Use of infrared spectroscopy to assess secondary structure of human growthhormone within biodegradable microspheres

The purpose of this study was to test the utility of infrared (IR) spectros copy to determine protein secondary structure in biodegradable microspheres . Encapsulation of proteins within biodegradable polymers, [e.g, poly(lacti c-co-glycolic acid) (PLGA)] for controlled drug release has recently been t he subject of intense research effort. The ability to assess protein integr ity after microsphere production is necessary to successfully produce micro spheres that release native proteins. We used IR spectroscopy, a noninvasiv e method-as opposed to conventional organic solvent extraction or in vitro release at elevated temperature-to assess the secondary structure of recomb inant human growth hormone (rhGH) within dry and rehydrated microspheres. P LGA microspheres containing rhGH with different excipients were prepared by a conventional double-emulsion method. The protein IR spectra indicated th at the encapsulation process could perturb the structure of rhGH and that e xcipients could inhibit this damage to varying degrees. A strong positive c orrelation was found between intensity of the dominant a-helical band in th e spectra of rhGH in rehydrated microspheres and the percent monomer releas ed from microspheres during incubation in buffer. We also studied microsphe res prepared with zinc-precipitated rhGH. The addition of Zn2+ during micro sphere processing partially inhibited protein unfolding and fostered comple te refolding of rhGH upon rehydration. In conclusion, IR spectroscopy can s erve as a valuable tool to assess protein structure within both dried and r ehydrated microspheres.