Using cell size kinetics to determine optimal harvest time for Spodoptera frugiperda and Trichoplusia ni BTI-TN-5B1-4 cells infected with a baculovirus expression vector system expressing enhanced green fluorescent protein

Abstract

Infecting insect cells with a baculovirus expression vector system (BEVS) is an increasingly popular method for the production of recombinant proteins. Due to the lytic nature of the system, however, determining the optimal harvest time is critical for maximizing protein yield. We found that measuring the change in average diameter during the progress of infection with an automated cell analysis system (Cedex HiRes, Innovatis AG) could be used to determine the time of maximum protein production and, thus, optimal harvest time. As a model system, we use insect cells infected with a baculovirus expressing enhanced green fluorescent protein (EGFP). We infected two commonly used insect cell lines, Spodoptera frugiperda (Sf-9) and Trichoplusia ni BTI-TN-5B1-4 (Hi5) with an Autographa californica nuclear polyhedrosis virus (AcNPV) encoding EGFP at various multiplicities of infection (MOI). We monitored the progress of infection with regard to viability, viable cell density and change in average cell diameter with a Cedex HiRes analyzer and compared the results to the EGFP produced. Peak protein production was reached one to two days after the point of maximum average diameter in all conditions. Thus, optimal harvest time could be determined by monitoring the change in average cell diameter during the course of an infection of a cell culture.

Monitoring of the effects of transfection with baculovirus on Sf9 cell line and expression of human dipeptidyl peptidase IV

“…Hence, the results indicate that there has been an opposite correlation between cell density and infection, namely protein production in infected cells. Whereas Nishikawa et al (2003) have supported the data, Sander and Harrysson (2007) reported no strong connection between the cell density and infection.…”

“…7). Sander and Harrysson (2007) reported the increased diameter of infected cell as 28 % with MOI = 0.5 at the same time. It is clearly understood that the cell size of Sf9 could be considered as one of the infection signs.…”

Protein production using the baculovirus‐insect cell expression system

“…The Sf9, Sf21, HighFive™, and other lepidopteran cell lines reproduce well both in adherent cultures and suspension cultures and can be adapted to serum‐free media . The HighFive™ cell line is said to achieve higher titers of recombinant proteins compared with the Sf9 cells on a per cell basis . However, the Sf9 cells (and also the Sf21) commonly achieve higher densities in culture than do the HighFive™ cells under the typically used culture conditions and therefore provide higher volumetric productivities than the HighFive™ …”

Genetic modification of a baculovirus vector for increased expression in insect cells

“…For many proteins, it is critical to harvest the cells and extract the protein at the point of peak production before proteolysis starts having an effect on the protein yield. Both β-galactosidase and EGFP are very stable proteins, and so, they may not be good models for testing recombinant protein stability (Sander and Harrysson 2007). To assess if Ac chiA−/cath−/p10− improved protein stability, we expressed a kinase, PAK5.…”