Name: comparison of tobacco host cell protein removal methods by blanching intact plants or by heat treatment of extracts
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We would like to acknowledge Dr. Thomas Rademacher, Alexander Boes and Veronique Bei&#223; 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 G&#252;ven Edg&#252; 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.

1. Cultivate the Tobacco Plants
<ol>
	<li>Flush each mineral wool block with 1 to 2 L of deionized water and subsequently with 1 L of 0.1% [w/v] fertilizer solution. Place one tobacco seed in each mineral wool block and gently flush with 0.25 L of fertilizer solution without washing away the seed 16.</li>
	<li>Cultivate the tobacco plants for 7 weeks in a greenhouse with 70% relative humidity, a 16 hr photoperiod (180 &#181;mol sec&#8722;1 m&#8722;2;&#160;&#955; = ...

<ol>
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The three methods for heat precipitation described above can effectively remove tobacco HCPs prior to any chromatographic purification step 16,17. They complement other strategies that aim to increase initial product purity, e.g., guttation 29, rhizosecretion 30 or centrifugal extraction 31,32, all of which are limited to secreted proteins. However, the heat-based methods can only be used in a meaningful way if the target protein to be purified can withstand the minimu...

Modern healthcare systems increasingly depend on biopharmaceutical proteins 1. Producing these proteins in plants is advantageous due to the low pathogen burden and greater scalability compared to conventional expression systems 2-4. However, the downstream processing (DSP) of plant-derived pharmaceuticals can be challenging because the disruptive extraction procedures result in a high particle burden, with turbidities exceeding 5,000 nephelometric turbidity units (NTUs), and host cell protein (HCP) concentrations often exceeding 95% [m/m] 5,6.
Elaborate clarification procedures are required to remove dispersed particles 7-9, but chromatography equipment is less expensive to operate in bind-and-elute mode during initial product recovery if there is an earlier step for the efficient removal of HCPs 10,11. This can be achieved either by precipitating the target protein using flocculants 12 or low pH 13,14, as well as by causing the HCPs to aggregate. The selective aggregation of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant HCP in green plants such as tobacco (Nicotiana tabacum), can be promoted by adding polyethylene glycol 15, but this is expensive and incompatible with large-scale manufacturing. Heat treatment has been shown to denature and precipitate more than 95% of tobacco HCPs, while protein malaria vaccine candidates such as Vax8 remain stable in solution 16-18.
Three different approaches were used to achieve the heat-induced precipitation of tobacco HCPs: (i) blanching, i.e., the immersion of intact leaves in hot liquid, (ii) a temperature-controlled stirred vessel, and (iii) a heat exchanger (Figure 1) 16. For intact leaves, blanching achieved the rapid and efficient precipitation of HCPs and was also easy to scale up and compatible with existing large-scale manufacturing processes that include an initial step to wash the plant biomass 19. In contrast, temperature-controlled vessels are already available in some processes and can be used for the thermal treatment of plant extracts 20, but their scalability and energy transfer rate are limited because the surface-to-volume ratio of the tanks is progressively reduced and becomes unsuitable at process scale. A heat exchanger is a technically well-defined alternative to heated stirred vessels but requires an abundant supply of heating and cooling media, e.g., steam and cold water, as well as a tightly controlled volumetric flow rate that is adapted to the heat exchanger geometry and media properties, e.g., the specific heat capacity. This article shows how all three methods can be used for the heat-induced precipitation of tobacco HCPs, and plant HCPs in general. The establishment and operation of each method in a laboratory setting can be used to evaluate their suitability for larger-scale processes. The major challenge is to identify adequate scale-down models and running conditions for each operation that resemble the devices and conditions used during process-scale manufacturing. The data presented here refer to experiments conducted with transgenic tobacco plants expressing the malaria vaccine candidate Vax8 and fluorescent protein DsRed 16, but the method has also been successfully applied to N. benthamiana plants transiently expressing other biopharmaceutical proteins 21.
A design-of-experiments (DoE) approach 22 can facilitate process development, and flocculants 23 can also be beneficial in this context as previously described 8. The main difference between blanching, heated vessels and heat exchangers is that blanching is applied to intact leaves early in the process whereas the others are applied to plant extracts (Figure 1).
<img alt="Figure 1" src="/files/ftp_upload/54343/54343fig1.jpg" /> 
Figure 1: Process Flow Scheme Illustrating the Implementation of Three Different Methods for Tobacco HCP Heat Precipitation. The plant material is washed and homogenized before clarification and purification. The equipment for the blanching step (red) can easily be added to the existing machinery. In contrast, using a stirred vessel (orange) and especially a heat exchanger (blue) requires one or several additional devices and tubing. <a href="https://www.jove.com/files/ftp_upload/54343/54343fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">CM5 chip</td>
			<td style="width:195px;">GE Healthcare</td>
			<td style="width:119px;">BR100012&#160;</td>
			<td>Chip for SPR measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Cuvette 10x10x45</td>
			<td style="width:195px;">Sarsted</td>
			<td style="width:119px;">67.754</td>
			<td>Cuvette for Zetasizer Nano ZS</td>
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		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Ferty 2 Mega</td>
			<td style="width:195px;">Kammlott</td>
			<td style="width:119px;">5.220072</td>
			<td>Fertilizer</td>
		</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>
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		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Twentey-loop heat exchanger (4.8 m length)</td>
			<td style="width:195px;">n.a. (custom design)</td>
			<td style="width:119px;">n.a.</td>
			<td>Heat exchanger</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>
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		<tr height="21">
			<td height="21" style="height:21px;width:360px;">K200P 60D</td>
			<td style="width:195px;">Pall</td>
			<td style="width:119px;">5302303</td>
			<td>Depth filter layer</td>
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		<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;">Lauda E300</td>
			<td style="width:195px;">Lauda Dr Wobser GmbH</td>
			<td style="width:119px;">Z90010</td>
			<td>Water bath thermostat</td>
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			<td height="21" style="height:21px;width:360px;">L/S 24</td>
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			<td style="width:119px;">SN-06508-24</td>
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			<td>Vax8 specific antibody</td>
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			<td style="width:195px;">Masterflex</td>
			<td style="width:119px;">HV-77921-75</td>
			<td>Peristaltic pump</td>
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			<td style="width:195px;">Labomedic</td>
			<td style="width:119px;">475855-1R</td>
			<td>Filter cloth</td>
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		<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;">Sodium disulfit</td>
			<td style="width:195px;">Carl Roth GmbH</td>
			<td style="width:119px;">8554.1</td>
			<td>Media component</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="42">
			<td height="42" style="height:42px;width:360px;">Stainless-steel vessel; 0.7-kg 2.0-L; height 180 mm; diameter 120 mm</td>
			<td style="width:195px;">n.a. (custom design)</td>
			<td style="width:119px;">n.a.</td>
			<td>Container for heat precipitation</td>
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		<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>
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		<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>
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			<td height="21" style="height:21px;width:360px;">Zetasizer Nano ZS</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Zetasizer Software v7.11</td>
			<td style="width:195px;">Malvern</td>
			<td style="width:119px;">n.a.</td>
			<td>Software to operate the Zetasizer Nano ZS device</td>
		</tr>
	</tbody>
</table>

