ABSTRACT
Due to the large size of proteins, as well as the importance of the three-dimensional protein fold held together by weak noncovalent forces, analytical characterization of a protein’s structural integrity is more complex than that of the most conventional drug molecules. In this chapter, we will discuss the most common methods to characterize proteins, as well as outline their applicability in a pharmaceutical development setting. The methods are briey summarized in Table 4.1. It is not possible to discuss any of the methods in detail or even all available methods, which means that the reader will have to refer to the cited literature if additional details
4.1 Introduction: The Challenge of Characterizing Proteins ............................... 49 4.2 Methods for Characterization ......................................................................... 55
4.2.1 X-ray Crystallography, NMR Spectroscopy, and Computational Methods ..................................................................... 55
4.2.2 Chromatography ................................................................................. 57 4.2.3 Electrophoresis ................................................................................... 61 4.2.4 Mass Spectrometry ............................................................................. 62 4.2.5 Analytical Ultracentrifugation ........................................................... 63 4.2.6 Spectroscopic Methods Using Radiation in the UV, Vis, and
Infrared Range .................................................................................... 63 4.2.7 Scattering Techniques ......................................................................... 67 4.2.8 Microscopy .........................................................................................68 4.2.9 Measuring the Thermodynamics of the Protein Fold .........................69 4.2.10 High-Throughput Methodology .......................................................... 70
4.3 Choosing the Proper Methodology ................................................................. 70 4.4 Concluding Remarks ...................................................................................... 73 References ................................................................................................................ 74
TA B
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are required. Moreover, this chapter will mainly focus on the characterization of the protein, rather than the formulation. We will end this chapter with a discussion on choosing the proper methodology.
