Name: procedure to evaluate the efficiency of flocculants for the removal of dispersed particles from plant extracts
Uploaded: 2016-04-09T13:00:00.000+00:00
Duration: 10 min 37 s
Description: Watch this Scientific Journal Video about Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts at JoVE.com

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.

1.制定一个适当的实验策略<ol><li>确定有关要建立或优化絮凝过程， 即它因素对絮凝作用最强的环境和工艺参数。通常情况下，有这样一个能源部方式作为最近描述20是必要的，因为缺乏机理模型的几个这样的参数。 <ol><li>根据文献资料12，先前的知识和经验与系统参数选择（的因素）。典型因素包括缓冲液，缓冲液的导电性，保温时间和温度，以及聚合物的类型和浓度15,16,27。 </li><li>使用相同的参考（见1.1）来定义每个数字的因素有意义的范围和级别分类的因素。 </li><li>限定了实验的结果（响应）进行监测，并用于评价絮凝的效率。根据系统的不同，这可以是滤液或上清液浊度，沉降速度，聚集体尺寸或后续滤波器16,23,28-30的容量。 </li><li>确保用于测量反应的检测是定量的，稳健的和可重复的/可再现的，使得所得的高质量的数据可与以后或由另一操作员进行的实验来扩充。 </li></ol></li><li>选择能源部类型的适合于进行调查的因子的数量和知识的程度已经积累有关系统。使用现有的文献，以确定合适能源部类型20。 <ol><li>如果有关于絮凝系统的信息很少进行调查，大量的参数必须屏蔽或知之甚少的参数意义的范围内选择一个筛选的设计。典型的筛选设计是充分，部分因子设计。包括在设计中心点如果参数预期具有非线性的影响， 例如 </em&gt;饱和高于特定阈值19。 </li><li>选择一个响应面方法（RSM）， 例如中心复合设计（CCD）31或最佳32,33，如果只有几个因素与知名的范围需要被精确地表征。 </li></ol></li><li>成立能源部相应的软件，以确保先天的质量标准，如设计空间的比例均达到20。 </li></ol> 2.准备絮凝试验<img alt="图1" src="/files/ftp_upload/53940/53940fig1.jpg" /> 图1:植物提取物絮凝工作流程:生产规模（左）和台式量表（右）。以下用含水缓冲液提取蛋白质，细胞碎片的分散粒子通过添加絮凝剂的凝聚。聚集体然后通过袋子和深度过滤的级联以及这些的容量除去与滤液浊度沿过滤器可以直接用于测量絮凝的效率。 <ol><li>开发一个通用实验工作流程（ 图1）。 <ol><li>使用提取设备，在最终的应用规模导致预期相同的粒度分布（或已经观察到的）， 例如 ，一个专门的均化器。如果可能的话，作为用于烟草的同质叶10描述设计提取的一个尺度缩小的模型。 </li><li>定义期间絮凝实验（这里20ml）中所用的提取物的卷。选择一个卷，允许颗粒的代表号码存在于反应容器中， 例如 ，在20毫升等份絮凝实验，得到含有〜7％[w / v的]的固体34和颗粒尺寸〜0.5烟草萃取物可再现和可扩展的结果μm至〜3毫米16。 </li><li>设计中的所有监控和絮凝后的操作，使得日EY用于展示最终应用规模的，具有相同的颗粒保留行为的那些在生产规模使用例如选择过滤器。 </li></ol></li><li>培育从中提取物将导出的植物。 <ol><li>使用相同的植物系，这里烟草 cv。微型哈瓦那SR1，并且将在生产过程中使用的培养条件预先所描述的33。 </li><li>如果其他原料将被处理，在此过程中准备这些原料，使它们代表最终应用的规模， 比如使用正品的缓冲区，考虑任何稀释步骤，坚持预期的pH值和电导率，这里pH值7.5，电导率30毫秒-1。 </li></ol></li><li>准备过滤工具，浊度监测设备和取样容器。 <ol><li>切过滤材料到所需的尺寸，这里15×15 cm 2时，如果它们不准备对我们Ë模块。确保所有的监测和分析设备的功能和标记所有样品管。 </li><li>尽管这些是简单的任务，在时间制备这些产品，以确保实际絮凝实验过程中，他们将不会造成中断。避免延迟，因为它们可以与给定该絮凝干扰结果是依赖于时间的过程。 </li></ol></li><li>准备絮凝剂股票方案。注意:处理絮凝剂时， 如手套穿戴合适的个人防护装备。该材料可能是危险的（根据欧洲UE 67/548 / CEE危险标志，1999/45 / CE包括N，习或Xn有害）。避免灰尘和参阅材料安全数据表。在通风橱下工作。 <ol><li>选择一个储备浓度为每絮凝剂，这里80.0克L -1为中使用的两个PEI絮凝剂。选择的浓度尽可能的高，以避免样品稀释添加絮凝剂时，这将减少粒子concentra化，从而影响絮凝效率。 </li><li>帐户为任何预稀释，反映了聚合物的制造商的制剂， 例如 ，如果将聚合物作为水50％[w / v的]溶液已经提供。的4-8％絮凝剂储备溶液[w / v的]被认为是最适合的为止。 </li><li>铭记上述讨论的要点，确保絮凝剂浓度不产生抑制移液，可能导致错误，因为最终的絮凝剂浓度调整不正确的高粘性溶液。 </li><li>使用相同的原料浓度对于所有絮凝剂如果可能的，因为这将有利于一个移液方案（2.5），从而减少了错误的可能性的设置。 </li><li>调整每个絮凝剂原液的pH和电导率，以匹配提取物，这里的pH 4-10和电导率15-55毫秒-1的条件。准备个股为如果m单絮凝剂矿石多于一组的pH和/或电导率条件为该聚合物测试。 </li><li>刚准备絮凝剂原液，最长不超过使用前48小时。尽管絮凝可以与贮存4周以上聚合物股被诱导，效率可在高或低pH值由于下降到聚合物水解。请参见制造商的更多详细信息的文档。 </li><li>确保选择用于美国能源部的参数可以精确地对在实验过程中的每个样本进行调整， 例如 ，确保有可调节的孵化温度，此处4℃，20℃和37℃加热/冷却浴。如果絮凝过程中混合的实验的一部分，确保混合装置代表最终应用规模的关键参数，如电源输入方面。 </li></ol></li><li>转换能源部计划到移液方案。 <ol><li>转换不同的絮凝剂浓度是工商业污水附加费泰德到将被添加到提取物样品的原液卷:由原料浓度除以最终絮凝剂的浓度和在絮凝实验中使用的样品体积相乘。确定最大得到的最终体积， 例如 ，如果在20 ml样品的与最大2毫升絮凝剂原液这将是22毫升混合。 </li><li>计算，以保持在基于絮凝剂股票的最大卷上的所有絮凝等分相同的最终体积所需体积的缓冲液中添加， 例如 ，如果0.75毫升絮凝剂股票必须加到样品然后1.25缓冲液是必需的，以保持将22毫升的最终样品体积（2.5.1）。 </li><li>综上所述的絮凝剂原液每种聚合物和絮凝状态来计算所需要的美国能源部原液绝对卷的卷。 </li></ol></li><li>采收烟叶，并准备提取。 <ol><li>卸下前六名LEAVES（或多达通过在过程中的指令指示）从适当的年龄， 例如 6周龄，并转移到一个合适的提取设备的烟草植物，如均化器或螺旋压力机。 </li><li>添加三卷提取缓冲液的每克生物量， 例如 300毫升每100克，并混合8分钟。 </li><li>用适当的缓冲液制备单独的提取物，如果不同pH和/或电导率被测试。这里，这涉及均质植物材料用于在混合器3×30秒或榨汁机34。 </li><li>或者，在一个方式是代表所研究的过程中制备的提取物。 </li></ol></li><li>分装提取并添加缓冲区。 <ol><li>整个过程，以确保样品是均匀的，均匀分布的颗粒的过程中手动和充分搅拌提取物。 </li><li>正是通过倾析分发预标记的反应管中的提取物，离开1毫升每艘船舶。自立50ml试管简化处理，非常适合20 1ml样品。 </li><li>加维持使用合适的吸移管每个单独的反应管的固定终体积所需的每种提取缓冲液的体积。 </li></ol></li></ol> 3.絮凝植物与不同的聚合物中提取<ol><li>吸管絮凝剂原液所需的体积，这里0.1-2.0毫升，由美国能源部的随机运行顺序依次指示的样本。加入絮凝剂剧烈手动振荡正是20秒后立即每个样品充分混合。 <ol><li>如果需要，调整其他原料的晃动时间，以确保充分混合，但严格确保所有的样品一致的混合时间。请记住，延长混合可能会导致絮体的不可逆的破坏和震动会扭曲絮凝结果不一致时所施加的力。 </li></ol></li><li>可选:修改上述用于两个或更多的絮凝剂的同时申请中所描述的过程。 <ol><li>选项​​1:相反的单一聚合物的添加两种或两种以上的絮凝剂，这里PEI和脱乙酰壳多糖的混合物。使用美国能源部规定的絮凝剂组合。合并能源部设置（1.1）中的比例和聚合物作为各个参数的绝对浓度。 </li><li>选项​​2:添加顺序地两种或多种聚合物的提取物。 <ol><li>使用个别的聚合物浓度，它们的类型，并在每次加入到提取物之间的温育时间，作为附加的因素能源部设置期间。 </li><li>还可以使用此方法来测试单个聚合物的重复加入能否改善絮凝。使用逐步加入到模拟缓慢加入絮凝剂， 例如四个步骤中，每个加入0.25毫升80克L -1絮凝剂库存到20ml的体积在4分钟内可以模仿在加入絮凝剂与0.25毫升分钟-1的流速。 </li><li>在所有的情况下，确定该设置一个新的最大最终样品体积通过将所有絮凝剂的最大体积的样品体积，这里仍然22毫升，并在必要时重新计算的絮凝剂储备溶液和缓冲液的所需的卷为音量调整。 </li></ol></li></ol></li><li>孵育样品在能源部的定义，一般3-30分钟的时间，让絮凝体形成。确保所有其他培养条件， 如温度，根据美国能源部的设置。 </li><li>观察和记录絮凝体形成。记录的絮凝体形成的进度要求， 例如作为絮状物毫米通过测量沉淀固体的高度每分钟沉淀。如果需要，延长了延长的时间周期絮凝如过夜。 </li><li>过滤絮凝提取物。 <ol><li>使用前面准备的过滤材料（2.3），以适当的澄清后的絮凝提取物通过过滤材料和到干净容器或反应管倾析絮凝样品孵育时间。 <ol><li>不重悬过滤前沉降絮 ​​凝物和应用该提取物在约300的速率毫升分钟-1到过滤器中，对应于3-4秒为20ml的样品。 </li><li>确认过滤步骤的性能，如果在台式的实验用不同的材料相比，在颗粒保留方面的最终处理， 例如 ，通过测量在两种类型的样品11的粒度分布。 </li></ol></li><li>分析在浊度和/或粒度分布根据需要使用合适的设备， 如浊度方面滤液。 </li><li>可选:延长后的培养时间重新分析， 例如 12-24小时，调查不稳定絮体的形成或改造。 </li></ol></li><li>在条款镨分析样品E-定义的响应（1.1.3）。 <ol><li>从滤液取样并分析它们对额外的反应参数， 例如不同的目标蛋白质或有价值的产物的浓度。 </li><li>可选:分析为相同的参数滞留（主要是固体），关闭该过程的质量平衡。具体地，通过比较样品处理或不与絮凝剂处理后质量剩余固体量化絮凝剂上液体回收的效果。 </li></ol></li><li>确认数据质量并把结果传送到能源部软件。 <ol><li>寻找在收集的响应数据的极端值， 例如意外的高值。 </li><li>确保所有响应数据正确及其相应的实验条件一致。 </li><li>将结果到能源部的软件，并确保标准和随机运行命令没有混淆。 </li></ol></li></ol> 4.评估美国能源部<ol><li>使用美国能源部软件的内置的数据分析工具如先前所述20开发预测模型。 <ol><li>如果需要的话，以促进建筑模型选择合适的数据转换模式，在这里记录10。一种用于与最大最小响应值的比例大于10表示一个数据转换可能需要。确定使用适当的统计工具最适合的改造， 例如一箱考克斯情节35。 </li><li>选择是:（i）适合于所选能源部（1.2）和（ii）用约基于方差（ANOVA）分析所研究的系统以前的知识能源部软件的工具， 例如一个二阶多项式同意一个基本模型常适合更好的孵育时间比一阶多项式观察到的效果。 </li><li>通过取消他们删除无关紧要的模型因子， 如 P&gt; 0.05，或因子相互作用迭代中，reby降低了模型的复杂性，并提高其预测能力。 </li><li>通过比较R 2，调整R 2，确认模型的质量，并与学生化残差的正态概率图沿预测R 2，残差-VS-来看，预测-VS-实际和Box-考克斯地块36。所有的R 2的值应为0.2的范围内。 </li><li>确保最终的模型，而没有负浓度预测的物理和热力学， 如基本的假设一致。 </li></ol></li><li>使用该模型预测是最有利的为所研究的系统，这里的低浊度的条件。 <ol><li>选择最重要的反应和他们的首选国家， 如最小的浑浊。在最大化满意度函数或类似的工具，这是内置的几个能源部软件包36功能结合这些选择。 </li><li>根据预期的应用ØF中的模型中，选择的确认运行如果条件预测为最有利的是不是最初能源部的一部分选择它们作为后续实验以验证最佳条件和/或在一般情况下， 例如，该模型的预测能力。 </li></ol></li></ol> 5.改进模型和验证的预测能力<ol><li>缩小初始设计空间，以最理想的操作条件下，在0-1,000浊度单位（NTU）范围如低浊度，基于所述模型预测（4.2）。 </li><li>建立一个新的DoE这些范围内时，仅包括那些已被标识为显著或有关因素（4.1.2））。 注:一个因素可以在模型分析，但不相关的进程而言显著， 即其对反应的影响是&lt;1％相比，其他因素。 </li><li>重复步骤1-5.2直到模型预测的质量requiremen匹配TS， 例如 ，由软件所指示的模型中的标准偏差是足以为预期的应用。如果需要，对于这些迭代， 例如 ，在细化只使用一个絮凝剂修改 ​​实验策略。 </li><li>传送和确认从尺度缩小的模型到中试规模或最终生产规模的结果。 </li></ol>

