Manufacturing Science and Technology, Swedish Orphan Biovitrum, Solna, Sweden.
AdBIOPRO, Competence Centre for Advanced Bioproduction by Continuous Processing, Stockholm, Sweden.
Biotechnol Bioeng. 2021 Sep;118(9):3533-3544. doi: 10.1002/bit.27806. Epub 2021 May 13.
Intensified bioprocesses have caught industrial interest in the field of biomanufacturing in recent years. Thanks to new technology, intensified processes can support high cell densities, higher productivities and longer process times, which together can offer lower cost of goods. In this study two different intensified process modes, high cell density perfusion and enhanced fed-batch, were evaluated and compared with a conventional fed-batch process for a difficult-to-express therapeutic enzyme. The intensified process modes were cultivated with a target cell density of 100 × 10 cells/ml and with alternating tangential flow filtration, ATF, as cell retention device. The processes were designed to resemble an established optimized fed-batch process using the knowledge of this process without new dedicated optimization for the intensified modes. The design strategy included decision of the ratio of feed concentrate to base medium and glucose supplementation, which were based on target cell-specific consumption rates of key amino acids and glucose, using a targeted feeding approach (TAFE). A difficult-to-express therapeutic enzyme with multiple glycosylation sites was expressed and analyzed in the different production processes. The two new intensified processes both achieved 10 times higher volumetric productivity (mg/L/day) with retained protein quality and minor changes to the glycan profile compared to the fed-batch process. The study demonstrates the potential of using intensified processes for sensitive complex enzymes. It is shown here that it is possible to transfer a developed fed-batch process into high cell density processes either in intensified fed-batch or steady-state perfusion without new dedicated optimization. The results demonstrated as well that these intensified modes significantly increase the productivity while maintaining the desired product quality, for instance the same amount of product was obtained in 1 day during the perfusion process than in a whole fed-batch run. Without any prior optimization of the perfusion rate, the high cell density perfusion process resulted in only 1.2 times higher medium cost per gram produced protein.
近年来，强化生物工艺引起了生物制造领域的工业关注。得益于新技术，强化工艺可以支持高细胞密度、更高的生产率和更长的工艺时间，这三者结合起来可以降低产品成本。在这项研究中，两种不同的强化工艺模式，高密度细胞灌流和增强分批补料，与传统的分批补料工艺一起，用于表达一种难表达的治疗性酶。强化工艺模式在目标细胞密度为 100×10 细胞/ml 下进行培养，并交替使用切向流过滤（ATF）作为细胞保留装置。这些工艺的设计类似于使用该工艺的知识，而无需针对强化模式进行新的专门优化，以建立一个已优化的分批补料工艺。设计策略包括确定补料浓缩液与基础培养基的比例以及葡萄糖补加量，这是基于关键氨基酸和葡萄糖的目标细胞特异性消耗率，采用靶向补料（TAFE）方法。使用不同的生产工艺表达和分析了一种具有多个糖基化位点的难表达治疗性酶。与分批补料工艺相比，这两种新的强化工艺都实现了 10 倍的更高体积产率（mg/L/天），同时保持了蛋白质质量和聚糖谱的微小变化。该研究证明了在敏感复杂酶方面使用强化工艺的潜力。结果表明，可以将已开发的分批补料工艺转化为高密度细胞工艺，无论是在强化分批补料还是稳态灌流中，而无需进行新的专门优化。结果还表明，这些强化模式在保持所需产品质量的同时，显著提高了生产率，例如在灌流工艺中，在 1 天内获得的产品量与整个分批补料运行相同。在没有对灌流率进行任何预先优化的情况下，高密度细胞灌流工艺仅使每克产生的蛋白质的培养基成本增加了 1.2 倍。
