Name: process development for the production and purification of adeno-associated virus (aav)2 vector using baculovirus-insect cell culture system
Uploaded: 2022-01-13T13:00:00.000+00:00
Duration: 10 min 31 s
Description: Watch this Scientific Journal Video about Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System at JoVE.com

We would like to thank Dr. Robert M. Kotin (National Heart, Blood and Lung Institute, NIH) for generously providing us the AAV plasmids and Danielle Steele and Rebecca Ernst (Cincinnati Children&#39;s Hospital) for their technical assistance. This work is supported by the Start-Up fund from Cincinnati Children&#39;s Research Foundation to M.N.

See Figure 1 for an illustration summarizing the protocol.
1. Generation of baculovirus-infected TIPS cells
<ol>
	<li>Thaw one vial of Sf9 cells and immediately seed them in 50 mL of insect cell culture medium. Grow Sf9 cells in an orbital shaker incubator at 125 rpm and 28 &#176;C in round bottom flasks with a loosely attached cap for exchange of air8,9. Propagate the cells in a 1 L flask containing 200 mL of medium to obtain enough cells for TIPS cells production.</li>
	<li>Add 50 mL of Sf9 cells at 2 x 106 cells/mL in two flasks: infect one flask of Sf9 cells with 0.5 mL of baculovirus (BV)-AAV2-GFP (or therapeutic gene) and the other flask of cells with 0.5 mL of BV-AAV2-rep-cap. 
	NOTE: AAV vector plasmids were generously provided by Dr. Robert M. Kotin (NIH). Baculovirus production from bacmid DNA (Bac-to-Bac System) has been described previously8,9. The baculovirus stability during the passage is a critical problem for the scale-up production of rAAV. It is better to use a lower passage number of baculovirus (up to passage number 4). Determine the optimal volume of the baculovirus supernatant empirically by checking the baculovirus infection efficiency using flow cytometry (Figure 2) during TIPS cell production.</li>
	<li>Monitor the baculovirus infection 3-4 days post-infection by staining baculovirus gp64 in infected Sf9 cells as follows.
	<ol>
		<li>Stain 2 x 105 Sf9 cells (both uninfected and baculovirus-infected cells) with 0.5 &#181;g of mouse anti-baculovirus gp64 antibody (1:200) containing a fluorescent dye in 100 &#181;L of blocking buffer for 30 min at ambient temperature.</li>
		<li>Wash the cells with 1 mL of PBS to remove the antibody and resuspend the stained cells in 300 &#181;L of PBS containing 0.5% bovine serum albumin.</li>
		<li>Analyze the baculovirus gp64 expression on cells using flow cytometry (Figure 2). 
		NOTE: Determine the gating strategy for flow cytometry acquisition and analysis empirically. A total of 10,000 single live cells is acquired. Baculovirus gp64 expression on Sf9 cell surfaces indicates baculovirus infection. After 3-4 days, most of the Sf9 cells become infected with the baculovirus, as evidenced by baculovirus gp64 expression (Figure 2).</li>
	</ol>
	</li>
	<li>When the cells are 80%-90% viable with an increase in the diameter (Figure 3), harvest the cells and cryopreserve 1 x 107 cells in 1 mL of insect culture medium supplemented with 10% fetal bovine serum and 10% dimethyl sulfoxide in a slow-freezing container at -80 &#176;C. The next day, transfer the tube containing TIPS cells to a liquid nitrogen freezer for long-term storage. 
	&#8203;NOTE: Perform cell culture in a biosafety cabinet following the standard aseptic laboratory technique at Biosafety Level 2 (BSL-2).</li>
</ol>
2. Production of AAV vector
<ol>
	<li>Day 1: Co-culture Sf9 cells with the baculovirus-infected TIPS cells
	<ol>
		<li>Culture and grow naive Sf9 cells at 1 x 106 cells/mL at 28 &#176;C in several 2 L flasks containing 400 mL of insect cell culture medium in a shaker incubator that is needed for AAV production. After 3-4 days, the cell density is increased up to 5 x 106-6 x 106 cells/mL. 
		NOTE: CO2 and humidity are not important factors for the growth of Sf9 cells.</li>
		<li>Seed the Sf9 cells either in shake flasks or in a bioreactor as follows.
		<ol>
			<li>AAV production in flasks in an orbital shaker incubator: Use a pipette or a peristaltic pump to seed 400 mL of Sf9 culture at 2 x 106 cells/mL into a 2 L flask aseptically. Set up the orbital shaker at 125 rpm and 28 &#176;C. 
			&#8203;NOTE Use a peristaltic pump or standard pipette depending on the scale of the AAV production.</li>
			<li>AAV production in a bioreactor: Use a peristaltic pump to seed 1 L of Sf9 culture at 2 x 106 cells/mL into a 10 L bioreactor bag aseptically. Load the bag onto the Bioreactor unit for culturing at 28 &#176;C. Add 40% oxygen to the bioreactor bag at 0.1 psi. Set up the bioreactor unit at a 20&#176; angle and shake the bag at 25 rpm.</li>
		</ol>
		</li>
