Separating Empty and Full Recombinant Adeno‐Associated Virus Particles Using Isocratic Anion Exchange Chromatography

Dickerson, Ryan; Argento, Christopher; Pieracci, John; Bakhshayeshi, Meisam

doi:10.1002/biot.202000015

Cited by 58 publications

(80 citation statements)

References 24 publications

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“…In particular, when we applied a step protocol for buffer B, we achieved an almost baseline separation of the full rAAV and the empty capsid peaks. Other studies have also achieved full/empty separation for different AAV serotypes by AEX [ 8 , 24 , 34 , 37 , 38 , 39 , 40 ]. For determination of the degree of separation of full rAAVs from empty capsids, we used analytical AEX.…”

Section: Discussionmentioning

confidence: 99%

Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors

et al. 2021

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Recombinant adeno-associated virus (rAAV) vectors have evolved as one of the most promising technologies for gene therapy due to their good safety profile, high transduction efficacy, and long-term gene expression in nondividing cells. rAAV-based gene therapy holds great promise for treating genetic disorders like inherited blindness, muscular atrophy, or bleeding disorders. There is a high demand for efficient and scalable production and purification methods for rAAVs. This is particularly true for the downstream purification methods. The current standard methods are based on multiple steps of gradient ultracentrifugation, which allow for the purification and enrichment of full rAAV particles, but the scale up of this method is challenging. Here, we explored fast, scalable, and universal liquid chromatography-based strategies for the purification of rAAVs. In contrast to the hydrophobic interaction chromatography (HIC), where a substantial amount of AAV was lost, the cation exchange chromatography (CEX) was performed robustly for multiple tested serotypes and resulted in a mixture of full and empty rAAVs with a good purity profile. For the used affinity chromatography (AC), a serotype dependence was observed. Anion exchange chromatography (AEX) worked well for the AAV8 serotype and achieved high levels of purification and a baseline separation of full and empty rAAVs. Depending on the AAV serotype, a combination of CEX and AEX or AC and AEX is recommended and holds promise for future translational projects that require highly pure and full particle-enriched rAAVs.

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Section: Discussionmentioning

confidence: 99%

Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors

et al. 2021

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“…Triton X-100 lysates of sf9/BEV and HEK293 cells producing AAV2/8 were obtained from The University of Nantes, Center for Translational Therapy for Genetic Diseases, INSERM UMR 1089, Nantes, France. AAV8 was chosen particularly because AEC is known to separate empty and full capsids into separate peaks [ 6 , 9 , 10 , 16 ]. All were membrane filtered to 0.45 µm.…”

Section: Methodsmentioning

confidence: 99%

“…The 260/280 ratios for empty and full AAV capsids in a non-chromatographic study were reported to be 0.59 and 1.44, respectively [ 7 ]. AEC has been shown to separate empty and full capsids into separate peaks for serotypes 1, 2, 4, 5, 6, 8, and 9 [ 6 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. Empty capsid peaks separated from full capsid peaks by anion exchange (AEC) commonly exhibit ratios in the range of 0.6 to 0.7, while full capsids peaks commonly exhibit ratios in the range of 1.3–1.4.…”

Section: Introductionmentioning

confidence: 99%

Multiple-Monitor HPLC Assays for Rapid Process Development, In-Process Monitoring, and Validation of AAV Production and Purification

et al. 2021

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HPLC is established as a fast convenient analytical technology for characterizing the content of empty and full capsids in purified samples containing adeno-associated virus (AAV). UV-based monitoring unfortunately over-estimates the proportion of full capsids and offers little value for characterizing unpurified samples. The present study combines dual-wavelength UV monitoring with intrinsic fluorescence, extrinsic fluorescence, and light-scattering to extend the utility of HPLC for supporting development of therapeutic AAV-based drugs. Applications with anion exchange (AEC), cation exchange (CEC), and size exclusion chromatography (SEC) are presented. Intrinsic fluorescence increases sensitivity of AAV detection over UV and enables more objective estimation of empty and full capsid ratios by comparison of their respective peak areas. Light scattering enables identification of AAV capsids in complex samples, plus semiquantitative estimation of empty and full capsid ratios from relative peak areas of empty and full capsids. Extrinsic Picogreen fluorescence enables semiquantitative tracking of DNA with all HPLC methods at all stages of purification. It does not detect encapsidated DNA but reveals DNA associated principally with the exteriors of empty capsids. It also enables monitoring of host DNA contamination across chromatograms. These enhancements support many opportunities to improve characterization of raw materials and process intermediates, to accelerate process development, provide rapid in-process monitoring, and support process validation.

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“…Anion exchange chromatography (AEX) also achieves fractionation of empty and full capsids for many AAV serotypes [4][5][6][7][8][9][10][11][12][13][14][15]. The degree of separation varies among serotypes and does not correlate strictly with DGUC.…”

Section: Introductionmentioning

confidence: 99%

Multiple-parameter profiling of density gradient ultracentrifugation for characterization of empty and full capsid distribution in AAV preparations

Peljhan¹,

Štokelj²,

Prebil³

et al. 2021

Cell Gene Therapy Insights

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Ultracentrifugation (UC) is a well-known technique for fractionating adeno-associated virus (AAV) capsids according to their density, which is mainly a function of their encapsidated DNA mass. Empty capsids represent the lowest density subpopulation. Full capsids represent the highest density subpopulation, sometimes accompanied by partially full capsids of intermediate density. Fractions can be collected after sedimentation for analysis but the practice is laborious and discourages application of multiple monitoring techniques that might provide deeper insights into sample composition. Anion exchange chromatography (AEC) also achieves fractionation of empty and full capsids for many AAV serotypes. The degree of separation varies among serotypes and does not correlate strictly with UC. This is not surprising since separation by AEC is highly influenced by capsid surface charge, which is independent of the amount of DNA packaged within the capsids. Chromatography methods however present a significant analytical advantage in the ease of monitoring the column effluent, including with multiple detectors. UV absorbance at 260 nm and 280 nm permits estimation of empty and full capsid proportions in any given peak. Intrinsic fluorescence enables estimation of relative areas of empty capsid peaks and full capsid peaks. Light scattering does the same and permits the further determination of capsid size and mass. In this report, we merge UC with an HPLC monitoring array to simultaneously analyze dual wavelength UV, intrinsic fluorescence, and light scattering through cesium chloride density gradient strata. Limitations of each monitoring method are discussed. UC results are compared with chromatography profiles to highlight distinction between separation methods.

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Separating Empty and Full Recombinant Adeno‐Associated Virus Particles Using Isocratic Anion Exchange Chromatography

Cited by 58 publications

References 24 publications

Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors

Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors

Multiple-Monitor HPLC Assays for Rapid Process Development, In-Process Monitoring, and Validation of AAV Production and Purification

Multiple-parameter profiling of density gradient ultracentrifugation for characterization of empty and full capsid distribution in AAV preparations

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