title: The indirect effects of multiplicity of infection on baculovirus expressed proteins in insect cells: secreted and non-secreted products.
abstract: The baculovirus expression vector system was employed to produce human apolipoprotein E and β-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 culture and infection parameters, such as the cell density at time of infection and the time of harvest, allowed determination of the direct and indirect influences of multiplicity of infection on recombinant protein synthesis and degradation in insect cells. Under non-limited, controlled conditions, the direct effect of multiplicity of infection (10(-1)-10 pfu/cell) on specific recombinant product yields of non-secreted β-galactosidase was found to be insignificant. Instead, the observed increased in accumulated product was directly correlated to the total number of infected cells during the production period and therefore ultimately dependent on an adequate supply of nutrients. Only the timing of recombinant virus and protein production was influenced by, and dependent on the multiplicity of infection. Evidence is presented in this study that indicates the extremely limited predictability of post-infection cell growth at very low multiplicities of infection of less than 0.1 pfu/cell. Due to the inaccuracy of the current virus quantification techniques, combined with the sensitivity of post-infection cell growth at low MOI, the possibility of excessive post-infection cell growth and subsequent nutrient limitation was found to be significantly increased. Finally, as an example, the degree of product stability and cellular and viral protein contamination at low multiplicity of infection is investigated for a secreted recombinant form of human apolipoprotein E. Comparison of human apolipoprotein E production and secretion at multiplicities of infection of 10(-4)-10 pfu/cell revealed increased product degradation and contamination with intracellular proteins at low multiplicities of infection.
authors.affiliation: Geneva Biomedical Research Institute, Geneva, 1228, Switzerland.
authors.forename: K M
authors.initials: KM
authors.lastname: Radford
authors.forename: C
authors.initials: C
authors.lastname: Cavegn
authors.forename: M
authors.initials: M
authors.lastname: Bertrand
authors.forename: A R
authors.initials: AR
authors.lastname: Bernard
authors.forename: S
authors.initials: S
authors.lastname: Reid
authors.forename: P F
authors.initials: PF
authors.lastname: Greenfield