Monitoring the production of AAV vectors in insect cells by fluorescence spectroscopy

Adeno-associated viruses (AAV) are among the most promising viral vectors for gene therapy, since they can transduce non-dividing cells from several tissues while maintaining a long-term gene expression. Besides, AAVs possess low immunogenicity compared to other viral vectors, and are physically resistant, which makes them resilient to industrial manufacturing conditions, long-term storage, and in vivo administration. One of the systems available for large scale production of AAVs is the insect cell-baculovirus expression vector system (IC-BEVS). Insect cells grow in suspension to high cell densities with modest growth requirements and without the need of serum supplementation. Consequently, scaling up the production in order to achieve the large number of AAV needed for clinical trials is more straight‑forward than with transfection-based systems. However, methods for online monitoring of AAV production are still lacking. Such methods would allow determination of the best time of harvest in real-time, thus allowing recovery of AAV as soon as its concentration medium was higher. Here we apply Fluorescence Spectroscopy to baculovirus-infected insect cell cultures producing adeno‑associated virus vectors, correlating the spectra to critical process parameters like cell concentration, viability and AAV concentration. Sf9 cells were co-infected with two baculovirus (expressing AAV rep and cap and a CMV-GFP transgene) at low or high multiplicities of infection (MOI), and the culture was followed by Fluorescence Spectroscopy in situ through a bioreactor probe. After an exploratory calibration using data from only one bioreactor, we attested the aptitude of this technique to capture overall data trend: using a 3 component PLS model, we have obtained a calibration NRMSE of 2.9% for total AAV particles per cell, 5.9% for viable cell density and 0.9% for viability). Additional bioreactor productions using different infection parameters (CCI, MOI, time of infection) allowed testing the robustness of fluorescence monitoring to process variability. With this dataset, we tested several pre-treatment methods for the raw spectra, as well as different regression algorithms in order to establish a good predictive model. Ultimately, fluorescence spectroscopy provides a simple tool for online monitoring of key process variables in baculovirus-infected insect cell cultures. Acknowledgments: Funding from Fundação para a Ciência e a Tecnologia, projects EXPL/BBBBIO/1129/2013 and Daniel Pais’ PhD research grant PD/BD/105873/2014

Dissecting insect cell heterogeneity during influenza VLP production using single-cell transcriptomics

Funding Information: This work has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 813453 and the grant agreement N° 730964, and by Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior (FCT/MCTES, Portugal) through the following initiatives: “Investigador FCT” Program (IF/01704/2014), Exploratory Research and Development Project (IF/01704/2014/CP1229/CT0001), PhD fellowships (SFRH/BD/134107/2017), iNOVA4Health (UIDB/04462/2020 and UIDP/04462/2020) and the Associate Laboratory LS4FUTURE (LA/P/0087/2020). Funding from INTERFACE Programme, through the Innovation, Technology, and Circular Economy Fund (FITEC), is gratefully acknowledged. Publisher Copyright: Copyright © 2023 Silvano, Virgolini, Correia, Clarke, Isidro, Alves and Roldão.The insect cell-baculovirus expression vector system (IC-BEVS) has been widely used to produce recombinant protein at high titers, including complex virus-like particles (VPLs). However, cell-to-cell variability upon infection is yet one of the least understood phenomena in virology, and little is known about its impact on production of therapeutic proteins. This study aimed at dissecting insect cell population heterogeneity during production of influenza VLPs in IC-BEVS using single-cell RNA-seq (scRNA-seq). High Five cell population was shown to be heterogeneous even before infection, with cell cycle being one of the factors contributing for this variation. In addition, infected insect cells were clustered according to the timing and level of baculovirus genes expression, with each cluster reporting similar influenza VLPs transgenes (i.e., hemagglutinin and M1) transcript counts. Trajectory analysis enabled to track infection progression throughout pseudotime. Specific pathways such as translation machinery, protein folding, sorting and degradation, endocytosis and energy metabolism were identified as being those which vary the most during insect cell infection and production of Influenza VLPs. Overall, this study lays the ground for the application of scRNA-seq in IC-BEVS processes to isolate relevant biological mechanisms during recombinant protein expression towards its further optimization.publishe

Dielectric spectroscopy monitoring of a bioreactor process for hiPSC expansion and differentiation

