Watson E, Shah B, Leiderman L, Hsu Y R, Karkare S, Lu H S, Lin F K
Amgen Inc., Amgen Center, Thousand Oaks, California 91320.
Biotechnol Prog. 1994 Jan-Feb;10(1):39-44. doi: 10.1021/bp00025a004.
Glycosylation heterogeneity in recombinant human tissue kallikrein (r-HuTK) produced by Chinese hamster ovary (CHO) cells from microcarrier culture and from a serum-free suspension cell recycle process has been compared. Significant differences in the degree of sialylation were observed in glycoform distribution and oligosaccharide heterogeneity. High-performance liquid chromatography with a pellicular anion-exchange column under low pH eluant conditions was used to characterize the number and types of N-linked complex type oligosaccharides present. The oligosaccharides were released by N-glycanase and, after reduction, were resolved into a number of peaks containing one, two, three, and four sialic acids with an additional subfractionation based on the nature of the antennary structure. The microcarrier process resulted in a reduced amount of sialylated oligosaccharide species as compared to the suspension cell process. Removal of sialic acid followed by chromatography of the asialooligosaccharides under high pH anion-exchange conditions indicated that the same antennary structures were present but in slightly different relative amounts. The oligosaccharide profiles are indicative of a highly complex array of microheterogeneity present, encompassing mono-, di-, tri-, and tetrasialylated complex type oligosaccharides.
对中国仓鼠卵巢（CHO）细胞在微载体培养以及无血清悬浮细胞循环工艺中产生的重组人组织激肽释放酶（r-HuTK）的糖基化异质性进行了比较。在糖型分布和寡糖异质性方面观察到唾液酸化程度存在显著差异。在低pH洗脱条件下，使用带有薄壳阴离子交换柱的高效液相色谱法来表征存在的N-连接复合型寡糖的数量和类型。寡糖通过N-糖苷酶释放，还原后，根据天线结构的性质进一步细分，分解为含有一个、两个、三个和四个唾液酸的多个峰。与悬浮细胞工艺相比，微载体工艺导致唾液酸化寡糖种类的数量减少。去除唾液酸后，在高pH阴离子交换条件下对去唾液酸寡糖进行色谱分析，结果表明存在相同的天线结构，但相对含量略有不同。寡糖谱表明存在高度复杂的微异质性阵列，包括单唾液酸、二唾液酸、三唾液酸和四唾液酸复合型寡糖。
