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 ( 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.
Oliveira
João Ezequiel
JE
Biotechnology Department, IPEN-CNEN, Av Prof Lineu Prestes 2242, Cidade Universitária, 05508-900, São Paulo, Brazil.
Damiani
Renata
R
Bartolini
Paolo
P
Ribela
Maria Teresa Carvalho Pinto
MT
D000818:Animals; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D015203:Reproducibility of Results; D013972:Thyrotropin / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D011994:Recombinant Proteins; D013972:Thyrotropin