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October 25, 2018

Four purification steps with buffer dilutions in one go? No problem!

By Dr. Shin Isogai, Researcher at Biozentrum Basel

Free up time by automating the protein purification and by reducing manual interactions. Learn how Shin Isogai at Biozentrum, Basel, harnesses automation to purify more proteins per week using ÄKTA pure, giving himself more time for other tasks.


Working with many repetitive sample runs in protein purification

At Biozentrum, Basel we are working on the structural analysis of a membrane protein (G-protein coupled receptor, GPCR) that requires a four-step protein purification protocol with dilutions in between steps. The purification typically takes three days. Because there are a number of manual steps needed, and having realized that the manual steps consume a lot of time in our daily routine, we started thinking about how to operate our ÄKTA pure chromatography system to free up time and eventually prevent us from having to go to the lab on weekends.

When we do the structural analysis of a protein, we often encounter situations that require repetitive purification of the same protein with minor changes, for example, point mutations and other modifications as a result of protein engineering. When working with repetitive sample preparations in multistep protein purification, it is generally true that we spend significant time interacting with the instrument and preparing the sample for the next step. We thought that reducing manual operations during the protein purification by increasing the level of automation has a lot of potential for improving our workflow.

Buffer dilution performed in-line

The protein purification protocol includes dilution before the second and fourth chromatography step. Developing the dilution steps was necessary to automate the entire protocol. We came up with two dilution methods, batch and in-line buffer dilutions, which were tested using the ÄKTA pure system.

Batch dilution was found to be relatively slow and required one inlet and outlet per sample. We wanted to run more samples in one run, so we decided to go further and established an in-line dilution. In-line dilution was found to be very responsive, easily controllable, and eliminated the use of the system’s inlets and outlets.

For our protocol, in-line buffer dilution was the way to go.

Combining multiple steps and in-line buffer dilution into one automation method

In the end, we built an automated purification method to include four chromatography and two in-line buffer dilution steps. All steps were performed without manual intervention and without storage of sample in a reservoir. The total run time per sample was about the same but the number of samples purified per week has increased from two to four. Furthermore, the increased level of automation means that we only have to perform manual interventions two days a week, compared to six previously. It means no more work on Saturdays! Finally, we get to enjoy the beautiful Nature in Switzerland.

Watch the video below to learn more.

Q&A from webinar

This application was presented during the webinar Gain time and add consistency with automated multistep protein purification, during the webinar questions were submitted. The Q&A related to the automated four-step application is listed below.

Question Answer
For the in-line dilution application that was presented, how generic is the developed purification protocols? Can it be applied on different targets? Or would additional development work be needed? In combination with an ion exchange chromatography (IEX) column, it can replace the loop collection in most 2D applications (pseudo-3D methods). By doing so, the method would be able to handle larger elution volume from the first column. To set up the method, please find an appropriate IEX column for your target. If your target is not stable under the low salt condition, please consider using [cation or anion exchange] multimodal columns (e.g., HiTrap Capto) without the in-line dilution.
Do you think that case two will replicate similarly at a large scale? The in-line dilution works without any major challenges in the range of 0.2 to 20 mL/min (total flow) on ÄKTA pure. When setting up the experiment, please beware of pump pulsing and pre-column pressure.
Is it possible to adjust pH and conductivity in in-line dilution at a large scale? By using the in-line dilution method, we can precisely control conductivity. Theoretically, we might be able to adjust pH in the same manner (by running acid, base, or buffer solutions from the sample pump).
Can you control the level of dilution that you achieve by in-line mixing, e.g., can you get a ratio of 5:1 buffer to sample? Or is it only possible to carry out a 50:50 mix? Yes, the dilution ratio can be adjusted by changing the rate of the pump flows. For example, one can achieve 5:1 (sample:diluent) by running the sample pump at 5 mL/min, and the system pump at 1 mL/min

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