TY - JOUR
T1 - Interferon-α 2b quantification in inclusion bodies using Reversed Phase-Ultra Performance Liquid Chromatography (RP-UPLC)
AU - Cueto-Rojas, H. F.
AU - Pérez, N. O.
AU - Pérez-Sánchez, G.
AU - Ocampo-Juárez, I.
AU - Medina-Rivero, E.
PY - 2010/4/15
Y1 - 2010/4/15
N2 - Interferon-α 2b (IFN-α 2b) is a recombinant therapeutic cytokine produced as inclusion bodies using a strain of Escherichia coli as expression system. After fermentation and recovery, it is necessary to know the amount of recombinant IFN-α 2b, in order to determine the yield and the load for solubilization, and chromatographic protein purification steps. The present work details the validation of a new short run-time and fast sample-preparation method to quantify IFN-α 2b in inclusion bodies using Reversed Phase-Ultra Performance Liquid Chromatography (RP-UPLC). The developed method demonstrated an accuracy of 100.28%; the relative standard deviations for method precision, repeatability and inter-day precision tests were found to be 0.57%, 1.54% and 1.83%, respectively. Linearity of the method was assessed in the range of concentrations from 0.05 mg/mL to 0.5 mg/mL, the curve obtained had a determination coefficient (r2) of 0.9989. Detection and quantification limits were found to be 0.008 mg/mL and 0.025 mg/mL, respectively. The method also demonstrated robustness for changes in column temperature, and specificity against host proteins and other recombinant protein expressed in the same E. coli strain.
AB - Interferon-α 2b (IFN-α 2b) is a recombinant therapeutic cytokine produced as inclusion bodies using a strain of Escherichia coli as expression system. After fermentation and recovery, it is necessary to know the amount of recombinant IFN-α 2b, in order to determine the yield and the load for solubilization, and chromatographic protein purification steps. The present work details the validation of a new short run-time and fast sample-preparation method to quantify IFN-α 2b in inclusion bodies using Reversed Phase-Ultra Performance Liquid Chromatography (RP-UPLC). The developed method demonstrated an accuracy of 100.28%; the relative standard deviations for method precision, repeatability and inter-day precision tests were found to be 0.57%, 1.54% and 1.83%, respectively. Linearity of the method was assessed in the range of concentrations from 0.05 mg/mL to 0.5 mg/mL, the curve obtained had a determination coefficient (r2) of 0.9989. Detection and quantification limits were found to be 0.008 mg/mL and 0.025 mg/mL, respectively. The method also demonstrated robustness for changes in column temperature, and specificity against host proteins and other recombinant protein expressed in the same E. coli strain.
KW - Inclusion body
KW - Interferon-α 2b
KW - UPLC
KW - Validation
UR - http://www.scopus.com/inward/record.url?scp=77950242026&partnerID=8YFLogxK
U2 - 10.1016/j.jchromb.2010.02.016
DO - 10.1016/j.jchromb.2010.02.016
M3 - Artículo
SN - 1570-0232
VL - 878
SP - 1019
EP - 1023
JO - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
JF - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
IS - 13-14
ER -