Characterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer

We observed differential infectivity and product yield between two recombinant chimpanzee adenovirus C68 constructs whose primary difference was genome length. To determine a possible reason for this outcome, we characterized the proportion and composition of the empty and packaged capsids. Both ana...

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Autores principales:	Elise K. Mullins, Thomas W. Powers, Jim Zobel, Kory M. Clawson, Lauren F. Barnes, Benjamin E. Draper, Qin Zou, Joseph J. Binder, Stanley Dai, Kun Zhang, Olga Friese, Herbert A. Runnels, Martin F. Jarrold, Lawrence C. Thompson
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	non-human primate adenovirus AEX-HPLC analytical ultracentrifugation differential centrifugation sedimentation charge detection mass spectrometry Biotechnology TP248.13-248.65
Acceso en línea:	https://doaj.org/article/f1f1e75dde024640af7aecb78aac433e
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spelling	oai:doaj.org-article:f1f1e75dde024640af7aecb78aac433e2021-11-04T05:15:40ZCharacterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer2296-418510.3389/fbioe.2021.753480https://doaj.org/article/f1f1e75dde024640af7aecb78aac433e2021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fbioe.2021.753480/fullhttps://doaj.org/toc/2296-4185We observed differential infectivity and product yield between two recombinant chimpanzee adenovirus C68 constructs whose primary difference was genome length. To determine a possible reason for this outcome, we characterized the proportion and composition of the empty and packaged capsids. Both analytical ultracentrifugation (AUC) and differential centrifugation sedimentation (DCS, a rapid and quantitative method for measuring adenoviral packaging variants) were employed for an initial assessment of genome packaging and showed multiple species whose abundance deviated between the virus builds but not manufacturing campaigns. Identity of the packaging variants was confirmed by charge detection mass spectrometry (CDMS), the first known application of this technique to analyze adenovirus. The empty and packaged capsid populations were separated via preparative ultracentrifugation and then combined into a series of mixtures. These mixtures showed the oft-utilized denaturing A260 adenoviral particle titer method will underestimate the actual particle titer by as much as three-fold depending on the empty/full ratio. In contrast, liquid chromatography with fluorescence detection proves to be a superior viral particle titer methodology.Elise K. MullinsThomas W. PowersJim ZobelKory M. ClawsonLauren F. BarnesBenjamin E. DraperQin ZouJoseph J. BinderStanley DaiKun ZhangOlga FrieseHerbert A. RunnelsMartin F. JarroldLawrence C. ThompsonFrontiers Media S.A.articlenon-human primateadenovirusAEX-HPLCanalytical ultracentrifugationdifferential centrifugation sedimentationcharge detection mass spectrometryBiotechnologyTP248.13-248.65ENFrontiers in Bioengineering and Biotechnology, Vol 9 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	non-human primate adenovirus AEX-HPLC analytical ultracentrifugation differential centrifugation sedimentation charge detection mass spectrometry Biotechnology TP248.13-248.65
spellingShingle	non-human primate adenovirus AEX-HPLC analytical ultracentrifugation differential centrifugation sedimentation charge detection mass spectrometry Biotechnology TP248.13-248.65 Elise K. Mullins Thomas W. Powers Jim Zobel Kory M. Clawson Lauren F. Barnes Benjamin E. Draper Qin Zou Joseph J. Binder Stanley Dai Kun Zhang Olga Friese Herbert A. Runnels Martin F. Jarrold Lawrence C. Thompson Characterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer
description	We observed differential infectivity and product yield between two recombinant chimpanzee adenovirus C68 constructs whose primary difference was genome length. To determine a possible reason for this outcome, we characterized the proportion and composition of the empty and packaged capsids. Both analytical ultracentrifugation (AUC) and differential centrifugation sedimentation (DCS, a rapid and quantitative method for measuring adenoviral packaging variants) were employed for an initial assessment of genome packaging and showed multiple species whose abundance deviated between the virus builds but not manufacturing campaigns. Identity of the packaging variants was confirmed by charge detection mass spectrometry (CDMS), the first known application of this technique to analyze adenovirus. The empty and packaged capsid populations were separated via preparative ultracentrifugation and then combined into a series of mixtures. These mixtures showed the oft-utilized denaturing A260 adenoviral particle titer method will underestimate the actual particle titer by as much as three-fold depending on the empty/full ratio. In contrast, liquid chromatography with fluorescence detection proves to be a superior viral particle titer methodology.
format	article
author	Elise K. Mullins Thomas W. Powers Jim Zobel Kory M. Clawson Lauren F. Barnes Benjamin E. Draper Qin Zou Joseph J. Binder Stanley Dai Kun Zhang Olga Friese Herbert A. Runnels Martin F. Jarrold Lawrence C. Thompson
author_facet	Elise K. Mullins Thomas W. Powers Jim Zobel Kory M. Clawson Lauren F. Barnes Benjamin E. Draper Qin Zou Joseph J. Binder Stanley Dai Kun Zhang Olga Friese Herbert A. Runnels Martin F. Jarrold Lawrence C. Thompson
author_sort	Elise K. Mullins
title	Characterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer
title_short	Characterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer
title_full	Characterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer
title_fullStr	Characterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer
title_full_unstemmed	Characterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer
title_sort	characterization of recombinant chimpanzee adenovirus c68 low and high-density particles: impact on determination of viral particle titer
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/f1f1e75dde024640af7aecb78aac433e
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Characterization of Recombinant Chimpanzee Adenovirus C68 Low and High-Density Particles: Impact on Determination of Viral Particle Titer

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