哺乳动物细胞仍然是生物生产宿主的主体。在工业中,栽培和收获策略以分批模式排序(例如,批处理,分批补料,集中补料分批和强化补料分批)和连续模式(例如,灌注)。为了获得更高的生产率和更好的产品质量,特别是对于容易碎裂的敏感产品,创新了具有各种修改的文化模式(例如,强化灌注培养[IPC])。在我们的研究中,我们证明了Fc融合产物(分子A)的片段化在传统补料分批(TFB)培养中是时间依赖性的。克隆A的碎片比例从3.8%增加到12.4%,从第10天至第14天,克隆B为0.8%至1.7%,克隆C为0.9%至2.0%。通过应用一种新的生物过程,IPC,这允许新鲜培养基的连续进料和使用过的培养基的恒定去除而不使细胞出血,以维持确定的恒定活细胞密度,片段减少到0.3%,而克隆A的生产率从2.96g/L增加到15.51g/L。为了验证片段减少是否对产品敏感,将携带两个其它Fc融合分子(分子B和分子C)的DNA序列的质粒转染到宿主中。结果表明,使用IPC具有一致的碎片减少效果。此外,培养规模扩大到50L和1000L。对于分子C,观察到低于0.1%的最小片段化水平。我们的研究揭示了IPC在减少Fc融合蛋白片段化方面的能力和当按比例放大同时保持高生产率时的再现性。
Mammalian cells remain the mainstay of biological production host. In industry, cultivating and harvest strategies are sorted in batch mode (e.g., batch, fed-batch, concentrated fed-batch and intensified fed-batch) and continuous mode (e.g., perfusion). To retrieve greater productivity and better product quality, especially for the sensitive products prone to fragmentation, culture modes with various modifications are innovated (e.g., intensified perfusion culture [IPC]). In our study, we demonstrated that the fragmentation of Fc-fusion product (Molecule A) is time-dependent in traditional fed-batch (TFB) culture. The fragmentation proportion increased from 3.8% to 12.4% for Clone A, 0.8% to 1.7% for Clone B and 0.9% to 2.0% for Clone C from Day 10 to Day 14. By applying a novel bioprocess, IPC, which allows continuous feeding of the fresh medium and constant removal of the spent medium without bleeding cells to maintain a defined constant viable cell density, the fragmentation was reduced to 0.3% while the productivity was increased from 2.96 g/L to 15.51 g/L for Clone A. To validate whether the fragmentation reduction is product-sensitive, plasmids carrying the DNA sequences of two other Fc-fusion molecules (Molecule B and Molecule C) were transfected into the host. The results showed consistent fragmentation reducing effect by using IPC. Furthermore, the cultivation scale was expanded to 50 L and 1000 L. A minimum fragmentation level below 0.1% was observed for Molecule C. Our study revealed the capability of IPC in reducing Fc-fusion protein fragmentation and the reproducibility when scaling up while maintaining high productivity.