A new nodavirus‐negative <i>Trichoplusia ni</i> cell line for baculovirus‐mediated protein production

Maghodia, Ajay B.; Geisler, Christoph; Jarvis, Donald L.

doi:10.1002/bit.27494

Cited by 7 publications

(4 citation statements)

References 74 publications

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“…Insect Cell Lines. Tn-nodavirus negative (Tn-NVN) 47 cells were used to isolate, amplify, and titer all BEVs used in this study. Tn-NVNΔFDL cells, a Tn-NVN subclone (Maghodia and Jarvis, unpublished) lacking an endogenous N-glycan trimming function (FDL; Figure 1), were used to produce all rH5 preparations used in this study except M3 (Table 1), which was produced in Tn-NVN cells because biosynthesis of this insect-type N-glycan requires FDL function.…”

Section: ■ Methodsmentioning

confidence: 99%

A New Bacmid for Customized Protein Glycosylation Pathway Engineering in the Baculovirus-Insect Cell System

Maghodia¹,

Geisler²,

Jarvis

2021

ACS Chem. Biol.

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One attractive feature of the baculovirus-insect cell system (BICS) is the baculoviral genome has a large capacity for genetic cargo. This enables construction of viral vectors designed to accept multigene insertions, which has facilitated efforts to produce recombinant multisubunit protein complexes. However, the large genetic capacity of baculovirus vectors has not yet been exploited for multistep pathway engineering. Therefore, we created PolyBac, which is a novel baculovirus shuttle vector, or bacmid, that can be used for this purpose. PolyBac was designed to accept multiple transgene insertions by three different mechanisms at three different sites within the baculovirus genome. After constructing and characterizing PolyBac, we used it to isolate nine derivatives encoding various combinations of up to eight different protein N-glycosylation pathway functions, or glycogenes. We then used these derivatives, which were designed to progressively extend the endogenous insect cell pathway, to assess PolyBac’s utility for protein glycosylation pathway engineering. This assessment was enabled by engineering each derivative to produce a recombinant influenza hemagglutinin (rH5), which was used to probe the impact of each glycoengineered PolyBac derivative on the endogenous insect cell pathway. Genetic analyses of these derivatives confirmed PolyBac can accept large DNA insertions. Biochemical analyses of the rH5 products showed each had distinct N-glycosylation profiles. Finally, the major N-glycan on each rH5 product was the predicted end product of the engineered N-glycosylation pathways encoded by each PolyBac derivative. These results generally indicate that PolyBac has utility for multistep metabolic pathway engineering and directly demonstrate that this new bacmid can be used for customized protein glycosylation pathway engineering in the BICS.

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Section: ■ Methodsmentioning

confidence: 99%

A New Bacmid for Customized Protein Glycosylation Pathway Engineering in the Baculovirus-Insect Cell System

Maghodia¹,

Geisler²,

Jarvis

2021

ACS Chem. Biol.

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“…As eukaryotes, insect cells can process complex secondary structures, are easy to culture and can grow in serum-free media, require less energy than mammalian cells and have low biosafety requirements [ 70 ]. The most frequently used insect cells derive from Spodoptera frugiperda and Trichoplusia ni and both offer GMP compatible cell lines [ 71 , 72 , 73 ]. By testing a variety of culture modes and culture medium supplementation strategies, production yields can be increased.…”

Section: The Eu-funded Addovenom Projectmentioning

confidence: 99%

ADDovenom: Thermostable Protein-Based ADDomer Nanoparticles as New Therapeutics for Snakebite Envenoming

Menzies,

Arinto-Garcia,

Amorim

et al. 2023

Toxins

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Snakebite envenoming can be a life-threatening medical emergency that requires prompt medical intervention to neutralise the effects of venom toxins. Each year up to 138,000 people die from snakebites and threefold more victims suffer life-altering disabilities. The current treatment of snakebite relies solely on antivenom—polyclonal antibodies isolated from the plasma of hyperimmunised animals—which is associated with numerous deficiencies. The ADDovenom project seeks to deliver a novel snakebite therapy, through the use of an innovative protein-based scaffold as a next-generation antivenom. The ADDomer is a megadalton-sized, thermostable synthetic nanoparticle derived from the adenovirus penton base protein; it has 60 high-avidity binding sites to neutralise venom toxins. Here, we outline our experimental strategies to achieve this goal using state-of-the-art protein engineering, expression technology and mass spectrometry, as well as in vitro and in vivo venom neutralisation assays. We anticipate that the approaches described here will produce antivenom with unparalleled efficacy, safety and affordability.

