Continuous culture of HEK293 producing the ECD-Her1. Existence steady states´ multiplicity at the same external conditions.Cell culture and metabolic datasets

Published: 5 September 2023| Version 4 | DOI: 10.17632/t9rcjv5362.4


The data correspond to the fermentation process in continuous mode of HEK293 cells producing the heterologous glycoprotein Her1, in a 2L bioreactor. Samples were taken daily from the culture for subsequent analysis of cell density, protein concentration and metabolites measurement. The data were acquired from different sources listed below: Viable cell density and viability, via microscope by trypan blue dye exclusion method. Extracellular domain Her1 concentration, via microplate photometer by ELISA sandwich method (GEN5 Reader Control Software). Briefly, 96-microwell plates were previously coated with 5 mg/mL of an anti-Her1 and incubated overnight at 4 °C. Later on, the samples and standard were added to the plates and incubated at 37 °C 1 h. An anti-EGFr antibody conjugated with biotin was added to the plate and incubated at 37 °C 1h. Then, Streptavidin-Peroxidase reagent was added to the plate and incubated at 37 °C 1 h. Finally, TMB substrate was added to the plate. After 20 minutes the reaction was stopped and the plates were read by a spectrophotometer at 450 nm. Metabolites concentration, via Liquid Chromatography-Mass spectrometry by derivatization method (TraceFinder software). Briefly, metabolites present in supernatant samples were extracted with extraction solvent (ACN:MeOH:H2O, 3:5:2) and transferred to LC-MS vials for separation and detection by employing the LC-MS equipment. Metabolites were firstly separated by the LC column with depend of their retention times. There was used as aqueous mobile phase solvent, 20 mM ammonium carbonate, adjusted to pH 9.4 with 0.1 % ammonium hydroxide solution (25 %), and 100 % Acetonitrile was used as organic mobile phase. A lineal gradient scheme was applied for the separation process at 200 mL/min taking the application around 15 minutes, and followed by an equilibration step. Column was kept in the oven at 45 °C, and samples were maintained at 4 °C prior to injection to the mass spectrometer. Finally, the metabolites were isolated by their mass/charge (m/z) with a mass accuracy below to 5 ppm for all metabolites with the Q-Exactive mass spectrometer. The metabolic data were autoscaling-standardized (ratio of centered mean and the standard deviation) prior the analysis.


Steps to reproduce

Continuous culture of HEK293 cells producing the extracellular domain Her1. 2L bioreactor: Culture environment conditions were set as follows: temperature at 37 ± 1 °C, dissolved oxygen at 40 ± 10 % and pH at 7.2 ± 0.2, as well as an agitation impeller tip speed kept at 1 m/s and aeration rate between 0.005 – 0.0075 vvd. Cells were cultured in continuous mode and three D (0,35 d-1; 0,4 d-1 and 0,45 d-1) were tested in order to transit the culture to different steady states. The same media formulation was used across the whole fermentation process, including Gluc and GLN contents. For further study, the continuous operation was started at 0,45 d-1 followed by 0,4 d-1 and later was reduced down to 0,35 d-1. Then, D was progressively increased to 0,4 d-1 and later 0,45 d-1 once more. Each steady state was kept for at least four days after the stabilization (which means coefficient of variation of cell density less than 20 %) and samples were collected from the culture each day to be subsequently tested.


Beatson Institute for Cancer Research, Centro de Inmunologia Molecular, Universidad de la Habana Facultad de Fisica


Metabolomics, Glycoprotein, Protein Expression, Liquid Chromatography Mass Spectrometry, Operation of Bioreactor, Continuous Cell Culture