<ol>
	<li>Godfray, H. C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+Security:+The+Challenge+of+Feeding+9+Billion+People.">Food Security: The Challenge of Feeding 9 Billion People.</a> Science. 327, 812-818 (2010).</li><li>Fischer, R., Schillberg, S., Buyel, J. F., Twyman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commercial+aspects+of+pharmaceutical+protein+production+in+plants.">Commercial aspects of pharmaceutical protein production in plants.</a> Curr. Pharm. Des. 19, 5471-5477 (2013).</li><li>Pastores, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Phase+3,+multicenter,+open-label,+switchover+trial+to+assess+the+safety+and+efficacy+of+taliglucerase+alfa,+a+plant+cell-expressed+recombinant+human+glucocerebrosidase,+in+adult+and+pediatric+patients+with+Gaucher+disease+previously+treated+with+imiglucerase.">A Phase 3, multicenter, open-label, switchover trial to assess the safety and efficacy of taliglucerase alfa, a plant cell-expressed recombinant human glucocerebrosidase, in adult and pediatric patients with Gaucher disease previously treated with imiglucerase.</a> Blood Cells Mol. Dis. 53, 253-260 (2014).</li><li>De Paepe, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+between+spiral-filter+press+and+belt+press+implemented+in+a+cloudy+apple+juice+production+process.">A comparative study between spiral-filter press and belt press implemented in a cloudy apple juice production process.</a> Food Chem. 173, 986-996 (2015).</li><li>Buyel, J. F., Twyman, R. M., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+downstream+processing+of+plant-derived+recombinant+proteins.">Extraction and downstream processing of plant-derived recombinant proteins.</a> Biotechnol. Adv. 33, 902-913 (2015).</li><li>Wilken, L. R., Nikolov, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+and+purification+of+plant-made+recombinant+proteins.">Recovery and purification of plant-made recombinant proteins.</a> Biotechnol. Adv. 30, 419-433 (2012).</li><li>Buyel, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Process+development+trategies+in+plant+molecular+farming.">Process development trategies in plant molecular farming.</a> Curr. Pharm. Biotechnol. 16, 966-982 (2015).</li><li>Hassan, S., Keshavarz-Moore, E., Ma, J., Thomas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breakage+of+transgenic+tobacco+roots+for+monoclonal+antibody+release+in+an+ultra-scale+down+shearing+device.">Breakage of transgenic tobacco roots for monoclonal antibody release in an ultra-scale down shearing device.</a> Biotechnol. Bioeng. 111, 196-201 (2014).</li><li>Hassan, S., van Dolleweerd, C. J., Ioakeimidis, F., Keshavarz-Moore, E., Ma, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+extraction+of+monoclonal+antibodies+targeted+to+different+subcellular+compartments+in+transgenic+tobacco+plants.">Considerations for extraction of monoclonal antibodies targeted to different subcellular compartments in transgenic tobacco plants.</a> Plant Biotechnol. J. 6, 733-748 (2008).</li><li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale-down+models+to+optimize+a+filter+train+for+the+downstream+purification+of+recombinant+pharmaceutical+proteins+produced+in+tobacco+leaves.">Scale-down models to optimize a filter train for the downstream purification of recombinant pharmaceutical proteins produced in tobacco leaves.</a> Biotechnol. J. 9, 415-425 (2014).</li><li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downstream+processing+of+biopharmaceutical+proteins+produced+in+plants:+the+pros+and+cons+of+flocculants.">Downstream processing of biopharmaceutical proteins produced in plants: the pros and cons of flocculants.</a> Bioengineered. 5, 138-142 (2014).</li><li>Gregory, J., Barany, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+and+flocculation+by+polymers+and+polymer+mixtures.">Adsorption and flocculation by polymers and polymer mixtures.</a> Adv. Colloid Interface Sci. 169, 1-12 (2011).</li><li>Zhou, Y., Franks, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flocculation+mechanism+induced+by+cationic+polymers+investigated+by+light+scattering.">Flocculation mechanism induced by cationic polymers investigated by light scattering.</a> Langmuir. 22, 6775-6786 (2006).</li><li>Runkana, V., Somasundaran, P., Kapur, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mathematical+modeling+of+polymer-induced+flocculation+by+charge+neutralization.">Mathematical modeling of polymer-induced flocculation by charge neutralization.</a> J. Colloid Interface Sci. 270, 347-358 (2004).</li><li>Hjorth, M., Jorgensen, B. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+flocculation+mechanism+in+animal+slurry+established+by+charge+neutralization.">Polymer flocculation mechanism in animal slurry established by charge neutralization.</a> Water Res. 46, 1045-1051 (2012).</li><li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flocculation+increases+the+efficacy+of+depth+filtration+during+the+downstream+processing+of+recombinant+pharmaceutical+proteins+produced+in+tobacco.">Flocculation increases the efficacy of depth filtration during the downstream processing of recombinant pharmaceutical proteins produced in tobacco.</a> Plant Biotechnol. J. 12, 240-252 (2014).</li><li>Buyel, J. F., Opdensteinen, P., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulose-based+filter+aids+increase+the+capacity+of+depth+filters+during+the+downstream+processing+of+plant-derived+biopharmaceutical+proteins.">Cellulose-based filter aids increase the capacity of depth filters during the downstream processing of plant-derived biopharmaceutical proteins.</a> Biotechnol. J. 10, 584-591 (2014).</li><li>Yasarla, L. R., Ramarao, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+Flocculation+of+Lignocellulosic+Hydrolyzates+by+Polymers.">Dynamics of Flocculation of Lignocellulosic Hydrolyzates by Polymers.</a> Ind. Eng. Chem. Res. 51, 6847-6861 (2012).</li><li>Montgomery, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Design and Analysis of Experiments. , (2007).</li><li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+complex+systems+using+the+design+of+experiments+approach:+transient+protein+expression+in+tobacco+as+a+case+study.">Characterization of complex systems using the design of experiments approach: transient protein expression in tobacco as a case study.