Our protocol demonstrates how to use a bench-scale acoustic wave separator for the primary separation of CHO cells from harvested culture fluid containing a model monoclonal antibody. Acoustic wave separation provides some advantages compared to traditional primary clarification methods, like centrifugation and filtration, such as small footprint, low contamination risk, and reduce filter fouling risk. Since acoustic wave separation provides a continuous flow of cell-free material for subsequent filtration and chromatography processes, there is potential for its application in continuous bioprocessing.
To prepare the acoustic wave separator, connect the turbidity cables to their respective ports and connect the chamber power BNC cables to the back of the acoustic wave separator system. Insert the turbidity probes into the turbidity meter and the thermometer housing and tighten the screws. Connect the feed tubing to the input of the feed turbidity port via the feed pump.
And connect the y-tubing from the output of the feed turbidity port to the inlet port of the acoustophoretic chamber. Connect the stage 1 tubing from the waste port of the acoustophoretic chamber to a cell collection vessel via the stage 1 pump, and connect the tubing from the permeate port of the acoustophoretic chamber to the input of probe 1 turbidity port. Then, connect the harvest tubing from out of the probe 1 turbidity port to a product collection vessel.
To prime the system with harvested cell culture fluid, turn on the acoustic wave separator and open the associated software program. In the Readings panel, click Start Test to initiate the data recording. And connect the feed tubing end into the harvested cell culture fluid vessel being stirred.
To start the feed pump, enter the pump rate and press Enter. Confirm that the pump direction arrow is correctly oriented to ensure that the cell fluid is pumped from the vessel into the acoustophoretic chamber, and click the triangle icon to start. Monitor the feed turbidity measurements in the Percent Reduction Panel during the filling of the acoustophoretic chamber.
If the harvested cell culture fluid is being mixed sufficiently within the cell fluid vessel, the turbidity values will be consistent during the loading of the chamber. Once the liquid is above the piezo transducer at the back of the acoustophoretic chamber, click Turn Off to stop the pump. And connect the other end of the BNC power cable to the acoustophoretic chamber.
Once the acoustophoretic chamber is filled and powered on, change the feed pump rate to the desired operating rate. To turn on the stage 1 piezo power, slide the bar in the power module to 10 watts and click Turn On.Once the cells begin to settle at the bottom of the acoustophoretic chamber, start the stage 1 pump with an appropriate rate based on the cell density and feed pump rate, while watching the acoustic wave separator chambers closely and adjusting the pump rates as necessary. To calculate the packed cell mass, tare a scale with an empty 15-milliliter tube and fill the tube with feed material.
After recording the total weight of the tube with feed, centrifuge the tube and decant the supernatant into a new container. Measure the weight of the tube with the cell pellet. Then, monitor the turbidity profile of the stage 1 turbidity as the overflow from the acoustophoretic chamber enters the turbidity probe 1.
When the run is over, stop the feed and stage 1 pumps, and turn off the power to the chamber. Disconnect the BNC power cable and collect the product harvest material for downstream analysis. To drain the remaining fluid from the acoustophoretic chamber, place the waste tubing into an empty vessel.
Disconnect the tubing from the acoustophoretic chamber inlet and permeate ports, and release the waste tubing from the pump head. When the chamber is empty, reconnect the tubing, place the waste tubing back into the pump head, and use a feed and stage 1 pump rate of 60 milliliters per minute to flush deionized water from the end of feed tubing for 15 to 20 minutes. At the end of the water rinse, pump 70%isopropyl alcohol through the tubing and chamber for 15 to 20 minutes, followed by an additional 15-to-20-minute deionized water rinse.
In this representative analysis, as the harvested cell fluid entered the acoustic wave separator chamber, the turbidity measurements from the feed turbidity probe remained consistent, around 1000 to 1100 NTU. And measurements from the probe 1 turbidity probe remained around 40 to 50 NTU. Keeping the harvested cell fluid within the chamber for longer time periods causes temperature increases, as in this example of a greater than six degrees Celsius temperature difference of the harvested cell fluid before entering the acoustophoretic chamber and after acoustic separation.
Another important consideration when running high cell density harvests is the saturation of the turbidity probes, as the turbidity measurements for the feed turbidity probe become saturated over 4600 NTU, likely resulting in an underestimation in the calculation of the cell removal efficiency. In addition, the cell removal efficiency decreases significantly from approximately 100%to 57%as the feed pump rate is increased, with a slower feed pump rate typically directly correlating with a better cell clarification. As manufacturers of biotechnology products move towards continuous manufacturing, the use of acoustic wave technology may have additional applications for continuous bioprocessing and profusion bioreactor operations.
