An optimization study for expression of the rabies virus glycoprotein (RVGP) in mammalian cell lines using the Semliki Forest virus (SFV).

The Semliki Forest virus (SFV) viral vector has been widely used for transient protein expression. This study aimed to analyze comprehensively the capacity of SFV vector to express rabies lyssavirus glycoprotein (RVGP) in mammalian cells. The assessed parameters were transfection strategy, multiplicity of infection (MOI), harvest time and mammalian cell host. Two transfection approaches, electroporation and lipofection were evaluated to obtain the recombinant SFV, and the electroporation was found to be the most effective. Viral quantification by RT-qPCR was performed to elucidate the relation between the amount of recombinant virus utilized in the infection process and the production levels of the heterologous protein. Four different multiplicities of infection (MOIs = 1; 10; 15; 50) were evaluated using five mammalian cell lines: BHK-21, HuH-7, Vero, L929, and HEK-293T. Protein expression was assessed at two harvest times after infection (24 and 48 h). The recombinant protein generated was characterized by western blot, dot blot, and indirect immunofluorescence (IIF), while its concentration was determined by enzyme-linked immunosorbent assay (ELISA). Similar expression patterns were observed in cell lines BHK-21, HEK-293T, L929, and Vero, with higher RVGP production in the first 24 h. The BHK-21 cells showed yields of up to 4.3 µg per 106 cells when lower MOIs (1 and 10) were used. The HEK-293 T cells also showed similar production (4.3 µg per 106 cells) with MOI of 1, while the L929 and Vero cell lines showed lower expression rates of 2.82 and 1.26 µg per 106 cells, respectively. These cell lines showed lower expression levels at 48 h after infection compared to 24 h. Controversially, in the case of the HuH-7 cell line, RVGP production was higher at 48 h after infection (4.0 µg per 106 cells) and using MOIs of 15 and 50. This work may contribute to optimize the RVGP production using SFV system in mammalian cells. This study can also substantiate for example, the development of approaches that use of SFV for applications for other protein expressions and suggests values for relevant parameters and cell lines of this biotechnique.

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

Mammalian cells are the most frequently used hosts for biopharmaceutical proteins manufacturing. Inoculum quality is a key element for establishing an efficient bioconversion process. The main objective in inoculation expansion process is to generate large volume of viable cells in the shortest time. The aim of this paper was to optimize the inoculum preparation stage of baby hamster kidney (BHK)-21 cells for suspension cultures in benchtop bioreactors, by means of a combination of static and agitated culture systems. Critical parameters for static (liquid column height: 5, 10, 15 mm) and agitated (working volume: 35, 50, 65 mL, inoculum volume percentage: 10, 30 % and agitation speed: 25, 60 rpm) cultures were study in T-flask and spinner flask, respectively. The optimal liquid column height was 5 mm for static culture. The maximum viable cell concentration in spinner flask cultures was reached with 50 mL working volume and the inoculum volume percentage was not significant in the range under study (10-30 %) at 25 rpm agitation. Agitation speed at 60 rpm did not change the main kinetic parameters with respect to those observed for 25 rpm. These results allowed for a schedule to produce more than 4 × 10(9) BHK-21 cells from 4 × 10(6) cells in 13 day with 1,051 mL culture medium.

Transient expression of rabies virus G-glycoprotein using BHK-21 cells cultured in suspension.

OBJECTIVE: To assess the expression of rabies virus G-glycoprotein (RVGP) expression using Semliki Forest virus as a vector in combination with BHK-21 cells cultured in suspension. RESULTS: A multilevel factorial design was used to quantify effects of temperature (33-37 °C), fresh medium addition after the viral adsorption step (100-200 % with respect to the initial cell suspension volume before infection) and harvest time (8-40 h) on RVGP production. Experimental runs were performed in 24-well cell culture plates at a multiplicity of infection (MOI) of 16. An additional experiment in spinner-flask was performed at MOI of 9, using the optimal conditions determined in cell culture plates. Values for temperature, fresh medium addition and harvest time of 33 °C, 100 % and 16 h, respectively, ensured the optimal RVGP production in culture plates. The volumetric yield (239 ng ml(-1)) in these conditions was higher than that reported previously for adherent cell culture. In spinner-flasks, the volumetric yield was improved (559 ng ml(-1)). CONCLUSION: These results establish the basis for designing bioprocess to produce RVGP.

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

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.