THE INDIRECT EFFECTS OF MULTIPLICITY OF INFECTION ON BACULOVIRUS-EXPRESSED PROTEINS IN INSECT CELLS - SECRETED AND NON-SECRETED PRODUCTS

The baculovirus expression vector system was employed to produce human apolipoprotein E and beta-galactosidase in order to study the effect of multiplicity of infection on secreted and non-secreted recombinant protein production. Prior knowledge of the influence of other cell cul ture and infection parameters, such as the cell density at time of inf ection and the time of harvest, allowed determination of the direct an d indirect influences of multiplicity of infection on recombinant prot ein synthesis and degradation in insect cells. Under non-limited, cont rolled conditions, the direct effect of multiplicity of infection (10( -1)-10 pfu/cell) on specific recombinant product yields of non-secrete d beta-galactosidase was found to be insignificant. Instead, the obser ved increased in accumulated product was directly correlated to the to tal number of infected cells during the production period and therefor e ultimately dependent on an adequate supply of nutrients. Only the ti ming of recombinant virus and protein production was influenced by, an d dependent on the multiplicity of infection. Evidence is presented in this study that indicates the extremely limited predictability of pos t-infection cell growth at very low multiplicities of infection of les s than 0.1 pfu/cell. Due to the inaccuracy of the current virus quanti fication techniques, combined with the sensitivity of post-infection c ell growth at low MOI, the possibility of excessive post-infection cel l growth and subsequent nutrient limitation was found to be significan tly increased. Finally, as an example, the degree of product stability and cellular and viral protein contamination at low multiplicity of i nfection is investigated for a secreted recombinant form of human apol ipoprotein E. Comparison of human apolipoprotein E production and secr etion at multiplicities of infection of 10(-4)-10 pfu/cell revealed in creased product degradation and contamination with intracellular prote ins at low multiplicities of infection.