Summary of Characterization Methods Offered
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 |
|
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 |
While we do not have the equipment ourselves, A.P.L. can also supply FTIR
spectroscopy through a collaboration with Tiansheng Li of
HTL Biosolutions, an IR expert with many
years of experience in the biotech industry, and fluorescence characterization through
collaboration with Integrity Biosolution,
a protein formulation and delivery firm.
- 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 |
- Fluorescence spectroscopy
| intrinsic fluorescence can be used to study changes in protein
conformation and monitor protein stability as a function of pH or
added denaturants |
| the hydrophobic fluorescent probe ANS is useful for
pre-formulation studies to assess changes in conformational stability
as a function of pH or
added salts |
|
