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; = 400 - 700 nm) and a 25/22 &#176;C light/dark temperature regime.</li>
	<li>Harvest all leaves except the four cotyledon leaves, which are located at the base of the plant stem.</li>
</ol>
2. Optional: Heat Precipitation by Blanching
NOTE: Carry out the steps described in steps 2.1 to 2.12 in order to precipitate tobacco HCPs by blanching. Skip the entire section 2, if the HCPs will be precipitated in a heated vessel (section 4) or using a heat exchanger (section 5).
<ol>
	<li>Set aside 50 g of plant material and carry out extraction without blanching (section 3). Take a sample of this extract as an internal control during subsequent analysis (section 7).</li>
	<li>Set up an 8-L working volume water bath, e.g., 50 x 40 x 40 cm, in a thermally-insulated polystyrene foam bucket or similar. Mount the adjustable thermostat on the water bath for subsequent temperature control (step 2.7).</li>
	<li>Transfer the entire assembly onto a magnetic stir plate and place a magnetic stir bar in the water bath. Ensure that the magnetic field is strong enough to rotate the stir bar.</li>
	<li>Surround the stir bar with at least four support tiles upon which a polypropylene basket (step 2.6) will be placed later during the blanching procedure. Make sure that the support tiles are made from a non-magnetic material (e.g., stainless steel alloy containing nickel) and that they are higher than the stir bar (Figure 2).</li>
	<li>Add 8 L of deionized water to the water bath. 
	NOTE: Using a buffer, e.g., 50 mM sodium phosphate (pH 7.5), can improve the target protein yields if low pH precipitation is an issue.</li>
	<li>Place a 23 x 23 x 23 cm polypropylene basket on the support tiles and make sure that the basket does not interfere with the stir bar rotation and that it is fully submerged in the liquid. If necessary, add additional water/buffer until the basket is fully submerged, then remove the basket again. 
	CAUTION: All subsequent steps up to 2.12 involve the handling of hot liquid. Wear appropriate personal protective equipment including thermally insulated gloves.</li>
	<li>Use the adjustable thermostat to bring the water bath to 70 &#176;C (or the temperature required for the experiments). Wait at least 15 min after the desired temperature is reached to ensure the entire assembly has reached a thermal equilibrium.</li>
	<li>Prepare 150 g aliquots from the harvested tobacco leaves. Place one aliquot in the basket while avoiding irreversible compression and damage to the leaves, e.g., by tearing. Avoid overfilling the basket with plant material or a dense packing of the latter.</li>
	<li>Carefully but quickly submerge the basket in the hot liquid and place it on the support tiles. Place a stainless steel block on top of the basket to prevent flotation.</li>
	<li>Incubate the leaves for 5 min in the blanching fluid, or select a time suiting the experimental design. Monitor the liquid temperature during the entire incubation period.</li>
	<li>Carefully remove the basket from the blanching fluid and let residual liquid drain from the leaves for 30 sec. Then take the plant leaves out of the basket, transfer them to the blender and immediately start the extraction (section 3). 
	NOTE: Plants can be kept for extended periods of time after blanching and before the start of the extraction, e.g., more than 30 min on ice or frozen at -20 &#176;C for several weeks has been successfully tested. However, product stability may decline with increasing storage times and thus immediate processing is recommended.</li>
	<li>Repeat steps 2.8 to 2.11 with fresh leaf material until the entire harvested biomass is processed.</li>
</ol>
3. Protein Extraction from Tobacco Leaves
CAUTION: The next steps involve a blender with rotating blades. Do not work in the blender bucket while it is mounted on the blender motor.
<ol>
	<li>Place 150 g (wet mass) of harvested (step 1.3) or blanched (step 2.11) leaves in the blender and add 450 ml of extraction buffer (50 mM sodium phosphate, 500 mM sodium chloride, 10 mM sodium disulfite, pH 8.0). 
	NOTE: The extraction buffer composition depends on the protein to be extracted and can thus require adjustment, e.g., use of another pH or buffer component such as Tris.</li>
	<li>Homogenize the leaves for 3 x 30 sec pulses with 30 sec interspersed breaks. Ensure that the leaves are homogenized and do not clog the blender bucket. Stop the blender and lift the leaves to prevent clogging if necessary, and then continue the homogenization.</li>
	<li>Take a 1 ml sample of each extract that is produced for subsequent analysis (section 7). If the plant material is blanched, continue to section 6. Otherwise continue with heat precipitation in a vessel (section 4) or heat exchanger (section 5), depending on the selected experimental approach.</li>
</ol>
4. Optional: Heat Precipitation in a Stirred Vessel
NOTE: Conduct the steps described in sections 4.2 to 4.11 in order to precipitate tobacco HCPs in a stirred vessel. Skip the entire section 4, if HCPs have been precipitated by blanching (section 2) or will be precipitated using a heat exchanger (section 5).
<ol>
	<li>Set up two 8-L working volume water baths, e.g., 50 x 40 x 40 cm, in thermally insulated polystyrene foam or similar. In the first bath, mount the adjustable thermostat for subsequent temperature control (step 4.6). In the second, add 5 L of deionized water and 2 kg of ice for subsequent cooling (step 4.9).</li>
	<li>Transfer the first water bath onto a magnetic stir plate, and place the 2-L stainless steel vessel into the water bath such that the center of the vessel is aligned to the center of the stir plate.</li>
	<li>Place a magnetic stir bar in the stainless-steel vessel. Ensure that the magnetic field is strong enough to rotate the stir bar.</li>
	<li>Fill the water bath with deionized water to 5 cm below the upper edge of the stainless steel vessel. Then fill the vessel with extraction buffer. Place a polystyrene foam lid on the vessel. 
	CAUTION: All subsequent steps up to 4.9 involve the handling of hot liquid. Wear appropriate personal protective equipment including thermally insulated gloves.</li>
	<li>Insert a thermometer into the stainless steel vessel through a suiting hole in the polystyrene foam lid. Set the water bath temperature to 78 &#176;C and incubate the entire assembly for 15 min to reach thermal equilibrium. Ensure that the temperature in the vessel is 70 &#176;C, approximately 8 &#176;C below the set point of the water bath.</li>
	<li>If the temperature in the stainless steel vessel differs from 70 &#176;C, adjust the temperature of the water bath accordingly and let the system equilibrate for another 15 min. Repeat this step until the temperature in the vessel is 70 &#176;C or as required for the experiment, and empty the stainless steel vessel.</li>
	<li>Pour 300 ml of extract (step 3.2) into the stainless-steel vessel while it is still in the water bath and start a timer. Stir the extract at 150 rpm and incubate for 5 min. Ensure that the extract reaches a temperature of 70 &#176;C for at least 2 min during this incubation period.</li>
	<li>Remove the hot water bath from the magnetic stirrer, take out the stainless steel vessel and place it in the ice-cold bucket. Place the latter onto the magnetic stirrer and remove the polystyrene foam lid.</li>
	<li>Ensure that the plant homogenate is well agitated at 150 rpm, place the thermometer in the extract and incubate until it reaches a temperature of 20 &#176;C or the temperature specified by the experimental design.</li>
	<li>Repeat steps 4.7 to 4.9 for all aliquots. Take a 1 ml sample of each heat precipitated extract once it has reached the final temperature, then proceed with section 6.</li>
</ol>
5. Optional: Heat Precipitation in a Heat Exchanger
NOTE: Conduct the steps described in sections 5.2 to 5.12 in order to precipitate tobacco HCPs using a heat exchanger. Skip the entire section 5, if HCPs have been precipitated by blanching (section 2) or in a heated vessel (section 4).
<ol>
	<li>Set up two 8-L working volume water baths, e.g., 50 x 40 x 40 cm, in thermally-insulated polystyrene foam buckets or similar. In the first bath, mount the adjustable thermostat for subsequent temperature control (step 5.3). In the second, add 5 L of deionized water and 2 kg of ice for subsequent cooling (step 5.10). 
	CAUTION: All subsequent steps up to 5.9 involve the handling of hot liquid. Wear appropriate personal protective equipment including thermally insulated gloves.</li>
	<li>Fill the first water bath with 8 L deionized water. Set the temperature to 74.5 &#176;C using the thermostat. Incubate the assembly for 15 min to reach thermal equilibrium.</li>
	<li>Prepare an insulated storage vessel by placing a 0.5-L plastic beaker into a 1-L plastic beaker and fill the gaps with cotton wool. Alternatively, use a dedicated thermally insulated vessel with a 0.5-L working volume.</li>
	<li>Connect the heat exchanger to the peristaltic pump at one end and to an outlet hose on the other end using L/S 24 tubing. Place both tubing ends along with a thermometer in the insulated storage vessel and fill it with 300 ml extraction buffer (Figure 2).</li>
	<li>Place the heat exchanger into the hot water bath and start the peristaltic pump at a rate of 300 ml min-1. Ensure that the resulting temperature in the vessel is 70 &#176;C, approximately 4.5 &#176;C below the set point of the water bath, after 3 min.</li>
	<li>If the temperature in the insulated-vessel differs from 70 &#176;C, adjust the temperature of the water bath accordingly and let the system equilibrate for another 15 min. Repeat this step until the temperature of the extraction buffer increases from ambient to 70 &#176;C in less than 3 min, or equals that required for the experiment, and empty the stainless-steel vessel.</li>
	<li>Discard the extraction buffer from the insulated vessel and prepare 300-ml aliquots of the plant extract. Fill the insulated vessel with one aliquot.</li>
	<li>Pump the plant extract (section 3.3) through the heat exchanger at 300 ml min-1 for 5 min. Ensure that the extract temperature is 70 &#176;C after 3 min, or equals the temperature defined in the experimental design.</li>
	<li>After 5 min, place the heat exchanger into the ice-cold water bath while the extract is still being pumped. Incubate in this setting until the temperature reaches exactly 20 &#176;C, or the temperature defined in the experimental design.</li>
	<li>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.</li>
