Insect cell entrapment, growth and recovering using a single-use fixed-bed bioreactor. Scaling up and recombinant protein production

Ventini-Monteiro, Daniella Cristina; Dubois, Stéphanie; Astray, Renato Mancini; Castillo, J.; Pereira, Carlos Augusto

doi:10.1016/j.jbiotec.2015.10.013

Cited by 13 publications

(10 citation statements)

References 16 publications

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“…Others have also found iCELLis bioreactor useful for retrovirus, 7 AAV, 8 Rabies, 9 Hepatitis-A, 9 and Chikungunya 9 vaccines, or for recombinant protein production in insect cells. 10 iCELLis 500 bioreactor is good manufacturing practice (GMP) compliant, fully disposable, and controlled system with perfusion capability. It supports adherent cell growth and high titer production.…”

Section: Introductionmentioning

confidence: 99%

Benchmarking of Scale-X Bioreactor System in Lentiviral and Adenoviral Vector Production

et al. 2020

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We have previously produced viral vectors (lentiviral vector, adenoviral vector, and adeno-associated viral vector) in small and in commercial scale in adherent cells using Pall fixed-bed iCELLis Ò bioreactor. Recently, a company called Univercells has launched a new fixed-bed bioreactor with the same cell growth surface matrix material, but with different fixed-bed structure than is used in iCELLis bioreactor. We sought to compare the new scale-XÔ hydro bioreactor (2.4 m 2) and iCELLis Nano system (2.67 m 2) to see if the difference has any effect on cell growth or lentiviral vector and adenoviral vector productivity. Runs were performed using parameters optimized for viral vector production in iCELLis Nano bioreactor. Cell growth was monitored by counting nuclei, as well as by following glucose consumption and lactate production. In both bioreactor systems, cells grew well, and the cell distribution was found quite homogeneous in scale-X bioreactor. Univercells scale-X bioreactor was proven to be at least equally efficient or even improved in both lentiviral vector and adenoviral vector production. Based on the results, the same protocol and parameters used in viral vector production in iCELLis bioreactor can also be successfully used for the production in scale-X bioreactor system.

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Section: Introductionmentioning

confidence: 99%

Benchmarking of Scale-X Bioreactor System in Lentiviral and Adenoviral Vector Production

et al. 2020

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“… 19 , 20 iCELLis allows scaling of adherent production in a controlled environment, and the highly integrated single-use equipment can be adapted to meet current good manufacturing practices requirements. Viral vaccines, 21 recombinant proteins, 22 adeno-associated viral vectors 23 and retroviral vectors 16 have been produced in iCELLis Nano. In addition, adenoviral vector production has been scaled-up to iCELLis 500.…”

Section: Introductionmentioning

confidence: 99%

Optimization of lentiviral vector production for scale-up in fixed-bed bioreactor

et al. 2017

Gene Ther

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Lentiviral vectors (LVs) are promising tools for gene therapy. However, scaling up the production methods of LVs in order to produce high-quality vectors for clinical purposes has proven to be difficult. In this article, we present a scalable and efficient method to produce LVs with transient transfection of adherent 293T cells in a fixed-bed bioreactor. The disposable iCELLis bioreactors are scalable with a large three-dimensional (3D) growth area range between 0.53 and 500 m2, an integrated perfusion system, and a controllable environment for production. In this study, iCELLis Nano (2.67–4 m2) was used for optimizing production parameters for scale-up. Transfections were first done using traditional calcium phosphate method, but in later runs polyethylenimine was found to be more reliable and easier to use. For scalable LV production, perfusion rate control by measuring cell metabolite concentrations in the bioreactor leads to higher productivity and reduced costs. Optimization of cell seeding density for targeted cell concentration during transfection, use of low compaction fixed-bed and lowering the culture pH have a positive effect on LV productivity. These results show for the first time that iCELLis bioreactor is scalable from bench level to clinical scale LV production.

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“…However, under our non-optimized testing conditions in shaking flasks, fragment yields were disappointing and would not allow us to carry out planned in vitro/in vivo experiments. Consequently, we turned to the insect S2 system allowing production of tens to hundreds of milligrams of secreted proteins per liter of media [53][54][55]. While bicistronic or IRES-containing plasmids can, in principle, be used for the co-expression of two genes in S2 cells [56], in the case of Fab production, we chose co-transfection of individual heavy-and light-chain encoding plasmids.…”

Section: Discussionmentioning

confidence: 99%

Engineered Fragments of the PSMA-Specific 5D3 Antibody and Their Functional Characterization

Nováková

Belousova

Foss

et al. 2020

IJMS

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Prostate-Specific Membrane Antigen (PSMA) is an established biomarker for the imaging and experimental therapy of prostate cancer (PCa), as it is strongly upregulated in high-grade primary, androgen-independent, and metastatic lesions. Here, we report on the development and functional characterization of recombinant single-chain Fv (scFv) and Fab fragments derived from the 5D3 PSMA-specific monoclonal antibody (mAb). These fragments were engineered, heterologously expressed in insect S2 cells, and purified to homogeneity with yields up to 20 mg/L. In vitro assays including ELISA, immunofluorescence and flow cytometry, revealed that the fragments retain the nanomolar affinity and single target specificity of the parent 5D3 antibody. Importantly, using a murine xenograft model of PCa, we verified the suitability of fluorescently labeled fragments for in vivo imaging of PSMA-positive tumors and compared their pharmacokinetics and tissue distribution to the parent mAb. Collectively, our data provide an experimental basis for the further development of 5D3 recombinant fragments for future clinical use.

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Insect cell entrapment, growth and recovering using a single-use fixed-bed bioreactor. Scaling up and recombinant protein production

Cited by 13 publications

References 16 publications

Benchmarking of Scale-X Bioreactor System in Lentiviral and Adenoviral Vector Production

Benchmarking of Scale-X Bioreactor System in Lentiviral and Adenoviral Vector Production

Optimization of lentiviral vector production for scale-up in fixed-bed bioreactor

Engineered Fragments of the PSMA-Specific 5D3 Antibody and Their Functional Characterization

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