Objective:

The following procedure describes the clarification of retroviral particles in crude harvested supernatants containing 10 % FBS by microfiltration using a dual HT Tuffryn^® polysulfone membrane (0.45/0.2 µm) capsule filtration device.

Materials:

Procedure:

1- Thaw retrovirus supernatants using water bath at 37ºC.

2- Pellet cells and cell debris by centrifugation at 10,000×g for 10 min. Decant the supernatant into a sterile bottle.(Note: Using this rotor the manufacturer informs that viruses with a sedimentation coefficient of ~700S would pellet in 90min at 15,000×g or 10,000rpm).

3- Aliquot starting material samples for analyses and measure the starting volume. Keep the supernatant at 4ºC until further use.

4- Set up the filtration system in the biological safety cabinet. Flush the microcapsule membrane with 500 mL of Milli-Q H₂O and empty the tubing.

5- Filter supernatants at constant flow rate (start at 10mL/min and gradually increase to 50 mL/min). Collect the permeate in sterile bottles. Check the pressure at all times. Using the capsule filtration device with an effective filtration area of 500 cm², 1-2 L of retrovirus vector supernatants can be filtered with low back pressure (below 0.01 MPa).

6- Aliquot clarified supernatant samples for analyses and measure the final volume.

7- Keep the clarified supernatant at 4ºC until the next downstream processing step or store at -80ºC along with the aliquoted samples.

Objective:

The following procedure describes the concentration of retroviral particles in clarified supernatants containing 10% FBS by ultra-diafiltration using Omega^TM polyethersulfone membrane disc filters (MWCO 300,000) and stirred cell units.

Materials:

Procedure:

1- Use the following table to select the stirred cell model and membrane size according to the application

2- Soak the membrane in a container with Milli-Q H₂O to remove trace amounts of glycerine humectants and sodium azide bacteriostat. The membrane should be kept wet at all times from this point on.

3- Clean and assembly the stirred cell unit. The glossy side of the membrane should be facing up.

4- Fill the stirred cell unit with Milli-Q H₂O. Prewash the membrane with Milli-Q H₂O and check the water flux at 10 psi to ensure the membrane is not broken or displaced. A water flux of 1-3 mL/min/cm² indicates the 300, 000 MWCO membrane is intact.

5- Prefoul the membrane with ~ 0.25 mL/cm² of DMEM + 10% FBS media (note: the media should not contain phenol red if the supernatants to be concentrated are produced in the absence of this pH indicator). Adjust the tip speed to 33.5 cm/sec and increase the nitrogen pressure to 30 psi. Be careful not to let the membrane dry. Add PBS if necessary.

6- Aliquot starting material clarified retrovirus samples for analyses and measure the starting volume. Considering this volume, calculate the final volume desired in order to attain a predetermined concentration factor at the end of the process.

7- Add the starting material into the stirred cell unit and carry out ultrafiltration at nitrogen constant pressure (30 psi) and tip speed (33.5 cm/sec). Collect the permeate in sterile bottles. Monitor the process at different times by determining the flow rate and volume of permeate. (Note: The flow rate should drop slowly during the process).

8- Once the desired volume of retentate is reached, add cold ultrafiltration buffer into the stirred cell unit (~1 mL/cm²) and carry out diafiltration at nitrogen constant pressure (30 psi) and tip speed (33.5 cm/sec). Repeat diafiltration 3 times in discontinuous mode. (Note: The permeates obtained during ultrafiltration and diafiltration can be collected separately for subsequent analyses).

9- The ultra/diafiltration process is stopped once the final desired volume of retentate is reached. Slowly resuspend the virus and gently wash the membrane with diafiltration buffer. Aliquot the concentrated supernatant samples for analyses and measure the final volume.

10- Store concentrated samples at -80ºC along with the previously aliquoted samples.

Objective:

The following procedure describes the purification of retroviral particles in 20-fold concentrated supernatants by affinity chromatography using a Fractogel^® EMD heparin affinity chromatography column

Materials:

Note: The HR5/5 filters should be replaced with a 10 μm mesh filter for virus purification purposes

Procedure:

1- Turn on the UV lamp and rinse the GradiFrac system thoroughly with Milli-Q H₂O

2- Install the 1-mL Fractogel^® EMD heparin column and remove the storage buffer with 10 column volumes (CV) of Milli-Q H₂O at 0.3 mL/min.

3- Rinse the lines A and B with the corresponding buffers and equilibrate the column with 10 CV of binding buffer (150 mM NaCl in 20 mM Tris-HCl buffer, pH 7.5) at 0.5 mL/min.

4- Thaw retrovirus 20-fold concentrated samples. Filter the samples using 0.45 µm pore size Acrodisc syringe-mounted filters with HT Tuffryn^® polysulfone membrane. Aliquot starting material samples for analyses.

