter Sterilize by autoclaving. After autoclaving the solution will separate into two layers. Allow the temperature to decrease to about 50 o C and agitate vigorously to mix the layers together. The solution should clear forming only one layer. Alternatively, the solutions can be put onto a slow shaker plate (such as the one in the fume hood in 7731) while cooling down Store at room temperature Just prior to using for adenoviral precipitation, add 100µL of 1M Tris-HCl ph 7.4 for every 100mL of PEGNaCl mM Tris-Cl/1mM MgCl 2, ph Into a 500mL volumetric flask, add 5mL of 1M Tris-Cl ph 8.0, 500µL 1M MgCl 2, and de-ionized water up to 500mL Filter sterilize with 0.22µm filter Store at room temperature g/ml, 1.30 g/ml and 1.40 g/ml CsCl CsCl (Roche Cat# ) is dissolved in autoclaved 10 mm Tris- HCl/1mM MgCl 2 ph8.0 and the density is adjusted by adding 10 mm Tris- HCl /1mM MgCl 2 ph8.0 until 1.0 ml weighs exactly 1.10 g, 1.30g/mL, or 1.40g/mL respectively. This is filtered through a 0.22 micron Nalgene filter unit to remove particulates. Densities should not deviate from the desired levels by more than 0.01 g/ml Roughly, the 1.10g/mL solution can be made by adding 11.93g CsCl to a 100mL bottle and bringing the weight up to 100g with 10mM Tris-HCl/1mM MgCl2 ph Roughly, the 1.30g/mL solution can be made by adding 31.24g CsCl to a 100mL bottle and bringing the weight up to 100g with 10mM Tris-HCl/1mM MgCl2 ph8.0.

3 Roughly, the 1.40g/mL solution can be made by adding 38.83g CsCl to a 100mL bottle and bringing the weight up to 100g with 10mM Tris-HCl/1mM MgCl2 ph Store at room temperature M Tris-HCl ph 7.4 (Cambrex Catalog #51237) Virus Diluting Buffer (old Dialyzing Solution) Solution A 80g NaCl 2g KCl 11.5g Na 2 HPO Bring up to 1 liter with de-ionized water in a volumetric flask Filter sterilize with 0.22µm filter Solution B 1g CaCl 2-2H 2 O Bring up to 100mL with de-ionized water in volumetric flask Filter sterilize with 0.22µm filter Solution C 1g MgCl 2-6H 2 O Bring up to 100mL with de-ionized water in volumetric flask Filter sterilize with 0.22µm filter Fill a 1 liter volumetric flask about halfway with de-ionized water Add 100mL of solution A; mix gently Add 10mL of solution B; mix gently Add 10mL of solution C; mix gently Bring volume up to 1 liter with de-ionized water Mix thoroughly Filter sterilize with 0.22µm filter Phosphate Buffered Saline (Invitrogen catalog # ) Trypsin EDTA, 0.05% Trypsin and 0.53mM (Invitrogen catalog # ) Tissue culture media For cell culture: DMEM, high glucose (Invitrogen catalog # ), 500 ml Fetal Bovine Serum, 55 ml X Penicillin/Streptomycin (Invitrogen Cat# ) For virus propagation: DMEM, high glucose (Invitrogen catalog # ), 1000 ml Fetal Bovine Serum, 30 ml X Penicillin/Streptomycin (Invitrogen Cat# ) X Glutamine (Invitrogen catalog # ), 10 ml Sterile glycerol Filter glycerol (Sigma Chemical catalog # G5516) through a sterile 0.22µm filter unit in a tissue culture hood. This may require overnight filtration because of the viscosity of the glycerol. Seal in a sterile container and store at room temperature.

