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首页> 外文会议>Asia-Pacific biochemical engineering conference;APBIOCHEC'97 >Characterization and improvement of interferon-gamma sialylation in chinese hamster ovary cell culture
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Characterization and improvement of interferon-gamma sialylation in chinese hamster ovary cell culture

机译：中国仓鼠卵巢细胞培养中干扰素-γ唾液酸化的表征及改良

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Since sialic acid content is known to be a critical determinant of the biological properties of glycoproteins, it is essential to characterize and monitor sialylation patterns of recombinant glycoproteins intended for therapeutic use. This study reports sialylation of human interferon-gamma (IFN-gamma) derived from Chinese hamster ovary (CHO) cell culture, which was quantified by reversed-phase HPLC separations of the site-specific pools of tryptic glycopeptides representing IFN-gamma's two potential N-linked glycosylation sites (i.e., Asn~25 and Asn~97). Although sialylation at each glycosylation site was found to be incomplete, glycans of Asn~25 were more heavily sialylated than those of Asn~97. Furthermore, Man(alpha 1-3) arms of the predominant complex biantennary structures were more favorably sialylated than Man(alpha 1-6) branches at each glycosylation site. Both intracellular and extracellular processes defined the sialic acid content of the final product. Furhtermore, the incomplete intracellular sialylation of IFN-gamma produced by CHO cell culture was minimized by supplementing the culture medium with N-acetylmannosamine (ManNAc), a direct intracellular precursor for sialic acid synthesis. By introducing 20 mM ManNAc into the culture medium, the percentage of incompletely sialylated IFN-gamm for the dominant binatennry glycan structure was reduced by nearly 50
机译：由于已知唾液酸含量是糖蛋白生物学特性的关键决定因素，因此表征和监测打算用于治疗用途的重组糖蛋白的唾液酸化模式至关重要。这项研究报告了源自中国仓鼠卵巢（CHO）细胞培养物的人干扰素-γ（IFN-γ）的唾液酸化作用，通过反相HPLC分离代表胰蛋白酶IFN-γ的两个潜在N的胰蛋白酶糖肽的位点特异性库进行了定量分析连接的糖基化位点（即Asn〜25和Asn〜97）。尽管发现每个糖基化位点的唾液酸化是不完全的，但是Asn〜25的聚糖比Asn〜97的聚糖的唾液酸化程度更高。此外，在每个糖基化位点，主要复杂双天线结构的Man（alpha 1-3）臂比Man（alpha 1-6）分支更容易被唾液酸化。细胞内和细胞外过程均定义了最终产物的唾液酸含量。此外，通过向培养基中补充唾液酸合成的直接细胞内前体N-乙酰甘露糖胺（ManNAc），将CHO细胞培养产生的IFN-γ的不完全细胞内唾液酸化程度降至最低。通过将20 mM ManNAc引入培养基中，显性唾液酸聚糖结构的不完全唾液酸化IFN-γ的百分比降低了近50％

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