5- Monitor UV absorbance at 280 nm. When stable baseline is achieved, load 3 mL of the concentrated virus sample and apply a step-wise gradient elution strategy that includes a wash step at 150 mM NaCl (19.5 CV) to remove the bulk of serum contaminating proteins, followed by virus elution at 350 mM NaCl (13 CV) and a final high-stringency wash step at 1200 mM NaCl (7.5 CV) to remove tightly bound contaminants. The process is carried out at room temperature at 0.5 mL/min (or 153 cm/h linear flow rate).

6- The virus particles elute at 350 mM NaCl in a defined peak. Pool virus-containing fractions and aliquot for analyses.

7- After each run, re-equilibrate the column with binding buffer (10 CV) at 0.5 mL/min or store the column in storage buffer (10 CV) at 0.3 mL/min.

8- Store semi-purified samples at -80ºC along with the previously aliquoted samples.

Objective:

The following procedure describes the purification of retroviral particles in 20-fold concentrated supernatants by rate zonal ultracentrifugation 10-30% continuous iodixanol gradient.

Materials:

Procedure:

1- Rotor and buckets should be pre-cooled in the ultracentrifuge to 4 ºC before the run.

2- Prepare a 50% iodixanol working solution (WS) in 1 mM EDTA, 0.85% NaCl, 20 mM Tris-HCl buffer pH 7.5 by mixing 5 parts of OptiPrep^TM stock with 1 part of diluent for WS.

2- Prepare a 30% iodixanol gradient solution in 1 mM EDTA, 0.85% NaCl, 20 mM Tris-HCl buffer pH 7.5 by mixing 1.5 part of WS with 1 part of buffer

3- Prepare a 10% iodixanol gradient solution in 1 mM EDTA, 0.85% NaCl, 20 mM Tris-HCl buffer pH 7.5 by mixing 1 part of WS with 4 parts of buffer

4- Thaw retrovirus 20-fold concentrated samples. Aliquot starting material samples for analyses. Keep the virus at 4ºC until use.

5- Fill one chamber of the gradient maker with 16 mL of the 30% iodixanol gradient solution and the other with 18 mL of the 10% iodixanol gradient solution. Form 10-30% continuous iodixanol gradients in 25 × 89 mm Beckman UltraClear^TM tubes.

6- Carefully load 3 mL of the concentrated virus stock on top of the 34 mL continuous iodixanol gradient. Insert the tubes inside of the SW28 buckets and balance the weight of the buckets using an analytical balance. The buckets must be well balanced to avoid spills and tube collapse during ultra centrifugation. Avoid mixing the formed gradient at all times.

7- Carefully place buckets in the SW28 rotor and spin at 24,000 rpm (100,000×g) for 4 h at 4ºC. (Note: Use acceleration at 1 when using swinging bucket rotors and set deceleration at 0).

8- Remove buckets from the rotor and open them inside the biological safety cabinet. A yellow broad band containing most serum proteins is observed at the top of the tube. The virus bands in the middle of the tube (from 1.06 to 1.09 g/mL). No virus band is visualized at the concentrations of retrovirus stock used.

9- Clean the outside of the tube with EtOH and collect 15 gradient fractions of 2.5 mL by puncturing the bottom of the tube with an 18G needle.The density of each fraction can be determined by weight of 200 μL aliquots on an analytical balance.

10- Aliquot and store purified samples at -80ºC along with the previously aliquoted samples for analyses.

Objective:

The following procedure describes the final purification of retroviral particles in heparin semi-purified fractions by size exclusion chromatography using a Sepharose CL-4B chromatography column

Materials:

Procedure:

1- Turn on the UV lamp and rinse the GradiFrac system thoroughly with Milli-Q H₂O

2- Install the Sepharose CL-4B column and remove the storage buffer with 10 column volumes (CV) of Milli-Q H₂O at 0.5 mL/min.

3- Rinse the lines A with running buffer and equilibrate the column with 5 CV at 0.5 mL/min.

4- Thaw retrovirus semi-purified samples. Filter the samples using 0.45 µm pore size Acrodisc syringe-mounted filters with HT Tuffryn^® polysulfone membrane. Aliquot starting material samples for analyses.

5- Monitor UV absorbance at 280 nm. When stable baseline is achieved, load 7.5 mL of the concentrated virus sample and perform isocratic elution with PBS running buffer (1.2 CV). The process is carried out at room temperature at 0.5 mL/min. (Note: Load <10% of bed volume for best peak resolution).

6- The virus particles elute in the void volume of the column. Pool virus-containing fractions and aliquot for analyses.

7- After each run, re-equilibrate the column with running buffer (5 CV) at 0.5 mL/min or store the column in storage buffer (5 CV) at 0.5 mL/min (or 15 cm/h linear flow rate).

8- Store purified samples at -80ºC along with the previously aliquoted samples.