4 3.0 Procedure 3.1 Preparation of infectious stock propagation of the virus At least one day prior to plating cells in a cell factory, the process for preparing an infectious stock must be started. Aspirate the medium from one 15cm of confluent 293-HEK cells using a sterile Pasteur pipet This and all subsequent Pasteur pipets need to be decontaminated (by bleaching for 15 minutes or autoclaving) prior to disposal in the glass waste container Replace the medium with 20 ml of DMEM + 2% FBS + P/S + L-glutamine Infect the flask with about 10 7 to 10 8 pfu of the virus to be purified. This is usually about µl of a plaque amplification from a virus transfection or cloning. Incubate the flask at 37 o C. Alternately, 10-25µL of purified virus can be added to the cells When the cells have detached from the flask (usually about 3 to 4 days) harvest the medium and cells with a sterile 10 ml pipet in the biological hood. Transfer to a 50 ml conical tube and seal the tube. This will be the cell lysate The solution will need to go through one round of freeze/thaw in order to liberate the viruses from inside any cells that have not yet lysed. This can be done by storing the 20 o C for a minimum of 4 hours, or by submerging the tube containing the solution in a dry ice/ethanol bath for a minimum of 20 minutes. 3.2 Plating of 293-HEK cells in a cell factory On the day prior to plating cells in a cell factory place a 1-liter bottle of DMEM in a 37 o C incubator to prewarm. Place a cell factory in an incubator at 37 o C to prewarm This and all following steps except the centrifugation should be performed in a biological hood. On the day of plating cells add 110 ml of fetal bovine serum and 11mL of P/S to one liter of DMEM. Thaw about 20 ml of trypsin. Prewarm PBS ph 7.4 to 37 o C Completely aspirate off the medium from 4-6 x 15cm plates of confluent 293- HEK cells plated 3 to 4 days previously The number of plates to be used will depend on how quickly you would like the cell factory to be ready for infection. If you plate with 4 x 15cm, it will typically take 5-7 days for the cell factory to become confluent. Using 6 or even 7 x 15cm plates will usually produce a confluent cell factory within 3 days Wash the cells with 5 ml of PBS ph 7.4. Aspirate off PBS and place 3 ml of trypsin on the cells in each flask and rock flasks to evenly distribute over monolayer. Rock flask gently every few seconds until cells are completely detached. This usually requires about 2-5 minutes When all of the cells are detached use a 10 ml sterile pipet to add 7 ml of DMEM + 10% FBS + P/S to each flask to inactivate trypsin. Transfer cell suspensions to 50 ml centrifuge tube using a sterile 10 ml pipet. Place flasks and pipets in biohazard bag to be autoclaved Centrifuge the tubes in the Eppendorf 5810R centrifuge at 4 o C for 3 minutes at 1000 rpm.

5 3.2.7 As soon as the centrifuge has stopped, transfer the tubes to the biological safety hood and remove the supernatant by aspiration with a sterile Pasteur pipet. Gently resuspend the pellets in 10 ml of DMEM + 10% FBS + P/S using a sterile 10 ml pipet. Break up the pellet and any clumps by gently pipetting up and down Transfer the cell suspension to 1 liter of pre-warmed DMEM + 10% FBS + P/S and mix until the cells are evenly distributed Remove the red cover from one of the ports of the pre-warmed cell factory. Remove the paper seal from the port plug Aseptically remove the red cover and plug from the second port of the cell factory and place it aside on a sterile surface. Attach the connector from a sterile 1liter aspirator bottle to the port Transfer the cell suspension to the aspirator bottle and elevate the bottle to allow the cell suspension to flow into the cell factory Disconnect the aspirator bottle from the cell factory port and aseptically place the plug back over the port to seal it Firmly insert a sterile filter unit into the first port opened If any cell suspension bubbled out of this port, wipe the port down with a Kimwipe and ethanol prior to attaching the filter unit Lay the cell factory on its side to allow the volume of cell suspension to distribute evenly between the cell factory layers Make sure to lay the cell factory on the side away from the port with the filter unit or the filter will get wet and the cell factory will not be able to breathe Place the cell factory in a 37 o C incubator with 5% CO Make sure to level it with paper towels so that the medium depth is the same in all parts of the cell factory Place the medium bottle in a biohazard bag to be autoclaved. Rinse the aspirator bottle and hose with copious amounts of deionized water. Invert to drain. 3.3 Infection of the cell factory Once the cell factory has become nearly confluent, it is ready for infection. On the day of infection of the cell factory, prewarm 800 ml of DMEM + 3% FBS + P/S + 1X L-glutamine by placing at 37 o C Thaw the tube of cell 37 o C and vortex vigorously for a few seconds to release virus from the cell fragments Centrifuge the lysate in the Eppendorf 5810R centrifuge for 10 minutes at 4000 rpm at 4 o C Add the supernatant (containing the majority of the adenoviral vector) to the prewarmed medium and mix Discard the tube with the pellet in a biohazard bag to be autoclaved, as it still contains a large amount of adenoviral vector Transfer the cell factory to a biological safety hood and aseptically remove the red cap and plug from the port being careful to preserve the sterility of the plug, which must be reused. Attach a sterile connector and drain hose to the port and elevate and tip the cell factory to allow the medium to drain out being careful not to wet the filter.

