A reversed phase HPLC method for the quantification of HIV gp145 glycoprotein levels from cell culture supernatants
| Autor: | Ignacio Pino, José A. Lasalde-Dominicci, Abel Baerga-Ortiz, Gloriner Morell, Ruth Almodóvar, José H. Rivera, Coral M. Capó-Vélez, Daniel Eichinger, Pearl Akamine, Javier Rivera, José A. González-Feliciano, Manuel Delgado-Vélez |
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| Rok vydání: | 2021 |
| Předmět: |
Resolution (mass spectrometry)
Clinical Biochemistry Cell Culture Techniques Human immunodeficiency virus (HIV) CHO Cells medicine.disease_cause 030226 pharmacology & pharmacy 01 natural sciences Biochemistry High-performance liquid chromatography Article Analytical Chemistry Absorbance 03 medical and health sciences Cricetulus 0302 clinical medicine Limit of Detection Cricetinae medicine Animals Chromatography High Pressure Liquid Detection limit Chromatography Reverse-Phase Chromatography Chemistry 010401 analytical chemistry env Gene Products Human Immunodeficiency Virus Reproducibility of Results Cell Biology General Medicine Repeatability Reversed-phase chromatography Recombinant Proteins 0104 chemical sciences Cell culture Linear Models |
| Zdroj: | J Chromatogr B Analyt Technol Biomed Life Sci |
| ISSN: | 1570-0232 |
| DOI: | 10.1016/j.jchromb.2021.122562 |
| Popis: | 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 (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. |
| Databáze: | OpenAIRE |
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