Fourier‐transform Infrared Spectroscopy Demonstrates that Lyophilization Alters the Secondary Structure of Recombinant Human Growth Hormone

The value of pharmaceutical proteins depends upon the retention of their native structure. A powerful technique to investigate the conformation of proteins is Fourier-transform infrared (FTIR) spectroscopy, which can be applied to both liquids and solids. Herein, using the model pharmaceutical protein, excipient-free recombinant methionyl human growth hormone (rhGH), we have examined the amide III band region, which is sensitive to changes in the secondary structure. Gaussian curve-fitting analysis of rhGH vibrational spectra in solution yields a high degree of α-helix content, comprising half of the overall secondary structure, and virtually no β-sheets, consistent with the four antiparallel α-helices in the molecule as elucidated from the crystal structure. The remainder of the secondary structure is comprised of extended chain and other disordered structures. The IR spectra of rhGH was also measured in the lyophilized solid pressed into pellets with KBr. The data indicate that rhGH undergoes a dramatic change in the secondary structure upon lyophilization, namely, α-helices decrease by half with increases in β-sheet and unordered structures. The data were not significantly influenced by the conditions used in processing the protein-in-KBr pellet. In addition, we measured the spectrum of the dried powder alternatively, using an FTIR microscope, and found the structure much more similar to that obtained for the protein-in-KBr pellet than for the aqueous solution. The secondary structural reorganization was reversible, as determined from the protein spectra following reconstitution in water.