April 1, 2006 (Vol. 26, No. 7)

Recombinant proteins, peptides, and antibodies could account for up to half of all new drug approvals in coming years. Meanwhile, many of the original recombinant proteins are coming off patent or have already, and companies are beginning to manufacture generic versions. Therefore, protein characterization has never been more important. Characterization is a broad term, closely related to protein separation and purification, and involves creating a profile, or fingerprint, of the protein molecule’s physical, chemical, and biological properties.

In the past, protein characterization tended to be incomplete, because analytical methods were crude and based mainly on biological methods. In vivo studies were the gold standard for safety and efficacy. But now characterization has been transformed by the advent of a new generation of technologies, from advanced electrophoresis and mass spectrometry systems to comprehensive bioinformatic tools for searching and integrating with proteomics databases.

It is far more challenging to characterize a protein than it is a small molecule. Proteins simply have more properties to investigate. They are large molecules with complex and varied structures, and they are sensitive to conditions like temperature and pH, tending to lose their higher order structure if these are suboptimal. Proteins also have a tendency to form complexes, aggregate (or even precipitate), and undergo post-translational modifications such as glycosylation, which affect their properties. Careful sample preparation is therefore an important part of protein characterization studies.

Characterization Strategies

When it comes to developing a strategy and selecting techniques for protein characterization, much depends on context. Characterization has a role at many stages in the drug discovery and development process. Target discovery and validation depend upon structural studies that utilize techniques such as x-ray crystallography, NMR (protein spectroscopy), and bioinformatics. The more that is known of the properties of a target protein, the more likely it is that a drug—small molecule or biologic—will be found to bind to it with high specificity.

In the context of manufacturing, good analytical data can help show pharmaceutical equivalence between products from different batches and between a generic protein drug and the pioneer protein. It can also be used to support changes in process development (by demonstrating equivalence). Many generic manufacturers hope that advanced protein characterization will convince the regulatory authorities that fewer (if any) clinical studies are needed, although this is still a matter of debate.

Other useful techniques include light-scattering studies, UV/Visible spectroscopy, and circular dichroism to show correct folding of the protein and its alpha helix and beta sheet content, and microcalorimetry to study binding. However, not all techniques are applicable to all proteins. One major drawback is that membrane proteins, which are important drug targets, are difficult to crystallize, so their three dimensional structures cannot readily be determined.

Mass spectrometry (MS), coupled with high performance liquid chromatography, is perhaps the ultimate analytical tool for a protein, now allowing masses of protein molecules to be determined to within one dalton—which is impressive, given that a typical protein will have a molecular mass of several thousand daltons. MS systems have come down in price and many companies will use more than one product, depending on application.

With so many different technologies, the trend is toward building a multidimensional assessment of mass, sequence, structure, hydrophobicity, and many other different properties. Companies in this area are either trying to develop faster, cheaper, and more accurate versions of existing separation, purification, and characterization technologies like MS or electrophoresis, or they are bringing out completely new platforms.

Dynamic Light-Scattering

According to Ulf Nobbmann, Ph.D., specialist in biophysical characterization at Malvern Instruments (www.malvern.co.uk), one useful way of defining protein characterization is the process of understanding the state of a protein in solution and finding out how it behaves under various conditions. This is relevant in terms of target discovery, structural studies, biological properties, manufacture, and formulation.

Malvern specializes in systems such as the Zetasizer Nano series, where light-scattering techniques are used to measure the size and molecular weight of protein molecules. Of course, the latter is more accurately (and more expensively) measured with MS, but the company says dynamic light scattering (DLS) is fast, accurate, and straightforward. Sizing with DLS reveals the oligomeric state and homogeneity of a protein (monomer, dimer, oligomer, or a mixture of these), which can be important in terms of its clinical application.

Investigating the homogeneity of a protein sample in this way is also important in determining optimal crystallization conditions ahead of x-ray crystallography studies of protein structure, which are essential for structure-based drug design.

