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Storage Stability of Freeze-Dried Proteins

Summary:

We are resolving critical measurement issues that prevent researchers from reliably predicting the chemical and physical storage stability of biomacromolecules (proteins and DNA) in hydrophilic glasses such as those used by the biopharmaceutical industry to produce injectable medications. The analytical methods and theory that we develop will lead to rational formulation of stabilizing glasses, which will, in-turn, facilitate development of drug candidates that are promising but currently unstable, reduce process development costs for stabilizing drugs generally. Additionally, the work will shed light on the physical processes that lead to transport in glassy materials in general, which will, for example, help to predict such things as rates of drug elution from stents.

Description:

Intended Impact

Work in this project will enable industry to develop rational formulation approaches for freeze-drying, thus facilitating the manufacture safer, longer lasting biopharmaceuticals, with less time and resource cost required for stabilization. Biopharmaceuticals are the fastest growing subsector in pharmaceuticals, and currently generates $120 billion in sales annually; half of new therapeutic proteins will need to be freeze-dried for release, but formulation for freeze-drying is still inefficient and empirical, with only a 60% success rate. Improved analytical methods for characterizing protein stability in freeze-dried glasses, and better theory for understanding degradation routes are sorely needed. We are developing measurements and models to underpin the design and development of freeze-dried glass media that enable the chemical and physical stability of proteins and DNA during storage

Objectives

  • Develop rigorous metrics and theory for predicting protein stability in freeze-dried solids
  • Develop bench-top surrogates; analytical methods that can be used to obtain reliable and controlled approximations to the more rigorous, and typically more complex, stability-predicting measurements
  • Work with industry and academia to apply the analytical techniques we have developed in realistic product test and development environments

Technical Approach

This project is an integral part of a cooperative effort led by NIST and partially funded by the National Institutes of Health's National Institute of Biomedical Imaging and Bioengineering. It includes the University of Chicago, the University of Colorado and the University of Connecticut. Owing to the complexity of the protein stabilization problem, we have assembled a multidisciplinary team with world-class standing in each of the research areas needed. We use theory, simulation and experimentation to develop a clear understanding of key relationships between glass properties and likely stability outcomes of proteins in the glasses. We develop analytical methods to unambiguously and precisely measure the glass properties of interest. We validate the theoretical models by carrying out stability studies of model compounds and of pharmaceutically important proteins in fully characterized hydrophilic glasses. The theory, simulation, physical characterization and pharmaceutical studies are coordinated so that

Major Accomplishments:

  • In 2004 we showed for the first time that the amplitude of local, high-frequency motions (on ns timescale, and Angstrom length scale) in hydrophobic glasses are key indicators of protein stability within those glasses. Previous to this work, alpha relaxation in glasses was used as the sole measure of stability-related dynamics, even though it was known to correlate poorly.
  • We have recently demonstrated that a simple fluorescent probe can be used to reliably probe the ns dynamics relevant to protein stability in freeze-dried formulations. The ease of use and amenability to spatially-resolved measurements will allow us to develop characterization approaches for tissue scaffolds, and high-throughput formulation methods for use in the bio-pharmaceutical industry.
  • We have shown in that by considering both dynamics and protein conformation in the bulk and at the surface of freeze-dried formulations, we can quantitatively account for protein degradation rates during storage
 stability correlation plot

Protein degradation rates track fast β relaxation dynamics from neutron scattering. Chemical degradation and aggregation rates plotted on the ordinate for a dozen proteins in trehalose or sucrose glasses at temperatures ranging from (40 to 60) °C. The inverse of <u2>, a measure of the fast β relaxation amplitude, is plotted on the abscissa. All the data fall on the same general trend, independent of protein molecular weight, glass composition and temperature. The data show clearly that an increase in fast β relaxation corresponds to an increase in degradation rate. Such universal scaling with degradation has never been shown before.


Publications

MT Cicerone, JF Douglas, β relaxation governs protein stability in sugar-glass matricies, Soft Matter 8 (10), 2983 (1012).

J Giri, W-J Li, RS Tuan, MT Cicerone, Stabilization of proteins by nanoencapsulation in sugar–glass for tissue engineering and drug delivery applications, Advanced Materials 23 (42),4861-4867 (2011).

MT Cicerone, Q Zhong, J Johnson, KA Aamer, M Tyagi, Surrogate for Debye–Waller factors from dynamic Stokes shifts, The Journal of Physical Chemistry Letters, 2(12), 1464-1468 (2011).

End Date:

ongoing

Lead Organizational Unit:

mml

Source of Extramural Funding:

  • NIH / NIBIB - 1 R01 EB006398-01A1 (Completed in 2013)
  • CRADA with Medimmune (pending)

Customers/Contributors/Collaborators:

Pfizer
Medimmune
Eli Lilly
Merck
Mike Pikal (University of Connecticut)
Ted Randolph (University of Colorado)
John Carpenter (University of Colorado)
Juan de Pablo (University of Chicago)

Contact

Marcus Cicerone
cicerone@nist.gov
301.975.8104
100 Bureau Drive
Gaithersburg MD 20899-8543