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2022, Number 2

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Biotecnol Apl 2022; 39 (2)

Production of the SARS-CoV-2 nucleocapsid-human CD154 extracellular domain fusion protein in the EscoVacciXcell-CelCradle™ benchtop bioreactor

Aragón H, Quintana M, Ferro W, Llamo A, Menéndez G, González M, García C, García J, Somoza M, Barrisonte Y, La O T, Carpio Y, Estrada MP, Valdés R

Language: English
References: 34
Page: 2211-2216
PDF size: 529.66 Kb.


ABSTRACT

The SARS-CoV-2 nucleocapsid protein (N) is abundantly expressed during the viral infection, and it can be used for vaccine development, serological assays and other purposes. Different strategies have been implemented for N-protein production in either suspension or anchorage cell systems. In this work, a fusion protein of the SARS-CoV-2 nucleocapsid to the extracellular domain of the human CD154 (N-hCD154) was produced in HEK-293 cells cultivated in the EscoVacciXcell CelCradle™ benchtop bioreactor, operated in continuous mode for 35 days. Effective operation parameters were: 0.88 ± 0.27 × 106 HEK-293 cells quantified per carrier (BioNOC™II), 547.9-898.2 × 106 cells/day, 89.4-98.2 % HEK-293 cell viability, 0.6 ± 0.01 pg/cell/day (specific secretion rate) and 22.38 ± 2.6 μg/mL N-hCD154 fusion protein concentration per harvest. A 75 % N-hCD154 fusion protein average purity was achieved in culture supernatant samples, and the downstream method combining ammonium sulfate precipitation and diafiltration step provided a N-hCD154 fusion protein with over 95 % of purity. The combination of the CelCradle™ benchtop bioreactor for HEK-293 cell culture operated in continuous mode with ammonium sulfate precipitation and a diafiltration step, made possible obtaining N-hCD154 fusion protein with a medium recovery and very high purity level as to be characterized for further research with medicinal purposes.


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