Methods Summary

Analytical ultracentrifugation (AUC) - sedimentation velocity
  • an excellent method for studying and quantifying protein aggregation

  • often used for cross-validation of SEC methods

  • usually allows measurements directly under formulation conditions

  • good for demonstrating conformational equivalence of material from different manufacturing processes (comparability protocols)


Analytical ultracentrifugation (AUC) - sedimentation equilibrium
  • the "gold standard" for measuring molecular mass in solution and studying protein self-association and protein-protein interactions

  • also studies interactions with small molecules when binding is linked to protein association


Circular dichroism (CD) spectroscopy
  • excellent tool for characterizing thermal stability of proteins and the effects of solution conditions and excipients on stability and reversibility of unfolding

  • good for demonstrating conformational equivalence of material from different manufacturing processes (comparability protocols)

  • provides basic characterization of overall secondary structure (percentage alpha helix and beta sheet)

  • good for determining whether novel proteins are properly folded

  • detects changes in tertiary structure around aromatic residues


Laser light scattering
  • "classical" light scattering, when used in conjunction with size-exclusion chromatography, measures the true mass of each peak, independent of the molecular shape or elution position, making SEC an absolute method

    • confirms quaternary structure for proteins that elute abnormally on SEC

    • provides enhanced sensitivity for detecting small amounts of aggregates and distinguishes dimers from unfolded monomers

    • can distinguish PEG and product MW for PEGylated products

  • "dynamic" light scattering determines hydrodynamic size by measuring the Brownian motion of proteins

    • excellent and direct method to characterize the hydrodynamic size of PEGylated proteins, nucleic acids, etc.

    • an excellent method for detecting trace amounts of aggregates (especially large ones) and differences in aggregation for different formulations or from different manufacturing lots, but fairly weak in quantifying the actual amount of aggregate and measuring absolute masses

    • generally allows measurements to be made directly in formulation buffers

    • good for monitoring stability over time; accelerated stability studies can be carried out with real-time monitoring in situ or by periodic sampling

    • samples can be studied at concentrations up to ~50 mg/ml


Differential scanning calorimetry (DSC)
  • direct measurement of thermal stability in solution

  • DSC thermograms are often included in the characterization package of a CMC section (characterization of higher order structure)

  • often used in comparability protocols (comparability of tertiary structure and stability)

  • results can sometimes predict relative long-term storage stability

  • can detect whether the domains within multi-domain proteins such as antibodies unfold independently


Native gel electrophoresis
  • a very useful (but often overlooked) technique for characterizing aggregation and protein-protein complexes

  • in our hands it can readily be applied to basic as well as acidic proteins

  • excellent for rapid evaluation of accelerated stability samples


Fluorescence spectroscopy
  • intrinsic fluorescence can be used to study changes in protein conformation, for comparability protocols (comparability of tertiary structure), and to monitor protein stability as a function of pH or added denaturants

  • hydrophobic fluorescent probes such as ANS detect changes in surface hydrophobicity and are useful for pre-formulation studies to assess changes in conformational stability as a function of pH or added salts as well as for comparability protocols

FTIR spectroscopy
  • uniquely able to provide secondary structure information for lyophilized or spray-dried powders or precipitates, as well as the liquid state

  • more sensitive than CD to details of beta-sheet structures

  • detects aggregation arising through formation of intermolecular beta sheets