		<li>Thaw one vial of each BV-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap TIPS cells. Dilute the cells with 20 mL of insect culture medium and perform a viability count of the cells using trypan blue. Inoculate both TIPS cells at a ratio of 1:10,000 relative to the naive Sf9 cells cultured in a shaker flask or bioreactor. 
		&#8203;NOTE: The survival of the TIPS cells is reduced due to cryopreservation, and the recovery rate is approximately 50% when cell viability is determined with the trypan blue staining. Perform a pilot experiment to empirically determine the ratio of TIPS cells and na&#239;ve Sf9 cells before large-scale rAAV production.</li>
	</ol>
	</li>
	<li>Day 2: Culture monitoring
	<ol>
		<li>Harvest 1 mL of Sf9 culture aseptically. Stain with the Trypan blue dye to analyze the cell count, viability, and morphology.</li>
	</ol>
	</li>
	<li>Day 3: Culture monitoring and feeding
	<ol>
		<li>Harvest 1 mL of Sf9 culture and analyze the cell count, viability, and morphology. Check the status of baculovirus infection after staining the Sf9 cells as mentioned in step 1.3. 
		NOTE: Increase in cell diameter and cytopathic effect are indications of baculovirus infection. The increase in diameter precedes a decrease in cell viability, and these parameters are used to determine the cell harvest time during AAV production. Determine the optimal time point empirically.</li>
		<li>Feed the Sf9 culture with fresh insect culture medium at a ratio of 1:5 aseptically.</li>
	</ol>
	</li>
	<li>Day 4: Culture monitoring
	<ol>
		<li>Harvest 1 mL of Sf9 culture and analyze the cell count, viability, and morphology. Disconnect oxygen supply from the bioreactor bag.</li>
	</ol>
	</li>
	<li>Day 5: Culture monitoring and harvest
	<ol>
		<li>Harvest 1 mL of Sf9 culture and analyze the cell count, viability, and morphology. Harvest the culture medium and cells when Sf9 cell viability has decreased to around 50% (Figure 4).</li>
		<li>Spin the cell suspension at 2100 x g for 15 min at 4 &#176;C. Collect the supernatant and the cell pellet, and store them at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Lysis of cells and release of AAV
<ol>
	<li>Thaw the AAV producer cell pellet. Add 100 mL of cell lysis buffer (20 mM Tris-HCl pH 8.0, 150 mM sodium chloride, 0.5 % Triton-X-100) to the cell pellet, mix vigorously, and incubate for 30 min at ambient temperature to release the AAV particles.</li>
	<li>Centrifuge at 2100 &#215; g for 30 min at 4 &#176;C and transfer the cell lysate to a new container. Mix the cell lysate and cell culture supernatant after thawing.</li>
	<li>Add 20 U/mL nuclease and 10 mM MgCl2 to the cell lysate to digest the DNA and RNA. Incubate for 2-4 h at 37 &#176;C.</li>
	<li>Filter the lysate through a 0.8 &#181;m and 0.2 &#181;mpolyethersulfone dual filtration system using a pump.</li>
	<li>Store the clarified cell lysate at 4 &#176;C overnight or purify the AAV immediately.</li>
</ol>
4. Purification of AAV vector using affinity column chromatography system
<ol>
	<li>Prepare the chromatography instrument by sequentially washing the sample and buffer lines with sterile water, 1 N sodium hydroxide, water, and phosphate-buffered saline (PBS, pH 7.4) at a flow rate of 50 mL/min.</li>
	<li>Mount the AVB Sepharose column (10.0 mL) into the chromatography system, and run 100 mL of PBS at a flow rate of 5 mL/min. 
	NOTE: Use a lower or higher volume of AVB&#160;Sepharose column depending on the scale of AAV production and set up the flow rate as suggested by the column or resin manufacturer (see Table of Materials).</li>
	<li>To collect the fractions of column pass-through solution, wash buffer, and elution buffer containing AAV particles, insert tubes into the fraction collector slots of the chromatography instrument.</li>
	<li>Equilibrate the column with PBS at a flow rate of 5.0 mL/min.</li>
	<li>Load the filtered cell lysate containing AAV particles onto the&#160;column using the chromatography machine equipped with a sample pump. Run the sample at a flow rate of 3.0 mL per min.</li>
	<li>Run PBS through the AVB&#160;Sepharose column at a flow rate of 3.0 mL/min until the ultraviolet (UV) absorbance curve (280 nm) returns to the baseline and becomes stable.</li>
	<li>Elute the AAV particles from the column with 50 mM sodium citrate (pH 3.0) buffer at a flow rate of 3.0 mL/min (Figure 5). 
	NOTE: While the AAV particles dissociate from the resin and pass through the UV detector, an elution peak of protein can be seen on the chromatogram.</li>
	<li>Immediately dilute the AAV supernatant with a one-fifth volume of 500 mM Tris-HCl (pH 8.0) to increase the pH of the AAV containing supernatant to approximately pH 5.5 to prevent pH-mediated degradation with acidic elution buffer. Further, dilute the AAV supernatant 10-fold with PBS to fully neutralize the pH. 
