RESUMO
The protective effects of amino acids on stabilizing protein secondary structure were evaluated using diffuse reflectance FTIR spectroscopy, and interactions between proteins and arginine were detected using solid-state NMR spectroscopy. Upon freeze-drying, excipient-free anti-CD11a and anti-IgE antibodies underwent significant changes in their secondary structures. For both antibodies, the amount of intermolecular beta-sheet substantially increased and the native conformation of intramolecular beta-sheet content decreased considerably. The addition of amino acids to the formulations reduced protein secondary structure alterations in a concentration-dependent manner. Histidine and arginine appeared to be the most protective excipients (of the amino acids studied) in inhibiting protein secondary structural changes. Solid-state NMR illustrated that non-covalent interactions (e.g., hydrogen bonding, ion-dipole interactions) were formed between the arginine side chain and the protein. Glycine is the least effective additive of those studied in preventing secondary structure changes upon freeze-drying. Despite secondary structural changes, freeze-dried protein in the presence and absence of amino acids refolded back into its native conformation upon reconstitution in water.
