CONTROLLED OVARIAN HYPERSTIMULATION WITH IMPROVED RECOMBINANT HUMAN FOLLICLE-STIMULATING HORMONE

Abstract

The present invention pertains to methods for controlled ovarian hyperstimulation in a female subject using improved recombinant human follicle-stimulating hormone (rhFSH). The methods result in a high number of fertilizable oocytes even at low amounts of FSH administered to the female subject.

Claims

1. A method for controlled ovarian hyperstimulation for stimulating the development of multiple ovarian follicles in a female subject, comprising (a) administering to a female subject a recombinant FSH preparation using a dosage regimen wherein the single doses sum up to an average amount of from about 35 to about 250 IU FSH per day; (b) triggering ovulation when there are multiple follicles with a mean diameter equal to or greater than 12 mm and/or when there is at least one follicle with a diameter of at least 17 mm; (c) obtaining multiple oocytes from the female subject, wherein on average at least 5 oocytes per female subject are obtained and/or at least 5 oocytes from the female subject are obtained; wherein the recombinant FSH in the preparation has a glycosylation pattern comprising the following characteristics: (i) a relative amount of glycans carrying bisecting N-acetylglucosamine (bisGlcNAc) of at least 20% of all glycans attached to the FSH in the preparation; and (ii) a relative amount of 2,6-coupled sialic acid of at least 40% of all sialic acid residues attached to the FSH in the preparation.

2. The method of claim 1, wherein in step (a) a dosage regimen is used, wherein the single doses sum up to an average amount of from about 50 to about 125 IU FSH per day; in step (b) ovulation is triggered when there is at least one follicle with a diameter of at least 17 mm; in step (c) at least 5 oocytes are obtained from the female subject in the form of cumulus oocyte complexes (COCs), and at least 4 of these oocytes are metaphase II oocytes; and wherein the recombinant FSH in the preparation has a glycosylation pattern comprising the following characteristics: (i) a relative amount of glycans carrying bisecting N-acetylglucosamine (bisGlcNAc) in the range of from about 25% to about 50% of all glycans attached to the FSH in the preparation; (ii) a relative amount of 2,6-coupled sialic acid in the range of from about 53% to about 80% of all sialic acid residues attached to the FSH in the preparation; (iii) a relative amount of sulfated glycans of at least 5% of all glycans attached to the FSH in the preparation; (iv) a relative amount of glycans carrying outer arm fucose of 5% or less of all glycans attached to the FSH in the preparation; (v) a relative amount of glycans carrying core fucose of at least 30% of all glycans attached to the FSH in the preparation; (vi) a relative amount of at least tetraantennary glycans of at least 16% of all glycans attached to the FSH in the preparation; (vii) a relative amount of glycans carrying one or more sialic acid residues of at least 88% of all glycans attached to the FSH in the preparation; and (viii) a Z number of at least 210.

3. The method of claim 1, wherein a dosage regimen is used in step (a), wherein the single doses sum up to an average amount of from about 50 to about 125 IU FSH per day.

4. The method of claim 1, wherein a dosage regimen is used in step (a), wherein about 50 to about 125 IU FSH are administered every day; or wherein about 100 to about 250 IU FSH are administered every second day; or wherein about 150 to about 375 IU FSH are administered every third day.

5. The method of claim 1, wherein a dosage regimen is used in step (a), wherein the single doses sum up to an average amount of from about 70 to about 250 IU FSH per day, and wherein the female subject is selected from the group consisting of female subjects having an age of at least 35 years, preferably in the range of about 37 years to about 50 years; female subjects having a serum level of anti-mullerian hormone (AMH) of 1.5 female subjects having an antral follicle count of 9 or less as the sum of both ovaries, preferably in the range of 4 to 8; female subjects having a body mass index (BMI) of at lest 25 kg/m.sup.2, preferably in the range of about 28 kg/m.sup.2 to about 45 kg/m.sup.2; and female subjects having undergone a previous conventional FSH stimulation cycle wherein the development of less than 4 oocytes was induced.

6. The method of claim 1, wherein a dosage regimen is used in step (a) wherein the recombinant FSH preparation is administered in an amount in IU which is 75% or less, preferably 50% or less of the amount recommended for recombinant FSH preparations produced by CHO cells, in particular Gonal-f, in the same therapeutic situation.

