A reverse phase HPLC method for the quantification of HIV gp145
glycoprotein levels in cell culture supernatants
Abstract
A reverse phase high performance liquid chromatography (RP-HPLC) method
was developed for the quantitative determination of recombinant HIV-1
gp145 produced in CHO-K1 cells, as measured directly in culture
supernatants. Samples were diluted in 50% D-PBS and 50% PowerCHO-2
(PC2) spent media, and resolved on a Zorbax 300SB-C8 Rapid Resolution
(2.1 x 50 mm, 3.5 µm) column, fitted with a C8 guard column (Zorbax
300SB-C8, 2.1 x 12.5 mm, 5µm), using 0.1% TFA and 2% n-propanol as
mobile phase A and 0.1% TFA, 70% isopropanol, and 20% acetonitrile as
mobile phase B. The column temperature was 80ºC, the flow rate 1 ml/min
and the absorbance monitored at 280 nm. The procedures and capabilities
of the method were evaluated against the present criteria for linearity,
limit of detection (LOD), accuracy, precision, and robustness of the
International Conference on Harmonization (ICH) guidelines. Two
different variants of the HIV-1 envelope protein (Env), CO6980v0c22
gp145 and SF162 gp140, were analyzed and their retention times were
found to be different. The methods showed good linearity (R2 = 0.9996),
a lower LOD of 2.4 µg/ml, and an average recovery of 101%. The analysis
includes measurements of accuracy, inter-user precision, and robustness.
Overall, we present a RP-HPLC method that could be applied for the
quantitation of cell culture titers for this and other variants of HIV
Env following ICH guidelines.