Exploring Development Options of a Polishing Chromatography Step for AAV7 and AAV8

Abstract

As impact of adeno-associated virus (AAV) empty capsids on drug product safety and quality remains inconclusive, downstream purification strategy has been focusing on empty capsid removal. Anion-exchange chromatography (AEX) has made significant progress in separating empty from full capsids in recent years. Still, achieving baseline resolution between different AAV subpopulations remains challenging due to subtle charge differences. With a certain AAV construct design, this difficulty is compounded when upstream packaging efficiency is low or when empty and full capsids of a particular serotype have similar electrostatic charge profiles. To improve separation and product purity, secondary interaction mechanisms using multimodal (mix-mode) chromatography are often introduced.

In this study, we present a case study on developing a polishing chromatography step to remove empty capsids from AAV7 and AAV8 preparations. To create a challenging feed material for the polishing step, we used small gene-of-interest (GOI) and poorly packaged starting materials. We investigated multiple critical process parameters, including buffer matrix, salt concentration, pH, peak fractionation strategies, and column chemistry (strong AEX vs. mix-mode). Mass photometry (MP) and charge detection mass spectrometry (CDMS) were used to characterize capsid populations. Optimized AEX conditions for AAV8 achieved 80% full capsids by MP and 90% GOI-containing capsids by CDMS. For AAV7, the mix-mode column demonstrated improved resolution compared with the standard AEX gradient method. These results demonstrate that mix-mode chromatography provides an alternative polishing option for serotypes where traditional AEX fails to achieve the desired separation.

Keywords

adeno-associated virus purification development empty capsid anion-exchange chromatography mix-mode chromatography

INTRODUCTION

Recombinant adeno-associated virus (rAAV) vectors have been used to provide genetic cures for human diseases. By 2024, among more than 4000 emerging gene-, cell-, and RNA-based therapies in development, over half were gene therapies utilizing viral vectors or genetically modified cells.¹ With the approval of rAAV-based gene therapy products, the field has significantly advanced in understanding the clinical and pharmacological implications of AAV. In principle, therapeutic benefit is realized through the successful transduction of genome-containing (“full”) AAV capsids and release of genetic cargo into target cells. Fully formed AAV capsids without cargo (“empty”) are typically generated during production and are removed during purification. The therapeutic benefits of empty capsids remain unclear, whereas their potential to elicit immune responses is well documented.^2–4 Excessive levels of empty capsids may reduce transduction efficiency by saturating receptor binding sites, leading to stronger immune responses and cytotoxic side effects. Conversely, other reports propose a “decoy” hypothesis in which empty capsids bind neutralizing antibodies, thereby protecting full capsids.⁵ Regardless of their clinical impact, empty capsids contribute to total viral load and must be controlled to ensure consistent product quality, efficacy, and safety.

One widely used approach to remove empty capsids at the production scale is anion-exchange (AEX) chromatography. While AEX has made significant progress over the past two decades,^6–11 isolating high-purity full capsids remains difficult for serotypes or constructs with minimal charge differences between empty and full particles.^12,13 To address these limitations, secondary interaction can be incorporated, resulting in the mix-mode (multimodal) chromatography, which combines charge, affinity, hydrophobic, hydrogen bonding, or size-exclusion interactions. Immobilized metal affinity chromatography has been used to purify adenovirus,¹⁴ and ceramic hydroxyapatite has been applied to AAV9.¹⁵ Although these approaches demonstrated alternative binding mechanisms, separation of empty and full capsids was not achieved. Other studies explored hydrophobicity and charge differences in AAV2 and AAV8,¹⁶ suggesting additional mechanisms for separation. PrimaS multimodal columns (BIA Separations, Sartorius), which incorporate hydrogen bonding, have shown promise in improving AAV8 peak separation.^10,17 However, most studies have been proof-of-concept, and process levers for optimizing resolution are not fully understood.

