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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</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;">Lauda E300</td>
			<td style="width:195px;">Lauda Dr Wobser GmbH</td>
			<td style="width:119px;">Z90010</td>
			<td>Water bath thermostat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">L/S 24</td>
			<td style="width:195px;">Masterflex</td>
			<td style="width:119px;">SN-06508-24</td>
			<td>Tubing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:360px;">mAb 5.2</td>
			<td style="width:195px;">American Type Culture Collection</td>
			<td style="width:119px;">HB-9148</td>
			<td>Vax8 specific antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:360px;">Masterflex L/S</td>
			<td style="width:195px;">Masterflex</td>
			<td style="width:119px;">HV-77921-75</td>
			<td>Peristaltic pump</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;">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>
		</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 and spectrometric plate reader</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>
		<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 ...

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