	<li>Repeat steps 5.7 to 5.10 for all aliquots. Take a 1-ml sample of each heat-precipitated extract once it has reached the final temperature, then pass on the extracts to bag filtration (section 6). 
	NOTE: After a first cycle through steps 5.7 to 5.10 there will be no extraction buffer to discard in step 5.7.</li>
</ol>
6. Bag Filtration of the Plant Extract
<ol>
	<li>Mount a bag filter into the corresponding support basket which is fitted into the filter housing and provides mechanical support for the flexible filter material. Place a 1-L vessel beneath the basket and apply the extract aliquots (section 3.3, 4.10 or 5.11 depending on the selected heat treatment) to the bag at a rate of 150 ml min-1.</li>
	<li>After filtration, measure the turbidity of a 1:10 dilution of filtrate in extraction buffer using the turbidimeter.</li>
	<li>Take a 1 ml sample and process the bag filtrate as defined in the experimental design, e.g., depth filtration and chromatography 7,10.</li>
</ol>
7. Sample Analysis
<ol>
	<li>Measure the quantity of total soluble protein (TSP) using the Bradford method 24,25.
	<ol>
		<li>In triplicate, pipette 2.5 &#181;l of each sample into the single wells of a 96-well plate. Include eight bovine serum albumin (BSA) standards in triplicate covering the range 0 - 2,000 &#181;g ml-1.</li>
		<li>Add 200 &#181;l Bradford reagent to each well and mix thoroughly by pipetting up and down but gently enough to avoid forming bubbles.</li>
		<li>Incubate for 10 min at 22 &#176;C and measure the absorbance at 595 nm in a spectrophotometer. Calculate the TSP concentration in the samples based on a standard curve through the BSA reference points.</li>
	</ol>
	</li>
	<li>Quantify Vax8 by surface plasmon resonance spectroscopy 26.
	<ol>
		<li>Setup the surface plasmon resonance instrument with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) running buffer (10 mM HEPES, 3 mM ethylenediaminetetraacetic acid (EDTA), 1,500 mM sodium chloride, 0.05% v/v polysorbate 20, pH 7.4) and mount a carboxymethylated dextran surface chip into the device. Use the prime function to flush the system and start a manual run with a flow rate of 30 &#181;l min-1 over flow cells 1 and 2. Inject 30 mM hydrogen chloride twice for 60 sec with a 60 sec interspersed injection of 25 mM sodium hydroxide.</li>
		<li>Prepare 200 &#181;l of a 500 &#181;g ml-1 mAb 5.2 solution in 10 mM sodium acetate (pH 4.0). Thaw 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) vials and centrifuge them at 16,000 x g for 1 min. Mix 70 &#181;l of EDC with 70 &#181;l of NHS and transfer to a 7-mm plastic vial.</li>
		<li>Activate the carboxymethylated dextran surface chip surface by injecting the EDC/NHS mixture over flow cells 1 and 2 for 10 min with a flow rate of 10 &#181;l min-1.</li>
		<li>Set the flow path to flow cell 2 only. Couple mAb 5.2 by injecting it for 15 min at a rate of 10 &#181;l min-1.</li>
		<li>Switch the flow path to flow cells 1 and 2 and inject ethanolamine for 7 min at a rate of 5 &#181;l min-1 to deactivate the surface. Then inject 30 mM hydrogen chloride twice for 60 sec with a 60 sec interspersed injection of 25 mM sodium hydroxide.</li>
		<li>Inject the samples and MSP1 - 19 standards at a rate of 30 &#181;l min-1 for 180 sec. Ensure that the Vax8 concentration is 50 - 1,000 ng ml-1. Prepare pre-dilutions in HEPES buffered saline containing EDTA and polysorbate 20 (HBSEP) if necessary.</li>
		<li>Subtract the peak signal of flow cell 1 from that of flow cell 2 and use the difference to calculate the Vax8 concentration in samples based on the signal of the MSP 1 - 19 injection with a known protein concentration of 500 ng ml-1.</li>
		<li>Regenerate the chip surface after each sample or MSP 1 - 19 injection by exposure to 30 mM hydrogen chloride for 60 sec at a flow rate of 30 &#181;l min-1.</li>
	</ol>
	</li>
	<li>Quantify DsRed by fluorescence spectrometry
	<ol>
		<li>In triplicate, pipette 50 &#181;l of each sample into the single wells of a black half-area 96-well plate. Include six DsRed standards covering the range 0 - 225 &#181;g ml-1.</li>
		<li>Measure the fluorescence in duplicate using a 530 &#177; 30 nm excitation filter and a 590 &#177; 35 nm emission filter in a spectrophotometer. Calculate the DsRed concentration in the samples based on a standard curve through the DsRed reference points.</li>
	</ol>
	</li>
	<li>Determination of particle size distributions
	<ol>
		<li>Wash a cuvette with 1 ml isopropanol and then with 2 ml of deionized water to remove dust particles. Pipette 850 &#181;l of the sample into the cuvette.</li>
		<li>Place the cuvette into the zeta potential and particle size analyzer. Open the &#34;Software&#34; and start a manual measurement with &#34;Protein&#34; as the material and &#34;PBS&#34; as the dispersant. Select a temperature of 25 &#176;C and an equilibration time of 180 sec.</li>
		<li>Choose the &#34;DTS0012&#34; cell and measure at 173&#176; backscatter. Check the &#34;auto&#34; function for the measurement duration and select three measurements with no interspersed delay.</li>
		<li>Investigate the particle size distribution peak profile once the measurement is complete by selecting the three measurements of the sample in the experiment view. Choose the &#34;Volume PSD&#34; tab.</li>
	</ol>
	</li>
</ol>

