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October 08, 2018

Multistep protein purification: gain time and consistency

By Lotta Hedkvist, Global Product Manager

Our live webinar on May 16, Gain time and add consistency with automated multistep protein purification, described the benefits gained by automation, how to set up an automated purification protocol, and different case scenarios using ÄKTA pure chromatography system.

Q&A: automated multistep purification

Are you finding it difficult to keep up with all tasks involved in protein research? Would you like to spend more time on analysis and characterization of your targets and less time on purifying proteins? Do you want to know how to effectively maximize yield and purity of your target protein?

Shin Isogaii, Jay Ye, Hoang Tran, and Lotta Hedkvist discussed automated multistep protein purification including the benefits gained, how to get started, and different case studies using ÄKTA pure chromatography system. Automated multistep purification (also called multidimensional purification) can be applied using different approaches. The Q&A session from the webinar, Gain time and add consistency with automated multistep protein purification, is relevant if you are a beginner in this area or if you need to know how to increase automation even further in your protein purification workflow.

Questions on how to set up a protein purification protocol

In automated multistep purification, the protocol first needs to be established. The protocol may combine chromatography techniques to achieve the desired purity and yield. All different protein purification techniques may be used when you purify protein in a multistep process. Once you have decided on your protein purification protocol, the ÄKTA system and the UNICORN purification method need to be set up and generated.

During the webinar, we were asked many questions on different aspects of multistep protein chromatography and how to automate multiple protein purification steps. The questions and answers are listed below:

How can we can handle gel filtration chromatography [GF, also known as size exclusion chromatography, SEC] throughout the purification in a system? Is it practical to add SEC in this purification system? SEC can be used as a polishing step in multistep purification, the eluted protein can be collected in a fraction collector.
I am using a HiTrap GST [GSTrap] column to purify my protein on an ÄKTA avant 25. I am also using a protocol that has been used for other GST-tagged protein purifications, which worked successfully, however, I don't seem to achieve the same results. I get a little of my protein in the second to fifth elution fractions (1 mL column eluted with 5 mL and collected in 0.5 mL fractions). Most of my protein ends up in the flowthrough or the in wash. Since the binding kinetics are relatively slow between the glutathione and GST, it is important to use a low flow rate during the sample application to achieve high-binding capacity. To improve the binding, the sample should be of the same composition as the binding buffer. Yields of the target protein can be increased by repeating the purification two to three times and pool the eluates.
Can you please provide his-tag IMAC [immobilized metal ion affinity chromatography] purification info on ÄKTA avant, thanks? A method for purifying his-tagged proteins can very easily be set up using the predefined affinity chromatography (AC) method in UNICORN. By selecting an IMAC column in the method settings phase, the method will be adapted with suitable run parameters. The purification is performed in a similar way on both ÄKTA pure and ÄKTA avant and an example can be found in the application note Purification of a miniature recombinant spidroin protein expressed in E. coli using AKTA pure

Questions related to automating a multistep protocol

Regarding an automated set up to purify proteins in a multistep approach, there were questions about time for implementation, commonly used techniques, and challenges during implementation.

How much time does it take to implement a typical multistep method? This will depend on how complicated your multistep method is. For a two-step method, we can set it up in a couple of hours (hardware and software). After the first two to three runs, some optimization of the method may be required. We have set up methods with four to five steps and they can take longer to setup and optimize.
Which chromatography operations are the most pertinent to automated multistep purification? The most frequently used application is affinity chromatography followed by SEC/desalting. In some cases, ion exchange chromatography (IEX) is used for the first step. As you have seen in the presentations the combination of two or more capture steps is also feasible.
Can you use a different type of column for a second purification step? The majority of affinity, IEX, desalting, or SEC columns can be used in a second step. It is important to use the appropriate size of columns throughout the purification steps. The volume of the collected protein in the first step should be applied in the next step without the risk of losing resolution and recovery.
What are the obstacles in multidimensional purification? I would say that applying a multistep protocol to some unknown target molecule is the main hindrance to using multistep protocols. Once we know our targets and have defined the protocol, it is fairly easy to apply an automated multistep setup. Other challenges during implementation are to balance peak elution volumes, column sizes (especially when using SEC), and flow rates used. Flexibility in software and the hardware facilitates adaptation to different protocols. Adding steps and intermediate operations will require additional method development work.
How are the columns equilibrated—once or separately? The columns can be equilibrated in each purification step or separately before the multistep purification. If a large SEC column is used in the second step, the recommendation is to equilibrate the SEC columns prior to multistep purification. This shortens the time for the protein in the transfer loop (eluted from the first step) because equilibration of large SEC columns takes time.
Is there any automated system for purification/wash of inclusion bodies for bacterial protein purification? We have purified inclusion bodies from bacterial cell cultures and performed our runs using automated protocols. Usually the first purification step is performed during denaturing conditions followed by a polishing step. To regain activity, the protein needs to be refolded after the purification.

Questions about ÄKTA systems and multistep purification

Several ÄKTA system models may be used for a multistep process; in this section you can find questions and answers about what systems that are suitable to use to get purified proteins in a multistep setup.

Is it possible to upgrade ÄKTA avant 150 system for automated multistep purification? Yes, it's possible to set up ÄKTA avant 25 and 150 for automated multistep purification. For the simplest two-step application, no additional upgrade is needed. The procedure described for use of ÄKTA pure in Getting started with automated two-step protein purification can also be applied on ÄKTA avant. To support more complex protocols, additional valves and monitors may be added.
What are the facility requirements for this system? There are no specific requirements for multistep setups. The facility requirements for the ÄKTA system is described in the user manual for the system.
What’s the procedure to validate the automated multistep purification system? When setting up the multistep protocol, the following is important to validate:
  • Ensure that column sizes match. The expected peak volume from one step needs to be aligned with the next step.
  • The elution buffer must be compatible with the subsequent column.
  • Select the loop size to match the expected peak volume. As a rule of thumb, do not fill the loop more than 70% of the loop volume to avoid sample loss.
  • Delay volumes in the flow path will affect the recovery of very small peak volumes and desired recovery needs to be considered.
  • Perform a test run to validate that the method performs as expected.
What kind of additional hardware is required for the full setup? This will depend on the specific protocol. Typically, versatile valves and loop valves are included but other optional components may also be used. Slide 11 in the presentation lists examples on how the components can be used. In slide 50, four different examples methods are described.
Where can ÄKTA pure system solvent compatibility data be found? A chemical resistance guide can be found in the ÄKTA pure user manual.
Is the in-line dilution compatible with ÄKTAxpress? In-line dilution as described in the webinar is not possible on the ÄKTAxpress. The batch dilution is possible but would require text programming of the method used.
Can we have a similar seminar on ÄKTAxpress? Thank you for the input. We have no current plan to set up a similar webinar that focuses on ÄKTAxpress.
Is there a standard way to dynamically control an ÄKTA using an external program, like Excel®, matlab, or a custom program etc.? There is an OPC interface in UNICORN that can be used to connect to external programs. Further details can be found in the UNICORN manuals.

Your first step towards multistep purification

To get you started with multistep purification, GE has designed example methods for multistep purification that can be used with ÄKTA pure. These methods were described in the webinar and there was also a question about them. There is also support for automated multistep protein purification in the latest ÄKTA pure instrument configuration software.

Does the set up described on slide 50 on the right requires additional loop valve, i.e., additional hardware? Yes, the example displayed to the right uses an additional loop valve connected to the injection valve.
Slide 50 shows four different examples of hardware configurations that are supported with predefined methods and phases from our website.

Learn more about automated multistep purification