In RoboColumn workflows, optimization of the buffer (e.g. pH) and sample composition (e.g. concentration) are often as important or more so than process factors (e.g. flow rate and load volumes).
In this tutorial you will learn:
To describe pre-made buffers and samples to load in RoboColumn DOE workflows
To describe buffers and samples to load by your desired composition in RoboColumn DOE workflows
To get started, create a basic process factor type RoboColumn DOE workflow. To learn how, click here.
How to describe pre-made buffers and samples in RoboColumn DOE workflows
Define your samples and buffers using the Define Liquids and Plates element
For example, here two buffers and two samples are defined:
2. In this example, enable Buffers/Samples to Load as DOE factors in the Equilibrate Run Chromatography Stage element by switching your workflow into DOE mode and toggling the parameter on in the instance panel.
3. Select the predefined liquids as factor levels in the factor editor. To learn how to define a categorical liquid factor, click here.
How to link buffers and samples to load across stages by a common property
Often you will want to be able to maintain specific liquid properties across multiple stages of your purification process, for example, you might want to use the same pH buffer at the equilibration, load, and wash stages. Follow these steps to achieve this:
Add a custom numeric factor of pH. To learn how to define custom numeric factors in more detail, click here.
Enable Buffers/Samples to Load as DOE factors in relevant Run Chromatography Stage elements. To learn how to define parameters as factors in more detail, click here.
Add a derived factor for each element’s Buffers/Samples to Load and match the factor level to the sample/buffer. To learn how to define derived categorical factors in more detail, click here.
How to describe buffers and samples to load by your desired composition in RoboColumn DOE workflows
Often you might want to investigate a combination of buffer properties at various stages of your purification process to understand which buffer conditions perform the best. Follow these steps to learn how to define the buffer compositions driven by DOE.
Add a Make Mixtures element before the Run Chromatography Stage of interest
Follow the instructions on describing mixture composition factors in more detail by clicking here. In this example, we vary the protein concentration and the buffer to use as diluent. Note that just like with any other liquid being dispensed into the robocolumns during the load stage, it is essential that the mixtures you create here have the "Role = Sample" metadata associated with them.
Ensure Buffers/Samples to Load is disabled as a DOE factor in the Run Chromatography Stage element that the liquids have been wired into.
Note: Synthace will automatically identify that the liquids provided to the Run Chromatography Stage element are used in DOE and will pair them up to the appropriate number of resin columns.
Calculating a DOE design
Once you have selected your factors and levels continue with these steps:
Click on the Calculate Design tab
Select your design of choice. To learn more about DOE designs, click here.
Assess its quality. To learn more about DOE design quality, click here.
Once you are happy, verify the runs in your design match your expectations.
Click Simulate With Design.
Navigate to the simulation preview.
Note: In the simulation preview, each RoboColumn in your RoboColumn plate will correspond to a run in your DOE design. Use the preview to help validate that your RoboColumns DOE is calculated as you would expect.
Well done on making it to the end of this tutorial.
To learn how to build RoboColumns workflows where the factors are process parameters, click here.
To learn how to structure your DOE data for analysis in external stats tools or how to analyze your Robocolumns with the chromatography analysis functionality in Synthace, click here.