<ol><li>PhRMA. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%222013+Report%3A+Medicines+in+Development+-+Biologics%22">2013 Report: Medicines in Development - Biologics</a>. , Pharmaceutical Research and Manufacturers of America. Washington, D.C., USA. (2013).</li>
<li>Buyel, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Process+development+strategies+in+plant+molecular+farming%5BTitle%5D)+AND+%22Curr.+Pharm.+Biotechnol%22%5BJournal%5D)">Process development strategies in plant molecular farming.</a> Curr. Pharm. Biotechnol. 16, 966-982 (2015).</li>
<li>Stoger, E., Fischer, R., Moloney, M., Ma, J. K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant+molecular+pharming+for+the+treatment+of+chronic+and+infectious+diseases%5BTitle%5D)+AND+%22Annu.+Rev.+Plant+Biol%22%5BJournal%5D)">Plant molecular pharming for the treatment of chronic and infectious diseases.</a> Annu. Rev. Plant Biol. 65, 743-768 (2014).</li>
<li>Melnik, S., Stoger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Green+factories+for+biopharmaceuticals%5BTitle%5D)+AND+%22Curr.+Med.+Chem%22%5BJournal%5D)">Green factories for biopharmaceuticals.</a> Curr. Med. Chem. 20, 1038-1046 (2013).</li>
<li>Buyel, J. F., Twyman, R. M., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extraction+and+downstream+processing+of+plant-derived+recombinant+proteins%5BTitle%5D)+AND+%22Biotechnol.+Adv%22%5BJournal%5D)">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/pubmed?term=((Recovery+and+purification+of+plant-made+recombinant+proteins%5BTitle%5D)+AND+%22Biotechnol.+Adv%22%5BJournal%5D)">Recovery and purification of plant-made recombinant proteins.</a> Biotechnol. Adv. 30, 419-433 (2012).</li>
<li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scale-down+models+to+optimize+a+filter+train+for+the+downstream+purification+of+recombinant+pharmaceutical+proteins+produced+in+tobacco+leaves%5BTitle%5D)+AND+%22Biotechnol.+J%22%5BJournal%5D)">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/pubmed?term=((Flocculation+increases+the+efficacy+of+depth+filtration+during+the+downstream+processing+of+recombinant+pharmaceutical+proteins+produced+in+tobacco%5BTitle%5D)+AND+%22Plant+Biotechnol.+J%22%5BJournal%5D)">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/pubmed?term=((Cellulose-based+filter+aids+increase+the+capacity+of+depth+filters+during+the+downstream+processing+of+plant-derived+biopharmaceutical+proteins%5BTitle%5D)+AND+%22Biotechnol.+J%22%5BJournal%5D)">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>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generic+chromatography-based+purification+strategies+accelerate+the+development+of+downstream+processes+for+biopharmaceutical+proteins+produced+in+plants%5BTitle%5D)+AND+%22Biotechnol.+J%22%5BJournal%5D)">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>Buyel, J. F., Woo, J. A., Cramer, S. M., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+quantitative+structure-activity+relationship+models+to+develop+optimized+processes+for+the+removal+of+tobacco+host+cell+proteins+during+biopharmaceutical+production%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">The use of quantitative structure-activity relationship models to develop optimized processes for the removal of tobacco host cell proteins during biopharmaceutical production.</a> J. Chromatogr. A. 1322, 18-28 (2013).</li>
<li>Holler, C., Vaughan, D., Zhang, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polyethyleneimine+precipitation+versus+anion+exchange+chromatography+in+fractionating+recombinant+beta-glucuronidase+from+transgenic+tobacco+extract%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">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/pubmed?term=((Downstream+processing+of+biopharmaceutical+proteins+produced+in+plants%3A+the+pros+and+cons+of+flocculants%5BTitle%5D)+AND+%22Bioengineered%22%5BJournal%5D)">Downstream processing of biopharmaceutical proteins produced in plants: the pros and cons of flocculants.</a> Bioengineered. 5, 138-142 (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/pubmed?term=((Considerations+for+extraction+of+monoclonal+antibodies+targeted+to+different+subcellular+compartments+in+transgenic+tobacco+plants%5BTitle%5D)+AND+%22Plant+Biotechnol.+J%22%5BJournal%5D)">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>Arfi, Z. A., Drossard, J., Hellwig, S., Fischer, R., Buyel, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polyclonal+antibodies+can+effectively+detect+tobacco+host+cell+proteins+after+RuBisCO+depletion+and+endotoxin+removal%5BTitle%5D)+AND+%22Biotechnol.+J%22%5BJournal%5D)">Polyclonal antibodies can effectively detect tobacco host cell proteins after RuBisCO depletion and endotoxin removal.</a> Biotechnol. J. , (2015).</li>
