Dissolved carbon dioxide (dCO(2)) accumulation during cell culture has been recognized as an important parameter that needs to be controlled for successful scale-up of animal cell culture because above a certain concentration there are adverse effects on cell growth performance and protein production. We investigated the effect of accumulation of dCO(2) in bioreactor cultures of expresSF+((R)) insect cells infected with recombinant baculoviruses expressing recombinant influenza virus hemagglutinins (rHA). Different strategies for bioreactor cultures were used to obtain various ranges of concentrations of dCO(2) (<50, 50-100, 100-200, and >200mmHg) and to determine their effects on recombinant protein production and cell metabolic activity. We show that the accumulation of dCO(2) at levels>100mmHg resulted in reduced metabolic activity, slowed cell growth, prolonged culture viability after infection, and decreased infection kinetics. The reduced rHA yields were not caused by the decrease in the extracellular pH that resulted from dCO(2) accumulation, but were most likely due to the effect of dCO(2) accumulation in cells. The results obtained here at the 2L scale have been used for the design of large-scale processes to manufacture the rHA based recombinant vaccine Flublok at the 2500L scale Biotechnol. Bioeng. 2015;112: 2267-2275. (c) 2015 Wiley Periodicals, Inc. Jamal Meghrous and colleagues at Protein Sciences (Connecticut, USA) describe the impact of dissolved CO2 accumulation on the hemagglutinin (HA) yield manufactured usinginsect cell culture. HA proteins are the active ingredient of Flublok (R), the only FDA approved recombinant influenza vaccine. The objective of the work is to increase productivity and scale to make this important new seasonal influenza vaccine more widely available.