Effect of shear stress on intrinsic CHO culture state and glycosylation of recombinant tissue-type plasminogen activator protein
Effect of shear stress on intrinsic CHO culture state and glycosylation of recombinant tissue-type plasminogen activator protein
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DOI：
10.1021/bp025715f
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发表时间：
2003-07-01
影响因子：
2.9
通讯作者：
Karim, MN
中科院分区：
文献类型：
--
作者：
Senger, RS;Karim, MN
研究了悬浮培养中的剪切应力，作为控制重组组织型纤溶酶原激活蛋白（r-tPA）糖基化的可能操作参数，该重组组织型纤溶酶原激活蛋白（r-tPA）由重组中国仓鼠卵巢（CHO）细胞培养物产生，在无蛋白培养基中生长。监测部分糖基化、II 型和完全糖基化、I 型 r-tPA 蛋白的最终分数，将其作为培养环境剪切特性的直接函数。通过控制生物反应器搅拌速度，检查了 CHO 培养物对低剪切应力水平（未获得指数生长）和高剪切应力水平（导致广泛细胞死亡）的剪切诱导响应。从可变位点占用的角度来看，表观和内在细胞生长、代谢物消耗、副产物和 r-tPA 产生以及 r-tPA 糖基化均受到全程监测。动力学分析揭示了剪切应力诱导的细胞稳态改变，导致代谢产量系数和内在细胞裂解动力学常数对剪切应力的非线性依赖性。使用剪切应力的破坏水平来研究细胞死亡和裂解的剪切依赖性，以及对培养物固有生长速率的影响。动力学模型也是根据文化的内在状态开发的，并与传统模型进行比较。 r-tPA 总产量在中等剪切条件下最大化，培养物的活 CHO 细胞密度也是如此。然而，H 型 r-tPA 产量和 H 型糖型产量比例在剪切应力破坏水平下最大化。对生物质产量系数的分析与内质网 (ER) 中聚糖添加的活塞流反应器模型相结合，提出了随着剪切应力的增加而减少 r-tPA 蛋白位点糖基化的总体机制。由于与剪切保护机制相关的蛋白质合成增加，r-tPA 在 ER 中的停留时间减少，建议限制 Asn184 位点与 ER 中膜结合寡糖转移酶的接触。
Shear stress in suspension culture was investigated as a possible manipulative parameter for the control of glycosylation of the recombinant tissue-type plasminogen activator protein (r-tPA) produced by recombinant Chinese hamster ovary (CHO) cell culture, grown in protein-free media. Resulting fractions of partially glycosylated, Type II, and fully glycosylated, Type I, r-tPA protein were monitored as a direct function of the shear characteristics of the culture environment. The shear-induced response of CHO culture to levels of low shear stress, where exponential growth was not obtained, and to higher levels of shear stress, which resulted in extensive cell death, were examined through manipulation of the bioreactor stirring velocity. Both apparent and intrinsic cell growth, metabolite consumption, byproduct and r-tPA production, and r-tPA glycosylation, from a variable site-occupancy standpoint, were monitored throughout. Kinetic analyses revealed a shear-stress-induced alteration of cellular homeostasis resulting in a nonlinear dependency of metabolic yield coefficients and an intrinsic cell lysis kinetic constant on shear stress. Damaging levels of shear stress were used to investigate the shear dependence of cell death and lysis, as well as the effects on the intrinsic growth rate of the culture. Kinetic models were also developed on the basis of the intrinsic state of the culture and compared to traditional models. Total r-tPA production was maximized under moderate shear conditions, as was the viable CHO cell density of the culture. However, Type H r-tPA production and the fraction of Type H glycoform production ratio was maximized under damaging levels of shear stress. Analyses of biomass production yield coefficients coupled with a plug-flow reactor model of glycan addition in the endoplasmic reticulum (ER) were used to propose an overall mechanism of decreased r-tPA protein site-occupancy glycosylation with increasing shear stress. Decreased residence time of r-tPA in the ER as a result of increased protein synthesis related to shear protection mechanisms is proposed to limit contact of site Asn184 with the membrane-bound oligosaccharyltransferase enzyme in the ER.