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

Paper ID Volume ID Publish Year Pages File Format Full-Text
22780 43384 2015 6 PDF Available
Title
Insect cell entrapment, growth and recovering using a single-use fixed-bed bioreactor. Scaling up and recombinant protein production
Abstract

•Our work with the single-use iCELLis nano fixed-bed bioreactor shows the establishment of non-adherent insect cell cultures.•Cell immobilization into the fixed-bed and easy perfusion allow a suitable nutrient supply to and metabolites removal from the cell environment.•Low hydrodynamic stress to cells inside the perfused fixed-bed contributes to a better environment for membrane associated recombinant proteins.•As a consequence of improved environmental conditions in iCELLis bioreactor, cell growth and recombinant protein production is of higher quality.•Established conditions of cell cultivation into the fixed-bed allow us to recover, at high densities and viability, the cells from the fixed-bed.

Insect cells are largely used for industrial production of vaccines, viral vectors and recombinant proteins as well as in research and development as an important tool for biology and bioprocess studies. They grow in suspension and are semi-adherent cells. Among the cell culture systems enabling scalable bioprocess the single-use fixed-bed iCELLis® bioreactors offer great advantages. We have established the conditions for Drosophila melanogaster Schneider 2 (S2) and Spodoptera frugiperda (Sf9) cells entrapment into the fixed-bed, cell growth and recover from the fixed-bed once high cell densities were attained. Our established protocol allowed these cells, at a cell seeding of 2 × 1E5 cells/microfiber carriers (MC) (3.5 × 1E6 cells/mL; 1.7 × 1E4 cells/cm2), to grow inside a 4 m2/200 mL fixed-bed attaining a concentration of 5.3 × 1E6 cells/MC (9.5 × 1E7 cells/mL; 4.7 × 1E5 cells/cm2) for S2 cells or 4.6 × 1E6 cells/MC (8 × 1E7 cells/mL; 4.1 × 1E5 cells/cm2) for Sf9 cells. By washing the fixed-bed, entrapped cells could then be recovered from the fixed-bed at a high rate (>85%) with high viability (>95%) by increasing the agitation to 1200/1500 rpm. Although the cell yields in the fixed-bed bioreactor were comparable to those obtained in a stirred tank (respectively, 1.3 × 1E10 and 2.5 × 1E10 total cells), S2 cells stably transfected with a cDNA coding for the rabies virus glycoprotein (RVGP) showed a 30% higher preserved rRVGP production (2.5 ± 0.1 and 1.9 ± 0.1 μg/1E7 cells), as evidenced by a conformational ELISA evaluation. These findings demonstrate not only the possibility to entrap, cultivate to high densities and recover insect cells using a single-use fixed-bed bioreactor, but also that this system provides suitable physiological conditions for the entrapped cells to produce a cell membrane associated recombinant protein with higher specific biological activity as compared to classical suspension cell cultures.

Keywords
Single-use fixed-bed bioreactor; Insect cells; Cell recovering; Cell associated recombinant rabies vírus glycoprotein-rRVGP; Scaling-up
First Page Preview
Insect cell entrapment, growth and recovering using a single-use fixed-bed bioreactor. Scaling up and recombinant protein production
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 216, 20 December 2015, Pages 110–115
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering