过程评价絮凝剂的效率的分散粒子的从植物提取物的去除
Summary
The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.
Abstract
Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.
Introduction
植物被广泛用于生产食品等商品的果汁，但它们也可以开发为高价值生物制药产品1-3的制造平台。在这两种情况下，下游处理（DSP）通常始于液体从组织如叶子或果实的萃取，接着澄清含颗粒的提取液4,5。用于生产生物药品，DSP的成本可占整体生产成本6,7-高达80％，这部分反映负担存在于由破坏性方法如基于刀片的均质8,9制备的提取物的高的颗粒。尽管滤光层的合理选择，以在提取物中的粒度分布相匹配能够提高过滤器的能力，降低成本10,11，其改进不能超过由必须每保留粒子的数量限定绝对容量天花板过滤面积的单位，以获得澄清。
如果较少的粒子到达在过滤火车上等过滤器的表面上的天花板可以解除，这样就可以，如果分散颗粒是公知的作为促进聚集形成大的絮凝物12絮凝剂聚合物混合来实现。这种絮凝物可以保留由较粗和较便宜的袋式过滤器进一步上行，降低粒子负担达到更细的和更昂贵的深度过滤器。该聚合物必须有适合他们的应用程序， 如生物制药，他们必须符合良好生产规范（GMP）的安全配置文件，通常他们必须有一个摩尔质量> 100 kDa的，可以是中性或带电荷的13。而中性絮凝剂通常通过交联分散颗粒引起它们的聚集和絮凝物直径，形成充当>1毫米11，电荷的聚合物中和代办的ispersed颗粒，减少它们的溶解度，从而引起沉淀14。
絮凝可以通过调节参数，例如缓冲液pH或电导率，聚合物的类型或浓度，以匹配所述提取物15,16的特性得到改善。对于具有0.5-5.0克L- -1聚乙烯亚胺（PEI）预处理烟草萃取物，在深度过滤器的容量大于2倍的增加报告了一个100-L中试规模的过程。该聚合物的成本小于€10公斤-1所以其引入过程导致的对过滤器和消耗品约6000€成本节省每批次16或纤维素基助滤剂17更时合并。即使如此，预测模型是必需的，以评估絮凝剂的先验经济效益，因为它们的加入可以要求的15-30分钟16,18保持步骤，产生用于存储进一步的投资成本坦克。然而，目前没有可用的机械模型，可以预测这样的实验的结果，由于絮凝的复杂性。因此，在这篇文章中所描述的更合适的设计中，试验设计（DOE）的方法19的开发。一个为广大能源部程序协议最近已经公布了20。
小规模的设备现在可用于絮凝条件21高通量筛选。然而，这些设备可能无法实际地植物的絮凝过程中模拟的条件中提取，因为反应容器（〜7个毫米上的96孔平板的孔）和颗粒或絮凝物的尺寸可以小于相距一个数量级。这可能会影响混合型态，因此该模型的预测能力。此外，也可以是难以缩小涉及沉淀由于在混合行为和沉淀STA非线性变化过程相容性22。因此，本文概述了吞吐量每天50-75个样本的台式规模的筛选系统，产生的结果是从最初的20ml的反应容积为100升试工艺16可扩展性。当与美国能源部的方法相结合，这允许将要使用的预测模型的过程的优化和文档作为质量逐个设计概念的一部分。
下面所描述的方法也可以适于在基于细胞培养的方法，其中絮凝剂也被认为是一种节约成本的工具23产生的生物药剂。它也可以用来作为一种纯化策略的一部分靶蛋白的沉淀从粗提取物进行建模，这表现为在油菜，玉米和大豆24,25产生β葡萄糖醛酸酶。絮凝剂性能的详细说明可以在其他地方找到16,26，并确保重要的是聚合物精矿ations要么无毒或低于在最终产品11的有害的水平。
Protocol
Representative Results
Discussion
最重要的方面考虑设立时美国能源部表征颗粒絮凝的是，设计必须在原则上能够检测并描述了预期的或可能产生的影响36,38， 如 pH值，聚合物类型和聚合物浓度16的影响。因此，在开始实际实验之前评估的设计空间（FDS）的馏分是重要的。该FDS是多维实验空间内，它是可以检测给定的已知变异性的系统， 例如 ，检测在250浊度的差两个实验结果之间的预先定义的差异?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
I would like to acknowledge Dr. Thomas Rademacher for providing the transgenic tobacco seeds and Ibrahim Al Amedi for cultivating the tobacco plants. I wish to thank Dr. Richard M Twyman for editorial assistance and Prof. Dr. Rainer Fischer for fruitful discussions. This work was funded in part by the European Research Council Advanced Grant ”Future-Pharma”, proposal number 269110, the Fraunhofer-Zukunftsstiftung (Fraunhofer Future Foundation) and the Fraunhofer-Gesellschaft Internal Programs under Grant No. Attract 125-600164.
Materials
| 2100P Portable Turbidimeter | Hach | 4650000 | Turbidimeter |
| 2G12 antibody | Polymun | AB002 | Reference antibody |
| Biacore T200 | GE Healthcare | 28-9750-01 | SPR device |
| BP-410 | Furh | 2632410001 | Bag filter |
| Catiofast VSH | BASF | 79002360 | Flocculating agent |
| Centrifuge 5415D | Eppendorf | 5424 000.410 | Centrifuge |
| Centrifuge tube 15 mL | Labomedic | 2017106 | Reaction tube |
| Centrifuge tube 50 mL self-standing | Labomedic | 1110504 | Reaction tube |
| Chitosan | Carl Roth GmbH | 5375.1 | Flocculating agent |
| Design-Expert(R) 8 | Stat-Ease, Inc. | n.a. | DoE software |
| Disodium phosphate | Carl Roth GmbH | 4984.3 | Media component |
| Ferty 2 Mega | Kammlott | 5.220072 | Fertilizer |
| Forma -86C ULT freezer | ThermoFisher | 88400 | Freezer |
| Greenhouse | n.a. | n.a. | For plant cultivation |
| Grodan Rockwool Cubes 10x10cm | Grodan | 102446 | Rockwool block |
| HEPES | Carl Roth GmbH | 9105.3 | Media component |
| K700P 60D | Pall | 5302305 | Depth filter layer |
| KS50P 60D | Pall | B12486 | Depth filter layer |
| Miracloth | Labomedic | 475855-1R | Filter cloth |
| MultiLine Multi 3410 IDS | WTW | WTW_2020 | pH meter / conductivity meter |
| Osram cool white 36 W | Osram | 4930440 | Light source |
| Phytotron | Ilka Zell | n.a. | For plant cultivation |
| Polymin P | BASF | 79002360 | Flocculating agent |
| POLYTRON PT 6100 D | Kinematica | 11010110 | Homogenization device with custom blade tool |
| Protein A | Life technologies | 10-1006 | Antibody binding protein |
| Sodium chloride | Carl Roth GmbH | P029.2 | Media component |
| Synergy HT | BioTek | SIAFRT | Fluorescence plate reader |
| TRIS | Carl Roth GmbH | 4855.3 | Media component |
| Tween-20 | Carl Roth GmbH | 9127.3 | Media component |
| VelaPad 60 | Pall | VP60G03KNH4 | Filter housing |
| Zetasizer Nano ZS | Malvern | ZEN3600 | DLS particle size distribution measurement |
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