Optimization of bioprocess conditions improves production of a CHO cell-derived, bioengineered heparin.
Optimization of bioprocess conditions improves production of a CHO cell-derived, bioengineered heparin.
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DOI：
10.1002/biot.201400665
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发表时间：
2015-07
影响因子：
4.7
通讯作者：
Sharfstein ST
中科院分区：
文献类型：
--
作者：
Baik JY;Dahodwala H;Oduah E;Talman L;Gemmill TR;Gasimli L;Datta P;Yang B;Li G;Zhang F;Li L;Linhardt RJ;Campbell AM;Gorfien SF;Sharfstein ST
肝素是当今世界上使用最广泛的抗凝药物。肝素目前是由动物组织，主要是猪肠生产的。最近的污染危机促使开发这种关键药物的非动物来源。我们假设，中国仓鼠卵巢（CHO）细胞可以代谢工程，以产生生物工程肝素，相当于目前的药物肝素。我们之前对CHO-S®细胞进行了工程改造，使其过表达来自肝素/硫酸乙酰肝素生物合成途径的两种外源酶，使抗凝活性增加约100倍，肝素/硫酸乙酰肝素产量增加约10倍。在这里，我们探讨了生物工艺参数对生物工程GAG的产量和抗凝活性的影响。使用专有的化学成分确定的补料进行的分批补料摇瓶研究导致整合活细胞密度增加约2倍，比生产率增加70%，导致产品滴度增加近3倍。将该工艺转移到搅拌罐生物反应器进一步提高了生产率，最终产品浓度约为90 µg/mL。不幸的是，产品组成仍然不同于药物肝素，这表明需要额外的代谢工程。然而，这些研究清楚地表明，生物工艺优化与代谢工程改进并行，将在开发生物工程肝素以取代目前的动物源性药物方面发挥重要作用。
Heparin is the most widely used anticoagulant drug in the world today. Heparin is currently produced from animal tissues, primarily porcine intestines. A recent contamination crisis motivated development of a non-animal-derived source of this critical drug. We hypothesized that Chinese hamster ovary (CHO) cells could be metabolically engineered to produce a bioengineered heparin, equivalent to current pharmaceutical heparin. We previously engineered CHO-S® cells to overexpress two exogenous enzymes from the heparin/heparan sulfate biosynthetic pathway, increasing the anticoagulant activity ~100-fold and the heparin/heparan sulfate yield ~10-fold. Here, we explored the effects of bioprocess parameters on the yield and anticoagulant activity of the bioengineered GAGs. Fed-batch shaker-flask studies using a proprietary, chemically-defined feed, resulted in ~two-fold increase in integrated viable cell density and 70% increase in specific productivity, resulting in nearly three-fold increase in product titer. Transferring the process to a stirred-tank bioreactor increased the productivity further, yielding a final product concentration of ~90 µg/mL. Unfortunately, the product composition still differs from pharmaceutical heparin, suggesting that additional metabolic engineering will be required. However, these studies clearly demonstrate bioprocess optimization, in parallel with metabolic engineering refinements, will play a substantial role in developing a bioengineered heparin to replace the current animal-derived drug.