	NOTE: The pH value is approximately 5.5 after neutralizing the rAAV solution. After neutralizing AAV, dilute the rAAV solution 10-fold with PBS (pH 7.4) so that AAV is in a physiologic buffer.</li>
	<li>Store 1 mL of each column run-through samples, wash buffer, and 100 &#181;L of the eluted AAV supernatant to evaluate the presence of AAV by infection of the target cells.</li>
	<li>Clean the AVB&#160;Sepharose column with 100 mL of 100 mM citric acid (pH 2.1) and 100 mL of PBS (pH7.4) at a flow rate of 5.0 mL/min. Rinse the column with 20% ethanol at a flow rate of 5.0 mL/min and store at 4 &#176;C.</li>
	<li>Clean the pipelines of the chromatography instrument with the column offline position as described in section 4.1. Finally, rinse the chromatography system with 200 mL of 20% Ethanol at a flow rate of 50.0 mL/min and store in 20% ethanol.</li>
</ol>
5. Concentration and diafiltration of AAV vector using tangential flow filtration (TFF)
<ol>
	<li>Set up the TFF system equipped with a polysulfone membrane cartridge (100 kDa Molecular Weight Cut Off) to concentrate the AAV.</li>
	<li>Equilibrate TFF module with 200 mL of PBS for 10 min.</li>
	<li>Load the AAV sample&#160;by controlling the pump&#39;s flow until the sample is reduced to the desired volume while maintaining a trans-membrane pressure at 2-3 psi.</li>
	<li>Diafiltrate the retentate with 100 mL of PBS, as used in this study, or empirically define&#160;alternate buffer that supports long-term stability of AAV.</li>
	<li>After concentrating the AAV sample to the desired volume, collect the AAV sample, filter it through a 0.2 &#181;m polyethersulfonefilter, aliquot, and then store it at -80 &#176;C.</li>
</ol>
6. Infection of AAV samples into the target cells to evaluate the presence of AAV in the purification steps
<ol>
	<li>Inoculate 7 x 104 HT1080 cells/well in a 24-well plate in 500 &#181;L of Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) supplemented with 1% L-glutamine, 1% sodium pyruvate, and 10% fetal bovine serum (FBS) the day before infection. Culture the cells in an incubator at 37 &#176;C and 5% CO2.</li>
	<li>Count the cells before infection. Add the diluted unpurified AAV bulk sample before the column run, column run-through samples, wash buffer, and eluted purified AAV sample. 
	NOTE: Count the Trypan blue-stained cells with a hemocytometer. Determine the dilution factor and volume (&#181;L) of the AAV samples empirically. The higher the AAV titers, the more dilution is needed. Ensure that the baculovirus particles of the unpurified bulk sample before the column run, column run-through samples, wash buffer are heat-inactivated at 50 &#176;C for 50 min before infecting the target cells to determine infectious titers of AAV.</li>
	<li>Two days after infection, analyze the protein expression (e.g., GFP or therapeutic gene) in the cells using a flow cytometer.</li>
	<li>Calculate infectious titers of AAV using the number of cells at the time of infection, dilution factor, and percentage of GFP+ cells with the formula: (Total number of cells x Percentages GFP+ cells x Dilution Factor) / Volume of AAV in milliliters. 
	NOTE:&#160;For example, the number of cells is 1 x 105, the percentage of GFP+ cells is 3.4%, the dilution factor is 100, and the volume of AAV added is 2 &#181;L. The titer of AAV is 1.7 x 108 infectious unit (IU)/mL. The total amount of purified AAV particles in 25 mL of the eluted fraction is 4.3 x 109 infectious units (IU)/mL (Figure 6). The total number of initial unpurified AAV particles is 1.09 x 1010 IU/mL, and purified AAV particles are 4.3 x 109 IU/mL. Therefore, the percentage of recovery is 39.4%. The percentage of GFP+ cells should be between 1%-30%, which corresponds to a linear range when used to calculate the infectious titer of AAV. If more concentrated AAV vector particles are infected into the target cells; there is a possibility that multiple copies of the AAV particles may infect a single cell that will show as a single copy of the vector. Therefore, dilute the AAV vector supernatant to obtain 1%-30% vector infection in the target cells. If the AAV vector does not have a reporter gene, stain the transgene or the therapeutic gene expressed by the AAV vector with an antibody containing a fluorescent dye that can be detected and quantitated using a flow cytometer. Not all AAV serotypes can efficiently infect HT1080 cells. Therefore, to perform the infectivity assay for other AAV serotypes, use different cell lines. Titer the AAV2-GFP particles before purification and after purification on HT1080 cells and measure the GFP expression by flow cytometry. Calculate the recovery (%) by the ratio of the number of purified AAV particles and the number of AAV particles prior to purification multiplied by 100.</li>
</ol>

