胞外唾液酸酶造成工程中国仓鼠卵巢细胞株所产人源重组促红素唾液酸含量降低

Extracellular sialidase degrades sialic acid in recombinant human erythropoietin produced by an industrial Chinese hamster ovary cell strain

摘要:

为了对工程中国仓鼠卵巢 (CHO) 细胞所产人源重组促红素 (rhEPO) 的N-糖基化特点进行考察，静置培养工程细胞后，通过等电聚焦和凝集素共沉淀对培养上清中的rhEPO进行分析，并对无血清培养上清中乳酸脱氢酶 (LDH) 和唾液酸酶活性进行检测，发现这株CHO细胞可以表达唾液酸含量较高的rhEPO蛋白。但是随着培养时间的延长，细胞的存活率逐渐降低，死亡的细胞将胞内的唾液酸酶释放到胞外，唾液酸酶的降解作用会造成N-糖链分枝末端的唾液酸占有率降低，导致rhEPO蛋白糖基化形态的变化。所使用的方法及得到的结果为进一步对工业过程进行分析提供了参考。

关键词:中国仓鼠卵巢细胞株，人源重组促红素，N-糖基化，细胞活性，唾液酸酶

Abstract:

To investigate the N-glycosylation characteristics of recombinant human erythropoietin (rhEPO) produced by an industrial Chinese hamster ovary (CHO) cell line that is currently used in a large scale manufacturing process, we cultured this cell strain in static mode. The produced rhEPO in the culture supernatant was analyzed using isoelectric focusing (IEF) and Ricinus communis agglutinin-I (RCA-I) lectin precipitation. The lactate dehydrogenase (LDH) and sialidase activity in the serum-free supernatant were assayed as well. The analyses revealed that this cell strain could produce rhEPO with high sialic acid content, but during prolonged culture, cell viability decreased with time whilst the activity of sialidase present in the supernatant increased. The loss in rhEPO quality was due to a decrease in terminal sialic acid on the N-glycans, caused by sialidase degradation. The methods and findings in this paper serve as basis for further investigation of industrial production process.

Key words:Chinese hamster ovary cell strain, recombinant human erythropoietin, N-glycosylation, cell viability, sialidase