Increased titer and reduced lactate accumulation in recombinant retrovirus production through the down‐regulation of HIF1 and PDK

糖酵解 细胞培养 生物 乳酸脱氢酶 生物化学 效价 磷酸戊糖途径 代谢工程 乳酸脱氢酶A 新陈代谢 病毒 病毒学 遗传学
作者
Ana Paula Leite Rodrigues,Miguel R. Guerreiro,A. S. Formas-Oliveira,Paulo Fernandes,Anne-Kareen Blechert,Yvonne Genzel,Paula M. Alves,Wei Shou Hu,Ana S. Coroadinha
出处
期刊:Biotechnology and Bioengineering [Wiley]
卷期号:113 (1): 150-162 被引量:9
标识
DOI:10.1002/bit.25691
摘要

Many mammalian cell lines used in the manufacturing of biopharmaceuticals exhibit high glycolytic flux predominantly channeled to the production of lactate. The accumulation of lactate in culture reduces cell viability and may also decrease product quality. In this work, we engineered a HEK 293 derived cell line producing a recombinant gene therapy retroviral vector, by down-regulating hypoxia inducible factor 1 (HIF1) and pyruvate dehydrogenase kinase (PDK). Specific productivity of infectious viral titers could be increased more than 20-fold for single gene knock-down (HIF1 or PDK) and more than 30-fold under combined down-regulation. Lactate production was reduced up to 4-fold. However, the reduction in lactate production, alone, was not sufficient to enhance the titer: high-titer clones also showed significant enrollment of metabolic routes not related to lactate production. Transcriptome analysis indicated activation of biological amines metabolism, detoxification routes, including glutathione metabolism, pentose phosphate pathway, glycogen biosynthesis and amino acid catabolism. The latter were validated by enzyme activity assays and metabolite profiling, respectively. High-titer clones also presented substantially increased transcript levels of the viral genes expression cassettes. The results herein presented demonstrate the impact of HIF1 and PDK down-regulation on the production performance of a mammalian cell line, reporting one of the highest fold-increase in specific productivity of infectious virus titers achieved by metabolic engineering. They additionally highlight the contribution of secondary pathways, beyond those related to lactate production, that can be also explored to pursue improved metabolic status favoring a high-producing phenotype.
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