Effect of the covalent modification with poly(ethylene glycol) on a-chymotrypsin stability upon encapsulation in poly(lactic-co-glycolic) microspheres

Effect of the covalent modification with poly(ethylene glycol) on a-chymotrypsin stability upon encapsulation in poly(lactic-co-glycolic) microspheres
November 2004
Ingrid J. Castellanos 1, Wasfi Al-Azzam 2, Kai Griebenow 1 *
1Department of Chemistry, University of Puerto Rico, R?o Piedras Campus, P.O. Box 23346, San Juan, Puerto Rico 00931-3346
2Department of Biology, University of Puerto Rico, R?o Piedras Campus, P.O. Box 23346, San Juan, Puerto Rico 00931-3346
Journal of Pharmaceutical Sciences, Volume 94, Issue 2, Date: February 2005, Pages: 327-340
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Abstract
The effectiveness of the covalent modification of a-chymotrypsin with methoxy poly(ethylene glycol) (PEG) to afford its stabilization during encapsulation in poly(lactic-co-glycolic) acid (PLGA) microspheres by a solid-in-oil-in-water method was investigated. a-Chymotrypsin was chemically modified with PEG (Mw = 5000) using molar ratios of PEG-to-chymotrypsin ranging from 0.4 to 96. Various conjugates were obtained and the amount of PEG modification was determined by capillary electrophoresis. In this investigation, only those conjugates with PEG/chymotrypsin molar ratios between approximately 1 and 8 were considered because higher levels of modification caused protein instability even before encapsulation. The stability and functionality of the chymotrypsin formulations were investigated before encapsulation by measuring enzyme kinetics, thermal stability, and tertiary structure intactness, and after the initial lyophilization process by determining the secondary structure content. These stability parameters were related to select ones after encapsulation in PLGA microspheres (specifically, the amount of insoluble aggregates, residual enzyme activity, and magnitude of protein structural perturbations). The results show that the more stable the protein conformation before encapsulation was, the higher was the retention of the specific activity after encapsulation. In contrast, no relationship was found between the protein stability before encapsulation and the magnitude of encapsulation-induced protein aggregation. Even the lowest level of modification (PEG-to-chymotrypsin molar ratio of 0.7) drastically reduced the amount of insoluble aggregates from 18% for the nonmodified protein to 4%. The results demonstrate that PEG modification was able to largely prevent chymotrypsin aggregation and activity loss upon solid-in-oil-in-water encapsulation in PLGA microspheres. It is demonstrated that it is essential to optimize the degree of protein modification to ascertain protein stability upon encapsulation. ? 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:327-340, 2005