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首页> 外文期刊>Biotechnology Progress >Temperature Effects on Product-Quality-Related Enzymes in Batch CHO Cell Cultures Producing Recombinant tPA
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Temperature Effects on Product-Quality-Related Enzymes in Batch CHO Cell Cultures Producing Recombinant tPA

机译：温度对生产重组tPA的分批CHO细胞培养物中产物质量相关酶的影响

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摘要

Culture conditions that affect product quality are important to the successful operation and optimization of bioreactors.Previous studies have demonstrated that enzymes,such as proteases and sialidases,accumulate in batch bioreactors.These enzymes are known to be detrimental to the quality of recombinant glycoproteins.Bioreactor temperature has been used to control cell growth and recombinant protein production rates.However,the effect of culture temperature on the production of proteases and sialidases has not been investigated.In this study,Chinese hamster ovary cells were cultured with a temperature profile that decreased from 37 to 34 °C over 8 days and with a constant temperature of 37 °C.Analysis of extracellular protease activity indicated that two major proteases were present(50 and 69 kDa).The 50 kDa protease activity decreased similarly with time for both culture conditions.The 69 kDa protease activity increased with time for both culture conditions.The constant-temperature cultures had significantly lower 69 kDa protease levels compared to the ramped-temperature cultures in the early stationary phase.Intracellular sialidase activity was present in both cultures.The intracellular sialidase activity increased dramatically for both culture conditions immediately after the cells we're inoculated into fresh medium.The initial peak in intracellular sialidase activity was followed by a first-order decay.The intracellular sialidase activities for the two culture conditions were not significantly different.The production of recombinant tissue type plasminogen activator was not significantly different for the two culture conditions.Thus,the previously hypothesized advantages that lower culture temperatures have reduced protease activity and improved productivity do not appear to be universal.
机译：影响产品质量的培养条件对于生物反应器的成功运行和优化很重要。以前的研究表明，诸如蛋白酶和唾液酸酶等酶会在成批的生物反应器中积累，这些酶对重组糖蛋白的质量是有害的。温度已经被用来控制细胞的生长和重组蛋白的产生速率。然而，尚未研究培养温度对蛋白酶和唾液酸酶产生的影响。在这项研究中，中国仓鼠卵巢细胞的培养温度从在8天内从37至34°C并在37°C恒温下进行分析。胞外蛋白酶活性表明存在两种主要蛋白酶（50和69 kDa）。两种培养条件下50 kDa蛋白酶活性均随时间下降在两种培养条件下，69 kDa蛋白酶活性均随时间增加。与固定培养初期的升温培养相比，它们的蛋白酶水平显着降低了69 kDa。两种培养物中都存在细胞内唾液酸酶活性。在将细胞接种到新鲜细胞后，两种培养条件下细胞内唾液酸酶活性都急剧增加。培养基中细胞内唾液酸酶活性的最初峰值是一阶衰减，两种培养条件下的细胞内唾液酸酶活性没有显着差异，重组组织型纤溶酶原激活剂的产生在两种培养条件下无明显差异。因此，先前假设的较低培养温度具有降低的蛋白酶活性和提高的生产率的优点似乎并不普遍。

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