The impact of formulation composition on the stability of freeze -dried proteins
Abstract
The broad objective of this study was to gain an improved understanding of the impact of formulation composition on both short-term stability (stability during freeze-drying) and long-term stability (stability during storage) of freeze-dried proteins. Studies were carried out using two model proteins, lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PDH). Formulation composition was systematically varied focusing on the sucrose/glycine system for short-term stability and on the roll of glass transition—associated mobility for long-term stability. All of the results are consistent with the interfacial denaturation of protein as a significant mechanism of protein activity loss during freeze-drying. This study extends the current body of literature on denaturation at the ice/freeze-concentrate interface to include denaturation at the interface generated by crystallization of glycine from sucrose/glycine excipient system. Structural collapse during freeze-drying does not appear to cause additional inactivation of model proteins. The glass transition temperature (Tg) of the sucrose/glycine formulation can be well below room temperature; however, no structural collapse was observed after storage above Tg, due to the support of crystalline glycine on the physical strength of the freeze-dried cake. No sucrose crystallization was observed in the sucrose/glycine formulation after storage above T g, probably because of the inhibition of sucrose crystallization by glycine. The Tg of the sucrose/glycine formulation was systematically varied by adding different concentrations of dextran and HSA. The data support the conclusion that Tg, by itself, is not predictive of the stability of freeze-dried proteins even in rank order fashion. The Tg of freeze-dried human serum albumin has been measured at 188°C by DSC. The unfolding temperature (Tm) of HSA was measured for a solid sample with high residual water content. The superiority of human serum albumin as a stabilizer is not due to its high Tg, but is partially due to its surfactant effect by keeping proteins away from the interface. Increasing of HSA concentration above the concentration of maximum protective effect does not affect protein stability. However, beyond the optimum concentration of Tween-80, the stability of protein decreases with increased concentration of Tween-80. This is believed to arise from destabilization of the native protein structure through hydrophobic interaction with surfactant micelles.
Degree
Ph.D.
Advisors
Nail, Purdue University.
Subject Area
Pharmaceuticals
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.