Enhancing rAAV biomanufacturing: Process intensification strategies for streamlined upstream and downstream processing

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Improving AAV manufacturing by integrated and continuous processing

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Towards an integrated continuous manufacturing process of adeno- associated virus (AAVs)

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Efficient adeno-associated virus serotype 5 capture with affinity functionalized nanofiber adsorbents

Funding Information: The authors acknowledge the funding of Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), projects PTDC/EQU-EQU/0142/2020 and EXPL/EQU-EQU/1567/2021; SN is the recipient of an FCT fellowship from the project PTDC/EQU-EQU/0142/2020. Publisher Copyright: Copyright © 2023 Neto, Mendes, Santos, Solbrand, Carrondo, Peixoto and Silva.Adeno-associated viruses (AAVs) are one of the most promising tools for gene therapy applications. These vectors are purified using affinity and ion exchange chromatography, typically using packed beds of resin adsorbents. This leads to diffusion and pressure drop limitations that affect process productivity. Due to their high surface area and porosity, electrospun nanofiber adsorbents offer mass transfer and flow rate advantages over conventional chromatographic media. The present work investigated the use of affinity cellulose-based nanofiber adsorbents for adeno-associated virus serotype 5 (AAV5) capture, evaluating dynamic binding capacity, pressure drop, and AAV5 recovery at residence times (RT) less than 5 s. The dynamic binding capacity was found to be residence time-dependent, but nevertheless higher than 1.0 × 1014 TP mL−1 (RT = 1.6 s), with a pressure drop variation of 0.14 MPa obtained after loading more than 2,000 column volumes of clarified AAV5 feedstock. The single affinity chromatography purification step using these new affinity adsorbents resulted in 80% virus recovery, with the removal of impurities comparable to that of bead-based affinity adsorbents. The high binding capacity, virus recovery and reduced pressure drop observed at residence times in the sub-minute range can potentially eliminate the need for prior concentration steps, thereby reducing the overall number of unit operations, process time and costs.publishersversionpublishe

Accelerated Development of AAV Purification Process Using a High-Throughput and Automated Crossflow System

Funding Information: This work was funded by the Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020), the Associate Laboratory LS4FUTURE (LA/P/0087/2020) and projects PTDC/EQU-EQU/0142/2020 and EXPL/EQU-EQU/1567/2021. Publisher Copyright: © 2024 by the authors.Adeno-associated viruses (AAV) are currently predominant viral transfer tools for gene therapy, and efforts are being made to design faster and more efficient methods and technologies for their manufacturing. The early selection of high-performing filters is essential for developing an ultrafiltration and diafiltration (UF/DF) process, especially when feed material is scarce, and timelines are short. However, few methods and technologies exist to enable process optimization with multiple variations in a single run. In this study, we explored the potential of Ambr® Crossflow for high-throughput, automated screening of different membrane materials, pore sizes and different process conditions for the UF/DF step of AAV8. The best overall performance was obtained with a 100 kDa PES flat sheet cassette. The UF/DF process was further transferred to a larger scale to the Sartoflow® Smart Tangential Flow Filtration (TFF) system using a 100 kDa PES Sartocon® Slice 200 cassette and compared to a 100 kDa PES hollow fiber. Virus recovery, permeate flux and total protein removal values of the flat sheet cassette were similar to those achieved in small-scale devices, and higher than those of the hollow fiber, thus demonstrating similar performance at a larger process scale. The high-throughput, automated method described herein allowed to screen multiple materials and process parameters of a UF/DF process in a time- and resource-efficient way, making it a useful tool to accelerate early-stage downstream process development of AAV.publishersversionpublishe