</a> J. Vis. Exp. , e51216 (2014).</li><li>Espuny Garcia Del Real, G., Davies, J., Bracewell, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale-down+characterization+of+post-centrifuge+flocculation+processes+for+high-throughput+process+development.">Scale-down characterization of post-centrifuge flocculation processes for high-throughput process development.</a> Biotechnol. Bioeng. 111, 2486-2498 (2014).</li><li>Rathore, A. S., Sofer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Process Validation in Manufacturing of Biopharmaceuticals, 3rd edn, Vol. 1. , (2012).</li><li>Kang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+Novel+and+Efficient+Cell+Culture+Flocculation+Process+Using+a+Stimulus+Responsive+Polymer+to+Streamline+Antibody+Purification+Processes.">Development of a Novel and Efficient Cell Culture Flocculation Process Using a Stimulus Responsive Polymer to Streamline Antibody Purification Processes.</a> Biotechnol. Bioeng. 110, 2928-2937 (2013).</li><li>Menkhaus, T. J., Eriksson, S. U., Whitson, P. B., Glatz, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+selection+as+a+downstream+strategy:+Polyelectrolyte+precipitation+of+beta-glucuronidase+from+plant+extracts.">Host selection as a downstream strategy: Polyelectrolyte precipitation of beta-glucuronidase from plant extracts.</a> Biotechnol. Bioeng. 77, 148-154 (2002).</li><li>Holler, C., Vaughan, D., Zhang, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethyleneimine+precipitation+versus+anion+exchange+chromatography+in+fractionating+recombinant+beta-glucuronidase+from+transgenic+tobacco+extract.">Polyethyleneimine precipitation versus anion exchange chromatography in fractionating recombinant beta-glucuronidase from transgenic tobacco extract.</a> J. Chromatogr. A. 1142, 98-105 (2007).</li><li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+polymers+are+more+effective+than+natural+flocculants+for+the+clarification+of+tobacco+leaf+extracts.">Synthetic polymers are more effective than natural flocculants for the clarification of tobacco leaf extracts.</a> J. Biotechnol. 195, 37-42 (2014).</li><li>Pearson, C. R., Heng, M., Gebert, M., Glatz, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zeta+potential+as+a+measure+of+polyelectrolyte+flocculation+and+the+effect+of+polymer+dosing+conditions+on+cell+removal+from+fermentation+broth.">Zeta potential as a measure of polyelectrolyte flocculation and the effect of polymer dosing conditions on cell removal from fermentation broth.</a> Biotechnol. Bioeng. 87, 54-60 (2004).</li><li>Buyel, J. F., Gruchow, H. M., Boes, A., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+a+host+cell+protein+heat+precipitation+step+simplifies+the+subsequent+purification+of+recombinant+proteins+from+tobacco.">Rational design of a host cell protein heat precipitation step simplifies the subsequent purification of recombinant proteins from tobacco.</a> Biochem. Eng. J. 88, 162-170 (2014).</li><li>Wang, S., Liu, C., Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+polymer+flocculants+on+coagulation-microfiltration+of+surface+water.">Impact of polymer flocculants on coagulation-microfiltration of surface water.</a> Water Res. 47, 4538-4546 (2013).</li><li>Menkhaus, T. J., Anderson, J., Lane, S., Waddell, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyelectrolyte+flocculation+of+grain+stillage+for+improved+clarification+and+water+recovery+within+bioethanol+production+facilities.">Polyelectrolyte flocculation of grain stillage for improved clarification and water recovery within bioethanol production facilities.</a> Bioresour. Technol. 101, 2280-2286 (2010).</li><li>Mune, M. A. M., Minka, S. R., Mbome, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimising+functional+properties+during+preparation+of+cowpea+protein+concentrate.">Optimising functional properties during preparation of cowpea protein concentrate.</a> Food Chem. 154, 32-37 (2014).</li><li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictive+models+for+transient+protein+expression+in+tobacco+(Nicotiana+tabacum+L.)+can+optimize+process+time,+yield,+and+downstream+costs.">Predictive models for transient protein expression in tobacco (Nicotiana tabacum L.) can optimize process time, yield, and downstream costs.</a> Biotechnol. Bioeng. 109, 2575-2588 (2012).</li><li>Buyel, J. F., Kaever, T., Buyel, J. J., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predictive+models+for+the+accumulation+of+a+fluorescent+marker+protein+in+tobacco+leaves+according+to+the+promoter/5'UTR+combination.">Predictive models for the accumulation of a fluorescent marker protein in tobacco leaves according to the promoter/5'UTR combination.</a> Biotechnol. Bioeng. 110, 471-482 (2013).</li><li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+juice+extractor+can+simplify+the+downstream+processing+of+plant-derived+biopharmaceutical+proteins+compared+to+blade-based+homogenizers.">A juice extractor can simplify the downstream processing of plant-derived biopharmaceutical proteins compared to blade-based homogenizers.</a> Process Biochem. 50, 859-866 (2014).</li><li>Anderson, M. J., Whitcomb, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DOE Simplified: Practical Tools for Effective Experimentation. Vol. 1. 1, (2000).</li><li>Anderson, M. J., Whitcomb, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Response Surface Methods Simplified. , (2005).</li><li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generic+chromatography-based+purification+strategies+accelerate+the+development+of+downstream+processes+for+biopharmaceutical+proteins+produced+in+plants.">Generic chromatography-based purification strategies accelerate the development of downstream processes for biopharmaceutical proteins produced in plants.</a> Biotechnol. J. 9, 566-577 (2014).</li><li>Myers, R. H., Montgomery, D. C., Anderson-Cook, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Response Surface Methodology: Process and Product Optimization Using Designed Experiments. , (2009).</li></ol>