In this work, we evaluated mix-mode chromatography against AEX for two AAV serotypes (AAV7 and AAV8) using CIMmultus, a brand name from Sartorius Q and PrimaS columns. We systematically investigated critical process parameters including pH, salt gradients, MgCl₂ concentration, buffer matrix, and peak fractionation. The optimized AEX process for AAV8 achieved 90% full capsids, while the mix-mode process demonstrated improved separation for AAV7, suggesting a complementary role to AEX for challenging serotypes.

MATERIALS AND METHODS

AAV production and purification

Suspension-adapted human embryonic kidney cells 293 cells were cultured in shake flasks at 37°C for seed expansion. Viable cell density and viability were monitored daily using a ViCell Blu analyzer (Beckman Coulter Life Science, USA) to guide inoculation. AAV was produced in Sartorius 2 L bioreactors via the triple plasmid transient transfection method. FectoVIR (Polyplus, France) was used as the transfection reagent. After production, cell lysis was performed chemically with surfactant, MgCl₂ and Benzonase (Millipore, USA) for DNA digestion before NaCl addition. Lysate was clarified by depth filtration (Millipore USA). All AAV materials used in subsequent chromatography studies were captured by POROS CaptureSelect AAVX (Thermo Fisher Scientific, USA) on an ÄKTA Avant system (Cytiva, USA). AAV8 contained a 2.1 kb GOI, while AAV7 contained a self-complementary 4.3 kb GOI.

Polishing step—AEX chromatography

The AEX process was performed using a salt-based gradient. The CIMmultus quaternary amine (QA) column (2 µm pores; BIA Separations) was operated at 2 memrbane volume (MV)/min for all AEX purification steps. The column was prepared by sanitizing with 20 column volumes (CV) of 0.5 M NaOH, charging with 10 CV of 20 mM Bis-Tris-Propane, 2 M NaCl, 0.001% Pluronic, pH 9.0 ± 0.2, and then equilibrated with the equilibration buffer (20 mM Bis-Tris-Propane, 1 mM MgCl₂, 10 mM NaCl, 0.001% poloxamer 188). Neutralized affinity eluate was diluted up to 30-fold with 20 mM Bis-Tris-Propane (Sigma-Aldrich Chemie GmbH, Germany), 1 mM MgCl₂, and 0.001% poloxamer 188 (Kolliphor P 188, BASF, Germany), pH 9.2, until the diluted material reached a conductivity below 2.5 mS/cm and pH 9.0 ± 0.2. This material was then loaded onto the CIMmultus QA column and washed using the Phase A elution buffer. Elution was performed with a linear gradient from 100% Phase A (20 mM Bis-Tris-Propane-based buffer with tested buffer components) to 100% Phase B (same buffer matrix but containing higher salt concentration) at an approximate slope of 1.25% B per CV. After elution, the column was stripped with the charging buffer for 10 CV, then regenerated and stored. Elution fractions were collected in 1 CV increments and manually pooled based on UV absorbance profiles.

Polishing step—Mix-Mode chromatography

The CIMmultus PrimaS (AAV) mixed-mode column (2 µm pores; BIA Separations) was operated at 4 CV/min for all experiments. The column was prepared by cleaning with 20 CV of 0.1 M NaOH and 1 M NaCl, followed by equilibration with 10 mM Tris, 10 mM Bis-Tris-Propane, 2 mM MgCl₂, 1% sucrose, and 0.001% poloxamer 188, pH 7.5 (conductivity ≈ 2.5 mS/cm). Neutralized affinity eluate was diluted up to 35-fold in a load-dilution buffer consisting of a 1:1 mixture of the equilibration buffer and water-for-injection until the conductivity was below 2.5 mS/cm. The diluted load was then applied to the column and washed with equilibration buffer.

For pH-gradient elution, a 0%–100% Phase B gradient was applied at 0.8% B per CV, where Phase B matched the equilibration buffer composition but included 14 mM NaCl at pH 10.0. The inclusion of NaCl in Phase B was intended to match the conductivity of Phase A, ensuring that elution was driven solely by pH. For linear-salt gradient elution, Phase B contained 500 mM NaCl, and the gradient proceeded at 0.625% B per CV from 0% to 100% B. Following elution, the column was regenerated with 100 mM acetate, 1 M NaCl, and 0.001% Pluronic, pH 5.0 ± 0.2, for 20 CV, then re-equilibrated and stored. Fractionation was carried out in 1 CV increments and pooled based on UV chromatogram results, consistent with the AEX procedure.

