A step-by-step protocol to determine dynamic binding capacity (DBC) of a chromatography resin to screen for the optimal resin and running conditions for your protein purification.
What is DBC?
In protein purification, DBC of a chromatography column describes the maximum amount of target protein that you can load onto your column without causing unnecessary loss (Fig 1). DBC is measured under realistic experimental conditions (default flow rate, real protein sample).
Fig 1. Exceeding the DBC of the column will end up in loss of target protein.
DBC is key for choosing the resin that will be the most suitable for purifying your protein sample but also to determine the safe loading density for the chosen column. DBC is available in the technical specifications for Cytiva columns and resins, for typical proteins and running conditions. This is good reference data; however, you will need to determine the DBC yourself to screen for the optimal resin and running conditions for your specific protein.
Determining resin DBC step-by-step
Material needed to determine the resin’s DBC:
- ÄKTA™ chromatography system with a sample pump or a Superloop™ assembly.
- UNICORN™ software 6.4 or higher.
- UNICORN evaluation classic software module.
- DBC calculation extension.
- Binding buffer.
- Elution buffer.
- Sample—protein at appropriate concentration in binding buffer.
- A chromatography column (prepacked or packed in the lab).
DBC measurements are usually carried out using pure protein in binding buffer.
Overview of DBC determination
- Manually prime the ÄKTA system with your buffers of choice and measure 100% breakthrough absorbance by injecting sample bypassing the column.
- Create a method for DBC measurement. Make sure the load volume/mass is large enough to overload the column.
- Run the method.
- Open the result in UNICORN evaluation classic module, with the DBC calculation extension activated, and calculate DBC values.
Detailed description
|
Step |
Description |
|
1 |
Prime the system with binding buffer. |
|
2 |
Set the UV monitor to an appropriate wavelength for your molecule. Typically, this will be 280 nm for proteins and 260 nm for DNA/RNA. Autozero UV. |
|
3 |
Fill the sample pump or Superloop assembly with the protein sample and inject the sample to the system, bypassing the column, until the UV absorbance from the sample reaches a stable level. This value recorded here is the 100% breakthrough absorbance (Amax). The percentage of breakthrough value can be calculated later with reference to this 100% breakthrough absorbance value. Note: Choose appropriate sample concentration and flow cell length for UV monitor so that the 100% breakthrough absorbance Amax signal is high but still within the linear range, which is normally under 1200 mAU. If the signal is outside this range, dilute the sample for example 5×, and then multiply the obtained UV value with five. Using a sample pump is convenient for sample loading, but a certain amount of sample will be wasted when filling the sample pump. If you have a limited amount of sample, use a Superloop assembly for sample loading. |
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4 |
Wash out the sample from the system, by running binding buffer through the system, until zero absorbance is observed. |
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5 |
Attach the chromatography column to the system on a suitable column position. Avoid trapping air in the column or its tubing. |
| 6 | Equilibrate the chromatography column with 5 to 10 column volumes (CV) of binding buffer, until the UV (or other signal such as pH and/or conductivity) is stable. |
| 7 |
Create a method which includes the following steps:
|
| 8 | Run the method. |
Calculation of resin DBC in UNICORN software
The DBC calculation extension provides the capability of DBC calculations in UNICORN software in an extension. It can be used in the UNICORN software with versions higher than 6.4. For help on installing the DBC calculation extension, please contact your local ÄKTA system specialist.
If you are using UNICORN 6.4 or higher, DBC can be estimated as described here:
|
Step |
Description |
|
1 |
Open a result in UNICORN evaluation classic module. |
|
2 |
Select in the menu "Extensions→DBC calculations→Analyze". |
|
3 |
Set the parameters for the run:
In the example below, we calculate DBC at 5%, 10%, and 30% breakthrough. Settings are shown in Figure 2, and the result is shown in Figure 3. |
Fig 2. Settings screen in the DBC calculation extension, with settings for DBC calculation at 5%, 10%, and 30%.
DBC result
The result window shows the DBC values at various percentages of breakthrough. In the example in Figure 3, resin DBC at 10% breakthrough (QB10%) is 60.56 mg sample per mL of packed resin.
Fig 3. Results screen for DBC calculation at 5%, 10%, and 30%.
Estimating DBC with UNICORN software versions lower than 6.4
A rough estimate of DBC is performed in the following way:
- Determine the contaminant offset value (Aoffset).
- In a chromatogram with X-axis showing volume and Y-axis showing UV absorbance, move the cursor along X-axis until Y-axis shows a value of A where (A - Aoffset) is 10% of (Amax - Aoffset). Record this volume as Vx.
DBC (QB10%) can be estimated as (Vx - Vdelay) × sample concentration.
This is an over-estimation of the DBC since the amount of protein that is leaked during sample loading is not subtracted from the calculation (Fig 4, green area).
Tips for chromatography resin DBC measurements
- The protein sample should be stable (e.g., do not precipitate) at the running conditions (pH, temperature, and salt concentration).
- Ensure that UV baseline is stable before loading the protein sample.
- DBC is dependent on the flow rate used during protein sample loading. For example, low flow rate gives longer residence time (the time the protein is in contact with the resin) and in the end higher DBC. For some resins, extended residence time is needed to utilize the full capacity. Typically, the maximum DBC of Cytiva resins is obtained at a residence time of 6 to 8 min.
- If the contaminant offset is too high, the DBC calculation will not work correctly. Thus, for crude samples, for example in a capture step, the sample should be prepurified to estimate the DBC.
In-depth theory of DBC calculation in UNICORN software extension
The calculation of DBC is carried out by a UNICORN software extension.
Fig 4. Graph of DBC calculation. Blue area is caused by delay volume. The orange area represents the amount of nonbinding proteins. The blue curve is the UV signal and the green area represents the amount of protein that has leaked at X% breakthrough. The total DBC at X% breakthrough is represented by the gray area.
Example
In the example shown in Figure 4, Aoffset is 50 mAu and Amax is 1000 mAu.
At volume 50 (Vx) the UV absorbance is 126 mAu.
A - Aoffset = 126 - 50 = 76 mAu
Amax - Aoffset = 1000 - 50 = 950 mAu
Percentage of breakthrough is calculated as (A - Aoffset)/(Amax - Aoffset)
Breakthrough (%) = 76/950 = 8%
Thus, the gray area in the graph represents DBC at 8% breakthrough, that is the QB8% value.
CY4858