Page 6 - PR 2014 2016 04 Biotechnology

92 Biotechnology | Progress Report

Human pituitary field, to assess potency determination of hu-
glycoprotein hormones man erythropoietin, a sialoglycoprotein that

stimulates the erythropoiesis, we investigated
Human pituitary glycoprotein hormones in- an in vitro cell proliferation assay, which was
clude thyrotropin (hTSH), follitropin (hFSH) and applied in conjunction with a RP-HPLC meth-
luteotropin (hLH), all heterodimers formed by odology for the determination of sialic acid
an alpha and a beta subunit. hTSH is related to content and compared to the results of an in
thyroid function and metabolism, and is used vivo bioassay (Machado FT et al., 2016). The in
in the diagnosis and therapy of thyroid cancer, vitro assay resulted in a non-significant lower
while hFSH and hLH are mostly used for the mean difference of the estimated potencies.
treatment of human infertility. These recom- Glycosylation sites of hTSH are also studied
binant products are among those with the since these sites are not always occupied and
highest aggregate value, their purified forms occupancy is related to folding, trafficking,
reaching prices up to US$ 12.000/mg! Consider- initiation of inflammation and host defense,
ing their carbohydrate moiety, which is strictly as well as congenital disorders of glycosyla-
related to their in vivo bioactivity, these pro- tion (CDG). For the first time, N-glycoprofiling
teins must be synthesized in mammalian cells. analysis was applied to the site-occupancy
Being CHO cells the most commonly used for determination of two native pituitary hTSH,
their industrial production. Our laboratory has in comparison with three recombinant prepa-
synthesized and characterized hTSH, having rations of hTSH. The results showed that the
also the know-how for synthesizing hFSH and occupancy and carbohydrate mass can be up
hLH. The effects of butyrate and manganese to 34-57% higher in recombinant hormones.
on productivity, sialylation, N-glycosylation We believe that this kind of comparison is
site occupancy and biological properties of extremely important when characterizing a
CHO-derived hTSH were evaluated, showing widely used recombinant biopharmaceutical.
no evidence of alterations in its bioavailabil- These results were recently published in the
ity, although increases in hTSH production International Journal of Molecular Sciences
(up to 3-fold), in sialylation (up to 14%) and (Ribela MTCP et al., 2017). During this period,
site occupancy (up to 3%) occurred. In 2014, we also started using a strain of human embry-
these data were presented in the European onic kidney cells (HEK293) for the synthesis of
Biotechnology Congress, in Italy, and published hTSH, since recombinant biopharmaceuticals
in the Journal of Biotechnology (Damiani R et produced in these appropriate human cell lines
al., 2014). During this period (2014-2016), the are expected to present glycosylation profiles
laboratory completed the studies concerning more similar to their human counterparts and
to the substitution of animal-based bioassays less immunogenicity. The produced hTSH-HEK,
by alternative methodologies for hFSH and compared to a CHO-derived recombinant and
hTSH potency assessment. A reserved-phase to a pituitary-derived preparation, was consid-
high performance liquid chromatography (RP- ered to be suitable for clinical applications, in
HPLC), developed by us, was compared to the in view of its human origin, biological activity
vivo bioassay for hTSH potency determination and particular carbohydrate composition (pa-
(Almeida BE et al., 2014). The results demon- per submitted to Applied Microbiology and
strated that this physical-chemical method Biotechnology).
is a novel and viable alternative for avoiding
or reducing animal use. In the same research A work was also carried out in collaboration

Instituto de Pesquisas Energéticas e Nucleares