笔者没有利益冲突的披露。

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（由设计因素， 例如 pH值被覆盖）的分数NTU给予125 NTU的变化。该FDS可以增加通过增加与附加运行的设计和应≥0.95为旨在指导过程控制36的设计。此外，如果运行的次数不允许吨在一天应进行他整个实验中，块应在能源部预先定义以考虑批与批和天到一天变性。当使用植物材料的工作，参照列入每个块中运行（ 例如 ，非处理对照）有助于补偿变异性，允许数据从几次运行每个标准化到它们相应的参考运行比较。在这种情况下，增加在美国能源部复制运行的次数也是有用的。 当聚合物的大量筛选，最好是使用絮凝剂， 如电荷密度和分子质量的各特性，作为离散数字因素而不是聚合物本身作为绝对因素。这减少实验次数，因为实验设计往往需要复制的绝对因素，而数值的附加因素水平只需要少量的额外奔跑。信息CON实验帐篷也增加，并允许提高絮凝聚合物的性质， 例如 ，如在这里描述的实验中发现了高电荷密度的鉴定。 CCD和RSM实验设计是具有高预测力建立模型，允许的强大的处理条件的识别（ 例如 ，引导过程控制）有用，并且通常用于后续筛选设计。如果根据调查结果在美国能源部与超过400个单独的实验的因素和因素的水平的数量，这可能是可取的减少因子级别数或切换到其它设计类型的，因为这可以用该技术容易地处理样本的数目这里介绍的只限于〜100每天。 但从实验来看，聚合物必须保持在选定的实验条件下稳定， 例如 ，它们不能在低pH解聚。絮凝剂的精心准备在浓度计股也是必要的，以获得可再现的结果和高质量模式。在这方面，絮凝剂可能需要进行预处理， 例如肿胀倍或pH调节为几丁质，以确保完全溶解，从而得到一种均匀的溶液。高度应该避免粘性股因为聚合物转移到提取时这可能会导致移液误差。许多聚合物可以有较强的缓冲效果和股票有极端的pH值，pH值如 9.5〜8％[W / V] PEI。这可能会影响提取物的pH，如果股票未预调整和将扭曲的实验结果。例如，如果絮凝是在高pH和非pH调节的PEI库存更有效的使用，那么一个能源部可能表明，高的PEI浓度是更有效的。然而，这种效果会被更高的pH引起的溶液中加入该股票的较大体积而引起，而不是由增加聚合物浓度的pe- [R本身。股票使用浓度也应该类似于那些在大规模应用中使用，以避免不同的，可以影响颗粒浓度，从而絮凝鳞之间的稀释作用。一些基于粘土絮 ​​凝剂如高岭土包含大量微粒本身，其可以掩盖絮凝效果， 例如 ，初始过滤后浊度减少，而其它的反应，应选择以评估这些物质的效率， 例如 ，下游过滤器的能力。 用于数据分析它在极端值，偏差和普通的一致性， 例如 ，极端值可以指示一个复制粘贴误差，在小数位的移位或设备/分析设备的故障方面来评估所收集的结果是很重要的。透彻分析，将确保只有高质量的数据用于模型构建。在建立模型它不断地评估日是非常重要的Ë广泛的由美国能源部提供的软件质量指标。最基本的标准是，R 2，调节R 2和预测，R 2的值，但正常残差，残差-VS-运行和实际-VS-预测图（ 图4）是更重要的，因为它们提供了有关在每个运行信息实验而非总和的参数。此外，最终模型的一致性及其与絮凝的已知机制的预测应始终进行调查。可能出现的预测和科学的期望之间的差异主要是因为美国能源部模型只是描述性的，而不是机械的， 例如模型可以在设计空间体现了利用多项式拟合算法的边缘预测极值。 <img alt="图4" src="/files/ftp_upload/53940/53940fig4.jpg" /> 图4:美国能源部模型质量指标没有学生化残差的RMAL情节应该只有轻微的偏差（绿色箭头）的高品质车型可接受尽可能地（A）相似的直线。弯曲的外观（C）与理想线（红色）强偏差（红色箭头）表示差的模式， 比如由于缺少显著的因素。最终，预测和实验（实际）值应匹配（B）和再次遵循一条直线。从理想的线（红色圆圈和虚线）偏差表明差模型预测（D）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53940/53940fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 美国能源部方法可以帮助在复杂的原料，如植物提取物表征絮凝，即使没有现有数据。烟草萃取物的絮凝用2我们的工作负荷最优化EKS和〜500€耗材成本。这减少了对单个试规模批量60％，这实现了消耗品成本相应减少涉及〜2800升的植物提取物的所需深度过滤器的数量。 该絮凝剂也适用于不同的植物提取物和细胞培养匀浆。虽然同絮凝剂是有效的，所有这些原料的，聚合物浓度的，以便容纳不同的浓度分散颗粒进行调整。此外，一次有效的聚合物已被确定时，过滤和/或离心步骤可能需要进行调整，以匹配不同粒度分布11。 这里所描述的方法可以容易地适用于其它的原料，因此，也有相关科学家和工程师开发用于哺乳动物细胞培养物和食品/饲料生产过程澄清策略。 ESPecially基于植物的进程将来自中间样品体积受益这里建议，因为植物提取物可含有颗粒高达1毫米的直径，因为在混合动力不同这是不符合微孔板格式21中 ， 例如，由于粒径为容器直径比该是不是代表这个过程的规模。

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&gt; 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&gt;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。

