The indirect effects of multiplicity of infection on baculovirus expressed proteins in insect cells: secreted and non-secreted products |
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Authors: | Kathryn M Radford Catherine Cavegn Martine Bertrand Alain R Bernard Steven Reid Paul F Greenfield |
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Institution: | (1) Geneva Biomedical Research Institute, Geneva, 1228, Switzerland;(2) Department of Chemical Engineering, The University of Queensland, Queensland, 4072, Australia |
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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.
This revised version was published online in July 2006 with corrections to the Cover Date. |
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Keywords: | baculovirus multiplicity of infection time of infection time of harvest apolipoprotein E insect cells |
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