Fluorescence spectroscopy of the protein

Amino acid composition (Partial) amino acid sequence Accurate mass, chemical degradation Amino acid sequencing, chemical degradation Chemical degradation (various types), impurities, content of active compound and additives Molecular size, aggregation, hydrolysis Relative molecular charge, chemical degradation with changes in charge Integrity of affinity site, presence of multiple conformers with different affinities Molecular size, aggregation, hydrolysis Depending on matrix and set-up, same as the chromatographic methods above

3D structure of protein (in crystal) 3D structure of protein

Protein secondary structure, crystallinity of the dry formulation

Protein secondary structure (solution + solid state), local structure around certain absorbing amino acids, crystallinity of the dry formulation Protein secondary and tertiary structure in solution

Local structure around fluorophores, presence of fluorescent impurities

Easily automated Easily automated Requires expert personnel

Can be automated, but optimization and interpretation takes time Very versatile, can be automated, but protein analysis is generally not easy to optimize

Limited resolution

Limited resolution

Impossible to determine which structural changes have occurred

Milder technique than SEC

Requires very little sample, but less established than HPLC

Slow procedure, requires expert personnel Slow procedure, requires expert personnel Can measure any physical state (gas, solution, solid) but requires high concentrations. Small volume, complex mixtures possible (drug delivery systems). Same as FTIR, can even be used for in-process control

Accurate protein concentration required for secondary structure, can only measure solutions Easy to do, but interpretation difficult

Trp, tyr, and phe in the amino acid sequence required

Table 7 Useful Methods for Determination of Protein Structure and Stability as Well as Characterization of the Formulation (Continued)


Information obtained


DSC (solution)

Protein thermal stability

Protein thermal stability can also be

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