<li>Buyel, J. F., Gruchow, H. M., Boes, A., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rational+design+of+a+host+cell+protein+heat+precipitation+step+simplifies+the+subsequent+purification+of+recombinant+proteins+from+tobacco%5BTitle%5D)+AND+%22Biochem.+Eng.+J%22%5BJournal%5D)">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>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+juice+extractor+can+simplify+the+downstream+processing+of+plant-derived+biopharmaceutical+proteins+compared+to+blade-based+homogenizers%5BTitle%5D)+AND+%22Process+Biochem%22%5BJournal%5D)">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>Beiss, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heat-precipitation+allows+the+efficient+purification+of+a+functional+plant-derived+malaria+transmission-blocking+vaccine+candidate+fusion+protein%5BTitle%5D)+AND+%22Biotechnol.+Bioeng%22%5BJournal%5D)">Heat-precipitation allows the efficient purification of a functional plant-derived malaria transmission-blocking vaccine candidate fusion protein.</a> Biotechnol. Bioeng. 112, 1297-1305 (2015).</li>
<li>Ma, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulatory+approval+and+a+first-in-human+phase+I+clinical+trial+of+a+monoclonal+antibody+produced+in+transgenic+tobacco+plants%5BTitle%5D)+AND+%22Plant+Biotechnol.+J%22%5BJournal%5D)">Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants.</a> Plant Biotechnol. J. 13, 1106-1120 (2015).</li>
<li>Mahajan, P. V., Caleb, O. J., Singh, Z., Watkins, C. B., Geyer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Postharvest+treatments+of+fresh+produce%5BTitle%5D)+AND+%22Philos+T+R+Soc+A%22%5BJournal%5D)">Postharvest treatments of fresh produce.</a> Philos T R Soc A. 372, (2014).</li>
<li>Menzel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+blanching+reduces+the+host+cell+protein+content+and+substantially+enhances+the+recovery+and+stability+of+two+plant+derived+malaria+vaccine+candidates%5BTitle%5D)+AND+%22Front.+Plant+Sci%22%5BJournal%5D)">Optimized blanching reduces the host cell protein content and substantially enhances the recovery and stability of two plant derived malaria vaccine candidates.</a> Front. Plant Sci. , (2015).</li>
<li>Buyel, J. F., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+complex+systems+using+the+design+of+experiments+approach%3A+transient+protein+expression+in+tobacco+as+a+case+study%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">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>Buyel, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Procedure+to+evaluate+the+efficiency+of+flocculants+for+the+removal+of+dispersed+particles+from+plant+extracts%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Procedure to evaluate the efficiency of flocculants for the removal of dispersed particles from plant extracts.</a> J. Vis. Exp. , e53940(2016).</li>
<li>Simonian, M. H., Smith, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spectrophotometric+and+colorimetric+determination+of+protein+concentration%5BTitle%5D)+AND+%22Curr.+Protoc.+Mol.+Biol%22%5BJournal%5D)">Spectrophotometric and colorimetric determination of protein concentration.</a> Curr. Protoc. Mol. Biol. 76, (2006).</li>
<li>Buyel, J. F., Kaever, T., Buyel, J. J., Fischer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Predictive+models+for+the+accumulation+of+a+fluorescent+marker+protein+in+tobacco+leaves+according+to+the+promoter%2F5%27UTR+combination%5BTitle%5D)+AND+%22Biotechnol.+Bioeng%22%5BJournal%5D)">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>Piliarik, M., Vaisocherova, H., Homola, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surface+plasmon+resonance+biosensing%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">Surface plasmon resonance biosensing.</a> Methods Mol. Biol. 503, 65-88 (2009).</li>
<li>Kim, T. D., Ryu, H. J., Cho, H. I., Yang, C. H., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Thermal+behavior+of+proteins%3A+Heat-resistant+proteins+and+their+heat-induced+secondary+structural+changes%5BTitle%5D)+AND+%22Biochemistry-Us%22%5BJournal%5D)">Thermal behavior of proteins: Heat-resistant proteins and their heat-induced secondary structural changes.</a> Biochemistry-Us. 39, 14839-14846 (2000).</li>
<li>Kwon, S., Jung, Y., Lim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proteomic+analysis+of+heat-stable+proteins+in+Escherichia+coli%5BTitle%5D)+AND+%22Bmb+Rep%22%5BJournal%5D)">Proteomic analysis of heat-stable proteins in Escherichia coli.</a> Bmb Rep. 41, 108-111 (2008).</li>
