7-Part Series

Optimizing Viral Vector Production: Strategies and Innovations

Improve AAV Product Recovery for Gene Therapy with Anion Exchange Polishing

Poster

Improve AAV Product Recovery for Gene Therapy with Anion Exchange Polishing

Avoid low full capsid yields with a novel, industry-verified polishing procedure

Written byCytiva

The separation of full and empty AAV capsids is of utmost importance for gene therapy production due to its direct impact on treatment efficacy and safety, but the process remains problematic. Separating the full capsids that contain the therapeutic cargo, such as a transgene intended for delivery to target cells, from the empty capsids that lack this cargo, is technically challenging.

A novel chromatography procedure using a strong catIon exchanger with dextran surface extenders addresses the problem of empty and partial AAV capsid carryover. This process has been industry-verified to improve full capsid recovery. The precise separation of full and empty AAV capsids is a crucial step in optimizing the safety and efficacy of AAV-based gene therapy, ultimately enhancing the prospects for successful treatment outcomes.

Download this resource to view original data and results of industry testing and the protocol that drastically improves the scalability and cost-efficiency of AAV separation for gene therapy applications.

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Cytiva technical poster portrait Separation of full and empty AAV capsids by anion exchange resin with dextran surface extendersÅsa Hagner-McWhirter, Brigitta Németh, Marcus Kjellander, Hans Blom, Ann-Christin Magnusson, and Jean-Luc MaloiselAdeno-associated virus (AAV) is the main vector for gene therapy, and there is need for scalable, cost-efficient, and robust filtration and chromatography- based purification processes. Key for a successful process is high overall yields of full capsids and efficient impurity removal. Our AAV process can be seen in Figure 1. The affinity step does not discriminate between full and empty capsids, and a polishing step is required to remove as much as possible of the empty capsids. Separation of full and empty capsids can be achieved with ion exchange by utilizing a small difference in pI (Fig 2). Here we show how full and empty separation of AAV2, AAV5, AAV8 and AAV9 can be improved using anion exchange, dextran surface extenders, and optimized conditions.HarvestCell lysis and DNA fragmentation150mM NaCl, 0.5% Tween, Denarase 40U/mL, 1 mM MgCl2 Low pH treatmentClarification/NFFÄKTA flux™ 6 systemULTA™ 5 + 2 µm GF + 0.6/0.2 HCFig 2. Principle of ion exchange separation in the polishing step.Strong quaternary ammonium (Q)anion exchange ligandCapto QDextran surfaceextenderCryo TEM imaging. VironovaAB, Stockholm Sweden.Fig 1. AAV process overview.Highly crosslinked agaroseresin beadFig 3. Capto Q extender and ligand structure.Anion exchange polishingÄKTA pure 25 system Capto Q resinAffinity captureÄKTA pure™ 150 or 25 system Capto™ AVB resinConcentration and buffer exchangeTFF with HF (300 000 NMWC) 10X UF/5X DF20 mM Tris, 150 mM NaCl, pH 8Upstream productionXcellerex™ XDR-10 or ReadyToProcess WAVE™ 25 bioreactorsHEK293T sus/ HyCell™ TransFx-H AAV -GFP triple plasmid transfectionPrescreeningFig 5. Prescreening to identify optimal elution conductivity (%B)for Capto Q. Dotted line indicates selected %B buffer for elution of empty capsids (step 1).Two-step elutionAUC results for AAV5, AAV8, and AAV9120100% full% empty% partials806040200AAV5 AAV5 AAV5 AAV8 AAV8 AAV8 AAV9 AAV9 AAV9start peak 1 peak 2 start peak 1 peak 2 start peak 1 peak 2* No reliable data, % full expected to be > 15% from chromatogram peak areas.Sample related error (mix-up or sample prep)Fig 7. UV 260:280 ratios and analytical ultracentrifugation (AUC) results of Capto Q peaks 1 and 2.Fig 6. Final two-step elution protocol using optimal elutionconductivity (%B, 20 CV) for empty capsids (step 1) and step 2 (5 CV) to elute the full capsids using Capto Q.Table 1. Results summary for two-step elutionFig 8. Comparison of Capto Q vs Capto Q ImpRes separationperformance. Full AAV5 capsids purity obtained by analytical ultracentrifugation (AUC) of peak 2.NA = not analyzedThe first peak contained mainly empty capsids while the second peak contained the full capsids as indicated by the UV260:280 ratio of ~ 0.6-0.7 for empty capsid and >1.2 for full capsids, respectively (1). The qPCR:ELISA ratio in the peaks and the calculated viral genome (VG) confirmed good separation, as shown in Table 1. The AAV2 start material contained a very low level of full capsids