Viral genome titer quantitation by digital PCR

Genome titers were quantified using the Qiacuity 8 perfusion plate-based digital PCR system. Samples were diluted in a buffer prepared from 10× GeneAmp PCR buffer (ThermoFisher 4486221) adjusted to 1× final concentration, supplemented with 25 mM MgCl₂ (final 1.5 mM; ThermoFisher 4486225) and 10% (w/v) poloxamer 188 (final 0.05% w/v; ThermoFisher 10977-015). After dilution, samples were treated with DNase (New England Biolabs M0303L) in its supplied reaction buffer for 1 h at 37°C to remove residual DNA external to the capsids. Samples were then treated with proteinase K (Teknova P0701) in an ethylenediaminetetraacetic acid-containing buffer for 1 h at 55°C to inactivate DNase and digest capsids, thereby releasing the encapsidated DNA.

At least three sample dilutions, each with three replicates, were prepared in PCR plates using the same dilution buffer. PCR master mix was added, containing forward, reverse, and probe primers specific to the GOI. The reaction mixtures were transferred to 8.5 K nanoplates and perfused into ∼8000 droplet wells before sealing and thermal cycling on the Qiacuity 8 system.

PCR was performed for 40 cycles (15 s at 95°C denaturation, 30 s at 60°C annealing). Positive versus negative wells were determined using the auto-threshold function (range 15–40). A well-characterized plasmid containing the GOI was used as a positive control on every plate.

AAV capsid empty and full quantification by mass photometry

AAV empty and full percentages were determined by mass photometry (MP) using the TwoMP system (Refeyn, Oxford, UK). For each sample, 10–12 µL of Dulbecco PBS (ThermoFisher 14190-144) was applied to a clean glass carrier slide within a silicone gasket. The drop was positioned over the laser, and the 233 × 166-pixel field was examined to ensure minimal background and absence of bright artifacts. A 1.0–2.5 µL aliquot of the neat AAV sample was quickly added and mixed by gentle pipetting, and a 60 s video was recorded to capture light-scattering data. The intensity of scattered light correlates with molecular mass, generating a distribution profile of heavier (full) and lighter (empty) AAV capsids binding over time. Videos were analyzed using DiscoverMP software, and molecular masses were calibrated using a 50:50 mixture of AEX-purified empty and full AAV8 samples as standards.

Empty and full capsid analysis by charge-detection mass spectrometry

All charge-detection mass spectrometry (CDMS) measurements were performed by MegaDalton Solutions to confirm empty-to-full ratios as an orthogonal analytical method.¹⁸

Definition of empty, partially filled, full, over-packaged, and ambiguous capsids

Due to construct-specific characteristics, we redefined partially filled, full, and over-packaged capsids. For AAV8, the full product contains a 2.1 kb GOI. Capsids containing DNA less than 2.1 kb were classified as “partially filled,” while those above 2.1 kb were categorized as “over-packaged.” For AAV7 (self-complementary DNA, scDNA), partially filled capsids contained DNA less than 4.3 kb, which include truncated GOI, single-copy GOI, or two-copy GOI with truncation. Only AAV7 capsids containing two complete GOI copies in self-complementary form were designated as “full.” Anything contained above 4.3 kb was labeled as “over-packaged.” Due to the resolution limits of MP, any signal between empty and full peaks was categorized as “ambiguous.” For AAV8, ambiguous species represented capsids packaging truncated DNA less than 2.1 kb. For AAV7, ambiguous species corresponded to capsids containing less than two GOI copies.

RESULTS AND DISCUSSION

Baseline assessment of an AEX protocol for AAV7 and AAV8

AEX chromatography performed under a conductivity gradient resolved AAV8 into three major peaks (Fig. 1a). Peaks 1 and 3 were identified as empty capsids, while peak 2 corresponded to full capsids. Genome titer measurements and full capsid percentage analyses confirmed this classification (Fig. 1c). However, the overall full capsid purity was 55%, below the targeted 70%, likely due to the minimal charge difference between empty and full AAV8 capsids, which is a result of small GOI. Eighty percent of genome was recovered in the full product peak. The starting material contained around 25% full capsids (data not included).

