Friday, 3 October 2014

Resins & Protein Binding Capacities

Compare & Contrast 
When choosing what resin to use for purifying a protein, one of the most important characteristics to look at is protein binding capacity.  Protein binding capacity is a critical parameter for chromatography media because it determines how much media is needed in order to purify a certain amount of protein. 
This in term determines the column size needed, flow-rates of your chromatography system, and ultimately the total costs for purifying your protein. But, can capacity data reported from different vendors always be directly compared?

The short answer is "No" and in this post we will try to explain what protein binding capacity is, how it can be measured, and what to look out for when looking at your vendor’s specification sheets.

Firstly, the resin capacity for different proteins is often specified by suppliers based on

  • different modes of measurements (dynamic or static)
  • experimental conditions (pH, salt/conductivity, protein concentration)
  • reference (capacity per milliliter wet resin or g dry resin)
Naturally, what protein has been used to determine the binding capacity is vital information in order to be able to compare specs. Unfortunately, in many cases, the method for determination of the binding capacity is not stated.
This makes it very difficult to compare the resins based on tabular values from the vendors.

What are the differences between static and dynamic capacity?
The static binding capacity (SBC, also called total protein capacity) is normally measured in batch mode in a beaker and is usually referred to as the maximum amount of protein bound to a chromatography medium at given solvent and protein concentration conditions. The size of SBC   varies significantly depending on the protein loaded. In these experiments, an excess of protein is loaded to give a maximum binding capacity. Protein loss is often over 50 %.

Dynamic binding capacity (DBC), on the other hand, is the binding capacity under operating conditions (i.e. in the packed affinity chromatography column during the sample application and washing procedures). The DBC of a chromatography medium is the amount of target protein that binds to the medium  under given flow conditions before a significant breakthrough of unbound protein occurs.
DBC is determined by loading a sample containing a known concentration of the target protein. The load of the protein sample on the column is monitored and will bind to the medium to a certain break point before unbound protein will flow through the column. From the breakthrough curve (see fig.1) at a loss of for example 10 % protein (named QB10),  the DBC is found and the experiment is stopped. As this parameter reflects the impact of mass transfer limitations that may occur as flow rate is increased, it is much more useful in predicting real process performance than a simple determination of saturated or static capacity.

In most instruction manuals from GE Healthcare, you can find information on the DBC and how it has been determined.

How does GE determine its resin binding capacity?
GE Healthcare uses the DBC as the measurement for media packed in columns since it takes the flow rate and bed height of the column into consideration. It also takes into account ligand density, size of protein, and the porosity of the media which are the only factors included in the measurement of SBC.
SBC can be used to determine the binding/total capacity of bulk media.
However, as DBC is measured under operating conditions you will also get information on what the maximum load of your target protein to your column should be in order to avoid unnecessary loss.

If you have any questions please ask away via the comments section or for more detail check out our Protein Skills Handbook which covers this and many other aspects of protein science