6 Collect the medium in a beaker. The medium can be poured directly down the drain while rinsing with copious amounts of tap water Rinse the connector and hose with large amounts of deionized water, drain and air dry Attach the connector from a sterile 1liter aspirator bottle to the port. Carefully pour the medium containing the virus into the aspirator bottle. Recap and seal the medium bottle, clearly label it with the name of the virus and the date and place at 4 o C. This bottle will be used to collect the virus from the cell factory Elevate the aspirator bottle to allow the medium + virus to flow into the cell factory Carefully detach the virus - contaminated connector from the cell factory, replace foil on top of the bottle, place the connector back into the 50 ml tube and place the bottle and hose in a polypropylene tray to be autoclaved. Reseal the cell factory port with the plug and red cap and lay the cell factory on its side (on the side away from the filter unit) to allow the medium to distribute evenly. Elevate the end of the cell factory with the ports and turn it so that it is upright Carefully transfer the cell factory to a biohazard-labeled 37 o C incubator with 5% CO 2. Level it. Incubate until all of the cells have detached. 3.4 Adenoviral purification Adenoviral purification consists of five steps. First, the infected cells are treated with detergent (NP-40) to release intracellular virus (about two thirds of adenovirus is typically within the cell and about one third in the supernatant). The correct concentration of NP-40 will lyse the outer plasma membrane without lysing the nuclear membrane. Second, the cell debris is removed by centrifugation. Third, the virus is concentrated by precipitation with PEG-NaCl. Fourth, the mature virus is purified away from viral and cellular DNA, viral and cellular proteins and empty viral capsids by cesium gradient ultracentrifugation. Fifth, the cesium chloride is removed and the virus transferred to virus dilution buffer on a gel filtration column When all of the infected cells have detached from the monolayer and most clumps of cells have broken apart (usually 3-7 days) harvest cells and medium from the cell factory by pipetting 10 ml of 20% NP-40 into the port without the filter. Mix the NP-40 throughout the medium by inverting the cell factory and allowing the medium to flow between the levels, again being careful not to get the filter unit wet Incubate at room temperature for one hour Remove cell lysate from the cell factory using a sterile hose and connector into the medium bottle saved from step Evenly distribute the lysate into 4 x 250 ml Nalgene centrifuge bottles and carefully cap and seal bottles. Any spills should be treated with 1% chlorine bleach and wiped up with paper towels. Place virally contaminated hose and connector in polypropylene pan to be autoclaved. Place cell factory and any contaminated paper towels in biohazard bag, autoclave and discard Centrifuge 11,000 rpm, 10 minutes, 4 C in JA-14 rotor in the J2-21 centrifuge to pellet cell debris Supernatant can again be collected in the bottle from step

7 Squirt ~10mL of 1% bleach into old bottles with cell debris pellets and discard pellets into biohazardous waste. Place bottles in polypropylene pan to be autoclaved Add 0.5 volume 22% PEG 8000, 2.75M NaCl (final conc. 7.3% PEG, 0.92 M NaCl) to supernatant. Mix thoroughly by inverting the bottle several times Usually, this will mean adding ~400mL PEGNaCl to your ~800mL of supernatant. The 1L DMEM bottles from Gibco hold approximately 1200mL so this amount should just fit into the bottle Equally distribute virus/pegnacl solution to six clean and sterile 250 ml centrifuge bottles and carefully seal bottles. Incubate on ice for 60 minutes If PEGNaCl and Nalgene centrifuge bottles are overnight prior to this step, the incubation time can be cut down to 45 minutes Pellet virus by centrifugation at 11,000 rpm, 10 minutes, 4 C in JA-14 rotor Discard supernatant into bleach (concentrated bleach should be at least 10% of the total volume) After at least one hour pour disinfected mixture down drain with copious amounts of water Invert centrifuge bottles on several layers of paper towels to drain off remaining supernatant or aspirate remaining supernatant with a sterile Pasteur pipet. Do not allow pellets to dry completely. Dispose of paper towels in biohazard bag, autoclave and discard. Wipe down benchtop under the paper towels with 