Determination of Chinese hamster ovary cell-derived recombinant thyrotropin by reversed-phase liquid chromatography.
A reversed-phase high-performance liquid chromatography (RP-HPLC) methodology for the qualitative and quantitative analysis of human thyrotropin (hTSH) in CHO cell conditioned medium and in purified preparations has been set up and validated for accuracy, precision and sensitivity. A recovery test indicated a bias of less than 2% and intra-day and inter-day quantitative determinations presented relative standard deviations (RSD) always <7%, while sensitivity was 0.2 microg (RSD=5.6%). The novel methodology was applied to the study of the best cultivation conditions and was able to detect a significant difference in retention time (t(R)) between pituitary and recombinant hTSH, probably reflecting the influence of the heterogeneity of the carbohydrate moiety on the hydrophobic properties of the molecule.
de Oliveira
João Ezequiel
JE
Biotechnology Department, IPEN-CNEN, Travessa R 400, Cidade Universitária, 05508-900 São Paulo, Brazil.
de Mendonça
Fernanda
F
Peroni
Cibele N
CN
Bartolini
Paolo
P
Ribela
Maria Teresa C P
MT
D000818:Animals; D016466:CHO Cells; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006224:Cricetinae; D011994:Recombinant Proteins / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013972:Thyrotropin / Q000032:analysis*
D011994:Recombinant Proteins; D013972:Thyrotropin