- Poster Presentation
- Open Access
Analysis and characterization of different preparations of recombinant human follicle stimulating hormone (hFSH) and of its subunits
© Ribela et al; licensee BioMed Central Ltd. 2006
- Published: 10 October 2006
- Individual Subunit
- Laser Desorption Ionization Time
- Human Pituitary Gland
- Human Follicle Stimulate Hormone
- Dissociation Yield
Human follicle stimulating hormone (hFSH), synthesized by the human pituitary gland, is a biologically active glycoprotein composed of two noncovalently bound α- and β- subunits and is critically involved in the maturation of ovarian follicles and in spermatogenesis. Considerable heterogeneity associated with different hFSH preparations has been reported, mainly related to the presence of different glycoforms . The characterization of preparations of hFSH utilized as a therapeutic agent in reproductive medicine is therefore very important, especially considering that no specific monography has yet been published by the main pharmacopoeias.
In this work four hFSH preparations were analyzed, two of them being natural (pituitary- and urinary-derived) and the other two recombinant (CHO-derived). Studies were conducted to assess and compare hydrophobicity, molecular weight, charge heterogeneity and purity of the natural and recombinant heterodimeric preparations. These characteristics were examined by reversed-phase high performance liquid chromatography (RP-HPLC), matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF), isoelectric focusing and size-exclusion high performance liquid chromatography (HPSEC).
RP-HPLC analysis indicated a significant difference (p < 0.005) between the retention time (tR) of the pituitary and of the two recombinant FSH preparations. Urinary-derived hFSH was found more heterogeneous than the other three preparations. HPSEC analysis showed a significant difference (p < 0.001) between the tR of the urinary preparation and that of the pituitary or of the recombinant preparations. Urinary-derived hFSH presented the lowest HPSEC tR, in agreement with the highest molecular mass more accurately determined by MALDI-TOF mass spectrometry. The relative molecular mass (Mr) for the heterodimeric form of urinary, pituitary and recombinant hFSH preparations was 32527, 29176 and 28536 respectively.
Relative molecular mass (Mr) of the heterodimer (α+β) and related subunits of different hFSH preparations, determined by Maldi-Tof mass spectrometry
Retention times of heterodimeric hFSH before dissociation, of α-and β-subunits after dissociation and relative retention times (tRR) of the α and β subunits with basis on heterodimeric hFSH, determined on RP-HPLC (n = 2).
24.43 ± 0.156
26.98 ± 0.160
36.63 ± 0.198
r-hFSH Gonal F
25.19 ± 0.129
27.62 ± 0.235
38.86 ± 0.214
25.29 ± 0.070
27.85 ± 0.131
38.16 ± 0.127
Different isoforms were observed, by RP-HPLC, in the analysis of hFSH preparations of different origins (CHO, urinary and pituitary-derived). While the recombinant and pituitary hFSH preparations presented one main peak, the urinary-derived hFSH presented two major isoforms, one of which was equivalent to the major form of the other preparations. The other form could be an oxidized form of FSH present in this urinary preparation in high amount, as reported . The RP-HPLC characterization of the hFSH heterodimer and of individual subunits revealed differences in hydrophobicity in the following order: α-subunit > β-subunit > heterodimer. For the first time a quite satisfactory separation of the heterodimer from the dissociated β-subunit was attained.
Urinary-derived hFSH showed a higher Mr (11–14%) when compared with pituitary and recombinant hFSH, while pituitary hFSH showed a slightly higher Mr (~ 2%) in comparison with the recombinant preparation.
Supported by FAPESP and CNPq
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