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The authors declare no conflicts of interest.

The parameters used in this protocol for the process development of production, purification, and concentration of AAV vectors can be applied to both small and large-scale manufacturing of AAV vectors for gene therapy applications. The entire upstream and downstream process can be performed in a closed system compatible with the current Good Manufacturing Practices (cGMP). The major advantages of the Sf9-baculovirus system are scalability for large-scale GMP-grade AAV production at an affordable cost. The system does not need expensive plasmids and transfection reagents, which are needed for the production of AAV using HEK293 cells. It has been reported that both HEK293 cells and Sf9 cells yield similar quality AAV vectors (Eric D. Horowitz, ASGCT 2018, Abstract #100). The major challenge is that this system requires the generation of baculoviruses and TIPS cells that need a significant time and effort.
In this protocol, two types of TIPS cells (TwoBac system) were used: one TIPS cell containing baculovirus with AAV-gene transfer cassette and another TIPS cell containing AAV-rep-cap. The OneBac system2,16,17 can generate TIPS cells containing both AAV gene transfer cassette and rep-cap genes in a single baculovirus. The OneBac system provides an alternative approach to produce the rAAV using only one set of TIPS cells rather than two as described in the TwoBac system, which would further simplify the protocol.
This protocol describes AAV production in Sf9 cells. It is important to optimize the ratio of the baculovirus-infected TIPS cells to producer na&#239;ve Sf9 cells to obtain a good AAV yield. If this ratio is sub-optimal, the yield of AAV will be reduced11. For example, if more AAV ITR containing gene transfer vectors are generated than the capsids in the producer cells due to the sub-optimal ratio of TIPS and producer cells, all of the available gene transfer vectors will not get enough capsids to produce full AAV particles. On the other hand, if more capsids are generated than the AAV gene transfer vectors in the producer cells, all capsids will not get AAV ITR containing gene transfer vectors that result in empty AAV particles.
As the cells multiply, the nutrients of the culture medium become exhausted, and metabolic waste products accumulate. Therefore, supplementation of 20% fresh growth medium into the cell culture 2 days after co-culture of TIPS cells and Sf9 cells can increase AAV titers significantly (Amine A. Kamen, National Research Council Canada, personal communication).
The purity of AAV particles is a critical factor for achieving effective transduction of target cells without any cytotoxicity for both in vitro and in vivo studies3,5. Therefore, it is important to include a chromatography step that can selectively purify AAV particles and eliminate impurities such as host cell proteins and debris, genomic and baculoviral DNA, and aggregated and fragmented vectors. While loading AAV supernatant onto a AVB Sepharose column, it is critical to check the flow-through samples for the presence of AAV particles that may pass through the column without binding to the resin. If that is the case, (1) a lower run speed that results in a longer residence time will be necessary for binding the AAV particles, (2) the amount of loaded sample onto the column should be reduced, and/or (3) the volume of the AVB&#160;Sepharose should be increased, so that the binding capacity of the column never exceeds the number of AAV particles. The ability of AVB Sepharose resin to bind AAV particles at a high flow rate and with high affinity and capacity is important to reduce purification time. The major limitation of AVB&#160;Sepharose is that it binds the capsid of AAV serotypes 1, 2, and 5. Therefore, different chromatography resins should be tested and used for the purification of other AAV serotypes18. The downstream purification protocol described herein can also be used to purify AAV produced in HEK 293 cells.
This protocol can purify AAV from cell lysate but cannot remove empty particles. A few articles have described methods that can distinguish empty vs. full AAV particles in purified AAV stocks19,20. However, we believe that the AAV production should be optimized at the upstream process level to minimize the generation of empty particles. If the ratio of AAV gene transfer vector and capsid production in producer cells are not optimal, more empty particles may generate.
In addition to binding full AAV particles, AVB&#160;Sepharose medium binds fragmented AAV particles or capsid proteins which can be removed using TFF. Most of the low molecular weight particles are eliminated from AAV samples by including the TFF step downstream of the column chromatography. In addition, TFF is used to perform a buffer exchange/diafiltration and to concentrate the AAV10,21.
Although ultracentrifugation of the AAV lysate with cesium chloride or iodixanol gradient is the preferred method for small-scale and pre-clinical grade AAV purification, this method is not scalable and less suitable for large-scale purification of AAV21,22.
In conclusion, this protocol for the process development of production and purification of AAV will be useful for small-scale pre-clinical and large-scale manufacturing of recombinant AAV for the gene therapy of inherited genetic diseases.

