Influence of aeration–homogenization system in stirred tank bioreactors, dissolved oxygen concentration and pH control mode on BHK-21 cell growth and metabolism

Abstract

This work focused on determining the effect of dissolved oxygen concentration (DO) on growth and metabolism of BHK-21 cell line (host cell for recombinant proteins manufacturing and viral vaccines) cultured in two stirred tank bioreactors with different aeration-homogenization systems, as well as pH control mode. BHK-21 cell line adapted to single-cell suspension was cultured in Celligen without aeration cage (rotating gas-sparger) and Bioflo 110, at 10, 30 and 50 % air saturation (impeller for gas dispersion from sparger-ring). The pH was controlled at 7.2 as far as it was possible with gas mixtures. In other runs, at 30 and 50 % (DO) in Bioflo 110, the cells grew at pH controlled with CO2 and NaHCO3 solution. Glucose, lactate, glutamine, and ammonium were quantified by enzymatic methods. Cell concentration, size and specific oxygen consumption were also determined. When NaHCO3 solution was not used, the optimal DOs were 10 and 50 % air saturation for Celligen and Bioflo 110, respectively. In this condition maximum cell concentrations were higher than 4 × 10(6) cell/mL. An increase in maximum cell concentration of 36 % was observed in batch carried out at 30 % air saturation in a classical stirred tank bioreactor (Bioflo 110) with base solution addition. The optimal parameters defined in this work allow for bioprocess developing of viral vaccines, transient protein expression and viral vector for gene therapy based on BHK-21 cell line in two stirred tank bioreactors with different agitation-aeration systems.

Model Comparison to Describe BHK-21 Cell Growth and Metabolism in Stirred Tank Bioreactors Operated in Batch Mode

“…However, the yield of lactate from glucose was independent of the operational conditions (Table 3, Figures 2-4). The higher glucose maintenance coefficients with respect to those for glutamine in most of the assessed conditions mean that the cells need higher quantities of glucose for cell maintenance (Craven et al, 2013;Núñez et al, 2013).…”

“…BHK-21 (C-13) cells (Sigma-Aldrich ECACC Cell Lines, Lyon, France) adapted to single cell suspension culture were kindly supplied by Dr. Renaud Wagner from Ecole Superieure Biotechnologie de Strasbourg, France. The detailed culture medium composition was previously defined (Núñez et al, 2013). Briefly, the culture medium was composed of the following ingredients (volume/volume percent): Iscove's Modified Dulbecco Medium with glutamine and Phenol red 45.5%; High glucose Dulbecco's Modified Eagle Medium 45.5%; heat inactivated fetal bovine serum 5.0%; 10% m/v Pluronic F-68 aqueous solution 2%; and 4 mM glutamine aqueous solution 2 %.…”

“…Samples from culture broth were centrifuged at 750 × g for 4 minutes. Subsequently, supernatants were filtered and frozen for later analysis of nutrients and metabolites (Núñez, et al, 2013). Glucose, lactate, glutamine and glutamate concentrations from supernant samples were measured using enzyme-coupled reactions and electrochemical detection in a YSI 2700 Select Bioanalyzer (YSI Life Sciences, Yellow Springs, OH, USA).…”

“…On the other hand, the bioreactor configuration and process parameters, such as pH, temperature, dissolved oxygen concentration, inoculum quality and concentration, define the cell growth and yield in bioconversion vessels (Streefland et al, 2013;Núñez et al, 2013). Therefore, values for parameters associated with process models could change with modifications in both sources of cell environmental shifts.…”

Approach toward an efficient inoculum preparation stage for suspension BHK-21 cell culture

“…The culture medium used had the following composition by volume: Iscove's Modified Dulbecco Medium with glutamine and phenol red (IMDM, catalog number 12200-036, Gibco, Grand Island, NY, USA) 45,5 %, High glucose Dulbecco's Modified Eagle Medium (DMEM, catalog number 12100-046, Gibco) 45,5 %, heat inactivated fetal bovine serum (Catalog number 10082-147, Gibco) 5 %, 10 % m/v Pluronic F-68 (Sigma-Aldrich, St. Louis, MO, USA) aqueous solution 2 %, and 4 mM glutamine (SigmaAldrich) aqueous solution 2 % (Fernández Núñez et al 2013).…”