Culture media selection and feeding strategy for high titer production of a lentiviral vector by stable producer clones cultivated at high cell density

Abstract

The growing interest in the use of lentiviral vectors (LVs) for various applications has created a strong demand for large quantities of vectors. To meet the increased demand, we developed a high cell density culture process for production of LV using stable producer clones generated from HEK293 cells, and improved volumetric LV productivity by up to fivefold, reaching a high titer of 8.2 × 107 TU/mL. However, culture media selection and feeding strategy development were not straightforward. The stable producer clone either did not grow or grow to lower cell density in majority of six commercial HEK293 media selected from four manufacturers, although its parental cell line, HEK293 cell, grows robustly in these media. In addition, the LV productivity was only improved up to 53% by increasing cell density from 1 × 106 and 3.8 × 106 cells/mL at induction in batch cultures using two identified top performance media, even these two media supported the clone growth to 5.7 × 106 and 8.1 × 106 cells/mL, respectively. A combination of media and feed from different companies was required to provide diverse nutrients and generate synergetic effect, which supported the clone growing to a higher cell density of 11 × 106 cells/mL and also increasing LV productivity by up to fivefold. This study illustrates that culture media selection and feeding strategy development for a new clone or cell line can be a complex process, due to variable nutritional requirements of a new clone. A combination of diversified culture media and feed provides a broader nutrients and could be used as one fast approach to dramatically improve process performance.

Development, optimization, and scale‐up of suspension Vero cell culture process for high titer production of oncolytic herpes simplex virus‐1

“…[ 19 ] When HEK293 and A549 cultures are fed with these three feeds for a production of adenovirus or lentivirus, there is no delay in the infection kinetic or virus production in the cultures infected without medium replacement. [ 26 ] Instead, an increase in the virus production is usually obtained. Fed‐batch culture has been employed as a common process to improve the production of monoclonal antibody and also successfully used to increase the volumetric productivity of various viruses.…”

Optimization of Culture Media and Feeding Strategy for High Titer Production of an Adenoviral Vector in HEK 293 Fed-Batch Culture

“…Based on our recent findings that titers of other types of viruses can be increased using commercially available or in-house developed media [20,21], we seek to increase Ad5 titers by re-examining the fed-batch culture process. Through testing and customizing media, feed and regimen, we have succeeded in infecting HEK 293 fed-batch cultures at cell densities up to 5 × 10 6 cells/mL while maintaining the cell-specific productivity, resulting in 6-fold improvement in the volumetric titers up to 3 L bench scale bioreactor.…”