通过热烫完整植株或提取物热处理烟草宿主细胞蛋白去除方法的比较
Summary
之前的任何其他纯化步骤三个换热沉淀方法都能够有效地除去90％以上的烟草宿主细胞蛋白质（医疗专业人员）的提取物。植物医疗专业人员不可逆聚合在温度高于60℃。
Abstract
植物不仅对人类提供食物，饲料和原料，但也已经被开发作为一种经济的生产系统，用于生物制药的蛋白质，如抗体，疫苗候选和酶。这些必须从植物生物质进行纯化，但色谱法步骤是由高浓度的植物提取液中宿主细胞蛋白（医疗专业人员）的阻碍。然而，大多数医疗专业人员不可逆聚合在温度高于60℃，促进靶蛋白的随后的纯化。这里，被呈现给实现烟草医疗专业人员的热沉淀在任一完整叶片或提取物的三种方法。完整叶片的热烫可以容易地纳入现有过程，但可能对随后的过滤步骤产生负面影响。相反的是一种用于在搅拌容器叶片提取物的热析出，从而可以提高下游操作的性能虽然有在工艺设备设计的重大变化，如真均质几何。最后，一个热交换器设置是公特征的传热条件方面和易于规模，但清洗可能是困难和可能存在于过滤器的能力造成负面影响。设计的-实验的方法可以被用来识别影响HCP去除和产物回收最相关的工艺参数。这有利于在其他平台上的表达每一种方法和最合适的方法鉴定为给定的纯化策略的应用。
Introduction
现代医疗保健系统越来越依赖于生物制药蛋白质1。在植物中生产这些蛋白质相比于常规表达系统2-4由于低病原体负担和更大的可扩展性是有利的。然而，植物性药物下游处理（DSP）是很有挑战性的，因为颠覆性的提取程序导致高粒子负担，与浊度超过5000浊度单位（NTUs）和宿主细胞蛋白（HCP）含量往往超过95 ％[M / M] 5,6。
精细澄清过程需要以除去分散颗粒7-9，但色谱仪器是如果存在用于高效医疗专业人员去除10,11较早步骤初始产物回收过程中结合和洗脱模式运行成本更低。这可以通过沉淀使用floccul靶蛋白可以实现任蚂蚁12或低pH 13,14，以及通过使医疗专业人员聚集。核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco），最丰富的HCP在绿色植物如烟草（ 烟草 ），所述的选择性汇聚可以通过添加聚乙二醇15来促进，但是这是昂贵且具有大不相容-scale制造。热处理已显示变性和沉淀烟草医疗专业人员的超过95％，而蛋白质疟疾疫苗候选物如Vax8留在溶液中16-18稳定。
三种不同的方法被用来实现烟草医疗专业人员的热诱导析出：（ⅰ）热烫， 即，完整叶片在热液体的浸入，（ⅱ）一个温度受控的搅拌容器，和（iii）的热交换器（ 图1）16。对于完整叶片，漂烫实现医疗专业人员的快速，高效的降水，也容易扩大规模，并与现有的大型的制造工艺，其中包括一个初始步骤洗植物生物质19兼容。与此相反，温度控制的容器已经在一些工艺可用，并且可以用于植物的热处理提取20，但其可扩展性和能量转移率是有限的，因为罐的表面-体积比逐渐减少，并变得不适合于生产规模。的热交换器是一个在技术上明确替代加热搅拌容器，但需要加热和冷却介质， 例如，蒸汽和冷水的供应充足，以及其适合于热交换器的几何形状的严格控制的体积流动速率和媒体属性， 例如 ，比热容量。本文显示了所有三种方法可如何用于在普通烟草医疗专业人员和植物医疗专业人员的热诱导沉淀。建立和EAC的操作在实验室设置h方法可用于评估其对于大规模的工艺的适用性。主要的挑战是确定适当尺度缩小的模型和运行条件类似于过程大规模生产过程中使用的设备和条件的每个操作。此处呈现的数据是指与表达的疟疾疫苗候选Vax8和荧光蛋白DsRed的16转基因烟草植物进行的实验，但该方法也已成功地应用到N.植物本生瞬时表达等生物制药蛋白21。
一个设计-的-实验（DOE）接近22可促进过程发展，絮凝剂23也可以是在这种情况下有益的，如前所述8。热烫，加热的容器和热交换器之间的主要区别是，热烫施加到完整叶片在过程的早期，而OT她的被施加到植物提取物（ 图1）。
图 1： 工艺流程图图解三种不同方法进行烟草HCP热沉淀实施植物材料洗净，澄清和纯化前均化。用于漂白步骤（红色）的设备可以很容易地添加到现有的机械。相比之下，使用搅拌容器（橙色）和特别是换热器（蓝色）需要一个或几个额外的设备和管道。 请点击此处查看该图的放大版本。
Protocol
1.培育烟草的研究冲洗的去离子水1〜2升，随后每矿棉块用1L 0.1％[w / v的]肥料溶液。将在每个矿棉块的一个烟草种子和0.25升的肥料溶液冲洗轻轻没有洗去种子16。 培育7周的烟草植株中，用70％相对湿度的温室中，16小时的光周期（180微摩尔仲丁基- 1米 – 2;λ= 400 – 700 nm），而一个25/22℃的光照/黑暗温度制度。 收获所有树叶以外的四个子?…
Representative Results
由热烫烟草宿主细胞蛋白质热沉淀 在第2节中描述的漂白过程已成功用于从烟草与70℃的叶，96±1％减少对TSP沉淀医疗专业人员（N = 3），而回收到的Vax8靶蛋白的51％，从而增加了它的为0.1％至1.2％的纯度的色谱分离16之前。它也可以恢复荧光蛋白DsRed的83±1％（N = 3），从3.3％增加其纯度64.1％。热烫步骤容易地集成到一个标准的提取和澄清方案包括生物?…
Discussion
