Effects of Oxygen/Glucose/Glutamine Feeding on Insect Cell Baculovirus Protein Expression: A Study on Epoxide Hydrolase Production

Abstract

The recombinant protein yields for batch cultures of the insect cell baculovirus expression system have been significantly enhanced by oxygen, glucose, and glutamine feeding. The improvement in both volumetric and specific yields was based on influencing the metabolism of infected cells. Oxygen was absolutely required for viral replication and high protein expression in infected cells. Increases of 200% in volumetric yield and 100% in specific yield of recombinant epoxide hydrolase were achieved by controlling the dissolved oxygen (DO) level to near 35% saturation. An additional 100% increase was achieved by glucose and glutamine feeding. Results indicated that the intracellular metabolite pool was not adequate for recombinant protein overproduction. Finally, the specific protein yield, based on initial infection cell density, in high cell density spinner flasks and bioreactors of spent media with glucose and glutamine feeding was equivalent to that freshly diluted cultures.

Proteolytic activity in infected and noninfected insect cells: Degradation of HIV-1 Pr55gag particles

“…Thus, an infection performed close to the maximum concentration (as the infection in the late exponential phase) will most probably induce a situation of oxygen depletion and decrease the infection efficiency due to the increase in the specific oxygen uptake rate caused by cell infection (Agathos, 1996). Other authors have also reported reduced productivities and considerably lower rates of cell death for cells infected in the late exponential or in the stationary growth phase (Caron et al, 1990, Wang et al, 1993, in agreement with Figure 3.…”

“…The insect cell / baculovirus system has been widely used in the production of several other proteins (Agathos 1996;Wang et al, 1993). Its main advantages include the capacity to perform posttranslational modifications, essential for HIV core-and virus-like particle production (such as myristylation and phosphorilation in the case of Pr55gag [Overton et al, 1989]), high productivities (both for cells and expressed product), and the ability to produce proteins with complex quaternary structures.…”

Comparison ofTrichoplusia ni BTI-Tn-5B1-4 (high five™) andSpodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures

“…Many previous studies have been conducted in which nutrients were added a limited number of times (usually only once) over the course of the Sf-9 cell culture (Bedard et al, 1994;Drews et al, 1995;Nguyen et al, 1993;Reuveny et al, 1993b;Wang et al, 1993a). Wang et al (1993a) found that glutamine and glucose addition resulted in enhanced cell growth and recombinant protein productivity.…”

Enhancement of Sf9 Cells and Baculovirus Production Employing Grace’s Medium Supplemented with Milk Whey Ultrafiltrate

“…Many studies have been performed with the purpose of improving insect cell culture media formulation (Wilkie et al 1980;Mitsuhashi 1989;Wang et al 1993;Drews et al 1995;Schlaeger 1996;Vaughn and Fan 1997;Ikonomou et al 2001;Maranga et al 2003, Marteijn et al 2003. The efforts to eliminate bovine serum resulted in several commercially available serum-free media for insect cells, however, many of these media present very high costs and still require supplementation with other complex components such as protein hydrolysates.…”