No single assay possesses all of the attributes described above, but improvements in existing methods and the development
of novel technologies have provided the biopharmaceutical industry with better tools for protein characterization. With the
advent of biosimilars, regulatory agencies have become more rigorous in their analyses of comparability in terms of protein
structure and have demonstrated notable interest in higher order structures and protein aggregates. As a result, biomanufacturers
are incorporating more comprehensive structural analyses into their product characterization assay portfolios.
Higher Order Structures
A number of techniques have emerged as valuable tools for evaluating higher order structures of protein biopharmaceuticals,
including x-ray crystallography, light scattering, calorimetry, and spectroscopy. Advanced spectroscopic techniques such as
nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence spectroscopy (FS) have proven particularly useful
for structural analysis.
CD is a form of UV absorption spectroscopy, and typically is used to examine the secondary structure of proteins. CD often
is used to assess the conformational stability of a protein under stress, providing insight into a protein's stability at
various temperatures and pH levels and in the presence of denaturing agents. Using this method, appropriate buffer compositions,
stabilizers, and excipients can be identified to increase the melting temperature or the reversibility of thermal unfolding
for a given protein, which in turn result in enhanced shelf life for the final drug product. Circular dichroism also can determine
whether protein–protein or protein–ligand interactions have the potential to alter the conformation of the target protein,
as conformational changes produce a spectrum which differs from the sum of the individual components. CD is a very sensitive
technique for analyzing a protein's secondary structure, requiring only µLs of solution at concentrations as low as 50 µg/mL
protein, and the sample can be analyzed in any buffer that does not have a high absorbance in the far-UV region of the spectrum.9
Fluorescence spectroscopy, a form of UV excitation spectroscopy, is commonly used to study the tertiary structure of proteins.
The intrinsic fluorescence of a given molecule is determined by the presence and location of tryptophan, tyrosine, and phenylalanine
residues in the target molecule.10 Proteins also can be labeled with fluorophores and the extrinsic fluorescence used to determine tertiary structure. A commonly
used covalent probe is fluorescein 5'-isothiocyanate, which can be attached to lysine residues in the target protein. Fluorescence
spectroscopy is accurate, highly sensitive, and can be used even at very low concentrations of the target protein.
Another technique that has recently gained attention for its potential applications in higher order structure analysis is
hydrogen-deuterium exchange with mass spectrometry (H/DX–MS). This method exploits the natural tendency of hydrogen atoms
in a protein to exchange with hydrogen atoms in the surrounding solvent.11 If an isotope of hydrogen is used as the solvent, namely deuterium oxide, its heavier mass gets incorporated into the protein.
Because the protein now weighs more than normal, this change in mass can be monitored with high-resolution mass spectrometers.
The rate of exchange of hydrogen atoms is used to determine the protein structure. Compared with other techniques for analyzing
higher order protein structures, H/DX–MS is extremely sensitive, requiring only picomole quantities of protein. Further, the
technique can be applied to proteins of varying sizes, including large protein complexes. Perhaps most notably, the technique
can effectively analyze membrane proteins, which are very difficult to examine with most other protein characterization techniques.
Aggregates have been identified as having a potential causal link to increased immunogenicity in vivo. As a result, biomanufacturers are under increasing pressure from regulatory bodies to provide detailed information about
the quantity and nature of any aggregates present in a biopharmaceutical product. Aggregation can be particularly challenging
to evaluate, as it can occur during manufacture, storage, and handling. Information on aggregate formation therefore can be
of particular value during the development process in evaluating possible formulations for the drug product. Aggregate concentration
also is a critical parameter to be monitored in stability studies.
Sedimentation velocity analytical ultracentrifugation (AUC–SV) uses strong centrifugal force to separate various species in
a given sample mixture. This method can be used to study samples over a relatively wide range of pH and ionic strength conditions,
and at temperatures from 4 to 40 °C.12 Sample volumes are typically <1 mL, and the total mass of protein required is <1 mg. AUC–SV typically is used as an orthogonal
method to SEC, because of its accuracy, high resolution of aggregates, and ability to analyze aggregates in a wide range of
buffers. The technique is time-consuming, however, and requires specialized equipment, so it has not yet gained acceptance
as a standard test method for product release.
Asymmetric field flow fractionation (aFFF) also uses non-column technology to characterize aggregates, and therefore is also
appropriate for use as an orthogonal method to SEC. This method uses a semi-permeable membrane and two perpendicular fluid
flows in a channel to separate macromolecules based on molecular weight and hydrodynamic size.13 The technique is gentler to macromolecules than SEC, and is therefore less likely to change aggregate composition during
the analysis. Also, aFFF has a wider dynamic range than SEC, enabling detection of larger aggregates. The method does have
limitations, including the potential for molecular interactions between the proteins and the membrane, as well as a lack of
precision. Used in conjunction with other complimentary methods, aFFF can form part of a comprehensive evaluation of protein