<li>Komarnytsky, S., Borisjuk, N. V., Borisjuk, L. G., Alam, M. Z., Raskin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+recombinant+proteins+in+tobacco+guttation+fluid%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Production of recombinant proteins in tobacco guttation fluid.</a> Plant Physiol. 124, 927-933 (2000).</li>
<li>Drake, P. M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+rhizosecretion+as+a+production+system+for+recombinant+proteins+from+hydroponic+cultivated+tobacco%5BTitle%5D)+AND+%22FASEB+J%22%5BJournal%5D)">Development of rhizosecretion as a production system for recombinant proteins from hydroponic cultivated tobacco.</a> FASEB J. 23, 3581-3589 (2009).</li>
<li>Turpen, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tobacco+mosaic+virus+and+the+virescence+of+biotechnology%5BTitle%5D)+AND+%22Philos.+Trans.+R.+Soc.+Lond.%2C+Ser.+B%3A+Biol.+Sci%22%5BJournal%5D)">Tobacco mosaic virus and the virescence of biotechnology.</a> Philos. Trans. R. Soc. Lond., Ser. B: Biol. Sci. 354, 665-673 (1999).</li>
<li>Kingsbury, N. J., McDonald, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+Evaluation+of+E1+Endoglucanase+Recovery+from+Tobacco+Leaves+Using+the+Vacuum+Infiltration-Centrifugation+Method%5BTitle%5D)+AND+%22Biomed.+Res.+Int%22%5BJournal%5D)">Quantitative Evaluation of E1 Endoglucanase Recovery from Tobacco Leaves Using the Vacuum Infiltration-Centrifugation Method.</a> Biomed. Res. Int. , (2014).</li>
<li>Buyel, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Numeric+simulation+can+be+used+to+predict+heat+transfer+during+the+blanching+of+leaves+and+intact%5BTitle%5D)+AND+%22Biochem.+Eng.+J%22%5BJournal%5D)">Numeric simulation can be used to predict heat transfer during the blanching of leaves and intact.</a> Biochem. Eng. J. , (2015).</li>
<li>Mandal, M. K., Fischer, R., Schillberg, S., Schiermeyer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+protease+activity+by+antisense+RNA+improves+recombinant+protein+production+in+Nicotiana+tabacum+cv.+Bright+Yellow+2+%28BY-2%29+suspension+cells%5BTitle%5D)+AND+%22Biotechnol.+J%22%5BJournal%5D)">Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells.</a> Biotechnol. J. 9, 1065-1073 (2014).</li>
<li>Welty, J. R., Wicks, C. E., Wilson, R. E. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWelty%2C+JR+%22Fundamentals+of+momentum%2C+heat%2C+and+mass+transfer%22">Fundamentals of momentum, heat, and mass transfer</a>. , Wiley. (1976).</li>
<li>Lowe, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aggregation%2C+stability%2C+and+formulation+of+human+antibody+therapeutics%5BTitle%5D)+AND+%22Advances+in+protein+chemistry+and+structural+biology%22%5BJournal%5D)">Aggregation, stability, and formulation of human antibody therapeutics.</a> Advances in protein chemistry and structural biology. 84, 41-61 (2011).</li>
<li>Gong, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineered+human+antibody+constant+domains+with+increased+stability%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Engineered human antibody constant domains with increased stability.</a> J. Biol. Chem. 284, 14203-14210 (2009).</li>
<li>Rouet, R., Lowe, D., Christ, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stability+engineering+of+the+human+antibody+repertoire%5BTitle%5D)+AND+%22FEBS+Lett%22%5BJournal%5D)">Stability engineering of the human antibody repertoire.</a> FEBS Lett. 588, 269-277 (2014).</li>
<li>Stabel, J. R., Lambertz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficacy+of+pasteurization+conditions+for+the+inactivation+of+Mycobacterium+avium+subsp+paratuberculosis+in+milk%5BTitle%5D)+AND+%22J.+Food+Prot%22%5BJournal%5D)">Efficacy of pasteurization conditions for the inactivation of Mycobacterium avium subsp paratuberculosis in milk.</a> J. Food Prot. 67, 2719-2726 (2004).</li>
<li>Wichers, H. Ch. 12. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWichers%2C+H+%22Managing+Allergens+in+Food%22">Managing Allergens in Food</a>. Mills, C., Wichers, H., Hoffmann-Sommergruber, K. , Elsevier Science. 336(2006).</li>
<li>Davis, P. J., Williams, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+modification+by+thermal+processing%5BTitle%5D)+AND+%22Allergy%22%5BJournal%5D)">Protein modification by thermal processing.</a> Allergy. 53, 102-105 (1998).</li>
<li>Dubois, M. F., Hovanessian, A. G., Bensaude, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heat-shock-induced+denaturation+of+proteins.+Characterization+of+the+insolubilization+of+the+interferon-induced+p68+kinase%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Heat-shock-induced denaturation of proteins. Characterization of the insolubilization of the interferon-induced p68 kinase.</a> J. Biol. Chem. 266, 9707-9711 (1991).</li>
</ol>