<table><tbody><tr><td>2100P Portable Turbidimeter</td><td>Hach</td><td>4650000</td><td>Turbidimeter</td></tr><tr><td>2G12 antibody</td><td>Polymun</td><td>AB002</td><td>Reference antibody</td></tr><tr><td>Biacore T200</td><td>GE Healthcare</td><td>28-9750-01</td><td>SPR device</td></tr><tr><td>BP-410</td><td>Furh</td><td>2632410001</td><td>Bag filter</td></tr><tr><td>Catiofast VSH</td><td>BASF</td><td>79002360</td><td>Flocculating agent</td></tr><tr><td>Centrifuge 5415D</td><td>Eppendorf</td><td>5424 000.410</td><td>Centrifuge</td></tr><tr><td>Centrifuge tube 15 ml</td><td>Labomedic</td><td>2017106</td><td>Reaction tube</td></tr><tr><td>Centrifuge tube 50 ml self-standing</td><td>Labomedic</td><td>1110504</td><td>Reaction tube</td></tr><tr><td>Chitosan</td><td>Carl Roth GmbH</td><td>5375.1</td><td>Flocculating agent</td></tr><tr><td>Design-Expert(R) 8</td><td>Stat-Ease, Inc.</td><td>n.a.</td><td>DoE software</td></tr><tr><td>Disodium phosphate</td><td>Carl Roth GmbH</td><td>&amp;nbsp;4984.3&amp;nbsp;</td><td>Media component</td></tr><tr><td>Ferty 2 Mega</td><td>Kammlott</td><td>5.220072</td><td>Fertilizer</td></tr><tr><td>Forma -86C ULT freezer</td><td>ThermoFisher</td><td>88400</td><td>Freezer</td></tr><tr><td>Greenhouse</td><td>n.a.</td><td>n.a.</td><td>For plant cultivation</td></tr><tr><td>Grodan Rockwool Cubes 10 x 10 cm</td><td>Grodan</td><td>102446</td><td>Rockwool block</td></tr><tr><td>HEPES</td><td>Carl Roth GmbH</td><td>9105.3</td><td>Media component</td></tr><tr><td>K700P 60D</td><td>Pall</td><td>5302305</td><td>Depth filter layer</td></tr><tr><td>KS50P 60D</td><td>Pall</td><td>B12486</td><td>Depth filter layer</td></tr><tr><td>Miracloth</td><td>Labomedic</td><td>475855-1R</td><td>Filter cloth</td></tr><tr><td>MultiLine Multi 3410 IDS</td><td>WTW</td><td>WTW_2020</td><td>pH meter / conductivity meter</td></tr><tr><td>Osram cool white 36 W</td><td>Osram</td><td>4930440</td><td>Light source</td></tr><tr><td>Phytotron</td><td>Ilka Zell</td><td>n.a.</td><td>For plant cultivation</td></tr><tr><td>Polymin P</td><td>BASF</td><td>79002360</td><td>Flocculating agent</td></tr><tr><td>POLYTRON PT 6100 D</td><td>Kinematica</td><td>11010110</td><td>Homogenization device with custom blade tool</td></tr><tr><td>Protein A</td><td>Life technologies</td><td>10-1006</td><td>Antibody binding protein</td></tr><tr><td>Sodium chloride</td><td>Carl Roth GmbH</td><td>P029.2</td><td>Media component</td></tr><tr><td>Synergy HT</td><td>BioTek</td><td>SIAFRT</td><td>Fluorescence plate reader</td></tr><tr><td>TRIS</td><td>Carl Roth GmbH</td><td>4855.3</td><td>Media component</td></tr><tr><td>Tween-20</td><td>Carl Roth GmbH</td><td>9127.3</td><td>Media component</td></tr><tr><td>VelaPad 60</td><td>Pall</td><td>VP60G03KNH4</td><td>Filter housing</td></tr><tr><td>Zetasizer Nano ZS</td><td>Malvern</td><td>ZEN3600</td><td>DLS particle size distribution measurement</td></tr></tbody></table>

procedure to evaluate the efficiency of flocculants for the removal of dispersed particles from plant extracts

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.

用不同的聚合物的烟草萃取物的絮凝上述方法已成功使用的单克隆抗体（艾滋病毒中和抗体2G12）和荧光蛋白（DsRed的）（ 图1）16的制造过程中，以开发用于烟草萃取物的絮凝的方法，并已被转移其他蛋白质包括凝集素，疟疾疫苗候选和融合蛋白（未发表数据）。通常，絮凝剂的应用从〜6000 NTU（萃取后万NTU）至〜1000 NTU减少袋过滤的植物提取物的浊度。在初步筛选实验中，使用一个91运行的IV-优化设计，以测试在三个不同浓度的18种不同的聚合物（因为这个因素影响絮凝效率13,27），并观察到絮凝过〜12小时培养牙周D（ 图2A和B）。长潜伏期可能很重要，以确定絮凝过程有意义的时间框架。 4-8同样的pH值由于特定的靶蛋白13,25,27,37的性能测试，因为这可能会在未来的进程有关。在18测试聚合物，六人发现，以减少提取典型的袋式过滤与25毫秒-1的电导率后提取物浑浊。 该模型是通过排除在两次迭代所有无效的聚合物，然后，包括额外的工艺参数，如在15-45毫秒电导率精制-1范围内，5-75分钟，4-30℃的温度下的温育时间，以产生适合于更广泛的工艺条件的模型。该模型的预测能力每次迭代之后增加，导致高度可靠的模型（ 图3A）。 </p<html> &gt; 后四次迭代，高充电阳离子和支链聚合物的PEI被发现是最有效的对中的烟草萃取物的分散颗粒的聚集。但是，这种聚合物的效率随着提取电导率下降。属性分子大小，电荷，结构（支链或直），电荷密度和胺取代度（伯，仲，叔或季）进行了测试如在美国能源部因素和最后两个参数具有最大的效果。细节已在其他地方16日报道。基于从能源部结果聚合物性质的这方面的知识，选择具有相似的PEI分子特性（电荷密度&gt;毫当量克-1和季胺）等五聚合物。一个这五个聚合物表现出更高的电导率（ 图3B）11更大的絮凝效果。 核苷酸。"FO:保持-together.within页="1"&gt;如能源部方法的一部分，可以确认没有的PEI根据任何的测试条件的影响的产品回收率的确深度过滤器的能力，随后被用于除去剩余的分散的固体增加的3.2-5.7倍，达到〜110 L M -2取决于过滤器类型。这些结果也证实了在一个100升中试规模的过程，在用于其中絮凝剂的应用降低了澄清由&gt; 50％ - 相关生产成本，约20％的总生产成本。 <img alt="图2" src="/files/ftp_upload/53940/53940fig2.jpg" /> 图2:不同的工艺条件下，不同的絮凝剂的效率 （A）中 。 提取物样品絮凝和袋式过滤后直接还会出现浑浊。 （B）的沉淀数小时后，的浊度同一样品可以显著降低。但是，因为在大规模生产过程延长保持时​​间，可能无法在过滤后立即得到的浊度值通常优选的。当施加到具有螺旋压力机，而不是通过在50ml试管底部的清晰的红色液体作为指示的混合器产生的植物提取物（C）的絮凝也是有效（红色是由于荧光蛋白DsRed的存在下）。 （D）的不同絮凝剂的混合物也可以诱导絮凝。 <img alt="图3" src="/files/ftp_upload/53940/53940fig3.jpg" /> 图3:使用能源部方法建模絮凝 （A）的模型预测增加作为模型的聚合物的数目的准确度从初始筛选降低到细化即使工艺参数的数目从T增加WO五。 （B）的切换聚合物类型（这里从一个PEI到另一个），为在工艺参数的变化（这里电导率）的结果保持有效的颗粒絮凝和相比未处理的对照提取物（红色实线）对应的低滤液浊度。在A和B误差线表示模型预测的标准偏差。红色虚线线表示未经处理的提取物（N = 10）的标准偏差。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53940/53940fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 烟草提取物絮凝用螺旋压力机准备絮凝结果也从用均化制备对那些与一螺旋压力机，这在毫米尺寸范围内，但更产生较少的分散粒子制备烟草提取物转移颗粒在微米大小范围。在一台29运行的IV-优化设计，它表明PEI也是有效用于在类似浓度范围这类提取物和靶蛋白的回收不受影响（ 图2C）。这证明（1）确定用于一种类型的原料的可以是转移到其他原料在一定程度上是絮凝条件，工艺开发过程中节省时间，以及（ii）该能源部策略可以被用来确认这个转印不仅对个别的工艺条件，但在整个设计空间。 絮凝实验中絮凝剂混合物絮凝剂的组合可以比单个的聚合物， 如更有效的，由于颗粒12之间更增强桥接。因此，该方法如上所述是适于容纳另外两种聚合物（3.2）26。三个非合成聚合物被单独测试，在彼此组合或与PEI混合。烟草萃取物的最有效的絮凝物单独与PEI实现，但PE​​I和脱乙酰壳多糖或聚磷酸盐的组合可以减少的PEI所需的浓度。此外，美国能源部方法允许我们省略PEI时（伴或不壳聚糖和多磷酸盐），从而帮助确定在PEI与目标蛋白质不兼容的过程， 例如 ，由于降水量最佳絮凝条件确定最有效的聚合物组合，报告βglucuronidase24,25。此外，美国能源部能够表征复杂的设计空间没有机械模型是可用的量（ 图2D）。利用美国能源部软件的ANOVA工具有可能可靠的预测模型和管理不善，同行评价（ 图4）区别开来。

