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. 2002 Sep;30(3):175-85.
doi: 10.1006/biol.2002.0322.

Thermal stability: a means to assure tertiary structure in therapeutic proteins

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Thermal stability: a means to assure tertiary structure in therapeutic proteins

Mike Cauchy et al. Biologicals. 2002 Sep.

Abstract

To be both safe and effective, a therapeutic product must have the correct chemical structure and be free of harmful contaminants. Structure in protein therapeutic products, however, implies not only the correct sequence of amino acids (primary structure) but also the proper folding of that amino acid chain in three-dimensional space (tertiary structure). This work is part of a general strategy to develop a battery of physico-chemical methods that could give assurances of structure (and hence function) in formulated therapeutic proteins in the absence of in vivo data. It focuses on recombinant human growth hormone (rhGH), a well-characterized therapeutic protein, and examines the utility of thermodynamic parameters in assessing its tertiary structure. Resistance of solutions of formulated rhGH to thermal denaturation was followed using Fourier Transform Infrared Spectroscopy (FTIR) by observing decreases in total helicity and increases in intermolecular beta-sheet formation. Under conditions known to induce changes in the intra-molecular ionic and H-bonding patterns stabilizing the tertiary structure but not affecting the protein's secondary structure or global fold, we have observed upwards of a 12 degrees C shift in the melting temperature of the protein. Furthermore, our results indicated that the T(m) of unfolding of rhGH was sensitive to much more subtle changes in the protein structure. Thus, resistance to thermal denaturation may well be a useful means to measure structure in formulations of well-characterized therapeutic proteins.

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