1% bleach Resuspend pellets (which may be spread all over wall of bottle) in 10 mm Tris- Cl/1mM MgCl 2 ph 8.0 containing CsCl (density= 1.10 g/ml). Typically, 5 ml total volume is used if viral pellets are small and 15 ml for larger pellets. Place resuspended viral pellet in sealable centrifuge tubes and carefully resuspend pellets by brief vortexing. Do not over vortex If the pellets do not go into suspension easily allow them to sit at room temperature for a few minutes. The CsCl solution will soften the pellets and allow more material to be resuspended by brief vortexing. This may need to be repeated several times. If pieces of cell debris pellet from steps and are present then it may not be possible to completely resuspend the solids. Spin out the remaining debris in Eppendorf 5810R (4,000 rpm for 5 min) prior to loading virus supernatant onto step gradient. Centrifuge bottles should be placed in polypropylene pan to be autoclaved. Pipets should be placed in biohazard bag, autoclaved and discarded Prepare CsCl step gradient: for 5 ml of suspension use 1 x SW41 ultracentrifuge tube. Place 3.0 ml CsCl (density=1.30 g/ml) in bottom of tube. Carefully layer 2.0 ml (density=1.40 g/ml) CsCl under previous layer, without disturbing the interface. Mark interface on side of tube. For 15 ml of suspension use 1 x SW28 ultracentrifuge tubes. Add 13.0 ml CsCl (density=1.30 g/ml) to bottom of tube. Carefully layer 7.0 ml (density=1.40 g/ml) CsCl under previous layer, without

8 disturbing the interface. Mark interface on side of tube. Accurate densities are critical Carefully layer virus over CsCl step gradient. Place empty tubes in which virus was resuspended and contaminated pipets in autoclave bag to be steam sterilized and discarded Create a balance tube identical to the one holding the virus suspension, using 1.10g/mL CsCl in place of a virus solution. This blank gradient must be balanced with the virus-containing gradient to within 0.1g Place the two gradients in rotor tubes that are exactly opposite one another. Spin in an ultracentrifuge in SW41 or SW28 rotor at 20,000 rpm for 2 hours at 20 C Alternatively, the virus can be spun at 22,000 rpm for 90 min if you are in a hurry The tubes will usually contain 3 layers and 2-4 bands. There may be a band present at the very top of the tube; this consists of various cellular proteins. Closer to the interface that was marked, there will be at least 2 bands. The upper band, which is sometimes a double band, is usually cream-colored, more diffuse than the lower band and contains empty capsids (adenoviral capsids without the viral genome packaged inside) and viral and cellular proteins. The virus band (the lower band) is a creamy white, often more defined, band found approximately at the interface between the 1.40 g/ml and 1.30 g/ml layers There may be a band found below the virus band, and it may be clumpy. This is generally genomic material. If it is inadvertently aspirated with the virus band, the clumps should be removed with a Pasteur pipet prior to application on the sepharose column, as they will hinder flow Aspirate off the layers above the virus band with a sterile Pasteur pipet and transfer the aspirant into bleach Using a vacuum is suitable for the top-most layers, but it may prove to disturb the virus band, as you get closer to it. A rubber bulb should be used to better minimize disturbance of the virus band when aspirating the layer immediately above the virus band Collect the virus layer with a sterile cotton-plugged 9 Pasteur pipet and a rubber bulb. Pasteur pipets should be placed in polypropylene pan to be autoclaved and then disposed of in glass waste box. Liquid remaining in ultracentrifuge tubes should be combined with bleach. Tubes should be placed in biohazard bags, autoclaved and discarded Transfer virus with sterile 5mL pipet to top of a disposable Sepharose CL-4B column packed in a plastic pipet (use a 10 ml column for SW41 tubes and a 25 ml column for SW28 tubes) to desalt virus (i.e. to remove the CsCl from the viral suspension). Virus will be eluted in virus diluting buffer (VDB) As soon as the virus has been placed on top of the column begin collecting the first fraction. Collect fractions in sterile polystyrene 17 x 100 tubes with caps Polystyrene tubes are used because they are a clear plastic, not cloudy, and it makes it much easier to see the virus elution When the first hint of virus elutes begin collecting the second fraction.