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.

Heat precipitation of tobacco host cell proteins by blanching 
The blanching procedure described in section 2. was successfully used to precipitate HCPs from tobacco leaves with 70 &#176;C, reducing the TSP by 96 &#177; 1% (n = 3) while recovering up to 51% of the Vax8 target protein, thus increasing its purity from 0.1% to 1.2% before chromatographic separation 16. It was also possible to recover 83 &#177; 1% (n =3) of the fluorescent protein DsRed, increasing it...

Watch this Scientific Journal Video about Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts at JoVE.com

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

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.

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

The overall goal of this set of experiments is to precipitate plant host cell proteins using heat. Thus facilitating subsequent chromatographic purification of recombinant biopharmaceutical proteins. This method can help to streamline process development and increase product stability, for example by activating host proteases.The main advantage of this technique is that it is easy to implement into existing proteases and can be quickly adapted to different target proteins. Demonstrating the procedure will be Hannah Gruchow a master's student from my laboratory. To heat precipitate host cell proteins.For example from tobacco leaves. After culturing tobacco plants according to the text protocol set up an eight liter working volume water bath in a thermally insulated polystyrene foam bucket. Transfer the entire assembly onto a magnetic stir plate, and place a magnetic bar in the water bath.Then surround the stir bar with a nonmagnetic support tile topping the stir bar by approximately one centimeter. Upon which a polypropylene basket will be placed later during the procedure. Add eight liters of buffer to the water bath.Then place a 23 by 23 by 23 centimeter polypropylene basket on the support tiles. Ensuring that the support does not interfere with the stir bar rotation, and that the basket is fully submerged in the liquid. Then removed the basket.Use an adjustable thermostat to bring the water bath to 70 degrees Celsius. Wait at least 15 minutes after the desired temperature is reached to ensure the entire assembly has reached the thermal equilibrium. Next, prepare 150 gram aliquots from the tobacco leaves.Place one aliquot in the basket while avoiding irreversible compression and damage to the leaves. For example, by tearing. Carefully but quickly submerge the basket in the hot liquid and place it on the support tiles.Then place a stainless steel block on top of the basket to prevent flotation. Incubate the leaves for five minutes in the blanching fluid. Or select a time suitable for the experimental design.Monitor the liquid temperature during the entire incubation period. After the incubation, carefully remove the basket from the blanching fluid and let the leaves drain for 30 seconds. Then take the plant leaves out of the basket, transfer them to the blender, and immediately start the extraction according to the text protocols.To heat precipitate HCPs in a stirred vessel. Transfer a water bath onto a magnetic stir plate and place a two liter stainless steel vessel into the water bath such that the center of the vessel is aligned to the center of the stir plate. Place a magnetic stir bar in the stainless steel vessel.Fill the vessel with extraction buffer. Then with the deionized water. Fill the water bath to five centimeters below the upper edge of the stainless steel vessel.Use a polystyrene foam lid to cover it. Next, insert a thermometer into the stainless steel vessel through a soothing hole in the lid. Then set the water bath temperature to 78 degrees Celsius and incubate the entire assembly for 15 minutes to reach thermal equilibrium.When the assembly has reached equilibrium empty the vessel and pour 300 milliliters of extract into the vessel while it is still in the water bath and start the timer. Stir the extract at 150 rpm while incubating for five minutes. Ensure that the extract reaches a temperature of 70 degrees Celsius for at least two minutes during the incubation period.After the incubation removed the hot water bath from the magnetic stirrer. Take out the stainless steel vessel and place it in a bucket with ice cold water. Then place the bucket onto the stir plate.Ensure that the plant homogenate is well agitated at 150 rpm. Place the thermometer in the extract and incubate it until it reaches