7. The method of claim 1, wherein the method comprises only one cycle of controlled ovarian hyperstimulation.

8. The method of claim 1, wherein the oocytes are obtained in step (c) in the form of cumulus oocyte complexes (COCs), and/or wherein at least 4 of the oocytes obtained in step (c) are metaphase II oocytes.

9. The method of claim 1, wherein the method further comprises (d) fertilizing at least one oocyte obtained in step (c); and (e) transferring at least one fertilized oocyte or embryo derived therefrom into a female human patient.

10. The method of claim 9, wherein the method further comprises freezing or vitrificating at least one oocyte obtained in step (c) prior to step (d); or freezing or vitrificating at least one fertilized oocyte obtained in step (d) or at least one embryo derived therefrom prior to step (e).

11. The method of claim 9, wherein only a subset of the oocytes obtained in step (c) are fertilized in step (d), and/or wherein only a subset of the oocytes fertilized in step (d) are transferred into a female human patient in step (e), and wherein the oocytes not fertilized in step (d) or the fertilized oocytes or embryos not transferred into a female human patient in step (e) are optionally frozen or vitrificated for subsequent use.

12. The method of claim 1, wherein the administration of the recombinant FSH preparation in step (a) does not comprise the concurrent administration of another gonadotropin such as LH or hCG or another agent which induces or enhances follicle growth.

13. The method of claim 1, wherein the administration of the recombinant FSH preparation in step (a) comprises the preceding and/or concurrent administration of a GnRH agonist or a GnRH antagonist.

14. The method of claim 1, wherein the female subject undergoes assisted reproductive technology (ART), in particular including in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), gamete intrafallopian transfer (GIFT), zygote intrafallopian transfer (ZIFT), and/or embryo transfer.

15. The method of claim 1, obtainable by production in the human cell line GT-5s deposited under the accession number DSM ACC3078, or a cell line derived therefrom or a cell line homologous thereto.

16. The method of claim 1, wherein the glycosylation pattern comprises the following characteristics: (i) a relative amount of glycans carrying bisecting N-acetylglucosamine (bisGlcNAc) in the range of from about 25% to about 50% of all glycans attached to the FSH in the preparation; (ii) a relative amount of 2,6-coupled sialic acid in the range of from about 53% to about 80% of all sialic acid residues attached to the FSH in the preparation; (iii) a relative amount of sulfated glycans of at least 3% of all glycans attached to the FSH in the preparation; (iv) a relative amount of glycans carrying outer arm fucose of 5% or less of all glycans attached to the FSH in the preparation; (v) a relative amount of glycans carrying core fucose of at least 30% of all glycans attached to the FSH in the preparation; (vi) a relative amount of at least tetraantennary glycans of at least 16% of all glycans attached to the FSH in the preparation; (vii) a relative amount of glycans carrying one or more sialic acid residues of at least 88% of all glycans attached to the FSH in the preparation; and (viii) a Z number of at least 210.

17. A method for stimulating follicle maturation in a female subject, comprising (a) inducing or enhancing follicle growth in a female subject by administering a recombinant FSH preparation; and (b) subsequently triggering ovulation; wherein the recombinant FSH in the preparation has a glycosylation pattern comprising the following characteristics: (i) a relative amount of glycans carrying bisecting N-acetylglucosamine (bisGlcNAc) of at least 20% of all glycans attached to the FSH in the preparation; and (ii) a relative amount of 2,6-coupled sialic acid of at least 40% of all sialic acid residues attached to the FSH in the preparation; wherein triggering ovulation in step (b) is commenced at least 48 h after termination of the administration of the recombinant FSH preparation in step (a).

18. The method of claim 17, wherein triggering ovulation in step (b) is commenced about 60 h to about 120 h, preferably about 72 h to about 96 h after termination of the administration of the recombinant FSH preparation in step (a).

19. The method of claim 17, wherein triggering ovulation in step (b) is performed by administering hCG or a derivative thereof.

20. A method for controlled ovarian hyperstimulation for stimulating the development of multiple ovarian follicles in a female subject, comprising (a) administering to a female subject a recombinant FSH preparation using a dosage regimen wherein the recombinant FSH preparation is administered in an amount in IU which is 80% or less of the amount recommended for recombinant FSH preparations produced by CHO cells in the same therapeutic situation; (b) triggering ovulation when there are multiple follicles with a mean diameter equal to or greater than 12 mm and/or when there is at least one follicle with a diameter of at least 17 mm; (c) obtaining multiple oocytes from the female subject, wherein on average at least 5% more oocytes per female subject are obtained compared to a similar treatment with the amount recommended for recombinant FSH preparations produced by CHO cells in the same therapeutic situation; wherein the recombinant FSH in the preparation has a glycosylation pattern comprising the following characteristics: (i) a relative amount of glycans carrying bisecting N-acetylglucosamine (bisGlcNAc) of at least 20% of all glycans attached to the FSH in the preparation; and (ii) a relative amount of 2,6-coupled sialic acid of at least 40% of all sialic acid residues attached to the FSH in the preparation.

