Fed-batch culture of insect cells: a method to increase the yield of recombinant human nerve growth factor (rhNGF) in the baculovirus expression system.
A fed-batch method was developed which increased the density of insect cells (Spodoptera frugiperda, Sf-9 cells) in suspension culture and the feeding of nutrients improved the yield of a recombinant protein produced by a baculovirus expression system. Analysis of spent medium samples indicated that depletions of glucose and glutamine correlated with the retardation of cell growth. Feeding of a mixture of nutrients consisting of glucose, glutamine, yeastolate and lipids solution restored the growth rate. In fed-batch culture, cell density was increased from 3 x 10(6) cells per ml to 1.2 x 10(7) cells per ml and the increased cell density enhanced the yield of the desired recombinant product, in this case, human nerve growth factor (rhNGF). The optimal conditions for the production of rhNGF were also defined by selecting the appropriate viral multiplicity of infection (MOI). At a cell density of 5 x 10(6) ml-1, a MOI of 0.05 (plaque forming units per cell) gave the highest yield of rhNGF in culture fluid 3 d post-infection. The yield of rhNGF was 20 mg l-1. The fed-batch method was scaled up to 12 l stirred bioreactor.
Nguyen
B
B
Department of Molecular Biology, Syntex Discovery Research, Palo Alto, California 94303.
Jarnagin
K
K
Williams
S
S
Chan
H
H
Barnett
J
J
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000502:physiology; D001709:Biotechnology / Q000379:methods*; D002455:Cell Division; D002460:Cell Line; D003470:Culture Media; D006801:Humans; D009036:Moths; D009414:Nerve Growth Factors / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014779:Virus Replication
D003470:Culture Media; D009414:Nerve Growth Factors; D011994:Recombinant Proteins