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...

<ol>
	<li>Hildinger, M., Auricchio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+AAV-mediated+gene+transfer+for+the+treatment+of+inherited+disorders.">Advances in AAV-mediated gene transfer for the treatment of inherited disorders.</a> European Journal of Human Genetics. 12 (4), 263-271 (2004).</li><li>Wu, Z., Asokan, A., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+serotypes:+vector+toolkit+for+human+gene+therapy.">Adeno-associated virus serotypes: vector toolkit for human gene therapy.</a> Molecular Therapy. 14 (3), 316-327 (2006).</li><li>Nathwani, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-associated+virus+vector-mediated+gene+transfer+in+hemophilia+B.">Adenovirus-associated virus vector-mediated gene transfer in hemophilia B.</a> The New England Journal of Medicine. 365 (25), 2357-2365 (2011).</li><li>Wang, D., Tai, P. W. L., Gao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+vector+as+a+platform+for+gene+therapy+delivery.">Adeno-associated virus vector as a platform for gene therapy delivery.</a> Nature Reviews Drug Discovery. 18 (5), 358-378 (2019).</li><li>Mingozzi, F., High, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+responses+to+AAV+vectors:+overcoming+barriers+to+successful+gene+therapy.">Immune responses to AAV vectors: overcoming barriers to successful gene therapy.</a> Blood. 122 (1), 23-36 (2013).</li><li>Grieger, J. C., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Packaging+capacity+of+adeno-associated+virus+serotypes:+impact+of+larger+genomes+on+infectivity+and+postentry+steps.">Packaging capacity of adeno-associated virus serotypes: impact of larger genomes on infectivity and postentry steps.</a> Journal of Virology. 79 (15), 9933-9944 (2005).</li><li>Rabinowitz, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-dressing+the+virion:+the+transcapsidation+of+adeno-associated+virus+serotypes+functionally+defines+subgroups.">Cross-dressing the virion: the transcapsidation of adeno-associated virus serotypes functionally defines subgroups.</a> Journal of Virology. 78 (9), 4421-4432 (2004).</li><li>Nasimuzzaman, M., Lynn, D., vander Loo, J. C. M., Malik, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+baculovirus+vectors+using+heparin+affinity+chromatography.">Purification of baculovirus vectors using heparin affinity chromatography.</a> Molecular Therapy - Methods &amp; Clinical Development. 3, 16071 (2016).</li><li>Nasimuzzaman, M., vander Loo, J. C. M., Malik, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+Purification+of+Baculovirus+for+Gene+Therapy+Application.">Production and Purification of Baculovirus for Gene Therapy Application.</a> Journal of Visualized Experiments: JoVE. (134), e57019 (2018).</li><li>Sandro, Q., Relizani, K., Benchaouir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV+Production+Using+Baculovirus+Expression+Vector+System.">AAV Production Using Baculovirus Expression Vector System.</a> Methods in Molecular Biology. 1937, 91-99 (2019).</li><li>Cecchini, S., Virag, T., Kotin, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducible+high+yields+of+recombinant+adeno-associated+virus+produced+using+invertebrate+cells+in+0.02-+to+200-liter+cultures.">Reproducible high yields of recombinant adeno-associated virus produced using invertebrate cells in 0.02- to 200-liter cultures.</a> Human Gene Therapy. 22 (8), 1021-1030 (2011).</li><li>Urabe, M., Ding, C., Kotin, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+cells+as+a+factory+to+produce+adeno-associated+virus+type+2+vectors.">Insect cells as a factory to produce adeno-associated virus type 2 vectors.</a> Human Gene Therapy. 13 (16), 1935-1943 (2002).</li><li>Urabe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scalable+generation+of+high-titer+recombinant+adeno-associated+virus+type+5+in+insect+cells.">Scalable generation of high-titer recombinant adeno-associated virus type 5 in insect cells.</a> Journal of Virology. 80 (4), 1874-1885 (2006).</li><li>Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intron+splicing-mediated+expression+of+AAV+Rep+and+Cap+genes+and+production+of+AAV+vectors+in+insect+cells.">Intron splicing-mediated expression of AAV Rep and Cap genes and production of AAV vectors in insect cells.</a> Molecular Therapy. 16 (5), 924-930 (2008).</li><li>Smith, R. H., Yang, L., Kotin, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatography-based+purification+of+adeno-associated+virus.">Chromatography-based purification of adeno-associated virus.</a> Methods in Molecular Biology. 434, 37-54 (2008).</li><li>Aslanidi, G., Lamb, K., Zolotukhin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inducible+system+for+highly+efficient+production+of+recombinant+adeno-associated+virus+(rAAV)+vectors+in+insect+Sf9+cells.">An inducible system for highly efficient production of recombinant adeno-associated virus (rAAV) vectors in insect Sf9 cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (13), 5059-5064 (2009).</li><li>Wu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Popularizing+recombinant+baculovirus-derived+OneBac+system+for+laboratory+production+of+all+recombinant+adeno-associated+virus+vector+serotypes.">Popularizing recombinant baculovirus-derived OneBac system for laboratory production of all recombinant adeno-associated virus vector serotypes.</a> Current Gene Therapy. 21 (2), 167-176 (2021).</li><li>Adams, B., Bak, H., Tustian, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moving+from+the+bench+towards+a+large+scale,+industrial+platform+process+for+adeno-associated+viral+vector+purification.">Moving from the bench towards a large scale, industrial platform process for adeno-associated viral vector purification.</a> Biotechnology and Bioengineering. 117 (10), 3199-3211 (2020).</li><li>Joshi, P. R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+scalable+and+robust+AEX+method+for+enriched+rAAV+preparations+in+genome-containing+VCs+of+serotypes+5,+6,+8,+and+9.">Development of a scalable and robust AEX method for enriched rAAV preparations in genome-containing VCs of serotypes 5, 6, 8, and 9.</a> Molecular Therapy - Methods &amp; Clinical Development. 21, 341-356 (2021).</li><li>Dickerson, R., Argento, C., Pieracci, J., Bakhshayeshi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separating+empty+and+full+recombinant+adeno-associated+virus+particles+using+isocratic+anion+exchange+chromatography.">Separating empty and full recombinant adeno-associated virus particles using isocratic anion exchange chromatography.</a> Biotechnology Journal. 16 (1), 2000015 (2021).</li><li>Wright, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manufacturing+and+characterizing+AAV-based+vectors+for+use+in+clinical+studies.">Manufacturing and characterizing AAV-based vectors for use in clinical studies.</a> Gene Therapy. 15 (11), 840-848 (2008).</li><li>Tomono, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=highly+efficient+ultracentrifugation-free+chromatographic+purification+of+recombinant+AAV+serotype+9.">highly efficient ultracentrifugation-free chromatographic purification of recombinant AAV serotype 9.</a> Molecular Therapy - Methods &amp; Clinical Development. 11, 180-190 (2018).</li></ol>

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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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)&#160;</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&#160;</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&#8482; 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&#160;</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&#160;</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 (...

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 ...

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.

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8.7K Views.  Cincinnati Children's Hospital Medical Center. 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 imm...