Study of forced degradation, by HPLC-UV and electrophoresis, of a new synthetic therapeutic elastic protein formulated in isotonic saline solution

Introduction et objective
The Laboratoire de Biologie Tissulaire et Ingénierie Thérapeutique has developed a synthetic elastic protein (SEP). The aim of this recombinant protein is to restore elasticity to tissues altered by genetic elastinopathies by virtue of its molecular prosthetic action. SEP is an unstructured biotherapeutic agent whose action is not linked to its conformation and whose stability is not yet known. To date, there is only one validated method for dosing SEP, using high-performance liquid chromatography (HPLC) coupled to a UV detector.
The aim of this study is to carry out the first study of forced degradation of SEP in order to identify the conditions that cause its degradation and to verify the stability-indicating nature of the SEP dosage method.

Materials and Methods
Fifteen degradation conditions were tested over 7 exposure times ranging from 0.5 to 168 h : freeze-thaw cycles, exposure to temperatures of 37°C or 80°C; ultraviolet (UV) at 254 nm and 365 nm; acidic solutions (HCl) with pH 1, 2 4 and 6 at 80°C; basic solutions (NaOH) with pH 8, 10 and 13 at 80°C; solutions with pH 1 and 13 at 20°C; hydrogen peroxide solutions 3% at 80°C. Each condition was tested on 2 SEP samples and controlled by a 0.9% NaCl solution. After neutralization of the degradation factor tested, the UV absorption spectrum of the samples was determined by UV-visible spectrophotometry before analysis by HPLC-UV. Samples that appeared to be 5-20% degraded at the HPLC dosage were then analysed by SDS-PAGE (sodium dodecysulphate polyacrylamide gel electrophoresis) stained with Coomassie blue and by anti-Flag Western-Blot.

Results
Spectrophotometry allowed the determination of 2 analytical wavelengths, 210 and 265 nm, used in HPLC-UV. With the exception of UV exposure (254 or 365 nm), all conditions allowed 5-20% SEP degradation in 168 h or less. The chromatograms showed only one peak whose area under the curve decreased or broadened as a function of degradation conditions. Migration on gels revealed the presence of degradation products not observed by HPLC. SEP dosage does not clearly distinguish the protein from its degradation products and is therefore not a stability indicator method.

Discussions and Conclusions
This study has enabled us to identify the situations that cause SEP degradation. The dosage method needs to be improved in order to increase its resolution before degradation products can be identified by mass spectrometry.

See the poster