Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

This protocol will be useful for the investigators who want to develop methods for AAV production and purification using the baculovirus-insect cell culture system. AAV production using baculovirus-insect cell culture system is a cost-effective method that is easy to scale up and is compatible with the current good manufacturing practice. Some parts of the protocol will be demonstrated by Alan Heizer, a research assistant of my laboratory.

Begin by thawing one vial of Sf9 cells, and immediately seed them in 15 milliliters of insect cell culture medium. Grow Sf9 cells in an orbital shaker incubator at 125 RPM and 28 degrees Celsius in round-bottom flasks with a loosely-attached cap for exchange of air. Propagate the cells in a one-liter flask containing 200 milliliters of medium to obtain enough cells for TIPS cell production.

Add 50 milliliters of Sf9 cells at 2 times 10 to the 6th power cells per milliliter in two flasks. Infect one flask of Sf9 cells with 0.5 milliliters of baculovirus-AAV2-GFP, and the other flask of cells with 0.5 milliliters of baculovirus-AAV2-rep-cap. Three days later, harvest a small aliquot of baculovirus-infected Sf9 cells, also known as TIPS cells.

Stain 2 times 10 to the 5th power of both uninfected and baculovirus-infected Sf9 cells with 0.5 micrograms of mouse anti-baculovirus gp64 antibody containing a fluorescent dye in 100 microliters of blocking buffer for 30 minutes at ambient temperature in the dark. After washing the cells with PBS to remove the antibody, re-suspend the stained cells in 300 microliters of PBS containing 0.5 bovine serum albumin. Analyze the baculovirus gp64 expression on cells using flow cytometry.

When the cells are 80 to 90%viable, with an increase in diameter, harvest the cells and cryopreserve 1 times 10 to the 7th power cells in one milliliter of insect culture medium, supplanted with 10%fetal bovine serum and 10%dimethyl sulfoxide in a slow-freezing container at negative 80 degrees Celsius. The next day, transfer the tube containing TIPS cells to a liquid nitrogen freezer for long-term storage. Culture and grow naive Sf9 cells at 2 times 10 to the 6th power cell per milliliter at 28 degrees Celsius in several two-liter flasks containing 400 milliliters of insect cell culture medium in a shaker incubator that is needed for adeno-associated virus or AAV production.

After three to four days, the cell density is increased to up to 5 to 6 times 10 to the 6th power cells per milliliter. For AAV production in flasks in an orbital shaker incubator, use a pipette or a peristaltic pump to seed 400 milliliters of Sf9 culture at 2 times 10 to the 6th power cells per milliliter into a two-liter flask, aseptically. Set up the orbital shaker at 125 RPM and 28 degrees Celsius.

Thaw one vial of each baculovirus-AAV-GFP and baculovirus-AAV-rep-cap TIPS cells. Dilute the cells with 20 milliliters of insect culture medium, and perform a viability count of the cells using trypan blue. Inoculate both TIPS cells at a ratio of 1 to 10, 000 relative to the naive Sf9 cells cultured in a shaker flask or bio-reactor.

On day two, harvest one milliliter of Sf9 culture aseptically, and stain with the trypan blue dye to analyze the cell count, viability, and morphology. On day three, repeat the steps performed on day two, and check the status of the baculovirus infection after staining the Sf9 cells. On day five, repeat the steps performed on the second day, and then harvest the culture medium and cells when Sf9 viability has decreased to around 50%Collect the supernatant and the cell pellet after spinning, and store them at negative 80 degrees Celsius.

Once the AAV-producer cell pellet is thawed, add cell lysis buffer to it and mix vigorously. Incubate the re-suspended pellet for 30 minutes at ambient temperature to release the AAV particles. After centrifugation, transfer the cell lysate to a new container.

Mix the cell lysate and cell culture supernatant after thawing. Add 20 units per milliliter nuclease and 10-millimolar magnesium chloride to the cell lysate to digest the DNA and RNA, and incubate for two to four hours at 37 degrees Celsius. Filter the lysate through a 0.8 micrometer and 0.2 micrometer polyethersulfone dual-filtration system using a pump.

Store the clarified cell lysate at four degrees Celsius overnight, or purify the AAV immediately. Mount a 10-milliliter AVB Sepharose column onto the chromatography machine, and insert the pipeline into the AAV sample, wash buffer, and elution buffer. Insert tubes into the fraction collector slots of the chromatography machine to collect the fractions of column pass-through solution, wash buffer, and elution buffer containing the AAV particles.

Equilibrate the AVB Sepharose column with five column volumes of PBS at a flow rate of five milliliters per minute. Load the filtered cell lysate containing AAV particles onto the AVB Sepharose affinity column using the chromatography machine equipped with a sample pump. Run the sample at a flow rate of three milliliters per minute.

Run PBS through the column at a flow rate of three milliliters per minute until the ultraviolet absorbance curve at 280 nanometers returns to the baseline and becomes stable. Elute the AAV particles from the AVB Sepharose column with 50-millimolar sodium citrate buffer pH 3 at a flow rate of three milliliters per minute. Immediately dilute the AAV supernatant with a one-fifth volume of 500-millimolar Tris HCl pH 8.0 to neutralize the acidic elution buffer containing AAV.

This prevents the pH-mediated degradation that could occur with acidic pH 3.0 elution buffer. Then dilute the AAV supernatant tenfold with PBS, so that AAV particles are in a physiologic buffer. Store one milliliter of each column run-through samples, wash buffer, and 100 microliters of the eluted AAV supernatant to evaluate the presence of AAV by infection of the target cells.

Set up the tangential flow filtration system, equipped with a polyethersulfone membrane cartridge with a 100-kilodalton molecular weight cutoff to concentrate the AAV. Run 200 milliliters of PBS for 10 minutes to equilibrate the tangential flow filtration module. Load the AAV sample by controlling the pump's flow until the sample is reduced to the desired volume.

Diafiltrate the retentate with 100 milliliters of PBS. After concentrating the AAV sample to the desired volume, collect the AAV sample, filter it through a 0.2 micrometer polyethersulfone filter, and aliquot it. Then store the AAV sample at negative 80 degrees Celsius.

Most of the Sf9 cells became infected with the baculovirus in three to four days, due to the multiple rounds of infection, evidenced by baculovirus glycoprotein gp64 expression. Most of the cells also show an increase in diameter. The cells show an increase in diameter, cytopathic effect, and around half of the cells die in five days post-infection, which are the signs of completion of AAV production.

A peak of protein was seen while elution with an acidic buffer corresponding to the AAV fraction. The purified AAV samples show three distinct capsid proteins:VP1, VP2, and VP3, after SDS-PAGE and silver staining. The most critical step is harvesting the TIPS cells before cryopreservation, when most of the cells become infected with the baculovirus, with a minimum number of cell death.

We have described the production and purification of AAV vector of serotype 2 as a model here. However, the protocol can be applied for other AAV serotypes as well.