Gawlitzek M, Conradt H S, Wagner R
Abteilung Zellkulturtechnik, Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, D-38124 Braunschweig, Germany.
Biotechnol Bioeng. 1995 Jun 20;46(6):536-44. doi: 10.1002/bit.260460606.
The effect of different short-term controlled cell culture conditions on the product quality of a genetically engineered human interleukin-2 N-glycosylation variant protein expressed from a baby hamster kidney cell line (BHK-21) has been investigated. A perfused 2-L stirred tank reactor was used. Products purified from the culture supernatant of cells grown under experimentally initiated nutrient limitations (glucose, amino acids, pO(2)) were characterized by their HPLC-elution profile, SDS-PAGE and western blotting, amino acid sequencing as well as for their N-linked carbohydrates, using "HPAEC-PAD fingerprinting" and methylation analysis. The glycoprotein products secreted from cells under the different culture conditions (kept for 24 h, after an adaption time period of 48 h) showed an almost identical oligosaccharide pattern. By contrast, short-term changes of the culture condition led to considerable differences in the ratio of glycosylated to unglycosylated protein forms. Significant amounts of NH(2)-terminally truncated polypeptide forms were observed. They lacked proponderantly the first two amino acids; however, under certain culture conditions forms lacking up to eight NH(2)-terminal amino acids were detected. (
研究了不同短期可控细胞培养条件对从小仓鼠肾细胞系（BHK-21）表达的基因工程化人白细胞介素-2 N-糖基化变体蛋白产品质量的影响。使用了一个2升的灌注搅拌罐反应器。从在实验性引发的营养限制（葡萄糖、氨基酸、pO₂）下生长的细胞培养上清液中纯化的产品，通过其HPLC洗脱图谱、SDS-PAGE和western印迹、氨基酸测序以及使用“HPAEC-PAD指纹图谱”和甲基化分析对其N-连接碳水化合物进行了表征。在不同培养条件下（经过48小时的适应期后保持24小时）细胞分泌的糖蛋白产品显示出几乎相同的寡糖模式。相比之下，培养条件的短期变化导致糖基化与未糖基化蛋白形式的比例存在显著差异。观察到大量NH₂末端截短的多肽形式。它们主要缺少前两个氨基酸；然而，在某些培养条件下，检测到缺少多达八个NH₂末端氨基酸的形式。
