Design space determination to optimize DNA complexation and full capsid formation in transient rAAV manufacturing

Fu, Qiang; Lee, Yong Suk; Green, Erica A; Wang, Yongdan; Park, So Young; Polanco, Ashli; Lee, Kelvin H.; Betenbaugh, Michael J.; McNally, D.; Yoon, Seongkyu

doi:10.1002/bit.28508

Cited by 11 publications

(1 citation statement)

References 52 publications

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“…Meeting the high demand for rAAVs is impeded by the scalability of this production process and the challenges associated with large-scale transient gene expression (TGE) 4,5 . Optimisation is crucial but can be challenging due to the involvement of multiple parameters, including cell line, cell culture conditions such as culture media and cell density, and transfection conditions such as total plasmid DNA amount, the plasmid ratio employed in the transfection process and the total amount of transfection reagent 6–10 .…”

Section: Introductionmentioning

confidence: 99%

Unlocking DOE potential selecting the most appropriate design for rAAV optimization

Tzimou,

Catalán-Tatjer,

Nielsen

et al. 2024

Preprint

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The production of recombinant adeno-associated virus (rAAV) for gene therapy via triple transfection is a highly intricate process involving many cellular interactions. Each of the different elements encoded in the three required plasmids - pHelper, pRepCap, and pGOI - play a distinct role and affect different cellular pathways when producing rAAVs. The expression balance of these different elements emphasizes the critical need to fine-tune the concentration of all three plasmids and transfection reagents effectively. The use of design of experiments (DOE) to find optimal plasmid and transfection reagent ratios is a powerful method to streamline the process. However, the choice of the DOE method and the design construction is crucial to avoid misleading results. In this work, we examined and compared four distinct DOE approaches: a rotatable central composite design (RCCD), a Box-Behnken design (BBD), a face-centered central composite design (FCCD), and a mixture design (MD). We compared the ability of the different models to predict optimal ratios, interactions among the three plasmids and transfection reagent, and the essentiality of studying the variability caused by uncontrolled random effects using blocking. Our findings revealed that MD, when coupled with FCCD, outperformed all other tested models. This outcome underscores the importance of selecting a model that can effectively account for the biological context, ultimately yielding superior results in optimizing rAAV production.

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Section: Introductionmentioning

confidence: 99%

Unlocking DOE potential selecting the most appropriate design for rAAV optimization

Tzimou,

Catalán-Tatjer,

Nielsen

et al. 2024

Preprint

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Image‐based hybrid model incorporating initial spatial distribution for mesenchymal stem cell cultivation process design

Hirono,

Hayashi,

A. Udugama

et al. 2024

AIChE Journal

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This work presents an image‐based hybrid model incorporating the initial spatial distribution for mesenchymal stem cell (MSC) cultivation process design. First, three levels of seeding bias in static MSC cultivation were experimentally investigated, and the resulting initial distribution was quantified using phase contrast microscopy image analysis. Second, the observed spatial heterogeneity was defined as a new parameter by calculating the standard deviation of the seeded cell number fraction among numerical 8 × 8 tiles on two‐dimensional plates. Third, the parameter was incorporated into kinetic models to consider spatial growth limitation. The model was then applied to simulate MSC cultivation processes with experimental data. Using dynamic and stochastic simulation outputs, feasible ranges of cell‐harvesting time could be determined to satisfy a given requirement for the minimum cell number and acceptable confluency level. The developed hybrid model could serve as a basis for quantitative decision‐making in the design of MSC cultivation processes.

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Multidose transient transfection of human embryonic kidney 293 cells modulates recombinant adeno‐associated virus2/5 Rep protein expression and influences the enrichment fraction of filled capsids

Srinivasan,

Canova,

Sha

et al. 2024

Biotech & Bioengineering

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Recombinant adeno‐associated virus (rAAV) is a commonly used in vivo gene therapy vector because of its nonpathogenicity, long‐term transgene expression, broad tropism, and ability to transduce both dividing and nondividing cells. However, rAAV vector production via transient transfection of mammalian cells typically yields a low fraction of filled‐to‐total capsids (~1%–30% of total capsids produced). Analysis of our previously developed mechanistic model for rAAV2/5 production attributed these low fill fractions to a poorly coordinated timeline between capsid synthesis and viral DNA replication and the repression of later phase capsid formation by Rep proteins. Here, we extend the model by quantifying the expression dynamics of total Rep proteins and their influence on the key steps of rAAV2/5 production using a multiple dosing transfection of human embryonic kidney 293 (HEK293) cells. We report that the availability of preformed empty capsids and viral DNA copies per cell are not limiting to the capsid‐filling reaction. However, optimal expression of Rep proteins (<240 ± 13 ag per cell) enables enrichment of the filled capsid population (>12% of total capsids/cell) upstream. Our analysis suggests increased enrichment of filled capsids via regulating the expression of Rep proteins is possible but at the expense of per cell capsid titer in a triple plasmid transfection. Our study reveals an intrinsic limitation of scaling rAAV2/5 vector genome (vg) production and underscores the need for approaches that allow for regulating the expression of Rep proteins to maximize vg titer per cell upstream.

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Design space determination to optimize DNA complexation and full capsid formation in transient rAAV manufacturing

Cited by 11 publications

References 52 publications

Unlocking DOE potential selecting the most appropriate design for rAAV optimization

Unlocking DOE potential selecting the most appropriate design for rAAV optimization

Image‐based hybrid model incorporating initial spatial distribution for mesenchymal stem cell cultivation process design

Multidose transient transfection of human embryonic kidney 293 cells modulates recombinant adeno‐associated virus2/5 Rep protein expression and influences the enrichment fraction of filled capsids

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