9 Virus is very opalescent and can be readily visualized as it elutes from column if collected in clear tubes. The virus fraction will elute just after one void volume of VDB. The void volume is about one third of the total column volume Collect the virus until the milkiness of the eluted material has significantly decreased. Cap and seal the virus fraction Never collect a virus fraction that is more than one third of the total column volume or CsCl may elute and ruin the virus fraction Pasteur pipets should be placed in polypropylene pan to be autoclaved and then disposed of in glass waste box. Collect all the material that elutes after the virus fraction and decontaminate with bleach and discard. Discard column to an autoclave bag to be steam sterilized. All liquid eluted from column which does not contain enough virus to use should be combined with bleach and tubes should be placed in a biohazard bag to be autoclaved. 3.5 Virus titration and distribution into aliquots The concentration of virus in particles/ml is determined by measuring the OD 260 of the virus fraction. Purified adenovirus type 5 will give an OD 260 of 1.0 at a concentration of 1 X particles per ml. The virus diluting buffer has been found to be suitable for direct injection in animal studies. The glycerol serves as a stabilizing agent during cryo-storage of the virus, and may not be optimal for use in vivo. Virus stored at -80 o C in this solution has been stable for at least five years. The virus is distributed to cryovials in 20µL and 500µL aliquots The virus is initially titrated by measuring the OD 260. Turn on the spectrophotometer about 20 minutes before making the measurement. Determine the background by measuring the OD 260 of 0.5 ml of virus diluting buffer (VDB) in a quartz cuvette with a 1 cm light path. Blank the spectrophotometer with VDB. Completely empty the cuvette Dilute a small sample of the virus fraction 1:10 with VDB (50 µl of virus µl of VDB) or 1:20 (25 µl of virus µl of VDB). Transfer the diluted virus to the quartz cuvette and measure the OD 260 and record the result. Dispose of diluted virus by placing in bleach. Quartz cuvettes should be cleaned by soaking in diluted bleach (1 part bleach to 4 parts water) for 30 minutes and then rinsed with water and dried Calculate the particle titer by multiplying the OD 260 of the diluted virus by the dilution factor (DF) and multiply that number by particles. The result is the concentration of the virus per ml of undiluted virus fraction. OD 260 * DF * particles = virus particles per ml Calculate the volume glycerol that must be added to achieve a final concentration of 10% glycerol. Divide the total volume (TV) by 9 to obtain the volume of glycerol (GV) that must be added. Volume of glycerol (GV) = TV / 9

10 3.5.3 Mix virus + glycerol by gentle pipetting. Pipet 50 x 20 µl aliquots of virus into sterile 2.0 ml cryovials. Aliquot remaining volume into 500µL aliquots Dispose of virus contaminated tubes, pipets and pipet tips in autoclave bag to be steam sterilized Print cryolabels (toughtags), affix labels to vials and store at -80 C. Labels should contain the name of the viral construct, the volume of viral suspension in the vial and the date. Sample Label: Ad CMV-βgal 20µL MM/DD/YY Wipe down benchtop and tissue culture hood work areas with 1% chlorine bleach. Follow by wiping down with 70% ethanol. Discard paper towels in biohazard bag and autoclave.