The analysis of structure and stability in pharmaceutically relevant polypeptides.

摘要

As potential therapeutic molecules, polypeptides pose unique challenges for development, due to the fact that they may possess pharmaceutical instabilities tied to the higher order structure they adopt in solution. Thus, a greater understanding of the structure of a polypeptide can aid during formulation development. The main goal of this thesis is to demonstrate the use of biophysical techniques for analysis of polypeptide structure with the ultimate goal of increasing their pharmaceutical stability.;In the first section, we used two complimentary spectroscopic techniques, circular dichroism and nuclear magnetic resonance, to deduce the solution structure of the nonapeptide, leuprolide. Our results, the first complete spectroscopic structural study of a linear LHRH agonist analogue, are contrary to the accepted dogma regarding the structural characteristics these analogues, which has been gained solely by computational studies to this point.;In the next section, we used the control of higher order structure to slow the oxidative degradation of the two methionine residues of the peptide hormone, human corticotropin releasing factor (hCRF). By using a variety of biophysical techniques, we determined that hCRF associates into two distinct types of oligomeric structures, which can be controlled by adjusting solution conditions. The results of oxidation studies indicate that both oligomeric structures tend to protect the methionine residues from oxidation, albeit with different selectivity.;In order to aid during the process of formulation of freeze-dried protein products, Fourier transform infrared spectroscopy (FTIR) is frequently utilized. In the next study, we examined whether a common sampling technique, KBr processing, was causing changes in lyophilized protein spectra. By comparing spectra of two model proteins before and after KBr processing, we determined only minor changes were occurring upon processing.;In the final section, we used response surface methodology to examine the effects of combining one of three bulking agents with sucrose on the stability of a monoclonal antibody during lyophilization and storage. By measuring several lyophilized cake characteristics using biophysical methodologies, we found that certain combinations excipients resulted in a dried cake with unique stabilizing properties. This, in turn, resulted in lower levels of degradation of the antibody upon storage.