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.