Mapping Epitopes with H/D-Ex Mass Spec

Epitope mapping is taking the spotlight in the race to develop new therapeutic monoclonal antibodies, a market expected to reach $33 billion by 2010. Identification and characterization of an epitope is key to this development process, as well as for vaccine design and mapping ligand binding sites in receptors using mAbs. A more recent application involves protecting the epitope from competitors, since the data is often used in patent filings.
ExSAR’s (www.exsar.com) mass spectrometry platform, H/D-Ex (amide hydrogen/deuterium exchange MS), was originally developed at the University of California, San Diego. It was later enhanced with automated sample handling and software to generate reproducible measures of amide proton-exchange rates. Differences in the rate of exchange serve to highlight the epitope location. “What makes this unique is that it’s widely applicable, with medium resolution and throughput for protein characterization,” says Yoshi Hamuro, Ph.D., senior director of technical development.
A protein is mixed with deuterated buffer and incubated, at which time backbone amide hydrogens exchange with bulk solvent deuterons. The exchange rate of each backbone amide hydrogen is unique to its environment. After incubation, the exchange reaction is quenched by shifting the pH to 2 and lowering the temperature to 0°C.
The exchanged protein is then proteolyzed with proteases. Peptic fragments are chromatographically separated and their mass determined by MS. The experiment is repeated in the absence of deuterium, and the molecular weight difference of identical fragments attributed to deuteration. A deuteration build-up curve allows the rate of exchange to be calculated.
This is accomplished by exposing the protein to deuterated solvent over increasing time periods. Resulting data curves are generated by plotting deuteration levels as a function of on-exchange time. Since deuteration trends can be difficult when looking at more than 30 curves, the company assigns different colors—red signifies highly deuterated and blue, not highly deuterated. The deuterated levels of various sequence segments are represented in the appropriate color and plotted beneath the corresponding sequence.

Epitope Mapping

Currently, the most popular H/D-Ex application is epitope mapping. This process involves separation of the antigen, then on-solution exchange experiments with the antigen by itself to determine the best time frame for on-/off-solution column exchange. The antibody is immobilized, and the binding capacity of the column against the antigen is determined. Then an on-/off-solution column exchange experiment is conducted to determine the antibody binding site.
“Although most peptic fragments carry almost no deuterium atoms, there are a few peptic fragments that carry some deuterium atoms after the process,” Dr. Hamuro says. These segments are considered the H/D exchange identified binding site. The resolution of information is limited by the size of the peptic fragments generated by proteolysis— usually 5–15 amino acid-residues long. 
Although x-ray crystallography is considered the gold standard for epitope mapping and provides high resolution, it remains technically challenging. Other methods, such as phage display, overlapping peptides, and limited proteolysis, provide high throughput but lack reliability, especially for conformational epitopes.
“H/D-Ex is compatible with these and has been used to create many conformational eptitope maps,” adds Dr. Hamuro. These epitopes have more than one segment of the antigen sequence interacting with the antibody. The vast majority of antigen-antibody interactions have confirmational epitopes. The platform was recently used to identify the epitope of a neutralizing antibody to IL-17 A, a cytokine involved in several proinflammatory signaling pathways. The epitope was then validated via x-ray crystallography.
Some limitations to H/D-Ex include heavily glycosylated proteins in which it may be difficult to identify glycosylated peptides after digestion, and heavily di-sulfide-bonded proteins, which are difficult to digest and make it difficult to identify peptic fragments. Also, proteins larger than 100 kDa may have reduced sequence coverage.

Drug Discovery

The platform’s drug discovery applications include ligand binding-site identification, binding-site and pharmacology correlations (full agonist vs. partial antagonist), and enabling fragment-based drug discovery strategies in the absence of ligand-bound x-ray structures.

A biogeneric that has a different formulation or manufacturing process is suspected of being nonbioequivalent to the original. According to the company, H/D-Ex is able to detect subtle changes in protein structure within a few days. It recently identified a lot-to-lot difference in the protein dynamics of an antigen used by a client to generate therapeutic neutralizing antibodies.

A 20-residue region showed a differential rate of amide-hydrogen exchange in one of the lots. The lots were supposed to have exactly the same protein and the same manufacturing. The client expressed concern about unspecified modification and wanted additional data on equivalence. A difference was detected in hydrogen exchange rates, indicating a structure that had been compromised or changed.

Dr. Hamuro says that some of the future applications of the platform include epitope mapping of patients’ polyclonal antibodies against protein therapeutics when immunogenicity is observed. Another large potential area is the study of membrane protein-ligand interactions. “These are difficult to work with and we are just starting to see a few examples of using the H/D-Ex data with membrane proteins.”

In addition, the company sees opportunities for its technology in the biosimilars arena. “Our technology can be helpful to develop biosimilars when we want to see a direct comparison to the original,” Robert Johnston, president and CEO says.