In contrast, AAV7 displayed substantial peak overlap (Fig. 1b). Three peaks and one transitional region (P2) were identified across the continuous conductivity gradient. Peaks 1 and 4 corresponded to empty capsids, as indicated by A260/A280 nm ratios. Peaks 3 was characterized as full peak with the A260 dominant signal. Genome titer recovery and full capsid percentage were further determined for each section (Fig. 1d). Peak 3 showed modest full capsid enrichment, reaching approximately 40%. Note that the starting material had a full percentage of 10% (data not included). Only 30% recovery was achieved with peak 3 alone, suggesting significant peak overlapping and genome loss during full capsid enrichment.

Optimize AEX chromatography

To further improve the separation between empty and full capsids on AEX chromatography, we evaluated several critical process parameters.

Effect of MgCl₂ concentration

Previous studies have shown that MgCl₂ can enhance the separation of empty and full AAV capsids.⁸ We performed an MgCl₂ concentration screen with 1 mM, 8 mM, and 15 mM MgCl₂ added into elution buffers. As shown in Figure 2a, increasing MgCl₂ concentration improved the resolution between AAV8 peaks 1 (empty), 2 (full), and 3 (empty). At 15 mM MgCl₂, baseline separation was achieved. However, MP analysis indicated that full capsid enrichment was highest at 8 mM Mg²⁺ (Fig. 2b), likely due to suboptimal fraction pooling. In contrast, when Mg²⁺ was screened from 1–20 mM for AAV7, separation efficiency declined (Fig. 2c), with peaks 1 and 2 becoming highly overlapped. No fraction analysis was performed because of the poor resolution observed from each chromatogram. We hypothesize that Mg²⁺–capsid interactions occur via multiple mechanisms. Prior reports suggest that surface charge alteration by Mg²⁺ is minimal for certain serotypes.¹⁹ Furthermore, Mg²⁺ can act as a counter-ion that binds to the negatively charged encapsidated DNA, thereby modifying the overall capsid charge profile.²⁰ This effect may differ further when the packaged genome is self-complementary rather than single-stranded, as the DNA conformation changes due to its self-complementary nature. Given the unresolved mechanisms of Mg²⁺ altering AAV surface charge, we recommend evaluating Mg²⁺ effects empirically for each serotype.

Effect of buffer matrix

The buffer matrix is a critical element of resolution in AEX chromatography. To identify factors influencing separation, we evaluated several buffer components commonly employed in AAV purification, focusing on how mono- and divalent salts affect the charge-based interactions between empty and full capsids. In the AAV8 study, a continuous MgSO₄ gradient was compared with the baseline NaCl gradient (Fig. 3a–b). Sucrose was also included as a potential stabilizing additive (Fig. 3c). Our findings indicate that MgSO₄ improved separation relative to NaCl, with a substantial fraction of empty capsids removed during the MgSO₄ wash phase, increasing the percent full capsid by 10% (Fig. 2d). However, because SO₄²⁻ is a stronger displacer and earlier elution was observed, raising potential concerns about process robustness. UV absorbance from the sucrose run suggested that sucrose affected AAV charge heterogeneity during gradient elution but did not improve overall resolution. These results suggested the need for thorough evaluation of AAV stability, charge heterogeneity, and elution behavior during AEX development.

Effect of pH in a conductivity gradient

From the baseline evaluation of AAV7, we noted that AAV7 full and empty capsids exhibited minimal surface charge differences. To improve resolution, we investigated whether modulating pH could enhance separation by exploiting small charge disparities. Elution profiles generated using NaCl gradients at pH 8.5 and 9.5 (Fig. 3e–h) revealed improved separation at the higher pH. A more distinct peak 3 was observed, suggesting that elevated pH magnifies charge differences among certain AAV subpopulations. Nevertheless, the increased pH did not alter the charge difference between the empty and full AAV7 capsids, resulting in an identical separation profile between peaks 1 and 2.