The authors have no conflicts of interest to disclose.

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>

<table><tbody><tr><td>2100P Portable Turbidimeter</td><td>Hach</td><td>4650000</td><td>Turbidimeter</td></tr><tr><td>Amine Coupling Kit</td><td>GE Healthcare</td><td>BR100050&amp;nbsp;</td><td>SPR chip coupling kit</td></tr><tr><td>Autoclaving basket</td><td>Nalgene</td><td>6917-0230</td><td>Basket for leaf blanching</td></tr><tr><td>Biacore T200</td><td>GE Healthcare</td><td>28-9750-01</td><td>SPR device</td></tr><tr><td>Bio Cell Analyser BCA 003 R&amp;amp;D with 3D ORM</td><td>Sequip</td><td>n.a.</td><td>Particle size analyzer</td></tr><tr><td>Blender</td><td>Waring</td><td>800EG</td><td>Blender</td></tr><tr><td>BP-410</td><td>Furh</td><td>2632410001</td><td>Bag filter</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>CM5 chip</td><td>GE Healthcare</td><td>BR100012&amp;nbsp;</td><td>Chip for SPR measurements</td></tr><tr><td>Cuvette 10 x 10 x 45</td><td>Sarsted</td><td>67.754</td><td>Cuvette for Zetasizer Nano ZS</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>Twentey-loop heat exchanger (4.8 m length)</td><td>n.a. (custom design)</td><td>n.a.</td><td>Heat exchanger</td></tr><tr><td>HEPES</td><td>Carl Roth GmbH</td><td>9105.3</td><td>Media component</td></tr><tr><td>K200P 60D</td><td>Pall</td><td>5302303</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>Lauda E300</td><td>Lauda Dr Wobser GmbH</td><td>Z90010</td><td>Water bath thermostat</td></tr><tr><td>L/S 24</td><td>Masterflex</td><td>SN-06508-24</td><td>Tubing</td></tr><tr><td>mAb 5.2</td><td>American Type Culture Collection</td><td>HB-9148</td><td>Vax8 specific antibody</td></tr><tr><td>Masterflex L/S</td><td>Masterflex</td><td>HV-77921-75</td><td>Peristaltic pump</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>Sodium disulfite</td><td>Carl Roth GmbH</td><td>8554.1</td><td>Media component</td></tr><tr><td>Sodium chloride</td><td>Carl Roth GmbH</td><td>P029.2</td><td>Media component</td></tr><tr><td>Stainless-steel vessel; 0.7 kg 2.0 L; height 180 mm; diameter 120 mm</td><td>n.a. (custom design)</td><td>n.a.</td><td>Container for heat precipitation</td></tr><tr><td>Synergy HT</td><td>BioTek</td><td>SIAFRT</td><td>Fluorescence and spectrometric plate reader</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><tr><td>Zetasizer Software v7.11</td><td>Malvern</td><td>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 ...

comparison-tobacco-host-cell-protein-removal-methods-blanching-intact

Research

JoVE Journal

Biology

9.0K Views.  Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V..  Drie warmte precipitatiewerkwijzen gepresenteerd die effectief meer dan 90% van gastheerceleiwitten (HCP) van tabak extracten te verwijderen vóór alle andere zuiveringsstap. De plant HCPs irreversibel aggregeren bij temperaturen boven 60 ° C. 

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

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Genetics

A Rapid Laser Probing Method Facilitates the Non-invasive and Contact-free Determination of Leaf Thermal Properties

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be streamlined by heat treatment (blanching). A blanching apparatus can be designed more precisely if the thermal properties of the leaves are known in detail, i.e., the specific heat capacity and thermal conductivity. The measurement of these properties is time consuming and labor intensive, and usually requires invasive methods that contact the sample directly. This can reduce the product yield and may be incompatible with containment requirements, e.g., in the context of good manufacturing practice. To address these issues, a non-invasive, contact-free method was developed that determines the specific heat capacity and thermal conductivity of an intact plant leaf in about one minute. The method involves the application of a short laser pulse of defined length and intensity to a small area of the leaf sample, causing a temperature increase that is measured using a near infrared sensor. The temperature increase is combined with known leaf properties (thickness and density) to determine the specific heat capacity. The thermal conductivity is then calculated based on the profile of the subsequent temperature decline, taking thermal radiation and convective heat transfer into account. The associated calculations and critical aspects of sample handling are discussed.

