Practical reversed-phase high-performance liquid chromatography method for laboratory-scale purification of recombinant human thyrotropin

Journal of Chromatography. a
João Ezequiel OliveiraMaria Teresa Carvalho Pinto Ribela

Abstract

A small, semi-preparative C(4) RP-HPLC column was used to set up the conclusive laboratory-scale purification of Chinese hamster ovary-derived human thyrotropin (hTSH), after a preliminary concentration-purification of an extremely dilute and poorly ( approximately 0.6 microg hTSH/mL; mass fraction=0.35%) conditioned medium on a cation exchanger. Several fractions of this eluate were repeatedly injected on the semi-preparative column, obtaining, in a single run (<1h chromatographic time), a concentrated pool ( approximately 1.2 mg/mL) of highly purified hTSH that could be further concentrated to >3 mg/mL and then efficiently lyophilized. The overall recovery in the rapid RP-HPLC purification step, including concentration and lyophilization, was of the order of 80%. The final product, when tested via a precise, single-dose in vivo bioassay, confirmed that it did not suffer any loss of bioactivity. This same methodology can be easily adapted to the small-scale purification of other recombinant products, even when obtained from genetically modified organisms at extremely low concentrations and mass fractions.

References

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Feb 1, 1995·Thyroid : Official Journal of the American Thyroid Association·J East-PalmerB D Weintraub
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Jun 8, 2001·Biotechnology and Applied Biochemistry·A BeldarraínM Gil
Mar 26, 2003·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·João Ezequiel de OliveiraMaria Teresa C P Ribela
May 28, 2003·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Maria Teresa C P RibelaPaolo Bartolini
Oct 20, 2006·Journal of Chromatography. a·Renan Fernandes LoureiroMaria Teresa C P Ribela

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Citations

Jun 14, 2012·Applied Biochemistry and Biotechnology·Fernanda Santos ArthusoCarlos Roberto Jorge Soares
Sep 3, 2013·Applied Biochemistry and Biotechnology·Renata DamianiMaria Teresa C P Ribela
Jan 27, 2009·Journal of Chromatography. a·C M CarvalhoM T C P Ribela

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