Name: procedure to evaluate the efficiency of flocculants for the removal of dispersed particles from plant extracts
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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. Develop an Adequate Experimental Strategy
<ol>
	<li>Identify the environmental and process parameters that are relevant for the flocculation procedure to be established or optimized, i.e. which factors have the strongest effect on flocculation. Typically, there are several such parameters so a DoE approach as recently described 20 is necessary due to the lack of mechanistic models.
	<ol>
		<li>Select parameters (factors) based on literature data 12, prior knowledge and experience with ...

<ol>
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The most important aspect to consider when setting up a DoE to characterize particle flocculation is that the design must in principle be able to detect and describe the anticipated or possible effects 36,38, e.g. the influence of pH, polymer type and polymer concentration 16. Therefore, it is important to evaluate the fraction of design space (FDS) before starting the actual experiments. The FDS is the fraction of the multidimensional experimental space (covered by the design factors, ...

Plants are widely used to produce food commodities such as fruit juices, but they can also be developed as platforms for the manufacture of higher-value biopharmaceutical products 1-3. In both cases, downstream processing (DSP) often begins with the extraction of liquids from tissues such as leaves or fruits, followed by the clarification of particle-laden extracts 4,5. For the manufacture of biopharmaceuticals, the costs of DSP can account for up to 80% of the overall production costs 6,7 and this in part reflects the high particle burden present in extracts prepared by disruptive methods such as blade-based homogenization 8,9. Although the rational selection of filter layers to match the particle size distribution in the extract can increase filter capacity and reduce costs 10,11, the improvement can never exceed the ceiling of absolute capacity defined by the number of particles that must be retained per unit of filter area to achieve clarification.
The ceiling can be lifted if fewer particles reach the surface of the finest filters in the filtration train, and this can be achieved if dispersed particles are mixed with polymers known as flocculants that promote aggregation to form large flocs 12. Such flocs can be retained further upstream by coarser and less expensive bag filters, reducing the particle burden reaching the finer and more expensive depth filters. The polymers must have safety profiles suitable for their applications, e.g. for biopharmaceuticals they must be compliant with good manufacturing practice (GMP), and typically they must have a molar mass &#62;100 kDa and can either be neutral or charged 13. Whereas neutral flocculants generally act by cross-linking dispersed particles causing their aggregation and the formation of flocs with diameters &#62;1 mm 11, charged polymers neutralize the charge of dispersed particles, reducing their solubility and thus causing precipitation 14.
Flocculation can be improved by adjusting parameters such as buffer pH or conductivity, and the polymer type or concentration, to match the properties of the extract 15,16. For tobacco extracts pretreated with 0.5-5.0 g L-1 polyethylenimine (PEI), a greater than 2-fold increase in depth filter capacity was reported in a 100-L pilot-scale process. The cost of this polymer is less than &#8364;10 kg-1 so its introduction into the process resulted in cost savings of about &#8364;6,000 for filters and consumables per batch 16 or even more when combined with cellulose-based filter aids 17. Even so, predictive models are required to evaluate the a priori economic benefits of flocculants because their inclusion can require hold steps of 15-30 min 16,18, resulting in further investment costs for storage tanks. However, there are currently no mechanistic models available that can predict the outcome of such experiments due to the complex nature of flocculation. Therefore, a more appropriate design-of-experiments (DoE) approach 19 was developed as described in this article. A protocol for the general DoE procedure has recently been published 20.
Small-scale devices are now available for the high-throughput screening of flocculation conditions 21. However, these devices may not realistically simulate conditions during the flocculation of plant extracts because the dimensions of the reaction vessel (~7 mm for wells on a 96-well plate) and the particles or flocs can be less than an order of magnitude apart. This can affect mixing patterns and thus the predictive power of the model. Furthermore, it can be difficult to scale down processes involving precipitation due to non-linear changes in the mixing behavior and precipitate stability 22. Therefore, this article outlines a bench-top-scale screening system with a throughput of 50-75 samples per day, yielding results that are scalable from the initial 20 ml reaction volume to a 100 L pilot-scale process 16. When combined with a DoE approach, this allows the predictive models to be used for process optimization and documentation as part of a quality-by-design concept.
The method described below may also be adapted to biopharmaceuticals produced in cell culture-based processes, where flocculants are also being considered as a cost-saving tool 23. It can also be used to model the precipitation of target proteins from a crude extract as part of a purification strategy, as demonstrated for &#946;-glucuronidase produced in canola, maize and soybean 24,25. A detailed description of flocculant properties can be found elsewhere 16,26 and it is important to ensure that the polymer concentrations are either non-toxic or below harmful levels in the final product 11.

