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

In this work the proteolytic activity in the supernatant and inside insect cells in culture was evaluated for different multiplicities of infection (M O[) and times of infection (TOI). Several methods to detect proteolytic act ivity in insect cells were tested and that using fluorescein thiocyanite-ca sein as a substrate was chosen. It was observed that infection caused not o nly a reduction in the concentration of proteases by decreasing their synth esis but also an inhibition of the intracellular proteolytic activity by in creasing the intracellular ATP level (measured by in vivo nuclear magnetic resonance, NMR). The maximum proteolytic activity in the supernatant was ob served at 72 hpi except when the cells were infected in the late exponentia l growth phase or with very low MOI, yielding a nonsynchronous infection. T he proteolytic degradation of Pr55gag particles was studied during culture and after harvest. In this particular case it was concluded that the supern atant should be stored at low temperature or quickly purified, since the de gradation after 24 h is only 3% at 4 degrees C while at 27 degrees C this v alue rises to 23%. There is a complex relationship between MOI, TOI, proteo lytic activity, and product titer and quality. Thus, the optimal conditions for each case will be a compromise between the final product titer, the de sired product quality, and operational issues like process time and capacit y, requiring proper integration between bioreaction and downstream processi ng. (C) 1999 John Wiley & Sons, Inc.