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“…As such, rhabdovirus cannot replicate in human or monkey cell lines [101,104] ; therefore, it is deemed harmless, whereas the host range of nodavirus is not yet clear. Importantly, the team from Glyobac recently established and characterized rhabdovirus-negative Sf9 cell line [102] and nodavirus-negative Hi5 cell line, [105] which showed the absence of corresponding adventitious AAV production using IC-BEVS is a transient phenomenon. Because of the lytic nature of baculovirus infection, AAVs are recovered by harvesting the cell culture within 96 hpi, preferably at viability more than 70%, [43] to minimize potential degradation of AAV capsids by cellularproteases released as a result of cell apoptosis.…”

Section: Insect Cell and Baculovirus Engineeringmentioning

confidence: 99%

“…As such, rhabdovirus cannot replicate in human or monkey cell lines [ 101,104 ] ; therefore, it is deemed harmless, whereas the host range of nodavirus is not yet clear. Importantly, the team from Glyobac recently established and characterized rhabdovirus‐negative Sf9 cell line [ 102 ] and nodavirus‐negative Hi5 cell line, [ 105 ] which showed the absence of corresponding adventitious viruses overextended cell passages making these cell lines preferentially more suitable for biologics manufacturing than the current Sf9 and Hi5 cell lines in use because of improved product safety. Notably, none of these new cell lines have been reportedly used for AAV production and remain open to further investigation.…”

Section: Future Perspectivesmentioning

confidence: 99%

Advancements in molecular design and bioprocessing of recombinant adeno‐associated virus gene delivery vectors using the insect‐cell baculovirus expression platform

Joshi

Venereo-Sánchez

Chahal

et al. 2021

Biotechnology Journal

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Despite rapid progress in the field, scalable high-yield production of adeno-associated virus (AAV) is still one of the critical bottlenecks the manufacturing sector is facing. The insect cell-baculovirus expression vector system (IC-BEVS) has emerged as a mainstream platform for the scalable production of recombinant proteins with clinically approved products for human use. In this review, we provide a detailed overview of the advancements in IC-BEVS for rAAV production. Since the first report of baculovirus-induced production of rAAV vector in insect cells in 2002, this platform has undergone significant improvements, including enhanced stability of Bac-vector expression and a reduced number of baculovirus-coinfections. The latter streamlining strategy led to the eventual development of the Two-Bac, One-Bac, and Mono-Bac systems. The one baculovirus system consisting of an inducible packaging insect cell line was further improved to enhance the AAV vector quality and potency. In parallel, the implementation of advanced manufacturing approaches and control of critical processing parameters have demonstrated promising results with process validation in large-scale bioreactor runs. Moreover, optimization of the molecular design of vectors to enable higher cell-specific yields of functional AAV particles combined with bioprocess intensification strategies may also contribute to addressing current and future manufacturing challenges.

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A new nodavirus‐negative Trichoplusia ni cell line for baculovirus‐mediated protein production

Cited by 7 publications

References 74 publications

A New Bacmid for Customized Protein Glycosylation Pathway Engineering in the Baculovirus-Insect Cell System

A New Bacmid for Customized Protein Glycosylation Pathway Engineering in the Baculovirus-Insect Cell System

ADDovenom: Thermostable Protein-Based ADDomer Nanoparticles as New Therapeutics for Snakebite Envenoming

Advancements in molecular design and bioprocessing of recombinant adeno‐associated virus gene delivery vectors using the insect‐cell baculovirus expression platform

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