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			<td height="21" style="height:21px;width:360px;">Biacore T200</td>
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			<td style="width:119px;">28-9750-01</td>
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			<td height="21" style="height:21px;width:360px;">BP-410</td>
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			<td height="21" style="height:21px;width:360px;">Catiofast VSH</td>
			<td style="width:195px;">BASF</td>
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			<td height="21" style="height:21px;width:360px;">Centrifuge 5415D</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">5424 000.410</td>
			<td>Centrifuge</td>
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			<td height="21" style="height:21px;width:360px;">Centrifuge tube 15 mL</td>
			<td style="width:195px;">Labomedic</td>
			<td style="width:119px;">2017106</td>
			<td>Reaction tube</td>
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			<td height="21" style="height:21px;width:360px;">Centrifuge tube 50 mL self-standing</td>
			<td style="width:195px;">Labomedic</td>
			<td style="width:119px;">1110504</td>
			<td>Reaction tube</td>
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			<td height="21" style="height:21px;width:360px;">Chitosan</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">5375.1</td>
			<td>Flocculating agent</td>
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			<td height="21" style="height:21px;width:360px;">Design-Expert(R) 8</td>
			<td style="width:195px;">Stat-Ease, Inc.</td>
			<td style="width:119px;">n.a.</td>
			<td>DoE software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Disodium phosphate</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">&#160;4984.3&#160;</td>
			<td>Media component</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ferty 2 Mega</td>
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			<td style="width:119px;">5.220072</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Forma -86C ULT freezer</td>
			<td style="width:195px;">ThermoFisher</td>
			<td style="width:119px;">88400</td>
			<td>Freezer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Greenhouse</td>
			<td style="width:195px;">n.a.</td>
			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Grodan Rockwool Cubes 10x10cm</td>
			<td style="width:195px;">Grodan</td>
			<td style="width:119px;">102446</td>
			<td>Rockwool block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">HEPES</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">9105.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">K700P 60D</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">5302305</td>
			<td>Depth filter layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">KS50P 60D</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">B12486</td>
			<td>Depth filter layer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Miracloth</td>
			<td style="width:195px;">Labomedic</td>
			<td style="width:119px;">475855-1R</td>
			<td>Filter cloth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">MultiLine Multi 3410 IDS</td>
			<td style="width:195px;">WTW</td>
			<td style="width:119px;">WTW_2020</td>
			<td>pH meter / conductivity meter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Osram cool white 36 W</td>
			<td style="width:195px;">Osram</td>
			<td style="width:119px;">4930440</td>
			<td>Light source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Phytotron</td>
			<td style="width:195px;">Ilka Zell</td>
			<td style="width:119px;">n.a.</td>
			<td>For plant cultivation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Polymin P</td>
			<td style="width:195px;">BASF</td>
			<td style="width:119px;">79002360</td>
			<td>Flocculating agent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">POLYTRON PT 6100 D</td>
			<td style="width:195px;">Kinematica</td>
			<td style="width:119px;">11010110</td>
			<td>Homogenization device with custom blade tool</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Protein A</td>
			<td style="width:195px;">Life technologies</td>
			<td style="width:119px;">10-1006</td>
			<td>Antibody binding protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Sodium chloride</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">P029.2</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Synergy HT</td>
			<td style="width:195px;">BioTek</td>
			<td style="width:119px;">SIAFRT</td>
			<td>Fluorescence plate reader</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">TRIS</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">4855.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Tween-20</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">9127.3</td>
			<td>Media component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">VelaPad 60</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">VP60G03KNH4</td>
			<td>Filter housing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Zetasizer Nano ZS</td>
			<td style="width:195px;">Malvern</td>
			<td style="width:119px;">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.

Flocculation of tobacco extract with different polymers
The method described above was successfully used to develop a process for the flocculation of tobacco extracts during the manufacture of a monoclonal antibody (the HIV-neutralizing antibody 2G12) and a fluorescent protein (DsRed) (Figure 1) 16, and has since been transferred to other proteins including lectins, malaria vaccine candidates and fusi...

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.

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 ...