Another property of a protein that can be measured with light-scattering techniques is the so-called zeta potential, which is related to the charge on the molecule. “While established in other areas, this technique is still in its infancy for protein applications, but can be very relevant to developing a stable formulation for a protein drug,“ says Dr. Nobbmann.

Investigations of protein solubility are also important. Under certain solution conditions, proteins tend to precipitate. This may have important consequences for the safe administration of protein drugs. “Knowledge of solubility characteristics from DLS is vital in setting the conditions for manufacturing a better drug,“ explains Dr. Nobbmann. Well-characterized proteins also give higher yields in manufacturing, which is essential in a climate when driving down costs is top priority.

Meanwhile, DeltaDOT (www.deltadot.com) developed a technology called Label Free Intrinsic Imaging (LFII) that combines detection of the intrinsic optical absorption properties of a protein with advanced signal processing. The system is used in conjunction with capillary electrophoresis and, according to the company, allows high-resolution of proteins with similar molecular weights, quantitation of the molecules in a mixture, and gives output in a digital format.

The system involves a diode array and detector system, which picks up a characteristic absorption signal from each protein as it moves on the gel. CCO Frank Smith says, “The technology gives capillary electrophoresis an extra tool for higher resolution, sensitivity, and reproducibility. The real plus, though, is being label-free, which saves on time and cost.“

Smith sees a important role for LFII in screening proteins prior to full MS. “Mass spectrometry remains the method of choice but it is still expensive and needs a skilled operator. Our system could free up mass spectrometry time.“ LFII is currently being used in molecular diagnostics applications and is marketed for applications in drug discovery, process development, and manufacture.

2-D Gel Electrophoresis

Another interesting development that will aid protein www.nextgensciences.com), which recently launched the a2Deoptimizer, a new system that allows the automatic pouring of gradient gels for 2-D gel electrophoresis. “People have been saying 2-D electrophoresis is on its way out for years, but it still has an important part to play in protein separations,“ says Robert Mount, Ph.D., customer support director.

In 2-D electrophoresis, separation of a protein mixture is done first in one dimension using a technique called isoelectric focusing, which separates the molecules on the basis of their isoelectric points (the pH at which there is no net charge on the molecule) and then the gel is turned and the proteins are further separated by size using the standard SDS technique. In this way, the proteome of a cell can be characterized by the display of 2,000 to 4,000 different proteins. The 2-D approach gives a much cleaner product than 1-D.

It is also useful for exploring post-translational modification, which is hard to do with other techniques. Gradient gels give much better separation in 2-D electrophoresis than standard gels, but are very hard to make reproducible, if poured manually. “Researchers know gradient gels are better, but they have such difficulty in making them that they are rarely used,“ explains Dr. Mount.

The a2Deoptimizer now makes gradient gels much more feasible, according to the company. It also permits IEF to be carried out at 10,000 volts, which gives a great speed and resolution advantage. And the new system is compatible with the GE Healthcare Ettan DIGE (2-D fluorescence difference gel electrophoresis) system, in which each protein spot carries its own internal standard—an important development in removing gel-to-gel variation.


Finally, bioinformatics has an important role to play in protein characterization, both in the interpretation and storage of data and in the identification of proteins. GeneBio (www.genebio.com) brings together bioinformatics and proteomics in a range of useful solutions.

The latest product, Phenyx 2.0, is a software platform developed to keep pace with advances in MS, which generates increasing amounts of data output requiring analysis. Phenyx was developed in collaboration with the Swiss Institute of Bioinformatics and identifies peptides and proteins from MS analysis, including those with post-translational modifications.

Another GeneBio tool is the Melanie platform, which powers GE Healthcare’s ImageMaster 2D Platinum system for the analysis of 2-D electrophoresis gels (including DIGE gels) and shortens the path from gel data to protein information. These new technologies are leading the way to a better understanding of protein drugs through characterization at all stages of their development.

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