Biophysical Techniques for Protein Size Distribution Analysis
This chapter discusses the biophysical methods for size distribution analysis of biopharmaceutical protein products. Protein aggregates, conformational variants, reversible self-association, fragments, and glycosylation are common contributors to size heterogeneity in protein pharmaceuticals. Size distribution analysis is essential to product understanding and for ensuring product quality during development. The choice of specific techniques for determination of size heterogeneity may not be straightforward due to a broad range of possible sizes from protein fragments to visible particles. We discuss commonly used biophysical methods for the determination of protein size distribution including size-exclusion chromatography, field-flow fractionation, sedimentation, light scattering, microscopy, light obscuration, electrical zone sensing, and mass spectrometry. The advantages and limitations of each method are also presented. A combination of several methods is often necessary to obtain a comprehensive view of size heterogeneity.
KeywordsHydrodynamic Size Semipermeable Membrane Size Distribution Analysis Nanoparticle Tracking Analysis Size Heterogeneity
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