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

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.