Boost your research with automated parallel protein purification

Why automate your purification process?

Generating many samples from optimization of cell culture conditions or your clone selection process can create work overloads and strain capacity. Remove the need to manually set up each chromatography step separately and free up time for other tasks with automation. You will gain time back for data analysis and create a higher throughput without spending more time in the lab.

Benefits of automation:

• Higher throughput
• Efficient use of resources
• Free up time for other tasks

Automated purification of multiple proteins in parallel

Cytiva has created a solution to automatically run multiple samples in parallel. Connect up to three ÄKTA go™ chromatography systems and use the same computer screen to control your entire process (Fig 1). The full solution gives you the possibility to purify 15 samples in parallel. Space is saved by using just one monitor to control your purifications. Each system needs a separate PC with UNICORN™ client to run the Instrument server. A PC easily fits on the back of the system to minimize the footprint.

The small footprint of the ÄKTA go system, combined with fraction collector F9-T placed underneath the system, saves valuable bench or cold cabinet space.

The UNICORN software toolbox with My Instrument and UNICORN online show an overview of your process and the ability to control and interact with the systems even when you’re not in front of them.

Why would you need more than one step to purify your target molecule?

Purification protocols are typically challenged by two factors:

1. Capturing as many target molecules as possible without degrading the sample

2. Removing impurities, unwanted isoforms and minimizing aggregate content

To capture your target, an affinity chromatography step (AC) is preferably used. This step enables the isolation of your molecule from initial sample (e.g., serum, cell culture) and usually results in high purity levels. However, the target may not be in the optimal condition and a second step needs to be introduced to maintain a stable molecule.

Depending on your target molecule there are different techniques to solve the challenge of removing unwanted molecules. An antibody may need antibody aggregates and/or fragments removed to obtain monomeric antibodies. Then a size exclusion chromatography (SEC) step is recommended. For adeno-associated virus (AAV) purification protocols, the affinity step can be followed by an ion exchange chromatography (IEX) step to differentiate between empty and full capsid.

How to set up an automatic 2-step purification run?

Setting up an automatic 2-step purification run does not have to be difficult. Simply queue up methods, handle the intermediate peak, and allow the system and software to run from start to finish to enable automated multistep purification. By utilizing the Method Queue in UNICORN software, steps can be added and run in sequence.

ÄKTA go peak collect tubing offers a standardized way to redirect the peak for intermediate storage between two purification steps. In the two-step purification run, the intermediate peak is collected in the tubing attached to the injection valve by using the Peak to loop functionality in UNICORN software. You can leave your system to do other tasks and return when both purification steps are finished, and the collected fractions are ready to move on to your next step.

Example: Automated two-step purification process of cell culture batches

ÄKTA go system is a small and compact liquid chromatography system that allows you to perform routine protein purification with ease, and with an efficient use of bench and cold cabinet space.

In this method, Cytiva cultured CHO cells under different conditions in a parallel bioreactor to show a proof of concept for automatic purification. The bioreactor can create 12 cell culture batches of 250 mL each. This generates multiple samples to be purified and pushes the workflow bottleneck to the next step in the process. To purify all samples in an efficient way that optimizes both time and resources, Cytiva setup an automatic two-step process using ÄKTA go system.

The affinity step was performed using a HiTrap™ MabSelect SuRe™, 1-mL column and a HiPrep™ 26/10 desalting column was used for size exclusion. Both columns were connected to the system at the same time using the column valve V9-C with 5 column positions, and the ÄKTA go peak collect tubing was used for intermediate peak collection (Fig. 2).

Predefined methods simplified the purification

ÄKTA go system is fully supported by UNICORN software, which gives real-time control of the chromatography system. Automated methods can be created in minutes for most common chromatography techniques using predefined methods. In this application, the methods below were generated using predefined methods (Fig 3).

Cytiva used the Method Queue functionality in UNICORN software to automatically link together and run the methods in sequence (Fig 4).

ÄKTA go system was set up to direct the peak from the affinity step to the size exclusion step using the Peak to loop option in the Fractionate section in the Elution phase (Fig 5). This is set up in the Affinity method. In the size exclusion method, Reinject counted volume was chosen in the Sample Application phase to automatically load the collected peak as sample in the second purification step (Fig 6).

Results

Protein of interest eluted in a nice homogenous peak during the elution phase at pH 3.5. In the chromatogram there are set marks to indicate when the ÄKTA go system started to collect the sample into the tube and when it stopped (Fig 7).

The affinity purified sample was automatically loaded onto the SEC column and eluted in a clean peak, as expected at 1.6 min corresponding to the monomeric size of the mAb (150 kDa) (Fig 8).

The yield between the two steps was 98% as calculated from the peak area, (Table 1).

Tips for automated purification using ÄKTA go system

The default configuration of ÄKTA go system comes with Outlet valve V9-Os with waste, fraction collector and one outlet position. When you want to collect flow-through during your purification run, Cytiva recommends changing to Outlet valve V9-O with 10 outlet positions (art no 29012261) (Fig 9).

Summary and conclusion

• This technology enables parallel purification of up to three ÄKTA go systems allowing a user to purify up to 15 samples while saving valuable lab bench and cold cabinet space.
• ÄKTA go system can now run automatic 2 step purifications by introducing peak to loop functionality and taking advantage of the user-friendly features in UNICORN software.
• Cytiva confirmed the ease-of-use and proof of concept by running the automatic two-step purification consisting of an affinity step and a desalting buffer exchange step, for a mAb expressed in CHO cells.

Whether purifying antibodies or other target molecules — ÄKTA go system is the reliable and efficient choice for protein purification directly from cell cultures.

Additional resources:

Explore Two-step chromatography for efficient Fab purification.