Protein purification alternatives

Protein purification, often referred to as downstream processing, is the most costly and time-consuming process in the manufacture of bio-molecules. EU-funded researchers integrated materials science with process development to produce novel low-cost materials and methods for selective purification with a focus on chromatography, membrane separation and extraction.

Purification is somewhat like passing sand and pebbles through a sieve except that separation is not dependent on gravity and relative size of components and holes. Instead, separation depends on chemical and electrical interactions between the biological fluid and specific binders (ligands) through which it passes.

Among the many proteins purified by the pharmaceutical industry are human immunoglobulin G (IgG) and monoclonal antibodies (MAbs), both important in immunity and thus disease therapy. The most common method for purifying IgG and MAbs is the use of protein A resin. However, pharmaceutical companies are increasingly concerned about the supply of protein A materials.

The 'Advanced interactive materials by design' (AIMS) project thus sought to develop alternatives to protein A technology for the purification of proteins. The investigators developed excellent modelling tools enabling assessment of interactions among support, linker, ligand and product promoting efficient and effective design of new materials.

The researchers created a new SartoAims protein A affinity membrane with enhanced affinity for IgG, providing an important alternative to protein A for IgG purification. In addition, the investigators studied two alternatives to protein A technology for purification of MAbs, one using much less expensive ion exchange resins in a Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) form of chromatography and one using aqueous two-phase extraction.

The researchers also developed new materials for use in ion exchange chromatography, a technique that relies on charge interactions for separation. In fact, the chromatographic resin FractoAims demonstrated superior mechanical stability and can be tailor-made based on bead size, pore size, surface area and ligand density.

The new process concepts were tested in a mini-plant to evaluate performance with respect to protein A technology. A combination of two MCSGP units operating with different parameters enabled reduction in operating costs by a factor of three in total MAb purification costs.

The AIMS project outcomes will have significant impact on the protein purification process that has until now been the most costly part of bio-molecule development in the pharmaceutical, chemical and biotechnology industries. Commercialisation of the new technologies promises to improve the European position in the huge global chemicals and pharmaceuticals market.

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Getting to xenon: Scientists examine alternatives for pulling this rare, expensive element out of air samples

"The promise of NiDOBDC and similar metal-organic frameworks is that we will be able to make faster and more sensitive separation and monitoring equipment," said Grate, who has been working in materials and systems for sensing and nonproliferation applications for his entire career at PNNL.

Absorbents, like activated charcoal and MOFs, can selectively capture xenon and release it on demand. Making these absorbents more efficient could benefit sustainable processes and national security. Commercial uses of xenon include lighting, scientific instruments, and anesthesia. Security uses focus on nuclear processes. Nuclear reprocessing, weapons tests, and nuclear accidents, such as the 2011 catastrophe in Japan, release xenon radioisotopes into the atmosphere. Around the world, monitors track xenon for the Comprehensive Test Ban Treaty.

The research team compared the performance of three materials that capture xenon. Conventional technologies for capturing xenon use activated charcoal, which is fine black carbon powder processed to be porous, with an effective surface area of 500 square meters a gram. Charcoal also absorbs xenon from air samples and releases it on demand.

"Activated charcoal is a really old-fashioned material with little potential for improvement," said Grate. "We wanted to see if we could find a material that we could push, that we could get new capabilities from."

Metal-organic frameworks or MOFs were an obvious starting point. NiDOBDC, MOF-5, and other members of this new class of framework-based sorbents are extremely porous. The frameworks can achieve surface areas ~10 times greater than activated charcoal. Further, the modular nature of the MOF synthesis method and their internal chemistry lets scientists build materials with a range of structures and properties.

"Interest in MOFs has exploded in the past decade," said Thallapally, who is also conducting research for DOE's Office of Nuclear Energy using these same materials.

At PNNL, Thallapally was working with MOFs for carbon sequestration, but saw more potential for the materials. He met with Grate to discuss MOF possibilities for security applications. They believed PNNL had the right capabilities to synthesize and characterize the necessary materials. They applied for and received PNNL funding to examine how well MOFs worked in xenon capture.

Previously, some MOFs proved disappointing. The materials worked well in the lab, but their framework structures were unstable under environmental conditions as vapors were sorbed and desorbed.

Working in PNNL's Sigma 5, the researchers tested three different xenon absorbers: NiDOBDC, activated charcoal, and a prototypical MOF, known as MOF-5. They found NiDOBDC takes up xenon about as well as activated charcoal and significantly better than MOF-5. NiDOBDC is superior at lower pressures. At 1 bar, the pressure at sea level, NiDOBDC was able to take up significant amounts of xenon and release all of it when the conditions were right. They also found that NiDOBDC was more selective than charcoal for xenon over krypton, which is fairly similar to xenon.

Thallapally and Grate are continuing to investigate MOFs, including NiDOBDC. Their work will include studies on scaling the materials to study their performance in real gas streams.

"While there isn't much you can do to improve charcoal, there is a world of potential in MOFs," said Thallapally. "The defined nanostructured framework gives you a lot of opportunities synthetically to add more functionality."

More information: PK Thallapally, et al. 2011. "Facile Xenon Capture and Release at Room Temperature using a Metal-Organic Framework: A Comparison with Activated Charcoal." Chemical Communications. DOI: 10.1039/C1CC14685H

Provided by Pacific Northwest National Laboratory (news : web)