Adeno-associated viruses (AAV) are non-enveloped human parvoviruses containing a single-stranded DNA of 4.6 kb. AAV vectors have several advantages over other viral vectors for gene therapy applications1,2,3,4. AAVs are naturally replication-incompetent, thereby, require a helper virus and host machinery for replication. AAVs do not cause any disease and have low immunogenicity in the infected host3,5. AAV can infect both quiescent and actively dividing cells and may persist as episome without integrating into the genome of the host cells (AAV rarely integrate into the host genome)1,3. These features have made AAV a desirable tool for gene therapy applications.
To generate an AAV gene transfer vector, the transgene cassette, including the therapeutic gene, is cloned between two internal terminal repeats (ITRs), which are typically derived from the AAV serotype 2. The maximum size from 5&#39; ITR to 3&#39; ITR, including the transgene sequence, is 4.6 kb6. Different capsids may have a different cell or tissue tropism. Therefore, capsids should be chosen based on the tissue or cell type intended to be targeted with the AAV vector7.
Recombinant AAV vectors are commonly produced in mammalian cell lines such as human embryonic kidney cells, HEK293 by transient transfection of the AAV gene transfer vector, AAV rep-cap, and helper virus plasmids2,3. However, there are several limitations for large-scale AAV production by transient transfection of adherent HEK293 cells. First, a large number of cell stacks or roller bottles are needed. Second, high-quality plasmid DNA and transfection reagents are needed, which increases the cost of manufacturing. Finally, when using adherent HEK293 cells, the serum is frequently needed for optimal production, complicating downstream processing1,2,3. An alternative method of AAV manufacturing involves using the insect cell line, Spodoptera frugiperda (Sf9) cells, and an insect virus called recombinant Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV or baculovirus)8,9,10. Sf9 cells are grown in serum-free suspension culture that is easy to scale up and is compatible with current good manufacturing practice (cGMP) production at a large scale, which does not require plasmid or transfection reagents. Moreover, the cost of the AAV production using the Sf9-baculovirus system is lower than the cost of using transient transfection of plasmids into HEK293 cells11.
The original rAAV production system using baculovirus-Sf9 cells used three baculoviruses: one baculovirus containing gene transfer cassette, the second baculovirus containing rep gene, and the third baculovirus containing serotype-specific capsid gene12,13. However, the baculovirus containing rep construct was genetically unstable upon multiple rounds of passages, which prevented amplification of the baculovirus for the large-scale AAV production. To resolve this issue, a novel rAAV vector system was developed, which contained two baculoviruses (TwoBac): one baculovirus containing the AAV gene transfer cassette and another baculovirus containing the AAV rep-cap genes together which are genetically more stable than the original system and more convenient to produce rAAV because of using TwoBac instead of three14,15. The OneBac system uses the AAV gene transfer cassette and the rep-cap genes in a single baculovirus which is more convenient to produce the rAAV because of using one baculovirus instead of using TwoBac or ThreeBac2,16,17. In our study, the TwoBac system was used for optimization.
The baculovirus system for AAV production also has limitations: baculovirus particles are unstable for long-term storage in serum-free medium11, and if the baculovirus titer is low, a large volume of baculovirus supernatant is needed, which may become toxic to the growth of Sf9 cells during AAV production (personal observation). The use of titer-less infected-cell preservation and scale-up (TIPS) cells, or baculovirus-infected insect cells (BIIC), provides a good option for AAV production in which baculovirus-infected Sf9 cells are prepared, cryopreserved, and subsequently used for infection of fresh Sf9 cells. Another advantage is the increased stability of baculovirus (BV) in Sf9 cells after cryopreservation10,11.
Two types of TIPS cells are generated to enable AAV production: the first one by infection of Sf9 cells with the BV-AAV2-GFP or therapeutic gene, and the second one by infection of Sf9 cell with BV-AAV2-rep-cap. TIPS cells are cryopreserved in small and ready-to-use aliquots. AAV vectors are produced in serum-free suspension culture in a flask placed in an orbital shaker or Wave bioreactor by co-culturing TIPS cells that produce baculoviruses and fresh Sf9 cells. Sf9 cells are infected by baculoviruses that carry the AAV2-GFP vector and the rep-cap sequences to generate AAV. Four to five days later, when AAV yields are the highest, the producer cells are lysed with detergent to release the AAV particles. The cell lysate is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the lysate by AVB Sepharose column chromatography. Finally, AAV vectors are concentrated using TFF. The protocol describes the production of AAV at a small scale, useful for research and pre-clinical studies. However, the methods are scalable and compatible with manufacturing clinical-grade AAV vectors for gene therapy applications.