Watch this Scientific Journal Video about Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts at JoVE.com

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

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.

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

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 ...

procedure-to-evaluate-efficiency-flocculants-for-removal-dispersed

Universidad San Sebastian

Research

JoVE Journal

Biology

8.8K 次观看.  Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V.. 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.

视频: Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

A Small Volume Procedure for Viral Concentration from Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

小体积的方法从水中病毒浓度

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

科研

环境科学

Extraction and Characterization of Surfactants from Atmospheric Aerosols

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in the Earth&#39;s atmosphere, a central process in meteorology, hydrology, and for the climate system. But because specific extraction and characterization of these compounds have been lacking for decades, very little is known on their identity, properties, mode of action and origins, thus preventing the full understanding of cloud formation and its potential links with the Earth&#39;s ecosystems.
In this paper we present recently developed methods for 1) the targeted extraction of all the surfactants from atmospheric aerosol samples and for the determination of 2) their absolute concentrations in the aerosol phase and 3) their static surface tension curves in water, including their Critical Micelle Concentration (CMC). These methods have been validated with 9 references surfactants, including anionic, cationic and non-ionic ones. Examples of results are presented for surfactants found in fine aerosol particles (diameter &#60;1 &#956;m) collected at a coastal site in Croatia and suggestions for future improvements and other characterizations than those presented are discussed.

Methods are presented for the targeted extraction of surfactants present in atmospheric aerosols and the determination of their absolute concentrations and surface tension curves in water, including their Critical Micelle Concentration (CMC).

提取和表面活性剂的特性，从大气气溶胶

Surface-active compounds, or surfactants, present in atmospheric aerosols are expected to play important roles in the formation of liquid water clouds in ...

Determination of the Settling Rate of Clay/Cyanobacterial Floccules

The mechanisms underpinning the deposition of fine-grained, organic-rich sediments are still largely debated. Specifically, the impact of the interaction of clay particles with reactive, planktonic cyanobacterial cells to the sedimentary record is under studied. This interaction is a potentially major contributor to shale depositional models. Within a lab setting, the flocculation and sedimentation rates of these materials can be examined and measured in a controlled environment. Here, we detail a protocol for measuring the sedimentation rate of cyanobacterial/clay mixtures. This methodology is demonstrated through the description of two sample experiments: the first uses kaolin (a dehydrated form of kaolinite) and Synechococcus sp. PCC 7002 (a marine coccoid cyanobacteria), and the second uses kaolin and Synechocystis sp. PCC 6803 (a freshwater coccoid cyanobacteria). Cyanobacterial cultures are mixed with varying amounts of clay within a specially designed tank apparatus optimized to allow continuous, real-time video and photographic recording. The sampling procedures are detailed as well as a post-collection protocol for precise measurement of chlorophyll a from which the concentration of cyanobacterial cells remaining in suspension can be determined. Through experimental replication, a profile is constructed that displays sedimentation rate.

The interaction and sedimentation of the clay and bacterial cells within the marine realm, observed in natural environments, can be best investigated in a controlled lab environment. Here, we describe a detailed protocol, which outlines a novel method for measuring the sedimentation rate of clay and cyanobacterial floccules.

粘土/蓝藻 Floccules 沉降速率的测定

The mechanisms underpinning the deposition of fine-grained, organic-rich sediments are still largely debated. Specifically, the impact of the interaction ...

Combined Size and Density Fractionation of Soils for Investigations of Organo-Mineral Interactions

Combined size and density fractionation (CSDF) is a method used to physically separate soil into fractions differing in particle size and mineralogy. CSDF relies on sequential density separation and sedimentation steps to isolate (1) the free light fraction (uncomplexed organic matter), (2) the occluded light fraction (uncomplexed organic matter trapped in soil aggregates) and (3) a variable number of heavy fractions (soil minerals and their associated organic matter) differing in composition. Provided that the parameters of the CSDF (dispersion energy, density cut-offs, sedimentation time) are properly selected, the method yields heavy fractions of relatively homogeneous mineral composition. Each of these fractions is expected to have a different complexing ability towards organic matter, rendering this a useful method to isolate and study the nature of organo-mineral interactions. Combining density and particle size separation brings an improved resolution compared to simple size or density fractionation methods, allowing the separation of heavy components according to both mineralogy and size (related to surface area) criteria. As is the case for all physical fractionation methods, it may be considered as less disruptive or aggressive than chemically-based extraction methods. However, CSDF is a time-consuming method and furthermore, the quantity of material obtained in some fractions can be limiting for subsequent analysis. Following CSDF, the fractions may be analyzed for mineralogical composition, soil organic carbon concentration and organic matter chemistry. The method provides quantitative information about organic carbon distribution within a soil sample and brings light to the sorptive capacity of the different, naturally-occurring mineral phases, thus providing mechanistic information about the preferential nature of organo-mineral interactions in soils (i.e., which minerals, what type of organic matter).