a temperature of 20 degrees Celsius or the temperature specified. After setting up two eight liter working volume water baths according to the text protocol, use deionized water to fill the first bath.Set the thermostat to 74.5 degrees Celsius and incubate the assembly for 15 minutes to reach thermal equilibrium. Prepare an insulated storage vessel by placing a 0.5 liter plastic beaker into a one liter plastic beaker. And use an isolation material such as cotton wool to fill the gaps.Using LS24 tubing, connect the heat exchanger to a peristaltic pump at one end, and to an outlet hose on the other end. Place both tubing ends along with the thermometer in the insulated storage vessel. And fill it with 300 millimeters of extraction buffer.Next, place the heat exchanger into the hot water bath, and start the peristaltic pump at a rate of 300 milliliters per minute. After three minutes, ensure that the resulting temperature in the vessel is 70 degrees Celsius. Approximately 4.5 degrees Celsius below the set point of the water bath.Discard the extraction buffer from the insulated vessel, and prepare 300 milliliter aliquots of the plant extract. Then with one aliquot, fill the insulated vessel. Now pump the plant extract through the heat exchanger at 300 milliliters per minute for five minutes.Then ensure that the extract temperature is 70 degrees Celsius after three minutes or equals the temperature to find in the experimental design. After five minutes, transfer the heat exchanger into the ice cold water bath. Make sure the temperature of the extract is below 30 degrees Celsius before proceeding.Remove the inlet hose connected to the peristaltic pump from the insulated vessel. And continue pumping to collect residual heat precipitated plant extract from within the heat exchanger. Then stop the pump.To carry out bag filtration of the plant extract, mount a bag filter into the corresponding support basket fitted into the filter housing. Place a one liter vessel beneath the basket and apply the extract aliquots to the bag at a rate of 150 milliliters per minute. After filtration, use a turbidimeter to measure the turbidity of a one to 10 dilution of filtrate in extraction buffer.Aliquot a one milliliter sample and process the bag filtrate. For example, through depth filtration and chromatography. Further analyze the sample according to the text protocol.Heat precipitation of host cell proteins by blanching reduce the total soluble protein or TSP by 96 plus or minus one percent. Blanching recovered up to 83 plus or minus one percent of DsRed and 51 percent of the Vax8 target protein. Increasing its purity from 0.1 percent to 1.2 percent before chromatographic separation.The filter capacity declined at blanching temperatures greater than 63 degrees Celsius. Which can be addressed by adding flocculants after protein extraction and filter aids after bag filtration. A stirred vessel for heat precipitation removed a maximum of 84 plus or minus one percent HCPs.Achieving a purity of 0.33 plus or minus 0.02 percent for Vax8 and 20.2 plus or minus 1.4 percent for DsRed. In contrast to blanching and the heat exchanger setup HCP precipitation in a vessel increase the capacity of downstream depth filtration by 2.5-fold. Reflecting the lower sheer forces in the vessel compared to pumping extract through the heat exchanger or homogenization after blanching.Approximately 88.3 plus or minus 0.7 percent of the HCP content was consistently removed from the extract using a heat exchanger between 60 and 70 degrees Celsius. Achieving a purity of 0.31 plus or minus 0.01 percent for Vax8. And 27.6 plus or minus 2.0 percent for DsRed.Finally, the heat exchanger also achieved the lowest downstream depth filter capacity. Clarifying only 13.5 plus or minus 6.0 liters per square meter before clogging. Once mastered this technique can be done in less than 50 minutes plus sample if it's performed properly.While attempting this method it's important to remember to ensure that the target protein is heat stable. Following this procedure other methods like chromatography can be performed in order to further purify the product to the required level. After its development this technique paved the way for researchers in the field of plant by a technology to explore the purification of vaccine candidate proteins and nicotiana species.After watching this video you should have a good understanding of how to precipitate plant host cell proteins using blanching, stirred vessel or heat exchanger.

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