<table><tbody><tr><td>1 N Sodium Hydroxide</td><td>Sigma-Aldrich</td><td>1.09137</td><td>For Akta Avant cleaning</td></tr><tr><td>2 L flasks</td><td>ThermoFisher Scientific</td><td>431281</td><td>Flask for suspension culture</td></tr><tr><td>50 ml Conical tube</td><td>ThermoFisher Scientific</td><td>14-959-49A</td><td>For collection of supernatants</td></tr><tr><td>24-well plate</td><td>ThermoFisher Scientific</td><td>07-200-80</td><td>Adherent cell culture plate</td></tr><tr><td>250 mL flasks</td><td>ThermoFisher Scientific</td><td>238071</td><td>Flask for suspension culture</td></tr><tr><td>Akta Avant 150 with Unicorn Software</td><td>Cytiva</td><td>28976337</td><td>Chromatography system</td></tr><tr><td>AVB Sepharose High Performance</td><td>Cytiva</td><td>28411210</td><td>Chromatography medium</td></tr><tr><td>Baculovirus-AAV-2 GFP</td><td>In-house</td><td>non-catalog</td><td>AAV transfer vector</td></tr><tr><td>Baculovirus-AAV-2 rep-cap</td><td>In-house</td><td>non-catalog</td><td>AAV packaging vector</td></tr><tr><td>Nuclease</td><td>Sigma-Aldrich</td><td>E1014</td><td>Enzyme to degrade DNA and RNA</td></tr><tr><td>Blocking buffer</td><td>Santa Cruz Biotechnologies</td><td>516214</td><td>Blocking to prevent non-specific antibody binding to cells</td></tr><tr><td>Cell lysis buffer</td><td>In-house</td><td>Non-catalog item</td><td>20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5 % Titron X-100</td></tr><tr><td>Cellbag, 2 L and 10 L</td><td>Cytiva</td><td>28937662</td><td>Bioreactor bag</td></tr><tr><td>Cleaning buffer</td><td>In-house</td><td>Non-catalog item</td><td>100 mM citric acid (pH 2.1)&amp;nbsp;</td></tr><tr><td>Cryovial</td><td>Thomas Scientific</td><td>1222C24</td><td>For cryopreservation</td></tr><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D6429</td><td>Growth media for cell lines</td></tr><tr><td>Elution buffer</td><td>In-house</td><td>Non-catalog item</td><td>50 mM sodium citrate buffer (pH 3.0)</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>E7073</td><td>For disinfection and storage of the chromatography</td></tr><tr><td>Filtration unit&amp;nbsp;</td><td>Pall Corporation</td><td>12941</td><td>Membrane filter</td></tr><tr><td>HT1080 cell line</td><td>ATCC</td><td>CCL-121</td><td>Fibroblast cell line</td></tr><tr><td>HyClone&amp;trade; SFX-Insect culture media</td><td>Cytiva</td><td>SH30278.02</td><td>Serum-free insect cell growth medium</td></tr><tr><td>Peristaltic Pump</td><td>Pall Corporation</td><td>Non-catalog item</td><td>TFF pump</td></tr><tr><td>MaxQ 8000 orbital shaker incubator&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>Non-catalog item</td><td>Shaker for suspension culture</td></tr><tr><td>Microscope</td><td>Nikon</td><td>Non-catalog item</td><td>Cell monitoring and counting&amp;nbsp;</td></tr><tr><td>Mouse anti-baculovirus gp64 PE antibody</td><td>Santa Cruz Biotechnologies</td><td>65498 PE</td><td>Monitoring baculovirus infection in Sf9 cells</td></tr><tr><td>Oxygen tank</td><td>Praxair</td><td>Non-catalog item</td><td>40 % Oxygen supply is needed for Sf9 cell growth</td></tr><tr><td>PBS</td><td>ThermoFisher Scientific</td><td>20012027</td><td>Wash buffer</td></tr><tr><td>Silver Staining kit</td><td>ThermoFisher Scientific</td><td>LC6100</td><td>Staining AAV capsid proteins</td></tr><tr><td>Sf9 cells</td><td>ThermoFisher Scientific</td><td>11496-015</td><td>Insect cells</td></tr><tr><td>Steile water</td><td>In-house</td><td>Non-catalog item</td><td>For Akta Avant cleaning</td></tr><tr><td>Table top centrifuge</td><td>ThermoFisher Scientific</td><td>75253839/433607</td><td>For clarification of Baculovirus supernatant</td></tr><tr><td>Tangential Flow Filtration (TFF) cartridge</td><td>Pall Corporation</td><td>OA100C12</td><td>TFF cartridge to concentrate the AAV</td></tr><tr><td>Tris-HCl, pH 8.0</td><td>ThermoFisher</td><td>15568025</td><td>Alkaline buffer to neutralize the eluted AAV</td></tr><tr><td>WAVE Bioreactor System 20/50</td><td>Cytiva</td><td>28-9378-00</td><td>Bioreactor</td></tr></tbody></table>

process development for the production and purification of adeno-associated virus (aav)2 vector using baculovirus-insect cell culture system

