In this video, the users introduced into the micro bioreactor software with planning and execution of design of experiments or DOE. The analyzers auxiliary valves are also demonstrated to optimize the process condition. The incorporation of modet into the automated micro bioreactor software has been official for the data analysis.
A large number of experiments can be planned and executed simultaneously in small scale. Demonstrating the procedure will be Tamanna Nagraik, A PhD student from my laboratory. Begin with the pre-culture procedure in main cultivation as described in the text protocol.
To create a new experiment, open the Amber self-culture software, and in the introduction tab, click on, create new experiment. In the new experiment tab, enter the name of the experiment along with the date on which it will be conducted. Activate the checkpoint for the culture station in the vessels to be used during the cultivation.
The auto-add DOE tags will also be activated for an easy transition during the programming of the DOE experiment. Click on next"to switch to the next tab. To set information about addition of media into the vessel, along with anti-foam, inoculum, feed, and glucose, activate the add media plate"checkpoint.
Define the plate type, name, and location of the plate containing the medium. Click on add media to vessels. Enter the volume of the media to be added into the vessels.
Define the mapping of media transfer from the plate to the vessels. Click on next"to switch to the next tab. After the media information has been fed into the software, assign the cultivation conditions.
Click on condition media, and fill in the temperature, target DO, upper PH limit, and stirring RPM. Then click Up stirring"or down stirring. To set addition of inoculums into the vessels, activate add cell plate.
Define the plate type, name, and location of the plate containing the medium. Click on add cells to vessels. Enter the time of inoculation and the volume of the media to be added to the vessels.
Define the path traveled by the liquid handler to transfer the cell from the plate to the vessels. Click on next"to switch to the next tab. To set addition of feed, glucose, and the anti-foam, activate the add feed plate, and define the plate type, name, and location.
Click on add feed to vessels, and enter the volume of the feed to be added to the vessels. Depending on the cultivation, add the number of feed addition. For this cultivation, the reactor is fed after 72 hours every 24 hours.
Manually add the time delay between the feeding by entering the data into delay from cells added. The first day of feeding is after 72 hours of inoculation, the next one is after 96 hours, and so on. Define the mapping of the transfer of the feed from the plate to the vessels.
To set sampling during the cultivation, activate the add sample plate"and define the plate type, name, and location. Check take sample from vessels"and enter the volume of the sample to be removed from the vessels. Ensure that the volume does not decrease below 10 mililiters during the entire course of cultivation.
Add the number of samples to be taken during the cultivation. Similar to feeding, add the time of the sample being removed from the vessel for each input sample point. Save the process.
It is now ready for execution. Finally, define the mapping of the transfer of the sample from the vessels to the plate. Open the Amber 15 DOE Software.
Click on investigation"and select new. Enter the name of the new DOE investigation in the create investigation"dialogue box. In order to assign an experiment to the DOE investigation, open the recipe created to study the different parameters.
Click on browse, and select the respective experiment. The vessel tags are already enlisted in the column. To define the desired DOE factor, select the parameter and click on the column labeled, DOE factor.
Select new"and add the units, abbreviation, as well as lower and upper limit of the factors. In the responses tab, define the values to be considered for the analysis of the data. Click on edit DOE responses, and define the name of the response, abbreviation, units, and minimum and maximum limits.
Once the responses are defined, select the amber variable for each response, and define. A response can be automatically associated with a microbe bioreactor variable. Choose the required variable from the dropdown list.
Change the equation for each response depending on the requirement. The choices between the minimum, maximum, first, last, and average data. To create a design, use the Start Design Wizard to select the type of experimental design and to add or remove the number of replicates and center points.
Select the objective, which determines the choice of designs and models. Finish and create work packets that can be imported into the Amber self-culture Software as described in the text protocol. In the experiment tab, click on create DOE experiment, and browse for the work packet created using the DOE software.
Initialize the process by clicking start. Once the experiment has been executed, export the data using export DOE results. The export DOE results"window opens, and the rows indicating the culture vessel and station are listed in the table.
Select the desired rows, and click on export selected rows"or export experimental data"to store all the results and save the file for further analysis. Import the data into the Amber DOE Module by switching to the results"tab and selecting import results. Browse for the desired data file, and click analyze results.
The cell growth in the automated microbe bioreactors is comparable with the multiuse bioreactors. In comparing the cell concentration from three different scales, it is observed that the 15 milliliter automated microbe bioreactor mimics the two liter glass bioreactor. The results from the shake flask are also compared to exhibit the benefit of Amber.
The influence of different stir speed and PH is studied in the automated microbe bioreactors. Shown here is a comparison of the viable cell concentration, or VCC, and monoclonal antibody concentration in the different microbe bioreactors. The negative influence of PH 6.9 on the VCC can be observed.
Additionally, the growth of the cells improved significantly under the culture at PH 7.3 compared with PH 7.1. Shown here are the response contour plots of VCC and monoclonal antibody concentration. The values are comparable in the vessels with the same PH and different stirrer speed, indicating that the stirrer speed collected for this process has no significant influence on the process output.
Every instruction given to the machine must be carefully written to avoid error during the execution of the experiment. The software is flexible and it can run a number of experiments at once, thereby reducing the amount of time taken to optimize a process. The design of experiment is useful in the field of bioprocess, as the results give knowledge-based process understanding, which can be extended to other organisms to a certain extent.
