Secondary structure characterization of microparticulate insulin powders.
The secondary structure content of microparticulate insulin powders produced by the supercritical antisolvent (SAS) precipitation technique was investigated via Raman spectroscopy. Precipitate samples were generated at 25 and 35 degrees C processing temperatures. Both precipitate samples gave amide I band spectra that were shifted roughly +10 cm-1 relative to the commercial powder. The corresponding secondary structure estimates had significantly increased beta-sheet contents with concomitant decreases in alpha-helix contents relative to the commercial protein; the sum of beta-turn and random coil content remained essentially unchanged. The magnitude of the perturbation was slightly greater for the 35 degrees C sample. Dissolution of the commercial powder and precipitates in 0.01 M HCl yielded solution structure estimates similar to that of the commercial powder. An analysis of insulin in dimethyl sulfoxide, the suspending solvent in the SAS process, indicated that some, but not all, of the structural change observed for the precipitate samples may be attributed to solvent exposure. These results are suggestive of extensive beta-sheet-mediated intermolecular interactions in precipitate states, consistent with analyses of irreversible protein aggregate/fibril states. Interestingly, unlike irreversible protein aggregates, the insulin powders recover essentially full biological activity on reconstitution.
Yeo
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Department of Chemical Engineering, Princeton University, NJ 08544-5263.
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D055598:Chemical Phenomena; D011232:Chemical Precipitation; D002626:Chemistry, Pharmaceutical / Q000379:methods; D002627:Chemistry, Physical; D004121:Dimethyl Sulfoxide; D005240:Feasibility Studies; D006851:Hydrochloric Acid; D007328:Insulin / Q000737:chemistry*; D010316:Particle Size; D011208:Powders; D017433:Protein Structure, Secondary; D012996:Solutions; D013059:Spectrum Analysis, Raman
D007328:Insulin; D011208:Powders; D012996:Solutions; D006851:Hydrochloric Acid; D004121:Dimethyl Sulfoxide