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Here, the representative results of process development for the production and purification of AAV vectors using the Sf9 insect cell system are shown. The method includes co-culture of Sf9 cells with baculovirus-infected TIPS cells, feeding the cells with growth medium, harvesting and lysis of the producer cells to release the AAV particles, clarification of the cell lysate with nuclease treatment, centrifugation and filtration, purification of AAV using AVB Sepharose affinity chromatography, and concentration with TFF (Figure 1).
TIPS cells are generated by infection of BV-AAV2-GFP or therapeutic gene and BV-AAV2-rep-cap into Sf9 cells separately. Most of the Sf9 cells become infected with the baculovirus in 3-4 days due to the multiple rounds of infection, evidenced by baculovirus glycoprotein gp64 expression in (Figure 2) and the cells show an increase in diameter (Figure 3). TIPS cells are harvested 3-4 days post-infection and cryopreserved. Sf9 cells are co-cultured with the TIPS cells that secrete baculovirus particles in the culture medium that infect naive Sf9 cells. Baculovirus is replication-competent; therefore, the number of infected cells rapidly increases by multiple round infections with newly produced baculovirus particles that are secreted into the culture medium8,9. The cells show an increase in diameter, cytopathic effect, and around half of the cells die in 5 days post-infection, which are the signs of completion of AAV production (Figure 4).
The AAV producer cells are harvested by low-speed centrifugation, lysed with buffer containing detergent to release the AAV into the cell lysate. This is then treated with nuclease for digestion of DNA and RNA to reduce viscosity, filtered through a 0.8 µm and 0.2 µm membrane, and subsequently purified and concentrated. The cell lysate is loaded onto a AVB Sepharose column using a chromatography system. The AVB Sepharose resin binds AAV2 particles due to its affinity to the capsid proteins. Wash buffer is run through the AVB Sepharose column to remove unbound and loosely bound materials until the ultraviolet (UV) absorbance curve (280 nm) becomes stable at the baseline. Since AAV particles strongly bind AVB Sepharose, no significant number of AAV2 particles is detected during the washing. The AAV particles are eluted with acidic buffer (pH 3.0), which dissociates the interaction between AAV particles and AVB Sepharose resin. To prevent the pH-mediated degradation of the AAV by the acidic solution, the eluant is neutralized with an alkaline buffer (pH 8.0). A peak of protein is seen while elution with acidic buffer corresponding to the AAV fraction (Figure 5 and Figure 6). Purified AAV is diluted 10-fold with PBS, concentrated and buffer exchanged with a TFF system. In this example, total AAV particles in cell lysate (560 mL) are 1.1 x 1014 vector genome (vg), after AVB Sepharose chromatography purification (25 mL) are 4.1 x 1013 vg, and after concentration with TFF (25 mL) are 2.4 x 1013 vg. The purified AAV samples show three distinct capsid proteins, VP1, VP2, and VP3, after SDS-PAGE and silver staining (Figure 7)
<img alt="AAV production process flowchart: co-culture, cell harvest, purification, filtration steps." class="xfigimg" src="/files/ftp_upload/62829/62829fig1v2.jpg" data-altupdatedat="1747736610000" data-alt="Figure 1"> 
Figure 1: A schematic diagram for the production and purification of AAV Vector. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62829/62829fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Flow cytometry diagram; Baculovirus gp64 analysis, Sf9 cell infection, viral protein detection." class="xfigimg" src="/files/ftp_upload/62829/62829fig02.jpg" data-altupdatedat="1747736610000" data-alt="Figure 2"> 
Figure 2: Flow cytometry analysis of baculovirus gp64 expression in Sf9 cells. Baculovirus infected Sf9 cells are stained with a mouse anti-baculovirus gp64 antibody containing a fluorescent dye, which is detected by flow cytometry. (A) Uninfected Sf9 cells. (B) Baculovirus infected Sf9 cells are showing gp64 expression in most of the cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62829/62829fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopy comparison of uninfected vs. Baculovirus-infected Sf9 cells, magnified 200x." class="xfigimg" src="/files/ftp_upload/62829/62829fig03.jpg" data-altupdatedat="1747736610000" data-alt="Figure 3"> 
Figure 3: Morphology of the baculovirus-infected Sf9 (TIPS) cells. Baculovirus is infected into the Sf9 cells to produce the TIPS cells. (A) Uninfected Sf9 cells. (B) Baculovirus infected Sf9 (TIPS) cells. Cells are shown at 200x magnification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62829/62829fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscope image, Sf9 cell comparison; uninfected vs AAV producer; cell morphology analysis, 200x." class="xfigimg" src="/files/ftp_upload/62829/62829fig04.jpg" data-altupdatedat="1747736610000" data-alt="Figure 4"> 
Figure 4: Morphology of the baculovirus-infected Sf9 cells during AAV production. TIPS cells secrete baculoviruses that infect co-cultured naïve Sf9 cells during AAV production. (A) Uninfected Sf9 cells. (B) Baculovirus infected Sf9 cells show an increase in diameter. Five days post-infection, almost half of the cells die (visualized under an inverted phase microscope after trypan blue staining). Red arrows indicate live cells, and blue arrows indicate dead cells. Cells are shown at 200x magnification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62829/62829fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Chromatography graph; protein purification; absorbance vs. volume; AAV peak; elution process." class="xfigimg" src="/files/ftp_upload/62829/62829fig05.jpg" data-altupdatedat="1747736610000" data-alt="Figure 5"> 
Figure 5: AAV purification using AVB Sepharose column chromatography. A chromatogram shows the absorbance of protein samples at 280 nm during sample loading on column, washing, and elution. The chromatogram has been modified to fit in the figure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62829/62829fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="AAV chromatography graph; protein purification; elution peak at B6; infectious unit analysis." class="xfigimg" src="/files/ftp_upload/62829/62829fig06.jpg" data-altupdatedat="1747736610000" data-alt="Figure 6"> 
Figure 6: The number of infectious AAV particles in the flow-through during loading onto the column, washing, and elution. A total of 560 mL of AAV samples are loaded on a 10 mL AVB  Sepharose column. The fraction volume for the column load is 50 mL, column wash is 50 mL, and elution is 25 mL. AAV titers are measured after infection of HT1080 cells with the column run-through samples while loading on column, washing, and elution to investigate the presence of AAV at each step of the purification. The total purified AAV yield is 4.3 x 109 infectious units. Each diamond-shaped symbol represents the infectious units (IU) of AAV in each fraction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62829/62829fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="AAV2 protein gel electrophoresis diagram; VP1, VP2, VP3 bands indicating protein sizes." class="xfigimg" src="/files/ftp_upload/62829/62829fig07.jpg" data-altupdatedat="1747736610000" data-alt="Figure 7"> 
Figure 7. SDS-PAGE and silver staining of pure AAV vector showing capsid proteins. The reduced AAV samples are run on SDS-PAGE, and silver staining is performed. Three distinct bands of AAV capsid proteins, VP1, VP2, and VP3, are visible. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62829/62829fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System at JoVE.com

