Retroviral vector production under serum deprivation: The role of lipids

病毒载体 传染性 载体(分子生物学) 效价 细胞培养 生物 生产力 病毒学 慢病毒 免疫学 病毒 生物技术 生物化学 重组DNA 基因 病毒性疾病 遗传学 宏观经济学 经济
作者
Ana F. Rodrigues,Marlene Carmo,Paula M. Alves,Ana S. Coroadinha
出处
期刊:Biotechnology and Bioengineering [Wiley]
卷期号:104 (6): 1171-1181 被引量:36
标识
DOI:10.1002/bit.22499
摘要

The use of retroviral vectors for gene therapy applications demands high titer preparations and stringent quality standards. However, the manufacturing of these vectors still represents a highly challenging task due to the low productivity of the cell lines and reduced stability of the vector infectivity, particularly under serum-free conditions. With the objective of understanding the major limitations of retroviral vector production under serum deprivation, a thorough study of viral production kinetics, vector characterization and cell growth and metabolic behavior was conducted, for 293 FLEX 18 and Te Fly Ga 18 producer cell lines using different serum concentrations. The reduction of serum supplementation in the culture medium resulted in pronounced decreases in cell productivity of infectious vector, up to ninefold in 293 FLEX 18 cells and sevenfold in Te Fly Ga 18 cells. Total particles productivity was maintained, as assessed by measuring viral RNA; therefore, the decrease in infectious vector production could be attributed to higher defective particles output. The absence of the serum lipid fraction was found to be the major cause for this decrease in cell viral productivity. The use of delipidated serum confirmed the requirement of serum lipids, particularly cholesterol, as its supplementation not only allowed the total recovery of viral titers as well as additional production increments in both cell lines when comparing with the standard 10% (v/v) FBS supplementation. This work identified lower production ratios of infectious particles/total particles as the main restraint of retroviral vector production under serum deprivation; this is of the utmost importance concerning the clinical efficacy of the viral preparations. Lipids were confirmed as the key serum component correlated with the production of infective retroviral vectors and this knowledge can be used to efficiently design medium supplementation strategies for serum-free production. Biotechnol. Bioeng. 2009; 104: 1171-1181. (c) 2009 Wiley Periodicals, Inc.

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