Protein purification is a sequence of processes to isolate and separate a protein from cells, tissues, or other complex compositions. Affinity chromatography and ion exchange chromatography are two methods of protein purification. At Purolite, we recommend performing purification in three stages: capture, intermediate purification, and polishing. In this Purolite How to Series, we’ll review how to measure the success of chromatographic protein purification methods.
Balancing Parameters in Protein Purification
As with any scientific procedure, standard parameters must be measured to gauge performance and compliance. For protein purification, five parameters should be considered: resolution, capacity, recovery, speed, and cost.
Looking at the three stages of protein isolation and purification - capture, intermediate purification, and polishing - each stage will require the prioritization of different parameters. Thus, the overall performance of a purification procedure is a balance between these five key parameters.
However, the balance between these parameters may vary depending on the protein purification techniques applied and the goals. For example, when purifying a protein for research purposes, the desired outcomes will differ when compared to the goals of protein purification for the development and commercialization of a therapeutic.
Below, each parameter is defined and recommendations for measurement are provided.
Resolution
Resolution is the separation of the target protein from other contaminants. This is displayed by peaks with the space between peaks representing a return to baseline.
A process’s resolution is measured by dividing the distance between peaks by the average peak width and depends on the efficiency (measure of peak width) and selectivity (distance between peaks) of the column.
When analyzing resolution, consider what process conditions obtain or maintain optimal resolution and what methods can be used to resolve contaminants that are related to the target molecule.
Capacity
Capacity relates to the amount of sample that can be loaded in a column. Resolution and capacity are two closely related parameters that must be balanced. For example, when evaluating protein purification techniques, consideration should be given to the impact of resolution on capacity. For example, how much can you load a column before you lose resolution and how would this loss affect process economics? Desired capacity will also impact the equipment used in the purification process. A low-capacity resin will need large columns, buffer volumes, and big skids or multiple cycles to process. These capacity changes will, in turn, impact production costs.
Recovery
Recovery reflects the yield of your target molecule. During each of the three stages of purification, strong consideration should be given to three key questions; (1) how much of your target protein is lost, is the activity of the target molecule being preserved, and (3) what conditions can be optimized to improve recovery.
Speed
Speed, or throughput, is a key parameter due to its impact on time-to-market and cost. When evaluating speed and its impact, consider the following questions:
- How much time does each step require?
- how fast are contaminates being removed?
- What impact does process speed have on the stability of your target molecule?
This final question will relate both the cost and, importantly, to the quality of the final product.
Cost
Cost is discussed throughout this blog as it’s directly impacted by almost all the four preceding parameters. However, there are multiple costs associated with the purification process that should be considered when developing or altering procedures. These costs include total operation cost, poor-quality output, equipment costs, sustainability costs, staff training costs, and labor costs, among others. Each cost and profit area should be weighed when making a process or product decision.
Explore Purolite’s product toolkit for Affinity Chromatography and Ion Exchange Chromatography.
