Peptide-level HDX MS
In a peptide-level HDX MS study, HD exchange for labeled soluble protein and aggregated protein samples is evaluated. The regions of a peptide involved in aggregate formation will show a different level of deuterium uptake from those regions in the free protein. Targeted electron-transfer dissociation (ETD) fragmentation of the peptides that exhibit deuterium uptake provides further detailed information. “Additional information on the dynamics of these interaction sites can be obtained by looking at uptake over a series of time points, where more tightly bound regions (less dynamic) will have a lower deuterium uptake profile than more transiently interacting sites that have more average solvent exposure,” observes Chakraborty.
HDX MS complements other technologies (e.g., nuclear magnetic resonance and X-ray crystallography) and has the advantages of requiring a minimal sample size, tolerance for many formulation components, and simplified sample preparation. It can also be used for larger proteins (hundreds of kilodaltons) and protein mixtures, and the presence of certain protein impurities is tolerated. The most important recent advances in this technology, however, have been in the development of informatics tools, such as Waters’ DynamX software, that enable much more rapid screening of the data produced during HDX MS analyses to determine the key regions of interaction. In addition, Waters has developed an integrated HDX MS system that has made HDX more routinely accessible and enabled automated data acquisition.
“With these two advances, it is now possible to rapidly reduce a very large quantity of analytical data very quickly to interpretable results,” Chakraborty states. “What used to take weeks can now be accomplished in a couple of days.” He also points to the use of the liquid chromatography/mass spectrometry technique LC/MSE for data-independent fragmentation and identification of hundreds of peptic peptides evaluated in sub-10-min HDX-MS runs and ion mobility LC/MSE (LC/HDMSE) for gas-phase separation of ions to achieve cleaner ion detection and better quantitation of deuterium uptake for individual peptides as being additional important developments. He would, however, like to see ETD fragmentation more widely used. To achieve that goal, Waters is working on peptide-specific optimizations, looking to improve HDX MS structural data generated for larger and more complex protein systems to develop new informatics tools to facilitate more efficient acquisition and interpretation of ETD fragmentation data for HDX MS experiments.
Evaluating the formulability of protein-based drugs
There have also been significant advances in analytical methods for the determination of protein aggregation, including high throughput capillary electrophoresis, immunoassays, and the optical imaging of aggregates for both quantitative and qualitative evaluations, according to Zurdo. Many of these methods, however, do not meet the requirements for very high-throughput analysis of potential drug formulations.
“The formulation readiness of protein-based drugs--or their formulability--can be an issue, particularly for biotherapeutics that must be administered in high concentrations. Because there are too many variables, there as yet have been no models developed to predict protein aggregation in a product formulation. Therefore, manufacturers must rely on physical analyses. As a consequence there is a significant need for inexpensive, simple, highly robust, and very high-throughput techniques for the determination of protein aggregation that give either yes/no or better/worse-type information that can be used to map the regions of experimental space where products appear to behave well,” says Zurdo.
The development of such methods is in the early stages. Some examples include modified immunoassays, self-interaction chromatography, and visual viscosity determination based on the diffusion of latex beads. “Unfortunately, there have been very few such techniques commercialized to date, and those that have been have not yet been fully validated. But progress is being made, and we expect to see some new methods being implemented in the not too distant future,” Zurdo notes.
The behavior of the formulated product once administered to patients must also be considered, because protein aggregation can occur in the human body as well (e.g., Alzheimer’s disease is caused by protein aggregation). There are ongoing studies aimed at developing sophisticated cell-based assays for the evaluation of the immune response to protein aggregates, but despite some preliminary links between in vitro data and clinical outcomes, to date there is not yet a definitive or absolute direct correlation between results obtained through this type of assay and the actual responses observed in patients.
It is a challenge to do this type of analysis because the final formulation of a therapeutic protein often does not contain the exact same composition that was used during clinical testing, and even the minor changes that are acceptable during final formulation can lead to protein modification or aggregation and thus promote immunogenic responses,” observes Zurdo.