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.

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

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera ...

process-development-for-production-purification-adeno-associated

Nanyang Technological University

Research

JoVE Journal

Biology

9.1K Views.  Cincinnati Children's Hospital Medical Center. Dit protocol zal nuttig zijn voor de onderzoekers die methoden willen ontwikkelen voor AAV-productie en -zuivering met behulp van het baculovirus-insectencelkweeksysteem. AAV-productie met behulp van baculovirus-insectencelkweeksysteem is een kosteneffectieve methode die gemakkelijk op te schalen is en compatibel is met de huidige goede productiepraktijken. Sommige delen van het protocol zullen worden gedemonstreerd door Alan Heizer, een onderzoeksassistent van mijn laboratorium.Begin ...

Video: Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

Production and Purification of Baculovirus for Gene Therapy Application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

Genetics

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

Biochemistry

Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 1010 infectious particles per ml and can be directly administrated in vivo.

These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro&#160;and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the ...

Immunology and Infection

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Neuroscience

Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. Clinical advancement of novel applications for AAV involves transitioning from small animal models, such as mice, to larger animal models, including dogs, sheep, and nonhuman primates. One of the limitations of administering AAV to larger animals is the requirement for large quantities of high-titer virus. While suspension cell culture is a scalable method for AAV vector production, few research labs have the equipment (e.g., bioreactors) or know how to produce AAV in this manner. Moreover, AAV titers are often significantly lower when produced in suspension HEK 293 cells as compared to adherent HEK293 cells. Described here is a method for producing large quantities of high-titer AAV using cell stacks. A detailed protocol for titering AAV as well as methods for validating vector purity are also described. Finally, representative results of AAV-mediated transgene expression in a sheep model are presented. This optimized protocol for large-scale production of AAV vectors in adherent cells will enable molecular biology laboratories to advance the testing of their novel AAV therapies in larger animal models.

Here we provide a detailed procedure for large-scale production of research-grade AAV vectors using adherent HEK 293 cells grown in cell stacks and affinity chromatography purification. This protocol consistently yields &#62;1 x 1013 vector genomes/mL, providing vector quantities appropriate for large animal studies.

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. ...

Purification of AAV Vectors by Single-Step Heparin Affinity Chromatography

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs.
This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

Author Spotlight: Improved Method for Production and Purification of Adeno-Associated Viral Vectors

This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. It presents a simple, scalable, and cost-effective approach, eliminating the need for ultracentrifugation. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical studies.

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. ...

Bioengineering

Efficient Adeno-Associated Virus Vector Production Using a Cell Factory Platform

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Author Spotlight: Advancing Gene Therapy Research with High-Titer Adeno-Associated Virus Vector Production

As the field of gene therapy continues to evolve, there is a growing need for innovative methods that can address these challenges. Here, a unique method is presented, which streamlines the process of generating high-yield and high-purity AAV vectors using a cell factory platform, meeting the quality standards for in vivo studies.

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. ...

Simplified and Efficient AAV Production Using HEK293 Suspension Culture

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 1011 genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 1012 genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 1012 GC in total, and TransIt yielded 2.4 x 1013 GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Author Spotlight: Advancing Gene Therapy with High-Yield AAV Vectors Through HEK293 Suspension Cell Cultivation

Here, a suspension HEK293 cell-based AAV production protocol is presented, resulting in reduced time and labor needed for vector production using components that are available for research purposes from commercial vendors.

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, ...

AAV Isolation by Double Iodixanol Density Gradient Centrifugation

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

Author Spotlight: Efficient Adeno-Associated Virus Isolation for Pre-Clinical Applications

Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use.

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

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