A new mechanism for decreasing aggregation of recombinant human interferon-γ by a surfactant: Slowed dissolution of lyophilized formulations in a solution containing 0.03% polysorbate 20
✍ Scribed by Serena D. Webb; Jeffrey L. Cleland; John F. Carpenter; Theodore W. Randolph
- Publisher
- John Wiley and Sons
- Year
- 2002
- Tongue
- English
- Weight
- 286 KB
- Volume
- 91
- Category
- Article
- ISSN
- 0022-3549
No coin nor oath required. For personal study only.
✦ Synopsis
To study the mechanisms by which Tween 20 (polysorbate 20) used in a reconstitution solution affects the aggregation of lyophilized recombinant human interferon-g (rhIFN-g), we used four types of buffered formulations containing 0.4± 5 mg/mL rhIFN-g in either 10 mM potassium phosphate or phosphate buffered saline:
(1) without excipients, (2) with 5% sucrose, (3) with 0.03% polysorbate 20, or (4) with the combination of 5% sucrose and 0.03% polysorbate 20. After lyophilization, infrared spectroscopy was used to analyze the secondary structure of the protein in the freezedried solid. Each solid showed structural perturbation of the protein. Each formulation was reconstituted with water or a 0.03% polysorbate 20 solution. Aggregation of rhIFNg after reconstitution was measured by optical density at A 350 , and recovery of soluble protein was determined by high-performance liquid chromatography and ultraviolet spectroscopy. After reconstitution with a 0.03% polysorbate 20 solution, aggregation levels in all formulations were either reduced or similar to those found after reconstitution with water. These results revealed the potential for recovery of native protein using the appropriate reconstitution conditions, even though the protein is non-native in the lyophilized state. Urea-induced unfolding with and without polysorbate 20 as measured by second-derivative ultraviolet spectroscopy indicated that a concentration of 0.03% polysorbate 20 lowered the free energy of unfolding for rhIFN-g (destabilizing). Polysorbate 20 also retarded refolding from urea solutions and increased aggregation. At a level of 0.03%, polysorbate 20 did not protect the protein against surface-induced aggregation during agitation. Dissolution times in water versus a 0.03% polysorbate 20 solution were measured using a rotating disk electrode for lyophilized formulations containing an electrochemically reactive species. The presence of 0.03% polysorbate 20 in the reconstitution solution nearly doubled the time required for dissolution of the phosphate buffered saline formulation, and the sucrose formulations dissolved 33±57% more slowly. Slowing the dissolution rates of lyophilized powders allows more time for