Implementation of a two-cycle AEX approach

During process development for constructs exhibiting low packaging efficiency, we determined that a single-cycle AEX method was insufficient for the removal of empty capsids. Accordingly, a two-cycle purification strategy was developed to enhance separation. The first cycle removed the majority of empty capsids, yielding a full product peak, which was subjected to a second AEX cycle to achieve additional removal of empty capsids. This approach provides additional mass-transfer opportunities to separate empty and full capsids with small charge differences, improving overall resolution. As illustrated in Figure 4, the initial cycle successfully separated most empty (first peak) and other non-product species (third peak) from the full AAV8 product peak (second peak). As we discovered previously, analysis of the full peak indicated co-elution of empty and full capsids (Fig. 4e), with most fractions—except fraction 2—falling below the 70% full specification. Re-injection of the cycle-one full peak under identical conditions produced a smaller empty peak and a third peak, supporting the hypothesis that mass-transfer limitations hinder separation when a high proportion of empty capsids are present during the first cycle. Subsequent fractionation confirmed enrichment of full capsids across the elution profile, achieving > 70% full in most fractions, with some reaching 80% by MP (Fig. 4f). From a recovery standpoint, pooling low-purity fractions from cycle one yielded about 80% genome recovery. After the second cycle, 55% of the total genome was recovered with 80% full capsid by MP. This was further confirmed by CDMS, where the full capsids were measured at 73% (Supplementary Fig. S2).

Characterization of partially filled capsids and overpackaged capsids

Because of the unique characteristics of small GOIs and self-complementary genomes, partially filled and overpackaged capsids were monitored. Due to the limited resolution of MP, these populations were not well-quantified. Overpackaged capsids were grouped with full capsids, while partially filled capsids were labeled as ambiguous species. CDMS provided additional insight into these species. For AAV8, 18% of capsids were overpackaged in addition to 73% of full capsids (Supplementary Fig. S2), with no separation between the full and overpackaged species, despite two cycles of AEX purification. Partially filled capsids were not noticeable, as short GOI has less chance of truncation. For AAV7, since only particles containing 2× GOI were defined as full, lower % full values were expected. As shown in Supplementary Figure S1, a significant portion of AAV7 capsids contained only 1× GOI. This explains the significant peak overlap between empty and full peaks because of the limited charge difference among empty, 1× GOI, and 2× GOI species. The observation raises questions about the definition of “full” capsids in a self-complementary AAV product, where a partially filled capsid (with 1x GOI) could have the desired therapeutic benefit.

Understanding critical parameters of a mixed-mode column

Mixed-mode chromatography offers enhanced resolving power by incorporating secondary interaction mechanisms alongside the primary ionic interaction. To determine key factors influencing AAV empty and full capsid separation on such systems, we conducted a series of studies using the PrimaS monolith column (Sartorius, Germany).

Effect of pH gradient

We first examined a linear pH gradient during elution to assess the resolution of AAV empty and full capsids using a 1 mL PrimaS column. For AAV8, three distinct peaks were obtained with baseline resolution—an improvement over the baseline process of the AEX column using a salt gradient (Fig. 5a vs. Fig. 1a). The full capsid peak eluted at pH 8.9, while the empty capsid peak centered at pH 8.5, providing a 0.4-unit pH window for separation. Fractionation analysis indicated that fractions 2–5 contained more than 70% full capsids based on MP. Pooled fraction analysis using both MP and CDMS confirmed efficient separation, yielding 77% full capsid and 81% GOI-containing capsids, respectively, outperforming the single-cycle AEX method (Fig. 5c,d). Total viral genome recovery reached 65%, consistent with the 60–80% recovery range typically observed for AEX chromatography.

For AAV7, chromatographic profiles showed modest improvement in separation among empty (peak 1), full (peak 2), and the mixed (peak 3) species. MP results indicated that the full-peak fraction from the PrimaS column contained ≈30% full capsids, compared with 20% from the AEX method (Fig. 1d). Although both methods exhibited limited enrichment, the PrimaS column provided a better starting point. Nevertheless, the pH difference between peak 1 (empty) and peak 2 (full) was only 0.2 unit, reflecting the peak overlap observed.

