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Accurate Quantification and Characterization of Adeno-Associated Viral Vectors

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...

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Autores principales: Dobnik, David, Kogovšek, Polona, Jakomin, Tjaša, Košir, Nejc, Tušek Žnidarič, Magda, Leskovec, Maja, Kaminsky, Stephen M., Mostrom, Janet, Lee, Hyunmi, Ravnikar, Maja
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650692/
https://www.ncbi.nlm.nih.gov/pubmed/31379763
http://dx.doi.org/10.3389/fmicb.2019.01570
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author Dobnik, David
Kogovšek, Polona
Jakomin, Tjaša
Košir, Nejc
Tušek Žnidarič, Magda
Leskovec, Maja
Kaminsky, Stephen M.
Mostrom, Janet
Lee, Hyunmi
Ravnikar, Maja
author_facet Dobnik, David
Kogovšek, Polona
Jakomin, Tjaša
Košir, Nejc
Tušek Žnidarič, Magda
Leskovec, Maja
Kaminsky, Stephen M.
Mostrom, Janet
Lee, Hyunmi
Ravnikar, Maja
author_sort Dobnik, David
collection PubMed
description 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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spelling pubmed-66506922019-08-02 Accurate Quantification and Characterization of Adeno-Associated Viral Vectors Dobnik, David Kogovšek, Polona Jakomin, Tjaša Košir, Nejc Tušek Žnidarič, Magda Leskovec, Maja Kaminsky, Stephen M. Mostrom, Janet Lee, Hyunmi Ravnikar, Maja Front Microbiol Microbiology 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. Frontiers Media S.A. 2019-07-17 /pmc/articles/PMC6650692/ /pubmed/31379763 http://dx.doi.org/10.3389/fmicb.2019.01570 Text en Copyright © 2019 Dobnik, Kogovšek, Jakomin, Košir, Tušek Žnidarič, Leskovec, Kaminsky, Mostrom, Lee and Ravnikar. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Dobnik, David
Kogovšek, Polona
Jakomin, Tjaša
Košir, Nejc
Tušek Žnidarič, Magda
Leskovec, Maja
Kaminsky, Stephen M.
Mostrom, Janet
Lee, Hyunmi
Ravnikar, Maja
Accurate Quantification and Characterization of Adeno-Associated Viral Vectors
title Accurate Quantification and Characterization of Adeno-Associated Viral Vectors
title_full Accurate Quantification and Characterization of Adeno-Associated Viral Vectors
title_fullStr Accurate Quantification and Characterization of Adeno-Associated Viral Vectors
title_full_unstemmed Accurate Quantification and Characterization of Adeno-Associated Viral Vectors
title_short Accurate Quantification and Characterization of Adeno-Associated Viral Vectors
title_sort accurate quantification and characterization of adeno-associated viral vectors
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650692/
https://www.ncbi.nlm.nih.gov/pubmed/31379763
http://dx.doi.org/10.3389/fmicb.2019.01570
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