Department of Chemical Engineering, University of Nebraska, 207 Othmer Hall, 820 North 16th Street, Lincoln, NE, 68588, USA.
Cytotechnology. 2007 May;54(1):57-68. doi: 10.1007/s10616-007-9074-3. Epub 2007 Jun 5.
A serum free medium was developed for the production of recombinant antibody against Botulinum A (BoNTA) using dihydrofolate reductase deficient Chinese Hamster Ovary Cells (CHO-DG44) in suspension culture. An initial control basal medium was prepared, which was similar in composition to HAM's F12: IMDM (1:1) supplemented with insulin, transeferrin, selenium and a lipid mixture. The vitamin concentration of the basal medium was twice that of HAM's F12: IMDM (1:1). CHO-DG44 cells expressing S25 antibody grew from 2 x 10(5) cells to maximum cell density of 1.04 x 10(6) cells/ml after 5 days in this control medium. A central composite design was used to identify optimal levels and interaction among five groups of medium components. These five groups were glutamine, Essential Amino Acids (EAA), Non Essential Amino Acids (NEAA), Insulin, Transferrin, Selenium (ITS), and lipids. Fifty experiments were carried out in four batches, with two controls in each batch. There was little effect of ITS and Lipid concentrations over the range studied, and glutamine concentration showed a strong interaction with EAA. The optimal concentrations of the variables studied were 2.5 mM Glutamine, 7.4 mM (2x) EAA, 1.4 mM (0.5x) NEAA, 1x ITS supplement, 0.7x Lipids supplement. The maximum viable cell density attained in the optimized medium was 1.4 x 10(6) cells/ml, a 35% improvement over the control culture, while the final antibody titer attained was 22 +/- 3.4 mug/mL, a 50% improvement.
开发了一种无血清培养基，用于在悬浮培养的二氢叶酸还原酶缺陷型中国仓鼠卵巢细胞（CHO-DG44）中生产针对肉毒梭菌 A（BoNTA）的重组抗体。最初制备了一种对照基础培养基，其组成与 HAM 的 F12：IMDM（1:1）相似，补充了胰岛素、转铁蛋白、硒和脂质混合物。基础培养基的维生素浓度是 HAM 的 F12：IMDM（1:1）的两倍。在该对照培养基中，表达 S25 抗体的 CHO-DG44 细胞从 2 x 10(5)个细胞增长到最大细胞密度 1.04 x 10(6)个细胞/ml，用时 5 天。使用中心复合设计来确定五个培养基成分组的最佳水平和相互作用。这五个组是谷氨酰胺、必需氨基酸（EAA）、非必需氨基酸（NEAA）、胰岛素、转铁蛋白、硒（ITS）和脂质。在四批实验中进行了 50 次实验，每批实验中有两个对照。在研究的范围内，ITS 和脂质浓度的影响很小，谷氨酰胺浓度与 EAA 之间存在强烈的相互作用。所研究变量的最佳浓度为 2.5 mM 谷氨酰胺、7.4 mM（2x）EAA、1.4 mM（0.5x）NEAA、1x ITS 补充剂、0.7x 脂质补充剂。在优化后的培养基中达到的最大活细胞密度为 1.4 x 10(6)个细胞/ml，比对照培养提高了 35%，而最终抗体滴度达到 22 +/- 3.4 mug/mL，提高了 50%。
