Interpreting protein binding capacities for chromatography media (resins)

Comparing protein binding capacities of media from different suppliers is not straightforward, because different methods may have been used and these methods are not always stated. In this post, I will explain what protein binding capacity is, how it can be measured, and what to look for on a manufacturer's specification sheet.

When choosing a chromatography medium to purify your protein of interest, protein binding capacity is one of the most important characteristics to consider. This parameter is critical, because it determines how much medium is needed in order to purify a certain amount of protein. Stated another way, protein binding capacity determines how much protein can be purified by a specific volume or mass of medium. So then a product with a higher reported binding capacity than another should be able to purify more of your protein, right? Not always.

In this post I will explain what protein binding capacity is, how it can be measured, and what to look for on a manufacturer’s specification sheet.

How is protein binding capacity determined?

The capacities that suppliers specify for their chromatography media may be based on different:

  • modes of measurement (static or dynamic)
  • experimental conditions (pH, salt/conductivity, protein identity and concentration)
  • units of measure (i.e., capacity per milliliter wet chromatography medium or per gram dry medium)

In order to be able to compare specifications between vendors, it is vital to have information on the conditions used to determine the binding capacity. Unfortunately, in many cases the method for obtaining the chromatography medium’s “binding capacity” is not stated. When inquiring about these conditions, it is worthwhile to ask the vendor whether the binding capacity was determined under static or dynamic conditions.

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. SBC is usually reported as the maximum amount of protein bound to a chromatography medium at given solvent and protein concentration conditions. In these experiments, an excess of protein is loaded to give a maximum binding capacity. Protein loss is often over 50%. The SBC value varies substantially depending on the specific protein that is loaded.

Dynamic binding capacity (DBC), on the other hand, is the binding capacity under operating conditions (i.e., in a packed affinity chromatography column during sample application). 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 and monitoring the flow through. The protein will bind to the medium to a certain break point before unbound protein will flow through the column.

A breakthrough curve is generated by graphing the amount of protein loaded versus the percent breakthrough. The DBC can be determined on the breakthrough curve at a loss of, for example, 10% protein (referred to as the QB10 value).

dynamic_ curve

Because the DBC is measured under operating conditions, this value provides information about the maximum amount of target protein that you can load onto your column in order to avoid unnecessary loss. In contrast, although the SBC reports the maximum protein quantity that a medium can bind, it does not state the amount of protein that failed to bind under these conditions, Therefore, you may experience substantial losses of protein to the flowthrough when using SBC as a guide for loading your protein.

low high dbc

How does GE determine protein binding capacity?

At GE Healthcare’s Life Sciences we measure the dynamic binding capacity. In order to obtain the DBC for a loose chromatography medium, the medium is packed into a column. Information on the DBC and how it has been determined can be found in most of the instruction manuals for GE’s prepacked columns and loose chromatography media.

how to check

8 Comments

GE\jehan.bahri's profile image

jehan.bahri

Hi Marianne, should you recommend what is the minimum height of the column to determine the DBC? Pre-packed column can work with it? Regards, Jehan.

November 05, 2016 Reply
GE\GE Protein purification team's profile image

GE Protein Purification Team

Dear Jehan, There is no lower limit when it comes to bed height when determining the DBC of a resin. However, the uncertainty increases with very short bed heights. Determining DBC in prepacked columns such as for example HiTrap columns (bed height 2.5 cm) works fine. Regards, Marianne

November 10, 2016
GE\sdy16's profile image

sdy16

Hi Marianne, Thanks for a nice article. How would one determine 100% to know 10%? Should protein be run without column to know 100% UV reading? Is it measured in other instruments to get 100% UV reading? I appreciate your clarification. Thanks SDY

October 28, 2016 Reply
GE\GE Protein purification team's profile image

GE Protein Purification Team

Hello SDY, To find out when the UV reaches 100 % saturation you should by-pass the column and load the sample containing the protein with the concentration you are going to use and run it through the UV detector. It is important to use a protein concentration that will be within the linear UV range of the UV detector otherwise you will not see the saturation. Regards, Marianne

November 02, 2016
GE\cscjlzz's profile image

cscjlzz

I was wondering is there a detail protocol on how to use AKTA FPLC to calculate DBC (10%) breakthrough?

June 11, 2016 Reply
GE\GE Protein purification team's profile image

GE Protein Purification Team

Hello, my apologies for a late reply. No, there is not a protocol in UNICORN 5 which is used with ÄKTA FPLC how to calculate DBC. You need to calculate the DBC after the run. In UNICORN 6 and 7 it is possible to calculate DBC in the software. Dynamic binding capacity at 10% breakthrough was calculated according to the following equation : DBC10% = (V10% - V0) C0/Vc where C0 = protein concentration (mg/ml), Vc = geometric total volume of the column (ml), and V0 = void volume of the column (ml). Regards, Marianne

November 02, 2016
GE\cscjlzz's profile image

cscjlzz

I was wondering is there a detail protocol on how to use AKTA FPLC to calculate DBC (10%) breakthrough?

June 11, 2016 Reply
GE\cscjlzz's profile image

cscjlzz

I was wondering is there a detail protocol on how to use AKTA FPLC to calculate DBC (10%) breakthrough?

June 11, 2016 Reply
GE\cscjlzz's profile image

cscjlzz

I was wondering is there a detail protocol on how to use AKTA FPLC to calculate DBC (10%) breakthrough?

June 11, 2016 Reply
GE\cscjlzz's profile image

cscjlzz

I was wondering is there a detail protocol on how to use AKTA FPLC to calculate DBC (10%) breakthrough?

June 11, 2016 Reply
GE\Ana's profile image

Ana

I like this explanation although is not easy to use directly in front of the customer. A very interesting learning.

April 01, 2016 Reply

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