Influence of intracellular nucleotide and nucleotide sugar contents on recombinant interferon-γ glycosylation during batch and fed-batch cultures of CHO cells
Influence of intracellular nucleotide and nucleotide sugar contents on recombinant interferon-γ glycosylation during batch and fed-batch cultures of CHO cells
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DOI：
10.1002/bit.21816
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发表时间：
2008-07-01
影响因子：
3.8
通讯作者：
Goergen, J. -L.
中科院分区：
文献类型：
--
作者：
Kochanowski, N.;Blanchard, F.;Goergen, J. -L.
在不同的分批和补料分批培养中，CHO细胞产生的重组人干扰素-γ的宏观异质性以及细胞内核苷酸和糖核苷酸的水平被表征。而PF-BDM培养基能在整个培养过程中保持较高比例的双糖化糖体，而在SF-RPMI分批培养过程中，单糖化和非糖化形式增加。与SF-RPMI相比，PF-BDM在整个分批培养过程中的细胞内UTP水平都较高。UDP-Gal仅在PF-BDM培养液中积累，可能是SF-RPMI培养液中UDP-GLC合成通量降低的结果。当分批补料培养重组CHO细胞时，含血清RPMI中的UTP水平保持在相对较高的水平，其滴度在补料阶段升高，表明生物合成过多。此外，还出现了UDP-Gal的积累。以上结果表明，在重组干扰素-γ的糖基化过程中，在SF-RPMI培养的CHO细胞中，UTP和UDP-GLC的合成可能受到限制。最后，对细胞在三个研究过程中的能量状态的测定表明，腺苷能荷与重组干扰素-γ的糖基化宏观异质性之间可能存在一定的关系。
Both the macroheterogeneity of recombinant human IFN-gamma produced by CHO cells and intracellular levels of nucleotides and sugar nucleotides, have been characterized during batch and fed-batch cultures carried out in different media. Whereas PF-BDM medium was capable to maintain a high percentage of the doubly-glycosylated glycoforms all over the process, mono-glycosylated and non-glycosylated forms increased during the batch culture using SF-RPMI medium. Intracellular level of UTP was higher in PF-BDM all over the batch culture compared to the SF-RPMI process. UDP-Gal accumulated only during the culture performed in PF-BDM medium, probably as a consequence of the reduced UDP-Glc synthesis flux in SF-RPMI medium. When the recombinant CHO cells were cultivated in fed-batch mode, the UTP level remained at a relatively high value in serum-containing RPMI and its titer increased during the fed-phase indicating an excess of biosynthesis. Besides, an accumulation of UDP-Gal occurred as well. Those results all together indicate that UTP and UDP-Glc syntheses in CHO cells cultivated in SF-RPMI medium in batch process, could be limiting during the glycosylation processes of the recombinant IFN-gamma. At last, the determination of the energetic status of the cells over the three studied processes suggested that a relationship between the adenylate energy charge and the glycosylation macroheterogeneity of the recombinant IFN-gamma may exist.