Optimization of pH in conductivity-gradient elution

We next applied a linear salt-gradient elution on the PrimaS column to investigate how secondary interactions influence empty/full capsid separation. This study aimed to incorporate elution-gradient pH and MgCl₂ concentration into the study design and evaluate their impact on viral genome recovery and % full capsid content using AAV8. Column loading was maintained at approximately 2 × 10¹³ vg/mL monolith, except for condition (a), which used a two-fold higher load (Fig. 6), as reflected by a larger chromatographic area. MP and CDMS were both used to assess capsid composition and separation efficiency.

Increasing the pH from 7.0 to 8.0 produced a small improvement in % full capsid (∼6% by MP). Unlike the AEX column, no significant differences were observed between the MgCl₂ concentrations tested, indicating that PrimaS resolving mechanisms rely less on ionic shielding and more on mixed-mode interactions. Among the tested conditions, only condition (d) (Fig. 6d) exhibited a notable decrease in viral genome recovery, likely resulting from the fractionation variations. It should be noted that fractionation was done at one column volume internal, which limited precise pooling of the full peak, introducing minor run-to-run variability.

CONCLUSION

Regular AEX salt gradient separation solely relyson a charge-based interaction and may not provide sufficient empty capsid removal. In this study, AAV8 full capsids were enriched above 80% by MP following the optimization of MgCl₂ concentration, buffer matrix, and the employment of a double cycle approach. The percentage of GOI-containing products was confirmed above 90% with CDMS. Genome recovery achieved 55% using the AAV8 construct with the developed process. An AAV7 AEX purification process was also presented. However, commonly used AEX chromatography levers that can improve empty and full separation failed to improve the purity profile, underlining the requirement of alternative solutions of empty capsid removal.

We also explored mix-mode chromatography as an alternative polishing step for AAV empty capsid removal. Leveraging secondary interactions at near physiological pH on the PrimaS monolith, AAV8 populations were consistently enriched above 70% full by MP in a single cycle. This offers more process flexibility for AAV constructs that are less stable under higher pH without compromising product purity. PrimaS also demonstrated separation between empty and full capsids with AAV7. However, the approach of pH and Mg²⁺ tuning did not offer any additional improvement of removing empty capsids, suggesting further investigations are required to elucidate the precise mechanism of multimodal separation and build robust enrichment of AAV7 capsids.

AUTHORS’ CONTRIBUTIONS

X.F.: Conceptualization, methodology, writing—original draft, visualization, and project admin. C.H.: Investigation, writing—original draft preparation, visualization, and data curation. D.B.: Writing—original draft and data curation. R.G.A.: Investigation and data curation.

Footnotes

ACKNOWLEDGMENTS

This development work was supported by Resilience Technology Development Group. The authors thank Allison Fleck, Owen Leeser, Danel Haq, and Patrick Starremans for providing analytical support; Oliver Meade, Nicole Nohilly, Catherine Siglin, and Yvette Valenzuela for cell culturing support; and Jeffrey Clark for purification support. The authors gratefully acknowledge Benjamin Draper for providing CDMS testing and data analysis.

FUNDING INFORMATION

This development work is solely funded by Resilience.

DISCLOSURE STATEMENT

No competing financial interests exist.

Supplemental Material

References

American Society of Gene & Cell Therapy and Citeline. Gene, Cell, + RNA Therapy Landscape Report. 2024.

Gao

, Li

, Zhong

, et al. Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects. Mol Ther Methods Clin Dev, 2014; 1(9):20139; doi: 10.1038/mtm.2013.9

Prasad

, Dimmock

, Greenberg

, et al. Immune responses and immunosuppressive strategies for adeno-associated virus-based gene therapy for treatment of central nervous system disorders: Current knowledge and approaches. Hum Gene Ther, 2022; 33(23–24):1228–1245; doi: 10.1089/hum.2022.138

Wright

. AAV empty capsids: For better or for worse? Mol Ther, 2014; 22(1):1–2; doi: 10.1038/mt.2013.268

