Alternating flow filtration as an alternative to internal spin filter based perfusion process: Impact on productivity and product quality.

This article reports the results obtained from comparison of internal spin filter (ISF) and alternating flow filtration (ATF) as cell retention systems, regarding cell growth, volumetric perfusion rate, cell specific perfusion rate and cell productivity in the fermentation process. As expected we were able to reach higher cell densities and to achieve longer runs since ATF systems are known to be less affected by fouling. Volumetric production of the reactor using the ATF system was 50-70% higher than the production achieved using the ISF due to higher cell density and a two-fold increase in the perfusion rate. On the other hand, downstream processing performances were evaluated regarding chromatographic steps yields and productivity and quality attributes of the purified materials. Similar results were obtained for all evaluated systems. The fact that we were able to achieve a 2 working volumes (WV)/day perfusion rate using an ATF system as cell retention device allowed us to virtually double the WV of a 25 L reactor. These results constitute valuable data for the optimization of recombinant protein production in perfusion processes since a two-fold increase in the average production of a manufacturing facility could be easily achieved as long as downstream scale up is possible. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1010-1014, 2017.

Statistical optimization of influenza H1N1 production from batch cultures of suspension Vero cells (sVero).

Efficient vaccine production requires the growth of large quantities of virus produced with high yield from a safe host system. Human influenza vaccines are produced in embryonated chicken eggs. However, over the last decade many efforts have allowed the establishment of cell culture-derived vaccines. We generated a Vero cell line adapted to grow in suspension (sVero) in a serum-free medium and evaluated it for its potential as host cell for influenza vaccine production. Initially we studied the capacity of sVero cells to grow in the presence of incremental concentrations of trypsin. In comparison with adherent Vero cells (aVero), we found that sVero cells maintain their growth kinetics even with a three-fold increase in trypsin concentration. The influence of the conditions of infection on the yield of H1N1 produced in serum-free suspension cultures of sVero cells was investigated by a 2(2) full factorial experiment with center point. Each experiment tested the influence of the multiplicity of infection (m.o.i.) and trypsin concentration, on production yields at two levels, in four possible combinations of levels and conditions, plus a further combination in which each condition was set in the middle of its extreme levels. On the basis of software analysis, a combination of m.o.i. of 0.0066TCID(50%)/cell and trypsin concentration of 5µg/1.0×10(6) cells with a desirability of 0.737 was selected as the optimized condition for H1N1 production in sVero cells. Our results show the importance of proper selection of infection conditions for H1N1 production on sVero cells in serum-free medium.

Suspension-Vero cell cultures as a platform for viral vaccine production.

Since Vero cells are currently considered as an acceptable cell substrate to produce a wide range of viruses, we developed a virus production platform using Vero cells adapted to grow in suspension in serum-free media. After adapting anchorage-dependent Vero cells to grow as a free-cell suspension, vesicular stomatitis virus, herpes simplex virus 1 and polio virus 1 production rates were evaluated in batch cultures using spinner flasks and perfused cultures in a bioreactor. The achieved results constitute valuable information for the development of a low-cost high-productivity process using a suspension culture of Vero cells to produce viral vaccines.