Analytical approaches to characterize AAV vector production &amp; purification: Advances and challenges

Dorange, Fabien; Bec, Christine Le

doi:10.18609/cgti.2018.015

Cited by 15 publications

(22 citation statements)

References 36 publications

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“…1 e). With the high precision of the SEC assay taken into account, the error is still within the variability range of the widely-used PCR and ELISA titering methods in samples with even up to ~ 50% light capsids 8 , 21 , 22 . Further, using UV absorbance, the relative percentage of light to heavy capsids was estimated from A260/A280 peak area ratios.…”

Section: Resultsmentioning

confidence: 99%

Comprehensive characterization and quantification of adeno associated vectors by size exclusion chromatography and multi angle light scattering

McIntosh

Berguig

Karim

et al. 2021

Sci Rep

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Adeno associated virus (AAV) capsids are a leading modality for in vivo gene delivery. Complete and precise characterization of capsid particles, including capsid and vector genome concentration, is necessary to safely and efficaciously dose patients. Size exclusion chromatography (SEC) coupled to multiangle light scattering (MALS) offers a straightforward approach to comprehensively characterize AAV capsids. The current study demonstrates that this method provides detailed AAV characterization information, including but not limited to aggregation profile, size-distribution, capsid content, capsid molar mass, encapsidated DNA molar mass, and total capsid and vector genome titer. Currently, multiple techniques are required to generate this information, with varying accuracy and precision. In the current study, a new series of equations for SEC-MALS are used in tandem with intrinsic properties of the capsids and encapsidated DNA to quantify multiple physical AAV attributes in one 20-min run with minimal sample manipulation, high accuracy, and high precision. These novel applications designate this well-established method as a powerful tool for product development and process analytics in future gene therapy programs.

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Section: Resultsmentioning

confidence: 99%

Comprehensive characterization and quantification of adeno associated vectors by size exclusion chromatography and multi angle light scattering

McIntosh

Berguig

Karim

et al. 2021

Sci Rep

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show abstract

“…This is especially important for validation of assays for in-process control, where the validation should be performed for each matrix individually. ddPCR can be run without prior DNA extraction, which enables fast analysis time and can thus beneficial for fast determination of yield of downstream processes (Dorange and Le Bec, 2018). We have seen that there is actually no matrix interference with ddPCR by downstream (Table 3) or upstream samples (experience from contract research activities).…”

Section: Discussionmentioning

confidence: 99%

“…The total capsid content may be as important as the vector genome titer and to date there is no harmonized method to assay total capsids or the ability to distinguish empty particles from genome containing particles. Separation of AAV particles and quantification of full/empty ratio can be achieved using techniques, such as enzyme linked immunosorbent assay (ELISA), electron microscopy, analytical ultracentrifugation (AUC), and high pressure liquid chromatography (HPLC), where each has its own advantages and limitations (D’Costa et al, 2016; Dorange and Le Bec, 2018).…”

Section: Introductionmentioning

confidence: 99%

Accurate Quantification and Characterization of Adeno-Associated Viral Vectors

et al. 2019

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One of the main challenges in the gene therapy viral vector development is to establish an optimized process for its large scale production. This requires optimization for upstream and downstream processes as well as methods that enable the step-by step analytical characterization of the virus, the results of which inform the iterative refinement of production for yield, purity and potency. The biggest problem here is a plethora of viral vector formulations, many of which interfere with analytical techniques. We took adeno-associated virus (AAV) as an example and showed benefits of combined use of molecular methods and transmission electron microscopy (TEM) for viral vectors’ characterization and quantification. Results of the analyses showed that droplet digital PCR (ddPCR) performs better than quantitative real-time PCR (qPCR), in terms of robustness and assay variance, and this was especially relevant for partially purified (in-process) samples. Moreover, we demonstrate the importance of sample preparation prior to PCR analysis. We evaluated viral structure, presence of aggregates and impurities with TEM analysis and found that these impacted the differences in viral titers observed by qPCR and ddPCR and could be altered by sample preparation. These results serve as a guide for the establishment of the analytical methods required to provide measures of identity and purity for AAV viral vectors.

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“…Due to the existence of empty capsids that do not possess therapeutic benefits, titration by viral genome is preferred for clinical dosing, manufacturing, and analytical testing. 2 , 3 , 4 Traditionally, AAV genome titer is determined by qPCR using a plasmid DNA standard. For the past two decades, extensive efforts have been made in improving qPCR standard preparation, sample treatment, and primer/probe design, etc.…”

Section: Introductionmentioning

confidence: 99%

A qPCR Method for AAV Genome Titer with ddPCR-Level of Accuracy and Precision

Wang

Menon

Shen

et al. 2020

Molecular Therapy - Methods & Clinical Development

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Recombinant adeno-associated virus (rAAV) is one of the main vectors used in gene therapy. An accurate genome titer is not only critical for clinical dosing, but also a prerequisite for many analytical assays for AAV product characterization. AAV genome titer is traditionally determined by qPCR; however, assay precision is not optimal despite extensive efforts. More recently, droplet digital PCR (ddPCR) emerged as a powerful alternative that offers excellent accuracy and precision. However, currently ddPCR is not as widely available as qPCR and operates at a lower throughput and a higher cost. In this paper, we introduce an improved qPCR method with two major optimizations: (1) using an AAV reference material as qPCR standard instead of plasmid DNA and (2) implementing a “digestion-free” method by adding 5% Tween 20 to standard and sample preparations. The new method has been extensively tested with AAV of different serotypes, purification status, and transgenes encapsidated and was found to be highly accurate, precise, and robust. This significantly improved and simplified assay can be easily adopted by researchers in the gene therapy field and further automated for high-throughput applications.

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Analytical approaches to characterize AAV vector production & purification: Advances and challenges

Cited by 15 publications

References 36 publications

Comprehensive characterization and quantification of adeno associated vectors by size exclusion chromatography and multi angle light scattering

Comprehensive characterization and quantification of adeno associated vectors by size exclusion chromatography and multi angle light scattering

Accurate Quantification and Characterization of Adeno-Associated Viral Vectors

A qPCR Method for AAV Genome Titer with ddPCR-Level of Accuracy and Precision

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