Bioprocessing strategies using 3D cell culturing approaches, such as cell aggregates, are promising solutions to achieve efficient and scalable bioprocesses for stem cell expansion and differentiation. However, tracking viable and total cell numbers in such culture systems is not straightforward. It requires cell detachment, disaggregation or disruption, which results in measurements that are laborious, biased and with high variability. In this work, we used a commercially available capacitance probe to explore the applicability of dielectric spectroscopy for in situ monitoring of a multistep process for expansion and differentiation of human induced pluripotent stem cells (hiPSC) cultivated as cell aggregates. After 5 days of cell expansion in a bioreactor, the hepatic differentiation step was integrated by addition of different levels of specific soluble factors at various stages of the process to promote growth and generate populations successively enriched for definitive endoderm, hepatoblasts, hepatocyte progenitors and mature hepatocytes. While this differentiation procedure has been previously validated for monolayer cultures, this was the first time it was carried out in a stirred tank bioreactor operated in perfusion mode. Phenotype analysis confirmed a marked increase in key hepatic differentiation markers culminating at day 21 of differentiation. Our data shows a good correlation between total volume of the cell aggregates and permittivity measured by the probe (R2 = 0.84). However, there was a delay between changes in cell concentration and the permittivity signal. This suggests that cell expansion requires a few days to result in increased volume of the cell aggregates and that each aggregate behaves as one overall inducible dipole. The β-dispersion curve shape also appears to change over culture time and could eventually be used as an indicator for differentiation progression. Dielectric spectroscopy has been used successfully to monitor viable cell concentration in different single-cell suspension cultures, but there are few published applications to 3D cultures. Our results demonstrate the potential of dielectric spectroscopy to monitor complex bioprocesses for human stem cell aggregates in stirred cultures. Acknowledgements: Funding provided by ERA-NET/E-Rare3 programme through research project ERAdicatPH (E-Rare3/0002/2015). The authors acknowledge Dr Juan Rodriguez-Madoz (University of Navarra, Spain) and Dr Anders Aspegren (Takara Bio Europe – Cellartis AB, Sweden) for helpful discussions on hepatic differentiation of hiPSC.

Enabling PAT in insect cell bioprocesses: A monitoring toolbox for rAAV production

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Transcriptome analysis of Sf9 insect cells during production of recombinant Adeno‐associated virus

The insect cell-baculovirus expression vector system (IC-BEVS) has emerged as an alternative time- and cost-efficient production platform for recombinant Adeno-associated virus (AAV) for gene therapy. However, a better understanding of the underlying biological mechanisms of IC-BEVS is fundamental to further optimize this expression system toward increased product titer and quality. Here, gene expression of Sf9 insect cells producing recombinant AAV through a dual baculovirus expression system, with low multiplicity of infection (MOI), was profiled by RNA-seq. An 8-fold increase in reads mapping to either baculovirus or AAV transgene sequences was observed between 24 and 48 h post-infection (hpi), confirming a take-over of the host cell transcriptome by the baculovirus. A total of 336 and 4784 genes were identified as differentially expressed at 24 hpi (vs non-infected cells) and at 48 hpi (vs. infected cells at 24 hpi), respectively, including dronc, birc5/iap5, and prp1. Functional annotation found biological processes such as cell cycle, cell growth, protein folding, and cellular amino acid metabolic processes enriched along infection. This work uncovers transcriptional changes in Sf9 in response to baculovirus infection, which provide new insights into cell and/or metabolic engineering targets that can be leveraged for rational bioprocess engineering of IC-BEVS for AAV production.publishersversionpublishe

Leveraging single-cell and bulk transcriptomics towards improved insect cell factories for biopharmaceuticals

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Exploring Metabolic Signatures of Ex Vivo Tumor Tissue Cultures for Prediction of Chemosensitivity in Ovarian Cancer

Predicting patient response to treatment and the onset of chemoresistance are still major challenges in oncology. Chemoresistance is deeply influenced by the complex cellular interactions occurring within the tumor microenvironment (TME), including metabolic crosstalk. We have previously shown that ex vivo tumor tissue cultures derived from ovarian carcinoma (OvC) resections retain the TME components for at least four weeks of culture and implemented assays for assessment of drug response. Here, we explored ex vivo patient-derived tumor tissue cultures to uncover metabolic signatures of chemosensitivity and/or resistance. Tissue cultures derived from nine OvC cases were challenged with carboplatin and paclitaxel, the standard-of-care chemotherapeutics, and the metabolic footprints were characterized by LC-MS. Partial least-squares discriminant analysis (PLS-DA) revealed metabolic signatures that discriminated high-responder from low-responder tissue cultures to ex vivo drug exposure. As a proof-of-concept, a set of potential metabolic biomarkers of drug response was identified based on the receiver operating characteristics (ROC) curve, comprising amino acids, fatty acids, pyrimidine, glutathione, and TCA cycle pathways. Overall, this work establishes an analytical and computational platform to explore metabolic features of the TME associated with response to treatment, which can leverage the discovery of biomarkers of drug response and resistance in OvC.publishersversionpublishe

Application of LDH assay for therapeutic efficacy evaluation of ex vivo tumor models