Mingozzi

, Anguela

, Pavani

, et al. Overcoming preexisting humoral immunity to AAV using capsid decoys. Sci Transl Med, 2013; 5(194):194ra92; doi: 10.1126/scitranslmed.3005795

Potter

, Lins

, Mietzsch

, et al. A simplified purification protocol for recombinant adeno-associated virus vectors. Mol Ther Methods Clin Dev, 2014; 1:14034; doi: 10.1038/mtm.2014.34

, Chen

W-C

, Argento

, et al. Analytical strategies for quantification of adeno-associated virus empty capsids to support process development. Hum Gene Ther Methods, 2019; 30(4):144–152; doi: 10.1089/hgtb.2019.088

Dickerson

, Argento

, Pieracci

, et al. Separating empty and full recombinant adeno‐associated virus particles using isocratic anion exchange chromatography. Biotechnol J, 2021; 16(1):e2000015; doi: 10.1002/biot.202000015

Kurth

, Li

, Hausker

, et al. Separation of full and empty adeno-associated virus capsids by anion-exchange chromatography using choline-type salts. Anal Biochem, 2024; 686:115421; doi: 10.1016/j.ab.2023.115421

10.

Rieser

, Koch

, Faccioli

, et al. Comparison of different liquid chromatography-based purification strategies for adeno-associated virus vectors. Pharmaceutics, 2021; 13(5):748; doi: 10.3390/pharmaceutics13050748

11.

El Andari

, Grimm

. Production, processing, and characterization of synthetic AAV gene therapy vectors. Biotechnol J, 2021; 16(1):e2000025; doi: 10.1002/biot.202000025

12.

Venkatakrishnan

, Yarbrough

, Domsic

, et al. Structure and dynamics of adeno-associated virus serotype 1 VP1-Unique N-terminal domain and its role in capsid trafficking. J Virol, 2013; 87(9):4974–4984; doi: 10.1128/JVI.02524-12

13.

Ibreljic

, Draper

, Lawton

. Recombinant AAV genome size effect on viral vector production, purification, and thermostability. Mol Ther Methods Clin Dev, 2024; 32(1):101188; doi: 10.1016/j.omtm.2024.101188

14.

Lee

D-S

, Kim

B-M

, Seol

D-W

. Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography. Biochem Biophys Res Commun, 2009; 378(3):640–644; doi: 10.1016/j.bbrc.2008.11.096

15.

, Wang

, Wu

, et al. Calcium-ion-modulated ceramic hydroxyapatite resin for the scalable purification of recombinant Adeno-Associated Virus serotype 9. J Chromatogr B Analyt Technol Biomed Life Sci, 2015; 990:15–22; doi: 10.1016/j.jchromb.2015.03.003

16.

Heldt

, Areo

, Joshi

, et al. Empty and full AAV capsid charge and hydrophobicity differences measured with single-particle AFM. Langmuir, 2023; 39(16):5641–5648; doi: 10.1021/acs.langmuir.2c02643

17.

Gagnon

, Leskovec

, Prebil

, et al. Removal of empty capsids from adeno-associated virus preparations by multimodal metal affinity chromatography. J Chromatogr A, 2021; 1649:462210; doi: 10.1016/j.chroma.2021.462210

18.

Barnes

, Draper

, Jarrold

. Analysis of thermally driven structural changes, genome release, disassembly, and aggregation of recombinant AAV by CDMS. Mol Ther Methods Clin Dev, 2022; 27:327–336; doi: 10.1016/j.omtm.2022.10.008

19.

Khanal

, Kumar

, Jin

. Adeno-associated viral capsid stability on anion exchange chromatography column and its impact on empty and full capsid separation. Mol Ther Methods Clin Dev, 2023; 31:101112; doi: 10.1016/j.omtm.2023.101112

20.

Sharifi

, Ghasemi

, Duran

, et al. Impact of salt on AAV8 capsid aggregation with single-stranded DNA: Insights from coarse-grained molecular dynamics simulations. Int J Pharm, 2025; 681:125867; doi: 10.1016/j.ijpharm.2025.125867

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.24 MB