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首页> 外文会议>Cell culture engineering conference >STUDY OF AN UNUSUALLY HIGH LEVEL OF N-GLYCOLYLNEURAMINIC ACID (NGNA) SIALYLATION ON A MONOCLONAL ANTIBODY EXPRESSED IN CHINESE HAMSTER OVARY CELLS
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STUDY OF AN UNUSUALLY HIGH LEVEL OF N-GLYCOLYLNEURAMINIC ACID (NGNA) SIALYLATION ON A MONOCLONAL ANTIBODY EXPRESSED IN CHINESE HAMSTER OVARY CELLS

机译：仓鼠卵巢细胞表达的单克隆抗体中异常高水平的N-糖基神经氨酸（NGNA）唾液酸化作用的研究

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Sialic or neuraminic acids of recombinant therapeutic glycoproteins produced in mammalian cells, including monoclonal antibodies, have significant impact on the half-life, stability, and biological activity of these proteins (Hossler et al., 2009; Ghaderi et al., 2012). The predominant sialic acid N-acetylneuraminic acid (NANA or Neu5Ac) is added from precursor CMP-NANA to galactose residues of W-linked glycoproteins by sialytransferases. In most mammals CMP-NANA can also be modified to its hydroxylated derivative CMP-NGNA by CMP-W-acetylneuraminic acid hydroxylase (CMAH). NGNA can then be added from CMP-NGNA to galactose residues of the N-linked glycoproteins, also by sialytransferases. However, humans cannot make functional CMAH due to an inactivating exon deletion mutation in CMAH gene (Okerblom and Varki, 2017), and therefore cannot convert CMP-NANA to CMP-NGNA. Consequently, when injected into human patients, NGNA sialic acid containing mAbs or other recombinant glycoproteins may induce immune responses, which could negatively impact pharmacokinetics or efficacy. Therefore high levels of NGNA on therapeutic mAbs or other recombinant glycoproteins are an undesirable product quality attribute. The level of total sialic acids of recombinant glycoproteins produced in Chinese hamster ovary (CHO) cells is dictated largely by the selected cell lines, upstream process, and to a lesser degree, downstream process. NGNA sialylation is generally rare in CHO cells (Kbnitzer et al., 2015). Hence, therapeutic glycoproteins manufactured in these cells are considered safe for human use. However, during a first-in-human (FIH) upstream process development for a novel mAb, an initially selected desirable cell line (A) was found to produce the mAb with an unexpectedly high level of the NGNA sialic acid (>30%). To the best of our knowledge such high level of NGNA sialylation on a mAb produced by CHO cells has not been reported. To mitigate potential risks associated with high NGNA in human patients, a new cell line (B) that produces the mAb with very low NGNA was selected as the manufacturing cell line for this project. In order to understand the molecular mechanism causing the high NGNA content in cell line A, we initiated comprehensive genetic gap analyses using next-generation sequencing technologies and determined the differences in genomic, transcriptomic, and miRnomic profiles of the two cell lines. The results indicate spontaneous upregulation of CMAH mRNA expression, at least 10 fold higher in cell line A compared to cell line B. In this talk we will summarize the results of our studies of this unusual sialylation behavior in CHO cells.
机译：在哺乳动物细胞中产生的重组治疗性糖蛋白的唾液酸或神经氨酸，包括单克隆抗体，对这些蛋白的半衰期，稳定性和生物学活性具有重大影响（Hossler等，2009； Ghaderi等，2012）。通过唾液酸转移酶将主要的唾液酸N-乙酰神经氨酸（NANA或Neu5Ac）从前体CMP-NANA添加至W-连接的糖蛋白的半乳糖残基。在大多数哺乳动物中，CMP-NANA也可以通过CMP-W-乙酰神经氨酸羟化酶（CMAH）修饰为其羟基化衍生物CMP-NGNA。然后也可以通过唾液酸转移酶将CMP-NGNA中的NGNA加入到N-连接糖蛋白的半乳糖残基中。但是，由于CMAH基因的外显子缺失失活突变，人类无法制造功能性CMAH（Okerblom和Varki，2017），因此无法将CMP-NANA转化为CMP-NGNA。因此，当注射到人类患者体内时，含有NGNA唾液酸的mAb或其他重组糖蛋白可能会诱导免疫反应，这可能会对药代动力学或功效产生负面影响。因此，治疗性单克隆抗体或其他重组糖蛋白上的NGNA含量高是不良的产品质量属性。中国仓鼠卵巢（CHO）细胞中产生的重组糖蛋白的总唾液酸水平主要取决于所选的细胞系，上游过程，而程度较小的下游过程。 NGNA唾液酸化通常在CHO细胞中很少见（Kbnitzer等人，2015）。因此，在这些细胞中产生的治疗性糖蛋白被认为对于人类使用是安全的。但是，在开发新型mAb的首次人类（FIH）上游工艺开发过程中，发现最初选择的理想细胞系（A）可以产生出乎意料的高水平NGNA唾液酸（> 30％）的mAb 。据我们所知，尚未报道CHO细胞产生的mAb上如此高水平的NGNA唾液酸化。为了减轻与人类患者高NGNA相关的潜在风险，该项目选择了一种新的细胞系（B），该细胞系产生的NGNA非常低。为了了解导致细胞系A中NGNA含量高的分子机制，我们使用下一代测序技术启动了全面的遗传缺口分析，并确定了两种细胞系在基因组，转录组和miRnomic谱方面的差异。结果表明，CMAH mRNA表达自发上调，在细胞系A中比细胞系B高至少10倍。在本次演讲中，我们将总结我们对CHO细胞这种异常唾液酸化行为的研究结果。

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