and the second peak, enriched with full capsids, is both smaller and had a lower UV260:280 ratio compared to the other serotypes. The results were further confirmed by analytical ultracentrifugation (AUC) with ~ 75% full capsids for AAV5 and AAV8 and ~ 88% full capsids for AAV9 in peak 2 (Fig 7). The partial capsids were reduced for AAV5 and AAV9 (Fig 7). The effect of the dextran surface extenders could also be confirmed by AUC. Capto Q resulted in ~ 75% AAV5 full capsids compared to ~ 35% using Capto Q ImpRes (Fig 8). The dynamic binding capacity was estimated to 1-3 x 1013 VP/mL Capto Q resin (results not shown).It is critical to dilute the eluate from affinity purification to reduce conductivity (~ 1-3 mS/cm depending on serotype), to ensure binding of full AAV capsids. Werecommend bypassing the mixer on the ÄKTA pure 25 system to reduce dead volume and ensure sharp conductivity steps (important in small scale) and to use a 10 mm path length UV detector cell for increased sensitivity. Make sure to set the pH of the buffer before adding MgCl2 and perform a wash with deionized water before CIP with NaOH to avoid precipitation.ExperimentalColumn: Capto Q resin packed in Tricorn™ 5/100, 2 mLSample load: ~ 1 × 1012 VP/mL resinFlow rate: 2 mL/minSystem: ÄKTA pure™ 25Buffer A: 20 mM BTP, pH 9.0, 2 mM MgCl2Buffer B: 20 mM BTP, pH 9.0, 2 mM MgCl2, 250 mM Na acetateEquilibration: Buffer A, 5 CV Wash: Buffer A, 5 CV Prescreening protocolGradient: Step elution, 5% increments, 3 CV eachTwo-step protocolGradient: Two-step elution for each serotype:rAAV2: Step 1 40% buffer B, 20 CVStep 2 100% buffer B, 5 CVrAAV5: Step 1 35% buffer B, 20 CVStep 2 100% buffer B, 5 CVrAAV8: Step 1 30% buffer B, 20 CVStep 2 100% buffer B, 5 CVrAAV9: Step 1 5% buffer B, 20 CVStep 2 30% buffer B, 5 CV%Cytiva, Björkgatan 30, 751 84 Uppsala, SwedenWe identified three critical parameters that enhance the full and empty capsid separation. Dextran surface extenders on the anion exchange resin (Fig 3), MgCl2 constant concentration in buffers, and step elution (Fig 4).Fig 4. Separation of AAV full and empty capsids using Tricorn 5/100 columns, (2 mL) 1. Capto Q ImpRes or Capto Q with dextran surface extenders (AAV8) or 2. Capto Q with constant 2 mM or 18 mM MgCl2 (AAV8) or 3. Capto Q with 18 mM MgCl2 and linear or step NaCl gradient (AAV5). Buffers used were: 20 mM BTP pH 9.5, 2 or 18 mM MgCl2 + additives (1% sucrose and 0.1% poloxamer 188) (A buffer) and A buffer + 400 mM NaCl (B buffer).We were able to develop an optimized step elution protocol that worked well for full and empty capsid separation of AAV2, AAV5, AAV8 and AAV9 using Capto™ Q with dextran surface extenders. We used 20 mM BTP, pH 9 containing 2 mM MgCl2 without any additives and replaced NaCl as an elution salt with sodium acetate, which is a softer (more kosmotropic) salt. The elution conductivity was identified in a prescreening procedure using incremental5% B steps (Fig 5). The % B for empty capsid elution was selected based on the UV 260:280 ratio. The step before the ratio started to increase was selected, and the first elution step was prolonged to 20 CV to maximize the empty capsid removal (Fig 6). This was followed by a second shorter (5 CV) step for elution of full capsids using 100% B or lower (Fig 6). It is possible (and can be an advantage for some serotypes) to adjust the conductivity to maintain the empty capsids in the flowthrough only allowing binding of full capsids.1. Dickerson R, Argento C, Pieracci J, Bakhshayeshi M. Separating empty and full recombinant adeno-associated virus AAV particles using isocratic anion exchange chromatography. BioTechnol J. 2021; 16(1):e2000015.Learn more about our end-to-end AAV production workflow: https://www.cytivalifesciences.com/solutions/cell-therapy/products-and- technology/gene-therapy/aav-vector-production-workflowCytiva and the Drop logo are trademarks of Life Sciences IP Holdings Corporation or an affiliate doing business as Cytiva.ÄKTA, ÄKTA flux, ÄKTA pure, Capto, HyCell, ReadyToProcess, ReadyToProcess WAVE, Tricorn, ULTA, and Xcellerex are trademarks of Global Life Sciences Solutions USA LLC or an affiliate doing business as Cytiva. Denarase is a trademark of c-LEcta GmbH. Tween is a trademark of the Croda Group of Companies.Any other third-party trademarks are the property of their respective owners.© 2022-2023 Cytiva.For local office contact information, visit cytiva.com/contact.CY40940-17Nov23-POWe acknowledge Akash Bhattacharya and Shawn Sternishaat Beckman Coulter Life Sciences for collaboration and for kindly performing the analytical ultracentrifugation.