The overall goal of the following experiment is to identify flocculants that can improve the removal of dispersed particles from plant extracts. This is achieved by setting up an experimental design to identify the most effective polymers and parameters controlling the aggregation of these dispersed particles. The second step defines the work flow, which corresponds to the unit operations used in a large-scale application.Next, leaf extracts are prepared for flocculants under various conditions and analyzed in order to measure turbidity and product recovery. The results show the optimal flocculation conditions and polymers based on a design of experiments model evaluation, which we shorten to DoE. The main advantage of this technique compared to traditional clarification using depth-filtration alone, is that the filter capacity can be increased several-fold, reducing overall process costs.Flocculation can increase the effective diameter of particles and facilitate their removal by centrifugation or filtration. Therefore, the environmental process parameters that are relevant for the flocculation procedure must be identified first. Typically, there are several such parameters.So a DoE approach is appropriate given the lack of mechanistic models. The DoE is set up by selecting parameters based on literature data and prior knowledge. Typical parameters include incubation time as well as polymer type and concentration.The experimental outcomes that are used to evaluate the efficiency of flocculation, such as filtratability, are also defined before the experiment. This information is then fed into a DoE type appropriate for the number of factors to be investigated and the degree of knowledge already accumulated about the system. A work flow is developed to encompass all steps relevant for the flocculation and laboratory-scale processing of plant extracts.The aliquot volume should be small enough to allow high sample throughput. The performance of the laboratory equipment should correlate with that of the process-scaled devices. Plants are cultivated in a manner that represents the final process.And all utilities are prepared before starting the extraction and flocculation experiments. The flocculant stock solutions are prepared carefully taking into account any pre-dilution by the manufacturer. Choosing the same stock concentration for all flocculants simplifies subsequent pipetting, reducing the likelihood of errors.The buffer components, pH, and conductivity for each fresh flocculant stock solution are prepared to match the conditions of the extracts. The DoE schedule is converted into a pipetting scheme setting out the volumes of stock solutions to be added to the plant extract samples. Plant extract samples are prepared from harvested tobacco leaves using the buffers matching the conditions to be tested in the DoE.While stirring the extract, aliquots are prepared, matching the pre-defined volume. This will ensure homogeneous particle distribution in all samples. Extraction buffer is added to each aliquot so that every sample has the same volume once a flocculant stock has been added.The volume of stock solution defined in the DoE is added to the sample sequentially as indicated by the randomized run order. The polymer is homogeneously distributed throughout the sample by shaking the tube immediately after adding the polymer, which will also ensure that the flocculation results are reproducible. The method can easily be modified to test combinations of flocculants or the repeated addition of the same flocculant.But this must be implemented in the DoE. Floc formation typically requires three to 30 minutes incubation time. Sticking to the incubation times defined in the DoE is necessary to achieve reproducible results.Either the settling velocity of the flocs or the turbidity of the filtrates and filter capacities can be used to evaluate the flocculant performance. Confirming that the polymers do not interfere with products such as proteins, is important too. Once the data quality is confirmed, information can be transferred into the DoE software.The experimental data are analyzed using the tools built into the DoE software including data transformation, factor selection, and quality control tools. As well as using these tools, the final model should also agree with the basic laws of physics and thermodynamics. Once the model quality is confirmed, the model can be used to predict the optimal conditions for flocculation.Based on the model predictions, the design space can be narrowed down to the most relevant region for which a new DoE can be set up, repeating the procedure described before. The results can then be confirmed at the pilot or production scale. In this representative study, the performance of 23 different synthetic and four natural flocculants was tested for their ability to flocculate tobacco leaf extracts using a DoE approach.Taking into account polymer-specific parameters such as polymer concentration and type, but also process parameters, such as incubation time, temperature, buffer pH, and conductivity. With the sequence of four nested IV-optimal designs, including a total of about 200 20 mL extract aliquots, we found that Polymin P was the most effective flocculant for tobacco extracts in the pH range four to eight, as long as the temperature was between four and 30 degrees Celsius. An incubation time of 15 minutes was sufficient for flocculation, but the effectiveness declined at conductivities greater than 40 mS per cm.However, the DoE approach showed that Catiofast VSH was complementary to Polymin P, achieving effective flocculation at conductivities greater than 40 mS per cm. When these flocculants were introduced into the downstream processing of tobacco extracts, the capacity of the deployed depth filters increased 3.2 to 5.7-fold, reducing the cost of goods for this step accordingly. We confirmed that the flocculants did not reduce the recovery of target proteins including a monoclonal antibody, a fluorescence protein, a lactin, and different vaccine candidates.The optimal flocculation conditions were also confirmed in a 100 L pilot scale batch. The results of the DoE were also successfully transferred to tobacco extracts prepared with a screw press rather than a blade-based homogenizer underlining how broadly applicable the collected information can be. We also adapted the method to test combinations of flocculants which worked equally well.After watching this video, you should understand how to set up flocculation experiments for plant extracts using a DoE approach.

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