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

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 (∼0.6 μg hTSH/mL; mass fraction = 0.35%) conditione...

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Bibliographic Details
Published in:Journal of Chromatography A 2007-09, Vol.1164 (1), p.206-211
Main Authors: Oliveira, João Ezequiel, Damiani, Renata, Bartolini, Paolo, Ribela, Maria Teresa Carvalho Pinto
Format: Article
Language:English
Subjects:
Analytical chemistry
Animals
Chemistry
CHO Cells
Chromatographic methods and physical methods associated with chromatography
Chromatography, High Pressure Liquid - methods
Cricetinae
Cricetulus
DNA recombinant
Exact sciences and technology
Humans
In vivo bioassay
Other chromatographic methods
Protein purification
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Reproducibility of Results
Reversed-phase HPLC
Thyrotropin
Thyrotropin - genetics
Thyrotropin - isolation & purification
Thyrotropin - metabolism
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Summary: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 (∼0.6 μg 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 (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.
ISSN:0021-9673
DOI:10.1016/j.chroma.2007.07.013