Combined size and density fractionation (CSDF) is a method to physically separate soil into fractions differing in texture (particle size) and mineralogy (density). The purpose is to isolate fractions with different reactivities towards soil organic matter (SOM), in order to better understand organo-mineral interactions and SOM dynamics.

土壤联合粒径与密度的分离研究

Combined size and density fractionation (CSDF) is a method used to physically separate soil into fractions differing in particle size and mineralogy. CSDF ...

Capture of Environmental DNA (eDNA) from Water Samples by Flocculation

The analysis of environmental DNA (eDNA) has become a widely used approach to problem solving in species management. The detection of cryptic species including invasive and (or) species at risk is the goal, typically accomplished by testing water and sediment for the presence of characteristic DNA signatures. Reliable and efficient procedures for the capture of eDNA are required, especially those that can be performed easily in the field by personnel with limited training and citizen scientists. The capture of eDNA using membrane filtration is widely used currently. This approach has inherent issues that include the choice of filter material and porosity, filter fouling, and time required on site for the process to be performed. Flocculation offers an alternative that can be easily implemented and applied to sampling regimes that strive to cover broad territories in limited time.

Capture of Environmental DNA eDNA from Water Samples by Flocculation

Environmental DNA (eDNA) is typically captured from water samples using alcohol precipitation, filtration, or flocculation. Presented here is an alternative protocol that uses lanthanum chloride to enable collection of dissolved and particulate-bound nucleic acids from water samples containing eukaryotic cells, prokaryotic cells, and virus.

The analysis of environmental DNA (eDNA) has become a widely used approach to problem solving in species management. The detection of cryptic species ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

活联甘蔗，用于亲和色谱树脂的快速发展 - 抗体捕获作为案例研究

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

生物化学

野生花生物种对黄曲霉毒素积累的抗性评价

Non-destructive SPE-UPLC-based Quantification of Aflatoxins and Stilbenoid Phytoalexins in Single Peanut (Arachis spp.) Seeds

Aflatoxins are highly carcinogenic secondary metabolites of some fungal species, particularly Aspergillus flavus. Aflatoxins often contaminate economically important agricultural commodities, including peanuts, posing a high risk to human and animal health. Due to the narrow genetic base, peanut cultivars demonstrate limited resistance to fungal pathogens. Therefore, numerous wild peanut species with tolerance to Aspergillus have received substantial consideration by scientists as sources of disease resistance.
Exploring plant germplasm for resistance to aflatoxins is difficult since aflatoxin accumulation does not follow a normal distribution, which dictates the need for the analyses of thousands of single peanut seeds. Sufficiently hydrated peanut (Arachis spp.) seeds, when infected by Aspergillus species, are capable of producing biologically active stilbenes&#160;(stilbenoids) that are considered defensive phytoalexins. Peanut stilbenes inhibit fungal development and aflatoxin production. Therefore, it is crucial to analyze the same seeds for peanut stilbenoids to explain the nature of seed resistance/susceptibility to the Aspergillus invasion. None of the published methods offer single-seed analyses for aflatoxins and/or stilbene phytoalexins.
We attempted to fulfill the demand for such a method that is environment-friendly, uses inexpensive consumables, and is sensitive and selective. In addition, the method is non-destructive since it uses only half of the seed and leaves the other half containing the embryonic axis intact. Such a technique allows germination and growth of the peanut plant to full maturity from the same seed used for the aflatoxin and stilbenoid analysis. The integrated part of this method, the manual challenging of the seeds with Aspergillus, is a limiting step that requires more time and labor compared to other steps in the method. The method has been used for the exploration of wild Arachis germplasm to identify species resistant to Aspergillus and to determine and characterize novel sources of genetic resistance to this fungal pathogen.

作者聚焦：定量分析花生种子中的黄曲霉毒素和植物抗毒素，以确定对曲霉菌的遗传抗性

We demonstrate a medium-throughput method for quantification of aflatoxins and stilbenoid phytoalexins in single peanut seeds using ultra performance liquid chromatography. This method was specifically developed for the analyses of wild Arachis species challenged by the aflatoxigenic Aspergillus species.

基于 SPE-UPLC 的单花生 （Arachis spp.） 中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子

Aflatoxins are highly carcinogenic secondary metabolites of some fungal species, particularly Aspergillus flavus. Aflatoxins often contaminate ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

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

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...

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

摘要

探索更多视频

此视频中的章节

小体积的方法从水中病毒浓度

提取和表面活性剂的特性，从大气气溶胶

粘土/蓝藻 Floccules 沉降速率的测定

土壤联合粒径与密度的分离研究

Capture of Environmental DNA (eDNA) from Water Samples by Flocculation

活联甘蔗，用于亲和色谱树脂的快速发展 - 抗体捕获作为案例研究

基于 SPE-UPLC 的单花生（Arachis spp.）中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子

基于 SPE-UPLC 的单花生（Arachis spp.）中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子

基于 SPE-UPLC 的单花生（Arachis spp.）中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子

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

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

摘要

探索更多视频

此视频中的章节

小体积的方法从水中病毒浓度

提取和表面活性剂的特性，从大气气溶胶

粘土/蓝藻 Floccules 沉降速率的测定

土壤联合粒径与密度的分离研究

Capture of Environmental DNA (eDNA) from Water Samples by Flocculation

活联甘蔗，用于亲和色谱树脂的快速发展 - 抗体捕获作为案例研究

基于 SPE-UPLC 的单花生 （Arachis spp.） 中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子

基于 SPE-UPLC 的单花生 （Arachis spp.） 中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子

基于 SPE-UPLC 的单花生 （Arachis spp.） 中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子

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

基于 SPE-UPLC 的单花生（Arachis spp.）中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子

基于 SPE-UPLC 的单花生（Arachis spp.）中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子

基于 SPE-UPLC 的单花生（Arachis spp.）中黄曲霉毒素和二苯乙烯类植物抗毒素的无损定量分析种子