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Chapter 7 Design new protocols
7
Prepare for the first trial run
Prepare for the first trial run
Complete the protocol definition including checking of speeds and triggers from similar processes and
cells. When these are not available, use the Rotea
behaviour of particles within the fluidized bed for any Step within the protocol including:
1. Estimate the percentage of cells retained or elutriated based on the input parameters, assuming a
normal distribution of particle size.
2. Estimate the theoretical cell size at the zero line (the transition point between cells being retained or
elutriated when the opposing forces on the cell are balanced).
If a scoping run is planned, use a recirculation step through an intermediate bag to enable in-process
samples to be drawn at different speeds.
Water runs
Set up a test Single-Use Kit and run the protocol with water to verify that steps, speeds and triggers
behave as you expected.
Use this opportunity to refine the settings, practice setting up the Single-Use Kit and practice with any
user interactions you have included.
First run with cells
For initial runs use small quantities of cells, e.g. 50 to 100 million cells, to verify loading speed and wash
behavior.
Carefully measure the cell count and viability of both the input and outputs. The focus should be on
viable cells only.
Analyze the outcome
Confirm the output cell count and viability.
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The CTS
Rotea
mass balance should be conducted:
• Mix and sample from every bag with fluid in it.
• Rinse the original cell supply bag to determine how many cells remain.
• Drain the kit and count cells.
• If cells are found in places other than the output, then refine the protocol to minimize the lost cells.
Refine the protocol to achieve target cell outcomes
Progressively increase the input volume to be representative of your final process and refine the
protocol to optimize the outcome.
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Counterflow Centrifugation System is a closed system. If recovery is <95% then a
CTS
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Process Model to better understand the potential
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Rotea
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Counterflow Centrifugation System Process Design User Guide
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