DataSheet1_Process Engineering of Biopharmaceutical Production in Moss Bioreactors via Model-Based Description and Evaluation of Phytohormone Impact.doc

The moss Physcomitrella is an interesting production host for recombinant biopharmaceuticals. Here we produced MFHR1, a synthetic complement regulator which has been proposed for the treatment of diseases associated to the complement system as part of human innate immunity. We studied the impact of different operation modes for the production process in 5 L stirred-tank photobioreactors. The total amount of recombinant protein was doubled by using fed-batch or batch compared to semi-continuous operation, although the maximum specific productivity (mg MFHR1/g FW) increased just by 35%. We proposed an unstructured kinetic model which fits accurately with the experimental data in batch and semi-continuous operation under autotrophic conditions with 2% CO2 enrichment. The model is able to predict recombinant protein production, nitrate uptake and biomass growth, which is useful for process control and optimization. We investigated strategies to further increase MFHR1 production. While mixotrophic and heterotrophic conditions decreased the MFHR1-specific productivity compared to autotrophic conditions, addition of the phytohormone auxin (NAA, 10 µM) to the medium enhanced it by 470% in shaken flasks and up to 230% and 260%, in batch and fed-batch bioreactors, respectively. Supporting this finding, the auxin-synthesis inhibitor L-kynurenine (100 µM) decreased MFHR1 production significantly by 110% and 580% at day 7 and 18, respectively. Expression analysis revealed that the MFHR1 transgene, driven by the Physcomitrella actin5 (PpAct5) promoter, was upregulated 16 h after NAA addition and remained enhanced over the whole process, whereas the auxin-responsive gene PpIAA1A was upregulated within the first 2 hours, indicating that the effect of auxin on PpAct5 promoter-driven expression is indirect. Furthermore, the day of NAA supplementation was crucial, leading to an up to 8-fold increase of MFHR1-specific productivity (0.82 mg MFHR1/g fresh weight, 150 mg accumulated over 7 days) compared to the productivity reported previously. Our findings are likely to be applicable to other plant-based expression systems to increase biopharmaceutical production and yields.

苔藓植物 Physcomitrella 作为重组生物制药的有趣生产宿主，备受关注。本研究中，我们成功生产了 MFHR1，一种合成的补体调节因子，该因子已被提出作为治疗与补体系统相关疾病的潜在治疗手段，是人体先天免疫的重要组成部分。我们对 5 升搅拌式光生物反应器中不同操作模式对生产过程的影响进行了研究。与半连续操作相比，采用连续补料或批量培养法，重组蛋白的总产量翻倍，尽管最大比生产率（mg MFHR1/g 干重）仅提高了 35%。我们提出了一种非结构化动力学模型，该模型在自养条件下，2% CO2 富集的批量及半连续操作中，与实验数据拟合准确。该模型能够预测重组蛋白生产、硝酸盐摄取和生物量增长，对于过程控制和优化具有重要意义。我们探讨了进一步提高 MFHR1 生产量的策略。虽然与自养条件相比，混合营养和异养条件降低了 MFHR1 的比生产率，但向培养基中添加植物激素生长素（NAA，10 µM）在摇瓶中将其提高了 470%，在批量及连续补料生物反应器中分别提高了 230% 和 260%。这一发现得到了生长素合成抑制剂 L-犬尿氨酸（100 µM）显著降低第 7 天和 18 天的 MFHR1 生产量 110% 和 580% 的支持。表达分析显示，由 Physcomitrella actin5（PpAct5）启动子驱动的 MFHR1 转基因在添加 NAA 后 16 小时上调，并在整个过程中保持增强，而生长素响应基因 PpIAA1A 在添加 NAA 后前 2 小时内上调，表明生长素对 PpAct5 启动子驱动的表达影响是间接的。此外，NAA 补充的日期至关重要，与先前报道的生产率相比，MFHR1 的比生产率（0.82 mg MFHR1/g 新鲜重，7 天内累积 150 mg）最高可提高 8 倍。我们的发现可能适用于其他基于植物的表达系统，以增加生物制药的生产量和产量。