Nguyen B, Jarnagin K, Williams S, Chan H, Barnett J
Department of Molecular Biology, Syntex Discovery Research, Palo Alto, California 94303.
J Biotechnol. 1993 Nov;31(2):205-17. doi: 10.1016/0168-1656(93)90161-f.
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.
开发了一种补料分批培养方法，该方法提高了悬浮培养中昆虫细胞（草地贪夜蛾，Sf-9细胞）的密度，营养物质的补料提高了杆状病毒表达系统产生的重组蛋白的产量。对用过的培养基样品的分析表明，葡萄糖和谷氨酰胺的消耗与细胞生长的迟缓相关。补加由葡萄糖、谷氨酰胺、酵母提取物和脂质溶液组成的营养混合物可恢复生长速率。在补料分批培养中，细胞密度从每毫升3×10⁶个细胞增加到每毫升1.2×10⁷个细胞，增加的细胞密度提高了所需重组产物的产量，在这种情况下是人类神经生长因子（rhNGF）。通过选择合适的病毒感染复数（MOI）也确定了生产rhNGF的最佳条件。在细胞密度为5×10⁶ ml⁻¹时，感染复数为0.05（每细胞空斑形成单位）在感染后3天的培养液中rhNGF产量最高。rhNGF的产量为20 mg l⁻¹。补料分批培养方法扩大到12升搅拌式生物反应器。
