In situ study of insulin aggregation induced by water-organic solvent interface.
OBJECTIVE
The aim of this study was to assess insulin stability by monitoring in situ time-course of insulin aggregation induced by a water-organic solvent (o/w) interface that occurs during the microencapsulation process.


METHODS
Insulin aggregation at a simple o/w interface was monitored spectrophotometrically by detecting the percentage of turbidity changes (%T) at 350 nm. The effects of protein concentration and agitation and the presence of poly (lactic-co-glycolic acid) (PLGA) in methylene chloride (MC) on insulin aggregation were observed. For the 0.72 mg/ml insulin in phosphate-buffered saline (PBS), the effect of nonionic (dodecyl maltoside [DDM]) and anionic (sodium dodecyl sulfate [SDS]) surfactant in PBS were also evaluated at various protein/surfactant mol ratios. The conformation of insulin protected by a 10-fold molar excess of SDS recovered after 1 h of contact with MC was evaluated via circular dichroism (CD) spectroscopy.


RESULTS
A typical turbidity-time profile was represented by a sigmoidal curve. Greater change in %T was observed with increasing insulin concentration, in the presence of PLGA in MC and in the presence of agitation. DDM failed to delay insulin aggregation at all ratios used, whereas a less than 10% change in %T was observed in 1 h when a 10- - approximately 20-fold excess of SDS was used. CD spectra indicated that the presence of insulin in SDS after 1 h of contact with MC qualitatively retained its secondary structure integrity.


CONCLUSIONS
An experimental method was designed for an in situ assessment of protein stability at the o/w interface.
Kwon
Y M
YM
Department of Pharmaceutics and Pharmaceutical Chemistry/CCCD, University of Utah, Salt Lake City 84112, USA.
Baudys
M
M
Knutson
K
K
Kim
S W
SW
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002942:Circular Dichroism; D003692:Delayed-Action Preparations; D004355:Drug Stability; D005079:Excipients / Q000737:chemistry; D006801:Humans; D007004:Hypoglycemic Agents / Q000737:chemistry*; D007328:Insulin / Q000737:chemistry*; D008863:Microspheres; D011487:Protein Conformation; D013501:Surface-Active Agents / Q000737:chemistry
D003692:Delayed-Action Preparations; D005079:Excipients; D007004:Hypoglycemic Agents; D007328:Insulin; D013501:Surface-Active Agents