Hashii Noritaka, Kawasaki Nana, Nakajima Yukari, Toyoda Masashi, Katagiri Yoko, Itoh Satsuki, Harazono Akira, Umezawa Akihiro, Yamaguchi Teruhide
Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, 1-18-1 Kamiyouga, Setagaya-ku, Tokyo 158-8501, Japan.
J Chromatogr A. 2007 Aug 10;1160(1-2):263-9. doi: 10.1016/j.chroma.2007.05.062. Epub 2007 May 25.
N-Glycolylneuraminic acid (NeuGc), an acidic nine-carbon sugar, is produced in several animals, such as cattle and mice. Since human cells cannot synthesize NeuGc, it is considered to be immunogenic in humans. Recently, NeuGc contamination was reported in human embryonic stem cells cultured with xenogeneic serum and cells, suggesting that possibly NeuGc may harm the efficacy and safety of cell therapy products. Sialic acids have been determined by derivatization with 1,2-diamino-4,5-methylenedioxybenzene (DMB) followed by liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS); however, the limited availability of cell therapy products requires more sensitive and specific methods for the quality test. Here we studied the use of nano-flow liquid chromatography/Fourier transformation ion cyclotron resonance mass spectrometry (nanoLC/FTMS) and nanoLC/MS/MS for NeuGc-specific determination at a low femtomole level. Using our method, we found NeuGc contamination of the human cell line (HL-60RG cells) cultured with human serum. Our method needs only 2.5x10(3) cells for one injection and would be applicable to the determination of NeuGc in cell therapy products.
N-羟乙酰神经氨酸（NeuGc）是一种酸性九碳糖，在牛和小鼠等多种动物体内产生。由于人类细胞无法合成NeuGc，因此它被认为在人体内具有免疫原性。最近，有报道称在用异种血清和细胞培养的人类胚胎干细胞中存在NeuGc污染，这表明NeuGc可能会损害细胞治疗产品的疗效和安全性。唾液酸已通过与1,2-二氨基-4,5-亚甲二氧基苯（DMB）衍生化，然后进行液相色谱/质谱（LC/MS）和液相色谱/串联质谱（LC/MS/MS）测定；然而，细胞治疗产品的有限供应需要更灵敏和特异的质量检测方法。在此，我们研究了使用纳流液相色谱/傅里叶变换离子回旋共振质谱（nanoLC/FTMS）和纳流液相色谱/串联质谱（nanoLC/MS/MS）在低飞摩尔水平特异性测定NeuGc。使用我们的方法，我们发现用人血清培养的人类细胞系（HL-60RG细胞）存在NeuGc污染。我们的方法每次进样仅需2.5×10³个细胞，可用于细胞治疗产品中NeuGc的测定。
