Quantitative correlation between transcriptional levels of ER chaperone, peroximal protein and FVIII productivity in human Hek-293 cell line

Rodrigues, Evandra Strazza; Picanço‐Castro, Virgínia; Espanhol, Marta Regina; Andrade, Luiz A. M; Palma, Patrícia Vianna Bonini; Kashima, Simone; Fontes, Aparecida Maria; Covas, Dimas Tadeu

doi:10.1186/2193-1801-2-328

Cited by 3 publications

(2 citation statements)

References 32 publications

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“…Nosso grupo de trabalho vem desenvolvendo, há 15 anos, pesquisas relacionadas à produção de fator FVIII e FIX de coagulação utilizando células humanas (PICANÇO et al, 2007;PICANÇO-CASTRO et al, 2008;RUSSO-CARBOLANTE et al, 2011;SWIECH et al, 2011;da ROSA et al, 2012;RODRIGUES et al, 2013;CORREA de FREITAS et al, 2014). Neste período excelentes resultados foram obtidos, o que resultou em 2 patentes para o FVIII (US2014/0051122 A1 e US20100172891 A1) e 1 para FIX (WO2011011841 A1).…”

Section: Discussionunclassified

Clonagem e expressão do fator VII de coagulação sanguínea em linhagens celulares humanas

Freitas¹

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Recombinant factor VII (FVIIr) has been the main therapeutic choice for hemophilic patients who develop inhibitors antibidies to conventional treatments (FVIII and FIX). Currently, the comercial product is produced in murine cells (BHK-21) which gives disadvantages considering the complexity of post-translational modifications of these proteins. The insertion of murine residues can be highly immunogenic in humans. Thus the production of proteins for therapeutic use in human cell lines appears as a promising alternative. In this context, the aim of this study was to clone and express the blood coagulation FVII in 3 human cell lines (HepG2, Sk-Hep-1, HKB-11) and select the best cell line for production of recombinant protein. The cells were modified with the lentiviral vector p1054-CIGWS containing the FVII gene and GFP gene marker. After cells modification we observed efficiency of transduction, in which 80% of BHK-21-FVIIr cells showed GFP expression, 73% of HepG2-FVIIr cells, 32% of HKB-11-FVIIr cells and 95% SK-Hep-FVIIr. Gene expression analysis by real-time PCR showed that the three modified human cell lines exhibited RNAm expression relative to FVIIr. When cells were treated with 5 ug/mL vitamin K in culture, the gene expression of FVIIr was similar in the three cell lines (HepG2: 164 563 URE, HKB-11: 119122 and SK-Hep URE: 124919 URE). For FVII activation, the main post translational modification is -carboxylation vitamin-K-dependent which envolves three enzymes, -carboxylase, VKORC1 and calumenina (inhibitor) . Gene expression of these enzymes was evaluated before and after treatment with vitamin K. It was observed that there was an increase in mRNA levels in human cells treated with vitamin K, suggesting that the treatment is capable of activating the enzymes of the vitamin K cycle. Cell growth kinetics showed that modified murine cells BHK-21 have a higher specific growth rate, around 25% more than human cells. However the kinetics of production of recombinant cell lines showed that human cells expressing rFVII 3-fold more rFVII than BHK-21 cells. Due the low rFVII production of murine cells, and the extremely slow growth profile of human cell line HepG2, the recombinant cell lines SkHep-1-rFVII and HKB-11-rFVII have been selected for cultivation tests in suspension using microcarriers in spinners flasks. Over 10 days of cultivation the HKB-11 cells showed a cumulative production of rFVII 152 ug, corresponding to 304 IU and SK-Hep-1 cells showed a rFVII production of 202.6 ug, corresponding to 405.2 IU. In summary, our data demonstrate that human cell lines are effective for producing recombinant factor VII. Using our production model, human cells were better than murine cells (BHK-21) used by the industry. Thus, these human cell lines can be used as a new platform for the FVII production, as well as for other recombinant proteins, with less risk of developing inhibitor antibodies.

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Section: Discussionunclassified

Clonagem e expressão do fator VII de coagulação sanguínea em linhagens celulares humanas

Freitas¹

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“…A FUNDHERP tem uma vasta experiência no desenvolvimento de pesquisas direcionadas à produção do FVIIIr utilizando linhagens celulares humanas (PICANÇO et al, 2007;PICANÇO-CASTRO et al, 2008;SWIECH et al, 2011;ROSA et al, 2012;RODRIGUES et al, 2013;CORREA et al, 2014) A linhagem celular Sk-Hep-1 é conhecida pelo seu alto potencial de expressar fatores de coagulação, tal como FVIIIr (ROSA et al, 2012). Outra característica é que esta linhagem pode ser adaptada ao crescimento em suspensão sem SFB (BIAGGIO et al, 2015).…”

Section: Comparação Entre As Diferentes Quantificações Analíticasunclassified

Utilização do monitoramento de reações múltiplas para quantificação de produtos de interesse biotecnológico

Filho¹,

Galdino²

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Cellular stress and coagulation factor production: when more is not necessarily better

Chen,

Herzog,

Kaufman

2023

Journal of Thrombosis and Haemostasis

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Quantitative correlation between transcriptional levels of ER chaperone, peroximal protein and FVIII productivity in human Hek-293 cell line

Cited by 3 publications

References 32 publications

Clonagem e expressão do fator VII de coagulação sanguínea em linhagens celulares humanas

Clonagem e expressão do fator VII de coagulação sanguínea em linhagens celulares humanas

Utilização do monitoramento de reações múltiplas para quantificação de produtos de interesse biotecnológico

Cellular stress and coagulation factor production: when more is not necessarily better

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