A method was developed to determine the specific heat capacity and thermal conductivity of leaf tissue by non-invasive, contact-free near infrared laser probing, which requires less than 1 min per sample.

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be ...

Biochemistry

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to possess medicinally relevant biological activity. However, thus far this potential has not translated into a plethora of triterpene-derived drugs in the clinic. This is arguably (at least partially) a consequence of limited practical synthetic access to this class of compound, a problem that can stifle the exploration of structure-activity relationships and development of lead candidates by traditional medicinal chemistry workflows. Despite their immense diversity, triterpenes are all derived from a single linear precursor, 2,3-oxidosqualene. Transient heterologous expression of biosynthetic enzymes in N. benthamiana can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products that are not naturally produced by this host. Agro-infiltration is an efficient and simple means of achieving transient expression in N. benthamiana. The process involves infiltration of plant leaves with a suspension of Agrobacterium tumefaciens carrying the expression construct(s) of interest. Co-infiltration of an additional A. tumefaciens strain carrying an expression construct encoding an enzyme that boosts precursor supply significantly increases yields. After a period of five days, the infiltrated leaf material can be harvested and processed to extract and isolate the resulting triterpene product(s). This is a process that is linearly and reliably scalable, simply by increasing the number of plants used in the experiment. Herein is described a protocol for rapid preparative-scale production of triterpenes utilizing this plant-based platform. The protocol utilizes an easily replicable vacuum infiltration apparatus, which allows the simultaneous infiltration of up to four plants, enabling batch-wise infiltration of hundreds of plants in a short period of time.

Transient Expression in Nicotiana Benthamiana Leaves for Triterpene Production at a Preparative Scale

Transient heterologous expression of biosynthetic enzymes in Nicotiana benthamiana leaves can divert endogenous supplies of 2,3-oxidosqualene towards the production of new high-value triterpene products. Herein is described a detailed protocol for rapid (5 days) preparative-scale production of triterpenes and analogs utilizing this powerful plant-based platform.

The triterpenes are one of the largest and most structurally diverse families of plant natural products. Many triterpene derivatives have been shown to ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Bioengineering

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.

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

Methods to Evaluate Cytotoxicity and Immunosuppression of Combustible Tobacco Product Preparations

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to increased susceptibility of smokers to microbial infections and tumor incidence. Ex vivo suppression of receptor-mediated immune responses in human peripheral blood mononuclear cells (PBMCs) treated with smoke constituents is an attractive approach to study mechanisms and evaluate the likely long-term effects of exposure to tobacco products. Here, we optimized methods to perform ex vivo assays using PBMCs stimulated by bacterial lipopolysaccharide, a Toll-like receptor-4 ligand. The effects of whole smoke-conditioned medium (WS-CM), a combustible tobacco product preparation (TPP), and nicotine were investigated on cytokine secretion and target cell killing by PBMCs in the ex vivo assays. We show that secreted cytokines IFN-&#947;, TNF, IL-10, IL-6, and IL-8 and intracellular cytokines IFN-&#947;, TNF-&#945;, and MIP-1&#945; were suppressed in WS-CM-exposed PBMCs. The cytolytic function of effector PBMCs, as determined by a K562 target cell killing assay was also reduced by exposure to WS-CM; nicotine was minimally effective in these assays. In summary, we present a set of improved assays to evaluate the effects of TPPs in ex vivo assays, and these methods could be readily adapted for testing other products of interest.

Using optimized human peripheral blood mononuclear cell (PBMC) ex vivo assays, we showed that a combustible tobacco product preparation markedly suppresses receptor-mediated intracellularly secreted cytokines and cytolytic ability of effector PBMCs. These rapid assays may be useful in product evaluation and understanding the potential long-term effects of tobacco exposure.

Among other pathophysiological changes, chronic exposure to cigarette smoke causes inflammation and immune suppression, which have been linked to ...

Immunology and Infection

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the flexible, low-cost production of high-quality, bioactive products.
In this study, we compared the expression of a target human recombinant protein in traditional fermenter-based cell cultures (bacterial and insect) with plant-based expression systems, both transient and stable.
For each platform, we described the set-up, optimization and length of the production process, the final product quality and the yields and we evaluated provisional production costs, specific for the selected target recombinant protein.
Overall, our results indicate that bacteria are unsuitable for the production of the target protein due to its accumulation within insoluble inclusion bodies. On the other hand, plant-based systems are versatile platforms that allow the production of the selected protein at lower-costs than Baculovirus/insect cell system. In particular, stable transgenic lines displayed the highest-yield of the final product and transient expressing plants the fastest process development. However, not all recombinant proteins may benefit from plant-based systems but the best production platform should be determined empirically with a case-by-case approach, as described here.

In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.

Plant-based systems are considered a valuable platform for the production of recombinant proteins as a result of their well-documented potential for the ...

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

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