N-Glycoprofiling Analysis for Carbohydrate Composition and Site-Occupancy Determination in a Poly-Glycosylated Protein: Human Thyrotropin of Different Origins.
Human thyrotropin (hTSH) is a glycoprotein with three potential glycosylation sites: two in the α-subunit and one in the β-subunit. These sites are not always occupied and occupancy is frequently neglected in glycoprotein characterization, even though it is related to folding, trafficking, initiation of inflammation and host defense, as well as congenital disorders of glycosylation (CDG). For the first timeN-glycoprofiling analysis was applied to the site-occupancy determination of two native pituitary hTSH, in comparison with three recombinant preparations of hTSH, a widely used biopharmaceutical. A single methodology provided the: (i) averageN-glycan mass; (ii) mass fraction of each monosaccharide and of sulfate; and (iii) percent carbohydrate. The results indicate that the occupancy (65%-87%) and carbohydrate mass (12%-19%) can be up to 34%-57% higher in recombinant hormones. The average glycan mass is 24% lower in pituitary hTSH and contains ~3-fold fewer moles of galactose (p <0.005) and sialic acid (p <0.01). One of the two native preparations, which had the smallest glycan mass together with the lowest occupancy and GalNAc, sulfate, Gal and sialic acid contents, also presented the lowest in vivo bioactivity and circulatory half-life. The methodology described, comparing a recombinant biopharmaceutical to its native equivalent, can be applied to any physiologically or clinical relevant glycoprotein.
Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP-Avenida Prof. Lineu Prestes, 2242-Cidade Universitária, 05508-000 São Paulo, Brazil. mtribela@ipen.br.
Maria Teresa C P
MT
Ribela
Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP-Avenida Prof. Lineu Prestes, 2242-Cidade Universitária, 05508-000 São Paulo, Brazil. renata.damiani@biosintesis.com.br.
Renata
R
Damiani
Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP-Avenida Prof. Lineu Prestes, 2242-Cidade Universitária, 05508-000 São Paulo, Brazil. felipedouglasichigo@gmail.com.
Felipe D
FD
Silva
Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP-Avenida Prof. Lineu Prestes, 2242-Cidade Universitária, 05508-000 São Paulo, Brazil. lima-eliana@hotmail.com.
Eliana R
ER
Lima
Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP-Avenida Prof. Lineu Prestes, 2242-Cidade Universitária, 05508-000 São Paulo, Brazil. jeolivei@ipen.br.
João E
JE
Oliveira
Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP-Avenida Prof. Lineu Prestes, 2242-Cidade Universitária, 05508-000 São Paulo, Brazil. cnperoni@ipen.br.
Cibele N
CN
Peroni
Hormone Laboratory, Oslo University Hospital, 0424 Oslo, Norway. pabusdal@online.no.
Peter A
PA
Torjesen
Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP-Avenida Prof. Lineu Prestes, 2242-Cidade Universitária, 05508-000 São Paulo, Brazil. crsoares@ipen.br.
Carlos R
CR
Soares
Biotechnology Center, Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP-Avenida Prof. Lineu Prestes, 2242-Cidade Universitária, 05508-000 São Paulo, Brazil. pbartoli@ipen.br.
Paolo
P
Bartolini
D000818:Animals; D016466:CHO Cells; D002241:Carbohydrates / Q000737:chemistry*; D003412:Cricetulus; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism* / Q000493:pharmacokinetics; D006031:Glycosylation; D006801:Humans; D051379:Mice; D011134:Polysaccharides; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013972:Thyrotropin / Q000737:chemistry* / Q000378:metabolism* / Q000493:pharmacokinetics
D002241:Carbohydrates; D006023:Glycoproteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D013972:Thyrotropin
MALDI-TOF-MS; N-glycans; N-glycoprofiling; carbohydrate site-occupancy; human thyrotropin (hTSH); pharmacokinetics