Determination of Particle Heterogeneity and Stability of Recombinant Adenovirus by Analytical Ultracentrifugation in CsCl Gradients

Yang, Xiaoyu; Agarwala, Shilpi; Ravindran, Sundari; Vellekamp, Gary

doi:10.1002/jps.21008

Cited by 17 publications

(14 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In contrast, analytic ultracentrifugation technology is a powerful tool for quantitative characterization of structural heterogeneity of rAAV preparations, allowing precise and selective observation of viral capsid sedimentation in real time. 27 , 28 For future studies, it may be necessary to use analytic ultracentrifugation for further characterization of compositions of clinical rAAV lots.…”

Section: Discussionmentioning

confidence: 99%

Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects

Gao

Zhong

et al. 2014

Molecular Therapy - Methods & Clinical Development

115

View full text Add to dashboard Cite

Empty virions are inadvertent by-products of recombinant adeno-associated virus (rAAV) packaging process, resulting in vector lots with mixtures of full and empty virions at variable ratios. Impact of empty virions on the efficiency and side-effects of rAAV transduction has not been well characterized. Here, we generated partially and completely empty AAV8 virions, fully packaged rAAV8 lots as well as mixtures of empty and fully packaged virions with variable ratios of empty virions (REVs). The aforementioned dosing formulations of rAAV8 expressing either cellular (EGFP or nuclear-targeted (n) LacZ) or secreted (human α1-antitrypsin, hA1AT) reporter genes were intravenously injected into two different mouse strains, followed by analyses of transgene expressions and serum alanine aminotransferase (ALT) levels at different time points. We found that addition of empty particles to the fixed doses of rAAV8 preparations repressed liver transduction up to 64% (serum hA1AT) and 44% (nLacZ) in C57BL/6 mice, respectively. The similar trend in inhibiting EGFP expression together with concurrent elevations of serum ATL levels were observed in the BALB/c mice, indicating that empty particles may also exacerbate side-effects of rAAV8EGFP transduction. Our results suggest that removal of empty particles from rAAV preparations may improve efficacy and safety of AAV in clinical applications.

show abstract

Section: Discussionmentioning

confidence: 99%

Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects

Gao

Zhong

et al. 2014

Molecular Therapy - Methods & Clinical Development

115

View full text Add to dashboard Cite

show abstract

“…Numerous techniques are available to provide this capsid content information (i.e., empty, partially packaged or fully packaged). The most commonly applied intact capsid approaches are dynamic light scattering (DLS) ( Kondylis et al, 2019 ), electron microscopy (EM) ( Kondylis et al, 2019 ), AUC ( Berkowitz and Philo, 2007 ; Berkowitz, 2008 ; Yang et al, 2008 ) and DCS ( Bondoc and Fitzpatrick, 1998 ; Shih et al, 2010 ). They can measure a range of viral particle populations from low density immature particles up to high mass aggregates.…”

Section: Introductionmentioning

confidence: 99%

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

Mullins

Powers

Zobel

et al. 2021

Front. Bioeng. Biotechnol.

View full text Add to dashboard Cite

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.

show abstract

“…Japanese encephalitis virus harvested from mouse brain was purified on a CsCl gradient by ultracentrifugation (9,10). The purified JEV was analyzed by SDS–PAGE and staining with Coomassie Brilliant blue.…”

Section: Resultsmentioning

confidence: 99%

“…Purified JEV was prepared in a cesium chloride (CsCl) gradient by ultracentrifugation (9,10). Murine monoclonal antibodies (mAbs) were prepared against Nakayama strain of JEV.…”

Section: Methodsmentioning

confidence: 99%

Development of an in vitro antigen‐detection test as an alternative method to the in vivo plaque reduction neutralization test for the quality control of Japanese encephalitis virus vaccine

Kim

et al. 2012

Microbiology and Immunology

View full text Add to dashboard Cite

Japanese encephalitis virus (JEV) causes diseases that attack the human central nervous system. Traditionally, the quality control for JEV vaccines, in which the plaque reduction neutralization (PRN) titer is measured by the national control laboratories before the vaccine batches are marketed, has required laboratory animal testing. However, classical animal tests have inherent problems, including the very fact that animals are used, ethical issues, and the possibility of error. In this study, JEV antigen was measured in an in vitro assay to assess the feasibility of replacing in vivo assays that measure the PRN titers of JEV vaccines. We constructed a double‐sandwich enzyme‐linked immunosorbent assay (DS‐ELISA) that could detect JEV envelope (E). Initially, monoclonal antibodies (mAbs) directed against the JEV E protein were generated and characterized. We isolated 18 mAbs against JEV E protein, and most were the IgG1 or IgG2a isotype. The mAbs (5F15 and 7D71) were selected as the most suitable mAb pair to detect JEV E protein. DS‐ELISA with this pair detected as little as approximately 3 μg/mL JEV E protein and demonstrated a relationship between the amount of JEV E protein and the PRN titer. From these results, we surmise that this DS‐ELISA may be useful, not only in terms of measuring the amount of JEV E protein, but also as a substitute for the PRN test for JEV vaccine evaluation.

show abstract

Determination of Particle Heterogeneity and Stability of Recombinant Adenovirus by Analytical Ultracentrifugation in CsCl Gradients

Cited by 17 publications

References 40 publications

Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects

Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects

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

Development of an in vitro antigen‐detection test as an alternative method to the in vivo plaque reduction neutralization test for the quality control of Japanese encephalitis virus vaccine

Contact Info

Product

Resources

About