Funding Information: This work was supported by AbbVie and by iNOVA4Health – UIDB/04462/2020, a program financially supported by Fundação para a Ciência e Tecnologia (FCT) / Ministério da Educação e Ciência, through national funds. RM and TFM were recipients of PhD fellowships funded by FCT (SFRH/BD/132163/2017 and PD/BD/128377/2017, respectively) and CB was funded by “The Discoveries Centre for Regenerative and Precision Medicine” (European Commission Horizon 2020 Research and Innovation programme, under the Grant Agreement 739572). Publisher Copyright: © 2021, The Author(s).The current standard preclinical oncology models are not able to fully recapitulate therapeutic targets and clinically relevant disease biology, evidenced by the 90% attrition rate of new therapies in clinical trials. Three-dimensional (3D) culture systems have the potential to enhance the relevance of preclinical models. However, the limitations of currently available cellular assays to accurately evaluate therapeutic efficacy in these models are hindering their widespread adoption. We assessed the compatibility of the lactate dehydrogenase (LDH) assay in 3D spheroid cultures against other commercially available readout methods. We developed a standardized protocol to apply the LDH assay to ex vivo cultures, considering the impact of culture growth dynamics. We show that accounting for growth rates and background release levels of LDH are sufficient to make the LDH assay a suitable methodology for longitudinal monitoring and endpoint assessment of therapeutic efficacy in both cell line-derived xenografts (xenospheres) and patient-derived explant cultures. This method has the added value of being non-destructive and not dependent on reagent penetration or manipulation of the parent material. The establishment of reliable readout methods for complex 3D culture systems will further the utility of these tumor models in preclinical and co-clinical drug development studies.publishersversionpublishe

Advancing manufacture of hiPSC-derived hepatocytes with improved functionality: A nature-inspired protocol

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Intensifying the manufacture of hiPSC therapy products through metabolic and process understanding

In vitro differentiation of human induced pluripotent stem cells into specific lineages such as cardiomyocytes (hPSC-CM) and hepatocytes (hPCS-Hep) is a crucial process to enable their application in cell therapy and drug discovery. Nevertheless, despite the remarkable efforts over the last decade towards the implementation of protocols for hPSC expansion and differentiation, there are some technological challenges remaining include the low scalability and differentiation yields. Additionally, generated cells are still immature, closely reminiscent of fetal/embryonic cells in what regards phenotype and function. In this study, we aim to overcome this hurdle by devising bioinspired and integrated strategies to improve the generation and functionality of these hiPSC-derivatives. We also applied robust multi-parametric techniques including proteomics, transcriptomics, metabolomics and fluxomics as complementary analytical tools to support bioprocess optimization and product characterization. We cultured hiPSC as 3D aggregates in stirred-tank bioreactors (STB) operated in perfusion and used a capacitance probe for in situ monitoring of cell growth/differentiation. After cell expansion, the hepatic differentiation step was integrated by addition of key soluble factors and controlling the dissolved oxygen concentration at various stages of the process to generate populations enriched for definitive endoderm, hepatocyte progenitors and mature hepatocytes. The analyses of hepatic markers expression throughout the stages of the differentiation confirmed that hepatocyte differentiation was improved in 3D spheroids when compared to 2D culture. Noteworthy, these hiPSC-HLC exhibited functional characteristics typical of hepatocytes (albumin production, glycogen storage and CYP450 activity). We also demonstrate the potential of dielectric spectroscopy to monitor cell expansion and hepatic differentiation in STB. For CM differentiation, we relied on the aggregation of hPSC-derived cardiac progenitors to establish a scalable differentiation protocol capable of generating highly pure CM aggregate cultures. We assessed if alteration of culture medium composition to mimic in vivo substrate usage during cardiac development improved further hPSC-CM maturation in vitro. Our results showed that shifting hPSC-CMs from glucose-containing to galactose- and fatty acid-containing medium promotes their fast maturation into adult-like CMs with higher oxidative metabolism, transcriptional signatures closer to those of adult ventricular tissue, higher myofibril density and alignment, improved calcium handling, enhanced contractility, and more physiological action potential kinetics. “-Omics” analyses showed that addition of galactose to culture medium and culturing the cells under perfusion improves total oxidative capacity of the cells and ameliorates fatty acid oxidation. This study demonstrated that metabolic shifts during differentiation/maturation of hPSC-CM are a cause, rather than a consequence, of the phenotypic and functional alterations observed. The metabolic-based strategy established herein holds technical and economic advantages over the existing protocols due to its scalability, simplicity and ease of application. Funding: This work was supported by FCT-funded projects NETDIAMOND (SAICTPAC/0047/2015), MetaCardio (Ref.032566) and FCT/ERA-Net (ERAdicatPH; Ref. E-Rare3/0002/2015). iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344) is also acknowledged