loading page

A reverse phase HPLC method for the quantification of HIV gp145 glycoprotein levels in cell culture supernatants
  • +9
  • José González-Feliciano,
  • Coral Capó-Vélez,
  • Pearl Akamine,
  • Manuel Delgado-Vélez,
  • Ruth Almodóvar,
  • Javier Rivera,
  • Ignacio Pino,
  • Gloriner Morell,
  • Daniel Eichinger,
  • José Rivera,
  • José Lasalde-Dominicci,
  • Abel Baerga-Ortiz
José González-Feliciano
University of Puerto Rico Molecular Sciences Research Center
Author Profile
Coral Capó-Vélez
University of Puerto Rico Molecular Sciences Research Center
Author Profile
Pearl Akamine
University of Puerto Rico Molecular Sciences Research Center
Author Profile
Manuel Delgado-Vélez
University of Puerto Rico Molecular Sciences Research Center
Author Profile
Ruth Almodóvar
CDI Laboratories
Author Profile
Javier Rivera
CDI Laboratories
Author Profile
Ignacio Pino
CDI Laboratories
Author Profile
Gloriner Morell
CDI Laboratories
Author Profile
Daniel Eichinger
CDI Laboratories
Author Profile
José Rivera
CDI Laboratories
Author Profile
José Lasalde-Dominicci
University of Puerto Rico Rio Piedras
Author Profile
Abel Baerga-Ortiz
University of Puerto Rico Medical Sciences Campus
Author Profile

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.

Peer review status:POSTED

14 Jul 2020Submitted to Biotechnology and Bioengineering
15 Jul 2020Assigned to Editor
15 Jul 2020Submission Checks Completed