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

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.

N-Glycoprofiling Analysis for Carbohydrate Composition and Site-Occupancy Determination in a Poly-Glycosylated Protein: Human Thyrotropin of Different Origins

“…The first detailed structural characterization of N-linked oligosaccharides, only from recombinant hTSH, was reported by Morelle et al [28], who identified 12 different N -glycans and also described their microheterogeneity at each glycosylation site. In previous work, our research group reported N -glycoprofilings of recombinant human thyrotropin obtained under different culture conditions, identifying 13–20 different structures [7,29] and of humanized recombinant hTSH (hlsr-hTSH), identifying 20 structures [30]. The comparison between the N -glycoprofilings of a native glycoprotein and of its equivalent recombinant form was carried out by our group only for G-hPRL.…”

“…The native hormone was obtained according to classical procedures by removing human pituitary glands at autopsy and storing them in liquid nitrogen until the extraction procedure [37,38,39]. Of the three recombinant preparations, one is a commercial biopharmaceutical (Thyrogen ® from Genzyme Corporation, Framingham, MA, USA) [27], while the other two were from our laboratory at IPEN-CNEN/SP (São Paulo, Brazil), prepared and characterized as previously described [29,30,35,36,40]. As mentioned the human-like sialylated recombinant human thyrotropin (hlsr-hTSH) was obtained in a particular CHO cell line, previously modified by the introduction of rat α2,6-sialyltransferase [30].…”

“…Human TSH IRMA was carried out by an in-house methodology, utilizing a secondary hTSH standard calibrated against the international standard of pituitary hTSH (WHO 80/558, 4.93 IU/mg), as described [12,29]. …”

Production of Lentiviral Vectors Encoding Recombinant Factor VIII Expression in Serum-Free Suspension Cultures

“…3B). Sodium butyrate (NaBu) was successfully used to enhance recombinant protein (Damiani et al 2013;Lee et al 2014) and lentivirus and retrovirus production (Karolewski et al 2003;Merten 2004). Transcriptional silencing of the LV-related transfected genes was demonstrated as a drawback in LV production ( Kafri et al 1999;Jaalouk et al 2000;Ni et al 2005).…”

6.2 High Cell Density Cultivation Process

“…In biotechnology industry, supplementation of cultures with sodium butyrate, an inhibitor of histone deacetylation, is one of the most successful and widespread strategies to increase glycoprotein synthesis, which is attributed to its ability to arrest the cell cycle progression (G 0 /G 1 ) and thus increase specific productivity of the product of interest [47]. Growth arrest can be also achieved by a decrease in culture temperature; this strategy, known as temperature shift, has also demonstrated an increase in protein specific productivity, although it is strictly dependent on the producer cell line [38,48].…”