An ultra scale‐down approach to assess the impact of the choice of recombinant P. pastoris strain on dewatering performance in centrifuges
An ultra scale‐down approach to assess the impact of the choice of recombinant P. pastoris strain on dewatering performance in centrifuges
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一种超小型方法，用于评估重组毕赤酵母菌株的选择对离心机脱水性能的影响
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发表时间：
2012
期刊：
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通讯作者：
E. Keshavarz‐Moore
中科院分区：
文献类型：
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作者：
A. Lopes;N. Khan;J. Liddell;E. Keshavarz‐Moore
巴斯德毕赤酵母正在成为生物制药工业中用于治疗剂生产的理想宿主。它在甲醇上生长至≥100 g DCW/L的高细胞密度，并以高滴度分泌外源蛋白。然而，达到高细胞密度的培养条件对用于细胞去除的初级回收操作（特别是离心）的可加工性提出了挑战。本工作旨在评估重组毕赤酵母菌株选择对离心脱水的影响。通常，巴斯德毕赤酵母重组菌株的选择基于最佳靶蛋白表达水平;然而，尚不清楚菌株的选择是否会影响离心操作的性能。为了实现这一目标，在这项工作中使用了先前开发的实验室超规模缩小（USD）方法，该方法成功地预测了中试规模盘式机器的离心脱水。两种重组巴斯德毕赤酵母菌株，即X-33和糖工程化毕赤酵母菌株，用于进行分泌不同产物的发酵。分析了所得收获的发酵培养物的性质，并使用USD方法评价了中试和大规模盘式离心机的脱水性能。发现巴斯德毕赤酵母菌株的选择对脱水性能具有相当大的影响，巴斯德毕赤酵母X-33菌株达到比糖工程化菌株更好的脱水水平。USD方法被证明是确定大型离心机需要操作的最佳条件的有用工具，减少了在治疗产品工艺开发早期阶段重复中试规模运行的需要。© 2012美国化学工程师学会生物技术。程序，28：1029-1036，2012
Pichia pastoris is becoming a desirable host in the biopharmaceutical industry for therapeutics production. It grows on methanol to high cell densities ≥100 g DCW/L and secretes foreign proteins at high titers. However, the culture conditions to reach high cell densities pose a challenge to the processability by primary recovery operations, in particular centrifugation, used for cell removal. This work aims to assess the impact of recombinant P. pastoris strain selection on centrifugal dewatering. Normally, the choice of P. pastoris recombinant strain is based on best target protein expression levels; however, it is unknown whether the choice of strain will have an impact on performance of centrifugation operation. To achieve this aim, a previously developed laboratory ultra‐scale down (USD) methodology that successfully predicted centrifugal dewatering of pilot‐scale disk‐type machines, was used in this work. Two recombinant P. pastoris strains, namely a X‐33 and a glycoengineered Pichia strain, were used to perform fermentations secreting different products. The resulting harvested fermentation culture properties were analyzed and the dewatering performances of a pilot‐ and a large‐scale disk‐type centrifuge were evaluated using the USD methodology. The choice of P. pastoris strain was found to have a considerable impact on dewatering performance, with P. pastoris X‐33 strain reaching better dewatering levels than the glycoengineered strain. The USD method proved to be a useful tool to determine optimal conditions under which the large scale centrifuge needed to be operated, reducing the need for repeated pilot‐scale runs during early stages of process development for therapeutic products. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1029–1036, 2012