Validation of a Quantitative Method for Detection of Adenovirus Aggregation

Shih, Shian-Jiun; Yagami, Machiko

doi:10.12665/j92.shih

Cited by 9 publications

(12 citation statements)

References 17 publications

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“…who used a SPA method , and for the characterization of adenovirus in the study of Shih et al. by a DCS disc centrifugation method , respectively. In the latter, an 8–24% sucrose gradient was compared to a conventional AUC method without sucrose gradient and only showed consistent minor reductions of the detected aggregate levels .…”

Section: Resultsmentioning

confidence: 99%

“…by a DCS disc centrifugation method , respectively. In the latter, an 8–24% sucrose gradient was compared to a conventional AUC method without sucrose gradient and only showed consistent minor reductions of the detected aggregate levels . This suggests that the addition of sucrose is not affecting the measured PSD via DCS disc centrifugation.…”

Section: Resultsmentioning

confidence: 99%

“…The implemented DCS method determines the sedimentation times of the particles of interest with unknown size but known buoyant density in a gradient fluid with known density to calculate their sizes based on Stokes’ law . Applications reported so far included the characterization of PSD of recombinant adeno VP , L1 virus‐like particles , and influenza VP . In addition, it was shown that a DCS method enables the determination of PSD with a higher resolution than other dynamic light scattering techniques .…”

Section: Introductionmentioning

confidence: 99%

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Specific ion effects on the particle size distributions of cell culture–derived influenza A virus particles within the Hofmeister series

Heyse

Wolff

Reichl

2016

Engineering in Life Sciences

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o r P e e r R e v i e w 2 PVC, polyvinyl chloride; RPM, revolutions per minute; RT, room temperature; SB, standard buffer; SPA, single particle approximation; VP, Virus particle. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 F o r P e e r R e v i e w 3 Page 2 of 31 Wiley-VCH Engineering in Life Sciences Practical applicationThis work shows the practical application of a differential centrifugal sedimentation (DCS) disc centrifugation method for virus particle size distribution screenings (PSDs). The high resolution and accuracy of the implemented PSD measurement method offered interesting insights on the (aggregation) behavior of influenza A/Puerto Rico/8/34 (H1N1) virus particles in different buffer systems. Therefore, the method's applicability for screening of other biological colloidal particle systems, like vaccine or viral vector formulations, is feasible. Considering the large impact of virus particle size and aggregation behavior on downstream processing yields as well as formulation and blending of vaccines and gene therapy vectors our results address important issues concerning process optimization and product quality. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 Page 3 of 31 Wiley-VCH Engineering in Life Sciences

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Specific ion effects on the particle size distributions of cell culture–derived influenza A virus particles within the Hofmeister series

Heyse

Wolff

Reichl

2016

Engineering in Life Sciences

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“…Electrophoretic light scattering (ELS) measurements of the rAAV samples were measured on a Zetasizer Ultra instrument (Malvern Panalytical Ltd., Malvern, UK) equipped with an He–Ne laser at a wavelength of 633 nm and maximum power of 10 mW. Measurements were performed using the diffusion barrier method (DBM) [ 37 , 43 , 44 , 45 , 46 ] in which 20 µL aliquots of the samples were introduced into a folded capillary cell (DTS1070 Malvern Panalytical Ltd., Malvern, UK) containing the PBS with 0.001% Pluronic F-68 buffer. The instrument settings were optimized automatically by means of the ZS XPLORER software.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques

Cole¹,

Fernandes²,

Hussain³

et al. 2021

Pharmaceutics

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Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies produced through controlled and economical manufacturing processes, multiple challenges need to be addressed starting from viral capsid design through identification of optimal process and formulation conditions to comprehensive quality control. Addressing these challenges requires fit-for-purpose analytics for extensive characterization of rAAV samples including measurements of capsid or particle titer, percentage of full rAAV particles, particle size, aggregate formation, thermal stability, genome release, and capsid charge, all of which may impact critical quality attributes of the final product. Importantly, there is a need for rapid analytical solutions not relying on the use of dedicated reagents and costly reference standards. In this study, we evaluate the capabilities of dynamic light scattering, multiangle dynamic light scattering, and SEC–MALS for analyses of rAAV5 samples in a broad range of viral concentrations (titers) at different levels of genome loading, sample heterogeneity, and sample conditions. The study shows that DLS and MADLS® can be used to determine the size of full and empty rAAV5 (27 ± 0.3 and 33 ± 0.4 nm, respectively). A linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 × 1011–8.7 × 1013 cp/mL for the nominally full rAAV5 samples and 3.4 × 1011–7 × 1013 cp/mL for the nominally empty rAAV5 samples with 3–8% and 10–37% CV for the full and empty rAAV5 samples, respectively. The structural stability and viral load release were also inferred from a combination of DLS, SEC–MALS, and DSC. The structural characteristics of the rAAV5 start to change from 40 °C onward, with increasing aggregation observed. With this study, we explored and demonstrated the applicability and value of orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples.

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“…Aggregation of individual virions has been observed for viruses of different species in the environment and cell culture preparations/buffer solutions 29 . Since viral aggregation increases resistance to disinfectants 30 and may interfere with high-quality production of vectors for gene therapy and vaccine 31 , this phenomenon has been studied for decades. Viral aggregates may vary from a few to hundreds of virions.…”

Section: Discussionmentioning

confidence: 99%

A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation

Stepanenko

Сосновцева

Valikhov

et al. 2021

Sci Rep

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Two-cycle cesium chloride (2 × CsCl) gradient ultracentrifugation is a conventional approach for purifying recombinant adenoviruses (rAds) for research purposes (gene therapy, vaccines, and oncolytic vectors). However, rAds containing the RGD-4C peptide in the HI loop of the fiber knob domain tend to aggregate during 2 × CsCl gradient ultracentrifugation resulting in a low infectious titer yield or even purification failure. An iodixanol-based purification method preventing aggregation of the RGD4C-modified rAds has been proposed. However, the reason explaining aggregation of the RGD4C-modified rAds during 2 × CsCl but not iodixanol gradient ultracentrifugation has not been revealed. In the present study, we showed that rAds with the RGD-4C peptide in the HI loop but not at the C-terminus of the fiber knob domain were prone to aggregate during 2 × CsCl but not iodixanol gradient ultracentrifugation. The cysteine residues with free thiol groups after the RGD motif within the inserted RGD-4C peptide were responsible for formation of the interparticle disulfide bonds under atmospheric oxygen and aggregation of Ad5-delta-24-RGD4C-based rAds during 2 × CsCl gradient ultracentrifugation, which could be prevented using iodixanol gradient ultracentrifugation, most likely due to antioxidant properties of iodixanol. A cysteine-to-glycine substitution of the cysteine residues with free thiol groups (RGD-2C2G) prevented aggregation during 2 × CsCl gradient purification but in coxsackie and adenovirus receptor (CAR)-low/negative cancer cell lines of human and rodent origin, this reduced cytolytic efficacy to the levels observed for a fiber non-modified control vector. However, both Ad5-delta-24-RGD4C and Ad5-delta-24-RGD2C2G were equally effective in the murine immunocompetent CT-2A glioma model due to a primary role of antitumor immune responses in the therapeutic efficacy of oncolytic virotherapy.

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Validation of a Quantitative Method for Detection of Adenovirus Aggregation

Cited by 9 publications

References 17 publications

Specific ion effects on the particle size distributions of cell culture–derived influenza A virus particles within the Hofmeister series

Specific ion effects on the particle size distributions of cell culture–derived influenza A virus particles within the Hofmeister series

Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques

A new insight into aggregation of oncolytic adenovirus Ad5-delta-24-RGD during CsCl gradient ultracentrifugation

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