以上所述的三种方法为热沉淀能有效地去除前的任何层析纯化步骤16,17烟草医疗专业人员。它们补充，旨在增加初始产品的纯度， 例如，吐水29，rhizosecretion 30或离心萃取31,32，所有这些都限于分泌蛋白的其他策略。但是，基于热的方法只能在一个有意义的方式使用，如果待纯化的目的蛋白可承受〜60℃的最低析出温度超过1分钟。因此，在任何的三种方法的?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We would like to acknowledge Dr. Thomas Rademacher, Alexander Boes and Veronique Beiß for providing the transgenic tobacco seeds, and Ibrahim Al Amedi for cultivating the tobacco plants. The authors wish to thank Dr. Richard M. Twyman for editorial assistance as well as Güven Edgü for providing the MSP1-19 reference. 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 Fraunhofer-Gesellschaft Internal Programs under Grant No. Attract 125-600164.
Materials
| 2100P Portable Turbidimeter | Hach | 4650000 | Turbidimeter |
| Amine Coupling Kit | GE Healthcare | BR100050 | SPR chip coupling kit |
| Autoclaving basket | Nalgene | 6917-0230 | Basket for leaf blanching |
| Biacore T200 | GE Healthcare | 28-9750-01 | SPR device |
| Bio Cell Analyser BCA 003 R&D with 3D ORM | Sequip | n.a. | Particle size analyzer |
| Blender | Waring | 800EG | Blender |
| BP-410 | Furh | 2632410001 | Bag filter |
| 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 |
| CM5 chip | GE Healthcare | BR100012 | Chip for SPR measurements |
| Cuvette 10x10x45 | Sarsted | 67.754 | Cuvette for Zetasizer Nano ZS |
| 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 |
| Twentey-loop heat exchanger (4.8 m length) | n.a. (custom design) | n.a. | Heat exchanger |
| HEPES | Carl Roth GmbH | 9105.3 | Media component |
| K200P 60D | Pall | 5302303 | Depth filter layer |
| KS50P 60D | Pall | B12486 | Depth filter layer |
| Lauda E300 | Lauda Dr Wobser GmbH | Z90010 | Water bath thermostat |
| L/S 24 | Masterflex | SN-06508-24 | Tubing |
| mAb 5.2 | American Type Culture Collection | HB-9148 | Vax8 specific antibody |
| Masterflex L/S | Masterflex | HV-77921-75 | Peristaltic pump |
| 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 |
| Sodium disulfit | Carl Roth GmbH | 8554.1 | Media component |
| Sodium chloride | Carl Roth GmbH | P029.2 | Media component |
| Stainless-steel vessel; 0.7-kg 2.0-L; height 180 mm; diameter 120 mm | n.a. (custom design) | n.a. | Container for heat precipitation |
| Synergy HT | BioTek | SIAFRT | Fluorescence and spectrometric plate reader |
| VelaPad 60 | Pall | VP60G03KNH4 | Filter housing |
| Zetasizer Nano ZS | Malvern | ZEN3600 | DLS particle size distribution measurement |
| Zetasizer Software v7.11 | Malvern | n.a. | Software to operate the Zetasizer Nano ZS device |
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Citazione di questo articolo
Buyel, J. F., Hubbuch, J., Fischer, R. Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts. J. Vis. Exp. (114), e54343, doi:10.3791/54343 (2016).