Sugar-frosting isn’t just for livening up boring bran flakes; it can
also preserve important therapeutic proteins. Researchers at the National
Institute of Standards and Technology (NIST) have developed a fast, inexpensive
and effective method for evaluating the sugars pharmaceutical companies use
to stabilize protein-drugs for storage at room temperature. The group presented
their findings* at the 236th American Chemical Society National Meeting and
Exposition.
In this image, a ribbon of protein stands in the foreground against a computer-simulation of a stress field in a glassy material (like a sugar-glass) in the background. Researchers at NIST have developed a fast, inexpensive and effective method for evaluating the sugars pharmaceutical companies use to stabilize protein-drugs for storage at room temperature. Credit: NIST
Protein-based drugs such as insulin and vaccines must be stabilized after manufacturing
in order to be used safely. For the past 30 years, researchers have been preserving
therapeutic proteins by freeze-drying them and coating them with a thin layer
of various formulations of glass-like sugars that act to stabilize their molecular
structures. This allows them to be safely stored for extended periods of time.
Pharmaceutical companies, though they have general guidelines, develop their
formulations essentially by trial and error and have to wait up to two years
to see if the glasses are suitable. The new methods will help pharmaceutical
companies make the best choice about which formulations to test and make it
easier to stabilize drugs at room temperature. Room-temperature storage is vital
when the pharmaceuticals are to be used in areas of the world where controlled
storage conditions are not available.
The new findings build upon previous work** at NIST in which the team used
neutron scattering to determine that rapidly solidified sugars preserve such
proteins best when they suppress molecular motions lasting a nanosecond or less.
Their latest experiments center on the hydrogen bonding that makes the sugars
rigid. They have shown that the lifetimes of these bond networks can be measured
directly with a fluorescent probe. This method is much more convenient than
using neutrons and could be used for routine formulation evaluation.
Hydrogen bonds are responsible for many of water’s properties; they make
water a liquid at room temperature. All biological fluids, which are composed
mostly of water, are also defined by their hydrogen bonds. Without these bonds,
proteins would unfold, and life as we know it would be impossible. Sugars used
to safeguard protein-based drugs act like cement, taking the place of water
by bonding to the proteins and locking them in place. By rapidly freezing liquid
sugar, its molecules have no time to form the usual orderly crystal patterns
typically found in sugars that are solids at room temperature. Lead NIST researcher
Marc Cicerone says that the randomly ordered sugar molecules fit the encased
proteins like a glove, “stiffening” molecular motions that cause
the proteins to chemically degrade.
Using the fluorescent probe, the team can now tell within minutes after freeze-drying
the protein whether the formulation will be stable, reducing the time and expense
associated with the “wait and see” method currently in use.
“Instead of needing relaxation measurements that require using neutron
scattering—a national facility with limited time availability—we
have developed a widely accessible solution in the form of readily available
steady-state fluorescence measurements,” Cicerone says. “This will
allow pharmaceutical companies to adopt the new metrology we’ve developed.”
When applied, the team’s findings should help to increase the availability
of viable medicines in places where refrigeration is scarce or unavailable.
* M. T. Cicerone and J. M. Johnson. Hydrogen bond network lifetime as an indicator
of protein stability in pharmaceutical preparations. Biophysical & Biomolecular
Symposium: Current Challenges in Protein Formulations. 236th ACS National Meeting,
Philadelphia, Penn., Monday, Aug. 18, 2008.