September 15, 2006 (Vol. 26, No. 16)

Technology Based on Hydrogen/Deuterium Exchange Has Applications for Biogenerics

The mass spectrometry platform developed by ExSAR ( reveals the conformational dynamics, or flexibility, of protein structures in solution at high resolution. Because the technology can detect small changes in protein structure, it is ideal for identifying drug-binding sites on target proteins and any changes in conformation due to ligand binding.

The company uses its technology for internal drug discovery and lead optimization to discover treatments for rare diseases caused by misfolded proteins. Called H/D Exchange (short for hydrogen/deuterium), the technology also could confirm that generic versions of biopharmaceuticals are structurally similar to brand-name biotherapeutics. “The time is right to introduce our technology to the FDA and the biopharmaceutical industry to show its capabilities for characterizing similarities of biogenerics,” says Bijan Almassian, Ph.D., president and CEO.

At least 60 recombinant protein and peptide drugs (also known as biotherapeutics, biomolecules, biopharmaceuticals, or biologics) are currently approved for sale. As their patents expire, manufacturers of biogenerics hope to produce cheaper versions. A major challenge facing generic manufacturers is whether biogenerics will be required to repeat extensive clinical trials to prove bioequivalence. An analytical tool that could demonstrate that a generic protein is biosimilar to a brand-name protein would speed the approval. “H/D Exchange could be that analytical tool,” says Patricia Weber, Ph.D., a structural biology consultant who advises ExSAR.

When the patent on a small molecule drug expires, regulations in the U.S. and Europe allow a generic version to be sold if the manufacturer can show that the generic drug is physicochemically identical to the original drug. In addition, bioequivalence must be demonstrated in a limited crossover clinical study of volunteers in which the generic drug must show similar pharmacokinetic and pharmacodynamic characteristics within certain limits.

Proving Bioequivalence

The regulatory climate for biogenerics is murkier. Unlike small molecules, protein drugs are not chemically synthesized, but produced in mammalian cells, microorganisms, or plants. The assessment of bioequivalence presents greater challenges.

Proteins are 100–1,000 times larger than small molecules, and even the most stringently controlled culture conditions can produce proteins with differences in glycosylation patterns or folding that influence biological activity or clinical properties. These complexities raise issues about how to prove bioequivalency.

“Experts at the FDA talk about finding an analytical tool that could show the similarity of proteins,” Dr. Weber says. “We feel that we have that tool.”

H/D Exchange could help biogenerics to reach the market. “ExSAR is capable of conducting high-throughput, automated H/D exchange experiments that provide high-resolution and comprehensive data about protein conformation and dynamics in solution. No other company has our particular technical expertise that can be applied to biogenerics,” says Dr. Almassian.

How it Works

H/D Exchange mimics the exchange of hydrogen molecules between water and proteins that occurs naturally. The H/D process measures the rate at which hydrogens located within each amino acid of the protein, called peptide amide hydrogens, exchange with hydrogens of the water in which a protein is dissolved. The rate of exchange depends on how exposed an amide hydrogen is to water in the folded protein. In unfolded regions, the amide hydrogens exchange more rapidly than do the amide hydrogens in the folded, structured portions of the protein. Amide hydrogen exchange rates can vary up to eight orders of magnitude in folded and unfolded protein regions.

Virgil Woods, M.D., developed high-resolution H/D Exchange methods at the University of California-San Diego, and ExSAR (Exchange Structure Activity Relationships), founded in 1999, holds exclusive rights to the technique.

Although people have studied aspects of H/D Exchange for more than 50 years, ExSAR “automated the mass spectrometry technology, increased the resolution to completely cover a protein, and created software for data analysis,” says Dr. Almassian.

Drug Optimization

In addition to the new emphasis on applying H/D Exchange to the upcoming regulatory issues associated with biogenerics, ExSAR uses the technology to measure drug/protein binding interactions for drug optimization. Subtle changes in protein structure can dramatically affect drug signaling and resistance. H/D Exchange can be applied directly to unlabeled samples of proteins in solution.

H/D Exchange identifies ligand-binding sites on protein targets, determines the structural consequences of ligand binding, and compares the conformations of native and recombinant proteins. Recently, ExSAR determined the interaction subsites for small molecules binding at the active site of p38 MAP kinase, which is drug target associated with inflammation and cancer. The experiments revealed that six subsites on the kinase were altered, and the findings helped to guide the synthesis of new compounds.

Genetic mutations alter protein folding, resulting in reduced protein levels and compromised protein function. A variety of illnesses are attributed to misfolded proteins, including cystic fibrosis; Alzheimer’s, Parkinson’s, and Huntington’s diseases; retinitis pigmentosa; and lysosomal storage disorders such as Tay-Sachs, Gaucher, Pompe, and Fabry’s diseases. Protein misfolding also is linked to prion disorders such as Creutzfeldt-Jakob and mad cow diseases. Structural comparisons of the native and misfolded proteins can identify drugs that promote proper folding or block the misshapen protein.

ExSAR in-licenses compounds to treat rare diseases caused by misfolded proteins. In late 2005, the company licensed NGT (N-acetyl-glucosamine thiazolide) from the Hospital for Sick Children in Toronto. NGT, a small molecule chaperone, controls a vital enzyme associated with adult Tay-Sachs disease. The FDA granted ExSAR Orphan Drug status for NGT. Compounds like NGT represent “a logical progression in a disease area where H/D technology can help to understand how compounds work,” says Dr. Weber.

ExSAR is seeking other compounds related to diseases caused by misfolded proteins. The company plans to advance them through preclinical and early clinical trials, then develop partnerships with larger companies, according to Dr. Almassian. ExSAR also performs contract services for major pharmaceutical and biopharmaceutical companies and biogeneric manufacturers.

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