Eluting Possibilities with Mixed-Mode Chromatography - Pharmaceutical Technology

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Eluting Possibilities with Mixed-Mode Chromatography
Mixed-mode chromatography sorbents and custom ligands aim to optimize protein purification.


Pharmaceutical Technology


Designing a ligand for a protein depends on the characteristics of the protein, and some general rules can be followed. "For example, if the pI is greater than 7, then because most contaminating proteins are usually acidic, then chances are you can quickly find an absorbent that would work," says Noel. "If it's right in the middle in terms of pI, then you would look to see how concentrated it is in your environment. If it stands out in concentration, such as 10 to 100 times more concentrated than any other protein, which in a recombinant system you would hope it is, then that helps."

There is increasing interest in using Protein A and then using a mixed-mode ligand in the next step. Traditionally, the protein A step is followed by two ion exchangers. "Using a mixed-mode ligand is giving an added advantage and effectively trying to replace those two ion exchangers with one step separation. So your whole process is just two steps," says Noel.

Recent improvement in mixed mode chromatography involves finding faster ways of identifying the best and most robust conditions. "People are taking one absorbent and looking at a wide range of conditions to get a fuller picture of how the absorbent is working in their system," says Noel. The practice has become popular in the past four years. "It gives the process engineer an understanding of how the absorbent is working in that process and where the no-go areas are, such as for pH, conductivity, or temperature."

Affinity ligands


Figure 2
Some process chromatographers point out that mixed-mode ligands have a broad range of applications, and thus tend to be unselective and provide relatively modest increases in purity. For this reason, bioscience firms are developing small chemical ligands for capturing specific target proteins (see Figure 2).

For example, ProMetic Biosciences, Ltd. (Cambridge, UK, http://www.prometic.com/) has designed a library comprising tens of thousands of affinity ligands (under its "Mimetic" trade name), which are more specific versions of mixed-mode ligands. Like mixed-mode ligands, these are small, synthetic compounds, but they differ greatly from what are generally understood as mixed-mode ligands because of their precise chemical composition and wide variety of chemical groups used. These affinity compounds are organized such that the orientation and position of chemical groups in a three-dimensional space is fixed. This fixed position enables interaction with specific and complementary groups on the protein, which in turn enables specific capture. The synthetic ligands are designed and developed specifically according to the target protein (e.g, monoclonal antibodies, albumin, glycoproteins, proteases, and plasma proteins).

"In many cases, we have already identified compounds that are effective in purifying particular proteins," says Steve Burton, CEO, ProMetic Biosciences, Ltd. "For example, we have some very good ligands that we have developed and scaled up for albumin purification. Sometimes we have clients approach us with a new protein that maybe has just been discovered. In this case we may not have anything currently developed for that new target, but within our ligand libraries we can usually find something that will bind specifically and can be used to purify it."

The affinity ligands are more complex than those used in ion exchange and HIC. Charge interactions, hydrophobic interactions, hydrogen bonding and van der Waals interactions plus specific three-dimensional geometries are incorporated into the media. "These are not generalized interactions," says Burton. With ion exchange, for example, the interaction is not normally on a specific site on the protein; its interaction is with the protein as a whole. "These small, synthetic ligands, however, are actually interacting with a very specific site on the protein. We engineer the ligands so they have complementary charge and hydrophobic groups, depending on the groups that are present in that specific region of the protein."

As Burton observes, one of the benefits of having small synthetic ligands is they are less expensive to produce than protein-based ligands such as Protein A. In addition, being small entities, they are stable, robust, and can be cleaned effectively with molar sodium hydroxide, for example, so they can be used for hundreds of cycles.

"Just a few years ago there was a lot of skepticism about whether one could actually use these small compounds in bioprocesses," says Burton. "Now people are realizing that there are some real benefits. They really do work well in many instances, and more time is being devoted to investigating their potential."


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