Gramer Michael J, Maas Jodi
BioVest International, Inc., 8500 Evergreen Boulevard, Minneapolis, Minnesota 55433, USA.
Biotechnol Prog. 2003 Nov-Dec;19(6):1762-6. doi: 10.1021/bp034102f.
An NSO/GS cell line secreting a humanized antibody was routinely propagated in a T-flask using 2% serum. For scale-up of antibody production, this cell line was inoculated into a hollow fiber system using the same serum concentration. The metabolic activity increased for a few days in the hollow fiber system, but invariably the activity dropped dramatically as the cells died by day 7. A hollow fiber micro-bioreactor was used as a screening tool to examine possible reasons for cell death in the large-scale system. As seen in the hollow fiber system, cells died when 2% serum was used either on the cell side only or on both sides of the fiber in the micro-bioreactor. In contrast, the use of 20% serum on the cell side of the fiber and basal medium on the non-cell side resulted in good cell expansion at high viability. Regardless of the cell side serum concentration, no further growth enhancements were seen when up to 20% serum was placed on the non-cell side of the fiber. These results suggest that a serum component that does not readily cross the fiber is limiting cell growth in the hollow fiber bioreactors. The addition of a cholesterol-rich lipid supplement resulted in better cell growth in the micro-bioreactor, while the addition of other non-cholesterol lipid supplements resulted in no growth enhancement. The growth-enhancing properties of the cholesterol supplement were more pronounced at lower serum concentrations, suggesting that poor growth at low serum concentration was due to suboptimal cholesterol levels. When the cell side serum concentration was increased to 20% in the hollow fiber system, cells grew and filled the bioreactor, allowing a 39-day production run. These results demonstrate that this NSO cell line requires an increased cell side serum concentration for optimal growth and that this requirement is likely due to the inherent cholesterol dependency of this cell line.
一种分泌人源化抗体的NSO/GS细胞系通常在T型烧瓶中使用2%血清进行传代培养。为了扩大抗体生产规模，将该细胞系接种到中空纤维系统中，使用相同的血清浓度。在中空纤维系统中，代谢活性在几天内有所增加，但到第7天时，随着细胞死亡，活性 invariably 急剧下降。使用中空纤维微型生物反应器作为筛选工具，以检查大规模系统中细胞死亡的可能原因。如在中空纤维系统中所见，当仅在细胞侧或微型生物反应器中纤维两侧使用2%血清时，细胞会死亡。相比之下，在纤维的细胞侧使用20%血清，在非细胞侧使用基础培养基，可使细胞在高活力下良好扩增。无论细胞侧血清浓度如何，当在纤维的非细胞侧放置高达20%血清时，未见进一步的生长增强。这些结果表明，一种不易穿过纤维的血清成分限制了中空纤维生物反应器中的细胞生长。添加富含胆固醇的脂质补充剂可使微型生物反应器中的细胞生长更好，而添加其他非胆固醇脂质补充剂则不会促进生长。胆固醇补充剂的生长促进特性在较低血清浓度下更为明显，表明低血清浓度下生长不佳是由于胆固醇水平次优所致。当在中空纤维系统中细胞侧血清浓度增加到20%时，细胞生长并充满生物反应器，可进行39天的生产运行。这些结果表明，这种NSO细胞系需要增加细胞侧血清浓度以实现最佳生长，并且这种需求可能是由于该细胞系固有的胆固醇依赖性。
