Table 2.
Overview of extended characterization program of Ovaleap® and comparability with Gonal-f®
| Characteristic | Method | Ovaleap® | Gonal-f® | |
|---|---|---|---|---|
| Molecular mass | ||||
| Molecular mass (Daltons) | Nonreduced | SDS-PAGE Coomassie staining | Main band with approximately 40 kDa | Main band with approximately 40 kDa |
| Reduced | Main band with approximately 20 kDa; additional faint bands between 16 and 20 kDa | Main band with approximately 20 kDa; additional faint bands between 16 and 20 kDa | ||
| α-Subunit (native) | MALDI-TOF MS | 14.6 kDa | 14.6 kDa | |
| β-Subunit (native) | 17.8/18.3 kDaa | 17.8/18.3 kDaa | ||
| Primary structure | ||||
| Peptide mapping | Peptide mapping using trypsin, endo Lys-C, and endo Glu-C followed by ESI-MS determination of peptides | All determined masses of peptides correspond with expected masses; 100% sequence coverage achieved | All determined masses of peptides correspond with expected masses; 100% sequence coverage achieved | |
| Similar peptide maps obtained with Ovaleap® and Gonal-f® | ||||
| Secondary structure | ||||
| Structural topology | Far-UV circular dichroism spectroscopy | Samples have identical structural conformations and folding, which are superimposable with those of FSH reference standard | Samples have identical structural conformations and folding, which are superimposable with those of FSH reference standard | |
| Isoform distribution | ||||
| Isoform distribution profile | Isoelectric focusing (Serva Violet 17 staining) | Nine isoforms detectable with pI values between 3.6 and 5.3 | Nine isoforms detectable with pI values between 3.6 and 5.3 | |
| High similarity with respect to isoform distribution between Ovaleap® and Gonal-f® | ||||
| Biological activity | ||||
| In vitro bioassay | Cell-based receptor binding assay | Similar specific activities and dose–response curves obtained for Ovaleap® and Gonal-f® | ||
| In vivo bioassay | Rat bioassay | Similar specific activities determined for Ovaleap® and Gonal-f® | ||
| Product-related impurities | ||||
| Nonmonomeric r-hFSH (multimers and aggregate) | SE-HPLC | No aggregates detectable | No aggregates detectable | |
| Nonreducing SDS-PAGE Western immunoblot | No aggregates detectable; expected main band of heterodimer, additional faint bands of free subunits | No aggregates detectable; expected main band of heterodimer, additional faint bands of free subunits | ||
| Ovaleap® truncated forms | Reducing SDS-PAGE Western immunoblot | No truncated forms detectable; expected bands of free subunits | No truncated forms detectable; expected bands of free subunits | |
ESI-MS electrospray ionization mass spectrometry, FSH follicle-stimulating hormone, kDa kilodalton, MALDI-TOF MS matrix-assisted laser desorption ionization time of flight mass spectrometry, pI isoelectric points, r-hFSH recombinant human FSH, SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis, SE-HPLC size exclusion high-performance liquid chromatography, UV ultraviolet
aAfter harsh denaturation and reducing conditions