A Digestion-free Method for Quantification of Residual Host Cell DNA in rAAV Gene Therapy Products

Wang, Yu; Cooper, Rebecca; Kiladjian, Albert; Bergelson, Svetlana; Feschenko, Marina S.

doi:10.1016/j.omtm.2019.05.005

Cited by 20 publications

(12 citation statements)

References 19 publications

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“…Determination of HEK cell DNA was done on a QuantStudio 5 system (Thermo Fisher Scientific, Dreieich, Germany) with a SYBR Green qPCR assay designed to amplify a 94-bp Alu sequence using the following primers: 5′-GAGGCGGGCGGATCA-3′ (forward) and 5′-CCCGGCTAATTTTTGTATTTTTAG-3′ (reverse) [ 26 ].…”

Section: Methodsmentioning

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: Methodsmentioning

confidence: 99%

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

et al. 2021

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“…For final product characterization, and as well as for mAbs, quantification of residual DNA from the producing cell line is always required for clinical lot release due to the stringent guidelines for this impurity (100 pg/dose < DNA < 10 ng/dose) [165]. Among the different methods used for host cell DNA quantification PicoGreen® assay, hybridization, Threshold™ technology, and qPCR, are the most used [165][166][167]. From the methods presented, Threshold™ (1-3 pg rDNA) and qPCR are the most sensitives.…”

Section: Analytics In Process Developmentmentioning

confidence: 99%

Current challenges in biotherapeutic particles manufacturing

Moleirinho

Silva

Alves

et al. 2019

Expert Opinion on Biological Therapy

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Introduction: The development of novel complex biotherapeutics led to new challenges in biopharmaceutical industry. The potential of these particles has been demonstrated by the approval of several products, in the different fields of gene therapy, oncolytic therapy, and tumor vaccines. However, their manufacturing still presents challenges related to the high dosages and purity required. Areas covered: The main challenges that biopharmaceutical industry faces today and the most recent developments in the manufacturing of different biotherapeutic particles are reported here. Several unit operations and downstream trains to purify virus, virus-like particles and extracellular vesicles are described. Innovations on the different purification steps are also highlighted with an eye on the implementation of continuous and integrated processes. Expert opinion: Manufacturing platforms that consist of a low number of unit operations, with higheryielding processes and reduced costs will be highly appreciated by the industry. The pipeline of complex therapeutic particles is expanding and there is a clear need for advanced tools and manufacturing capacity. The use of single-use technologies, as well as continuous integrated operations, are gaining ground in the biopharmaceutical industry and should be supported by more accurate and faster analytical methods.

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“…This work expanded the application of the “digestion-free method” we previously developed for residual DNA detection in AAV. 14 A simple dilution of AAV in TE buffer with 5% Tween 20 facilitates qPCR detection of encapsidated DNA and proteinase digestion of AAV capsid could be skipped. The addition of Tween 20 is important as other methods skipping proteinase treatment showed poor precision.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, we eliminated unnecessary sample treatments by employing a digestion-free method that has been demonstrated to improve qPCR quantification of residual DNA in AAV. 14 As a result, a simplified and more streamlined qPCR method for AAV genome titer has been developed, which is able to achieve ddPCR-level of accuracy and precision.…”

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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A Digestion-free Method for Quantification of Residual Host Cell DNA in rAAV Gene Therapy Products

Cited by 20 publications

References 19 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

Current challenges in biotherapeutic particles manufacturing

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

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