Merck Research Laboratories, P.O. Box 2000, 07065, Rahway, NJ, USA.
Cytotechnology. 1996 Jan;22(1-3):239-50. doi: 10.1007/BF00353944.
Investigations of biological effects of prolonged elevation of growth hormone in animals such as mice and rats require large amounts of mouse and rat growth hormone (GH) materials. As an alternative to scarce and expensive pituitary derived materials, both mouse and rat GH were expressed in NSO murine myeloma cells transfected with a vector containing the glutamine synthetase (GS) gene and two copies of mouse or rat GH cDNA. For optimal expression, the mouse GH vector also contained sequences for targeting integration by homologous recombination. Fed-batch culture processes for such clones were developed using a serum-free, glutamine-free medium and scaled up to 250 L production scale reactors. Concentrated solutions of proteins, amino acids and glucose were fed periodically to extend cell growth and culture lifetime, which led to an increase in the maximum viable cell concentration to 3.5×10(9) cells/L and an up to 10 fold increase in final mouse and rat rGH titers in comparison with batch cultures. For successful scale up, similar culture environmental conditions were maintained at different scales, and specific issues in large scale reactors such as balancing oxygen supply and carbon dioxide removal, were addressed. Very similar cell growth and protein productivity were obtained in the fed-batch cultures at different scales and in different production runs. The final mouse and rat rGH titers were approximately 580 and 240 mg/L, respectively. During fed-batch cultures, the cell growth stage transition was accompanied by a change in cellular metabolism. The specific glucose consumption rate decreased significantly after the transition from the growth to stationary stage, while lactate was produced in the exponential growth stage and became consumed in the stationary stage. This was roughly coincident with the beginning of ammonia and glutamate accumulation at the entry of cells into the stationary stage as the result of a reduced glutamine consumption and periodic nutrient additions.
为了研究生长激素在动物（如老鼠）中的长期升高的生物学效应，需要大量的鼠类生长激素（GH）材料。作为稀缺和昂贵的垂体衍生材料的替代品，我们使用含有谷氨酰胺合成酶（GS）基因和两个拷贝的鼠或大鼠 GH cDNA 的载体转染 NSO 鼠骨髓瘤细胞，表达了鼠和大鼠 GH。为了实现最佳表达，鼠 GH 载体还包含了通过同源重组靶向整合的序列。我们开发了用于此类克隆的 fed-batch 培养工艺，使用无血清、无谷氨酰胺的培养基，并将其放大到 250L 生产规模的反应器中。定期给浓缩的蛋白质、氨基酸和葡萄糖溶液，以延长细胞生长和培养时间，从而将最大活细胞浓度提高到 3.5×10(9)个细胞/L，并将最终的鼠和大鼠 rGH 滴度提高 10 倍，与分批培养相比。为了成功放大规模，在不同的规模下保持相似的培养环境条件，并解决了大规模反应器中的特定问题，如平衡供氧和二氧化碳去除。在不同的规模和不同的生产运行中，在 fed-batch 培养中获得了非常相似的细胞生长和蛋白质生产力。最终的鼠和大鼠 rGH 滴度分别约为 580 和 240mg/L。在 fed-batch 培养过程中，细胞生长阶段的转变伴随着细胞代谢的变化。在从生长到静止阶段的转变后，特定的葡萄糖消耗率显著下降，而在指数生长阶段产生乳酸，并在静止阶段消耗。这大致与氨和谷氨酸积累的开始时间相吻合，因为谷氨酰胺的消耗减少，并且周期性地添加营养物质。
