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Enhancement of Human Thyrotropin Synthesis by Sodium Butyrate Addition to Serum-Free CHO Cell Culture

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Abstract

The influence of sodium butyrate (NaBu) on the synthesis of recombinant human thyrotropin (r-hTSH) by CHO cells was investigated for the first time. A volumetric productivity of ~10 μg hTSH/mL was repeatedly obtained, with a 3.3-fold increase over a control culture carried out in the absence of NaBu. Since NaBu can induce CHO cell apoptosis and cell growth arrest, the increase in specific productivity was even higher, i.e., ca. 5-fold. Analysis of the N-glycan composition of r-hTSH obtained with the addition of NaBu to the culture medium showed an approximately 12 % increase in the amount of sialic acid, as well as in total carbohydrate, partly due to the increase in the site occupancy from 2.77 to 2.93 glycans per mole of hTSH. The two hormone preparations were characterized by N-glycan structural analysis, which showed that NaBu increased the bi-antennary structures by ca. 13 % while decreasing the tri-antennary structures by approximately the same amount. The in vivo biological activity and pharmacokinetic behavior (clearance) were found to be similar for the two hormone preparations.

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Abbreviations

NaBu:

Sodium butyrate

r-hTSH:

Recombinant human thyrotropin

CHO:

Chinese hamster ovary cells

r-hEPO:

Recombinant erythropoietin

β-IFN:

β-Interferon

CMP:

Cytodine monophosphate

t-PA:

Plasminogen activator

GP2:

Glycoprotein 2

RP-HPLC:

Reversed-phase high performance liquid chromatography

HPSEC:

Size exclusion high performance liquid chromatography

SA:

Sialic acid

OPD-2HCl:

o-Phenylenediamine dihydrochloride

GlcN:

N-acetyl-glucosamine

GalN:

N-acetyl-galactosamine

Gal:

Galactose

Fuc:

Fucose

Man:

Mannose

TFA:

Trifluoroacetic acid

SDS:

Sodium dodecyl sulfate

PNGase F:

Peptide-N-glycosidase F

DHB:

Dihydroxybenzoic acid

IRMA:

Immunoradiometric assay

MALDI-TOF-MS:

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry

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Acknowledgments

This work was supported by FAPESP, São Paulo, Brazil (11/07289-0) and by the Brazilian National Research Council (CNPq), Brasilia, Brazil (PQ 301615/2011-0 and PQ 300473/2009-5).

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Authors and Affiliations

  1. Biotechnology Department, IPEN-CNEN, Av. Prof. Lineu Prestes 2242, Cidade Universitária, 05508-900, São Paulo, Brazil

    Renata Damiani, Beatriz E. Almeida, João E. Oliveira, Paolo Bartolini & Maria Teresa C. P. Ribela

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  1. Renata Damiani
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  2. Beatriz E. Almeida
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  3. João E. Oliveira
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  4. Paolo Bartolini
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  5. Maria Teresa C. P. Ribela
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Correspondence to Maria Teresa C. P. Ribela.

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Damiani, R., Almeida, B.E., Oliveira, J.E. et al. Enhancement of Human Thyrotropin Synthesis by Sodium Butyrate Addition to Serum-Free CHO Cell Culture. Appl Biochem Biotechnol 171, 1658–1672 (2013). https://doi.org/10.1007/s12010-013-0467-9

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