Page 94 - 00. Complete Version - Progress Report IPEN 2014-2016

94 Biotechnology | Progress Report

Human pituitary mal use. In the same research field, to assess
glycoprotein hormones potency determination of human erythropoi-

etin, a sialoglycoprotein that stimulates the
Human pituitary glycoprotein hormones in- erythropoiesis, we investigated an in vitro
clude thyrotropin (hTSH), follitropin (hFSH) cell proliferation assay, which was applied in
and luteotropin (hLH), all heterodimers formed conjunction with a RP-HPLC methodology for
by an alpha and a beta subunit. hTSH is relat- the determination of sialic acid content and
ed to thyroid function and metabolism, and compared to the results of an in vivo bioassay
is used in the diagnosis and therapy of thy- (Machado FT et al., 2016). The in vitro assay re-
roid cancer, while hFSH and hLH are mostly sulted in a non-significant lower mean differ-
used for the treatment of human infertility. ence of the estimated potencies. Glycosylation
These recombinant products are among those sites of hTSH are also studied since these sites
with the highest aggregate value, their puri- are not always occupied and occupancy is re-
fied forms reaching prices up to US$ 12.000/ lated to folding, trafficking, initiation of in-
mg! Considering their carbohydrate moiety, flammation and host defense, as well as con-
which is strictly related to their in vivo bioac- genital disorders of glycosylation (CDG). For
tivity, these proteins must be synthesized in the first time, N-glycoprofiling analysis was
mammalian cells. Being CHO cells the most applied to the site-occupancy determination
commonly used for their industrial produc- of two native pituitary hTSH, in comparison
tion. Our laboratory has synthesized and char- with three recombinant preparations of hTSH.
acterized hTSH, having also the know-how for The results showed that the occupancy and
synthesizing hFSH and hLH. The effects of bu- carbohydrate mass can be up to 34-57% high-
tyrate and manganese on productivity, sialyla- er in recombinant hormones. We believe that
tion, N-glycosylation site occupancy and bio- this kind of comparison is extremely import-
logical properties of CHO-derived hTSH were ant when characterizing a widely used recom-
evaluated, showing no evidence of alterations binant biopharmaceutical. These results were
in its bioavailability, although increases in recently published in the International Jour-
hTSH production (up to 3-fold), in sialylation nal of Molecular Sciences (Ribela MTCP et al.,
(up to 14%) and site occupancy (up to 3%) oc- 2017). During this period, we also started us-
curred. In 2014, these data were presented in ing a strain of human embryonic kidney cells
the European Biotechnology Congress, in Ita- (HEK293) for the synthesis of hTSH, since re-
ly, and published in the Journal of Biotechnol- combinant biopharmaceuticals produced in
ogy (Damiani R et al., 2014). During this peri- these appropriate human cell lines are ex-
od (2014-2016), the laboratory completed the pected to present glycosylation profiles more
studies concerning to the substitution of an- similar to their human counterparts and less
imal-based bioassays by alternative method- immunogenicity. The produced hTSH-HEK,
ologies for hFSH and hTSH potency assess- compared to a CHO-derived recombinant and
ment. A reserved-phase high performance to a pituitary-derived preparation, was con-
liquid chromatography (RP-HPLC), developed sidered to be suitable for clinical applications,
by us, was compared to the in vivo bioassay for in view of its human origin, biological activ-
hTSH potency determination (Almeida BE et ity and particular carbohydrate composition
al., 2014). The results demonstrated that this (paper submitted to Applied Microbiology and
physical-chemical method is a novel and vi- Biotechnology).
able alternative for avoiding or reducing ani-

Instituto de Pesquisas Energéticas e Nucleares