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Individual Steps
In a chromatographic step there are four fundamental performance properties - resolution, speed, capacity, recovery.
In general, optimization of any one of these can only be realized at the expense of the others. Priority of these parameters depends on if the step is for capture, intermediate purification or polishing. In addition, the process must be optimized without compromising robustness.
Focus on the key objectives for each specific step: See figures.
Points to be considered during process optimization:
- Select important variables for optimization
Media volume/sample volume, column height, flow velocity (during sample application, washing, elution, re-equilibration, cleaning), pH and ionic strength for sample application, washing conditions, elution conditions. Consider the use of a step gradient or a linear gradient, consider the correct media particle size for the specific step.
- Define the proper dimensions of each individual steps
Consider robustness. Will the feed be handled in one cycle or multiple cycles? Take variations in sample properties and volumes into consideration and adapt your process conditions and column dimensions.
- Study the clearance of critical parameters
Check your sample whether the concentration of viruses, bacteria, fungi, host cell DNA, plasmid DNA, endotoxins, column leachables, modified target molecule, solvents and cleaning agents meets the specification set by the regulatory authorities and adjust your process according to the demands
- Challenge your process
Find acceptable range for critical parameters like variations in your sample vol./pH/cond./conc. etc
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