Sf9 Cell Metabolism Throughout the Recombinant Baculovirus and Rabies Virus-Like Particles Production in Two Culture Systems.

This work aimed to assess the Sf9 cell metabolism during growth, and infection steps with recombinant baculovirus bearing rabies virus proteins, to finally obtain rabies VLP in two culture systems: Schott flask (SF) and stirred tank reactor (STR). Eight assays were performed in SF and STR (four assays in each system) using serum-free SF900 III culture medium. Two non-infection growth kinetics assays and six recombinant baculovirus infection assays. The infection runs were carried out at 0.1 pfu/cell multiplicity of infection (MOI) for single baculovirus bearing rabies glycoprotein (BVG) and matrix protein (BVM) and a coinfection with both baculoviruses at MOI of 3 and 2 pfu/cell for BVG and BVM, respectively. The SF assays were done in triplicate. The glucose, glutamine, glutamate, lactate, and ammonium uptake or release specific rates were quantified over the exponential growth phase and infection stage. The highest uptake specific rate was observed for glucose (42.5 × 10-12 mmol cell/h) in SF and for glutamine (30.8 × 10-12 mmol/cell/h) in STR, in the exponential growth phases. A wave pattern was observed for assessed analytes throughout the infection phase and the glucose had the highest wave amplitude within the 10-10 mmol cell/h order. This alternative uptake and release behavior is in harmony with the lytic cycle of baculovirus in insect cells. The virus propagation and VLP generation were not limited by glucose, glutamine, and glutamate, neither by the toxicity of lactate nor ammonium under the conditions appraised in this work. The findings from this work can be useful to set baculovirus infection processes at high cell density to improve rabies VLP yield, purity, and productivity.

Biochemical monitoring throughout all stages of rabies virus-like particles production by Raman spectroscopy using global models.

This work aimed to quantify growth and biochemical parameters (viable cell density, Xv; cell viability, CV; glucose, lactate, glutamine, glutamate, ammonium, and potassium concentrations) in upstream stages to obtain rabies virus-like particles (rabies VLP) from insect cell-baculovirus system using on-line and off-line Raman spectra to calibrate global models with minimal experimental data. Five cultivations in bioreactor were performed. The first one comprised the growth of uninfected Spodoptera frugiperda (Sf9) cells, the second and third runs to obtain recombinant baculovirus (rBV) bearing Rabies G glycoprotein and matrix protein, respectively. The fourth one involved the generation of rabies VLP from rBVs and the last one was a repetition of the third one with cell inoculum infected by rBV. The spectra were acquired through a Raman spectrometer with a 785-nm laser source. The fitted Partial Least Square models for nutrients and metabolites were comparable with those previously reported for mammalian cell lines (Relative error < 15 %). However, the use of this chemometrics approach for Xv and CV was not as accurate as it was for other parameters. The findings from this work established the basis for bioprocess Raman spectroscopical monitoring using insect cells for VLP manufacturing, which are gaining ground in the pharmaceutical industry.