Efficient Protein Trans-Splicing at Low Vector Doses

Funding Information: Author Mariana V. Ferreira acknowledges Fundação para a Ciência e Tecnologia for PH.D. fellowship UI/BD/151256/2021 within the scope of the Ph.D. Program in Bioengineering—Cell Therapies and Regenerative Medicine. This work was funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through national funds to iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020) and the Associate Laboratory LS4FUTURE (LA/P/0087/2020). Publisher Copyright: © 2023 by the authors.Adeno-associated viral (AAV) vectors represent one of the leading platforms for gene delivery. Nevertheless, their small packaging capacity restricts their use for diseases requiring large-gene delivery. To overcome this, dual-AAV vector systems that rely on protein trans-splicing were developed, with the split-intein Npu DnaE among the most-used. However, the reconstitution efficiency of Npu DnaE is still insufficient, requiring higher vector doses. In this work, two split-inteins, Cfa and Gp41-1, with reportedly superior trans-splicing were evaluated in comparison with Npu DnaE by transient transfections and dual-AAV in vitro co-transductions. Both Cfa and Gp41-1 split-inteins enabled reconstitution rates that were over two-fold higher than Npu DnaE and 100% of protein reconstitution. The impact of different vector preparation qualities in split-intein performances was also evaluated in co-transduction assays. Higher-quality preparations increased split-inteins’ performances by three-fold when compared to low-quality preparations (60–75% vs. 20–30% full particles, respectively). Low-quality vector preparations were observed to limit split-gene reconstitutions by inhibiting co-transduction. We show that combining superior split-inteins with higher-quality vector preparations allowed vector doses to be decreased while maintaining high trans-splicing rates. These results show the potential of more-efficient protein-trans-splicing strategies in dual-AAV vector co-transduction, allowing the extension of its use to the delivery of larger therapeutic genes.publishersversionpublishe

Absence of First-order Transition and Tri-critical Point in the Dynamic Phase Diagram of a Spatially Extended Bistable System in an Oscillating Field

It has been well established that spatially extended, bistable systems that are driven by an oscillating field exhibit a nonequilibrium dynamic phase transition (DPT). The DPT occurs when the field frequency is on the order of the inverse of an intrinsic lifetime associated with the transitions between the two stable states in a static field of the same magnitude as the amplitude of the oscillating field. The DPT is continuous and belongs to the same universality class as the equilibrium phase transition of the Ising model in zero field [G. Korniss et al., Phys. Rev. E 63, 016120 (2001); H. Fujisaka et al., Phys. Rev. E 63, 036109 (2001)]. However, it has previously been claimed that the DPT becomes discontinuous at temperatures below a tricritical point [M. Acharyya, Phys. Rev. E 59, 218 (1999)]. This claim was based on observations in dynamic Monte Carlo simulations of a multipeaked probability density for the dynamic order parameter and negative values of the fourth-order cumulant ratio. Both phenomena can be characteristic of discontinuous phase transitions. Here we use classical nucleation theory for the decay of metastable phases, together with data from large-scale dynamic Monte Carlo simulations of a two-dimensional kinetic Ising ferromagnet, to show that these observations in this case are merely finite-size effects. For sufficiently small systems and low temperatures, the continuous DPT is replaced, not by a discontinuous phase transition, but by a crossover to stochastic resonance. In the infinite-system limit the stochastic-resonance regime vanishes, and the continuous DPT should persist for all nonzero temperatures

Bridge deck flutter derivatives: efficient numerical evaluation exploiting their interdependence

Increasing the efficiency in the process to numerically compute the flutter derivatives of bridge deck sections is desirable to advance the application of CFD based aerodynamic design in industrial projects. In this article, a 2D unsteady Reynolds-averaged Navier-Stokes (URANS) approach adopting Menter׳s SST k-ω turbulence model is employed for computing the flutter derivatives and the static aerodynamic characteristics of two well known examples: a rectangular cylinder showing a completely reattached flow and the generic G1 section representative of streamlined deck sections. The analytical relationships between flutter derivatives reported in the literature are applied with the purpose of halving the number of required numerical simulations for computing the flutter derivatives. The solver of choice has been the open source code OpenFOAM. It has been found that the proposed methodology offers results which agree well with the experimental data and the accuracy of the estimated flutter derivatives is similar to the results reported in the literature where the complete set of numerical simulations has been performed for both heave and pitch degrees of freedom