A reversed phase HPLC method for the quantification of HIV gp145 glycoprotein levels from cell culture supernatants.

Autor: González-Feliciano JA; Molecular Sciences Research Center, University of Puerto Rico, San Juan, PR 00926, USA; Clinical Bioreagent Center, University of Puerto Rico, San Juan, PR, 00926, USA., Capó-Vélez CM; Molecular Sciences Research Center, University of Puerto Rico, San Juan, PR 00926, USA; Clinical Bioreagent Center, University of Puerto Rico, San Juan, PR, 00926, USA., Akamine P; Molecular Sciences Research Center, University of Puerto Rico, San Juan, PR 00926, USA; Clinical Bioreagent Center, University of Puerto Rico, San Juan, PR, 00926, USA., Delgado-Vélez M; Molecular Sciences Research Center, University of Puerto Rico, San Juan, PR 00926, USA; Clinical Bioreagent Center, University of Puerto Rico, San Juan, PR, 00926, USA., Almodóvar R; CDI Laboratories, 12 W Méndez Vigo Ave. Mayagüez, PR 00680, USA; Clinical Bioreagent Center, University of Puerto Rico, San Juan, PR, 00926, USA., Rivera J; CDI Laboratories, 12 W Méndez Vigo Ave. Mayagüez, PR 00680, USA; Clinical Bioreagent Center, University of Puerto Rico, San Juan, PR, 00926, USA., Pino I; CDI Laboratories, 12 W Méndez Vigo Ave. Mayagüez, PR 00680, USA; Clinical Bioreagent Center, University of Puerto Rico, San Juan, PR, 00926, USA., Morell G; CDI Laboratories, 12 W Méndez Vigo Ave. Mayagüez, PR 00680, USA; Clinical Bioreagent Center, University of Puerto Rico, San Juan, PR, 00926, USA., Eichinger D; CDI Laboratories, 12 W Méndez Vigo Ave. Mayagüez, PR 00680, USA; Clinical Bioreagent Center, University of Puerto Rico, San Juan, PR, 00926, USA., Rivera JH; CDI Laboratories, 12 W Méndez Vigo Ave. Mayagüez, PR 00680, USA; Clinical Bioreagent Center, University of Puerto Rico, San Juan, PR, 00926, USA., Lasalde-Dominicci JA; Molecular Sciences Research Center, University of Puerto Rico, San Juan, PR 00926, USA; Department of Biology, University of Puerto Rico, Río Piedras Campus, San Juan, PR 00926, USA; Department of Chemistry, University of Puerto Rico, Río Piedras Campus, San Juan, PR 00925, USA; Clinical Bioreagent Center, University of Puerto Rico, San Juan, PR, 00926, USA., Baerga-Ortiz A; Molecular Sciences Research Center, University of Puerto Rico, San Juan, PR 00926, USA; Department of Biochemistry, University of Puerto Rico, Medical Sciences Campus, San Juan, PR 00936-5067, USA; Clinical Bioreagent Center, University of Puerto Rico, San Juan, PR, 00926, USA. Electronic address: abel.baerga@upr.edu.
Jazyk: angličtina
Zdroj: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences [J Chromatogr B Analyt Technol Biomed Life Sci] 2021 Mar 15; Vol. 1167, pp. 122562. Date of Electronic Publication: 2021 Jan 27.
DOI: 10.1016/j.jchromb.2021.122562
Abstrakt: A reversed 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 from culture supernatants. Samples were diluted in 50% D-PBS and 50% PowerCHO-2 (PC2) spent medium, and resolved on a Zorbax 300SB-C8 Rapid Resolution (2.1 × 50 mm, 3.5 µm) column, fitted with a C8 guard column (Zorbax 300SB-C8, 2.1 × 12.5 mm, 5 µm), using 0.1% TFA and 2% n-propanol in LC-MS water as mobile phase A and 0.1% TFA, 70% isopropanol, and 20% acetonitrile in LC-MS water as mobile phase B. The column temperature was 80 °C, the flow rate was 0.4 mL/min and the absorbance was monitored at 280 nm. The procedures and capabilities of the method were evaluated against the criteria for linearity, limit of detection (LOD), accuracy, repeatability, and robustness as defined by the International Conference on Harmonization (ICH) 2005 Q2(R1) 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 method showed good linearity (R 2  = 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.
(Copyright © 2021 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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