21. The method of claim 20, wherein a dosage regimen is used in step (a), wherein the recombinant FSH preparation is administered in an amount in IU which is 50% or less of the amount recommended for recombinant FSH preparations produced by CHO cells, in particular Gonal-f, in the same therapeutic situation.

22. The method of claim 20, wherein in step (c) on average at least 5% more metaphase II oocytes and/or at least 5% more cumulus oocyte complexes are obtained compared to a similar treatment with the amount recommended for recombinant FSH preparations produced by CHO cells, in particular Gonal-f, in the same therapeutic situation.

Description

FIGURES

[0201] FIG. 1 shows the relative change of the mean follicle size in healthy female volunteers after a single administration of the indicated amount of FSH (invention). The follicle size before administration is used as reference (100%).

[0202] FIG. 2 shows the relative change of the mean follicle size in healthy female volunteers after a single administration of placebo or 150 IU Bravelle or Gonal-f. The follicle size before administration is used as reference (100%).

[0203] FIG. 3 shows the relative change of the mean follicle size as an average of all subjects after a single administration of the indicated amount of FSH (invention) (A) or placebo, 150 IU Bravelle or 150 IU Gonal-f (B). The follicle size before administration is

[0204] FIG. 4 shows the concentration of FSH in the serum of healthy female volunteers during a multiple dose study with daily administration of FSH (invention) (also administration every 2.sup.nd day), Gonal-f or Bravelle. Similar amounts of FSH administered resulted in comparable FSH serum levels.

[0205] FIG. 5 shows the mean number of follicles with a diameter of 8.0 mm or more observed in healthy female volunteers (mean of 10 subjects) after daily dosing of 150 IU FSH (invention) for 7 days. The coloring of the bars indicates the follicle size.

[0206] FIG. 6 shows the mean number of follicles with a diameter of 8.0 mm or more observed in healthy female volunteers (mean of 10 subjects) after daily dosing of 150 IU Gonal-f for 7 days. The coloring of the bars indicates the follicle size.

[0207] FIG. 7 shows the mean number of follicles with a diameter of 8.0 mm or more observed in healthy female volunteers (mean of 10 subjects) after daily dosing of 150 IU Bravelle for 7 days. The coloring of the bars indicates the follicle size.

[0208] FIG. 8 shows the mean number of follicles with a diameter of 8.0 mm or more observed in healthy female volunteers (mean of 10 subjects) after daily dosing of 75 IU FSH (invention) for 7 days. The coloring of the bars indicates the follicle size.

[0209] FIG. 9 shows the mean number of follicles with a diameter of 8.0 mm or more observed in healthy female volunteers (mean of 10 subjects) after administration of 150 IU FSH (invention) every second day for 7 days. The coloring of the bars indicates the follicle size.

[0210] FIG. 10 shows the concentration of inhibin-B (A) and estradiol (B) in the serum of healthy female volunteers after a multiple dose study with administration of FSH (invention) (75 IU daily, 150 IU daily, 150 IU every 2.sup.nd day) or Gonal-f (150 IU daily) during days 1 to 7.

[0211] FIG. 11 shows the mean number of follicles with a diameter of 10.0 mm or more observed in healthy female volunteers (mean of 10 subjects) after administration of (A) 150 IU FSH (invention), (B) 150 IU Gonal-f or (C) 75 IU FSH (invention) daily or (D) 150 IU FSH (invention) every second day for 7 days. The coloring of the bars indicates the follicle size.

[0212] FIG. 12 shows the mean plasma FSH concentration versus time after the last of multiple doses of subcutaneously administered FSH.

[0213] FIG. 13 shows the cAMP release of isolated granulosa cells stimulated with different concentrations of the improved recombinant human FSH (FSH (invention); preparation 1: open squares, preparation 2: closed triangles) or FSH obtained from CHO cells (Gonal F; closed diamonds).

[0214] FIG. 14 shows the estradiol synthesis of isolated granulosa cells stimulated with different concentrations of the improved recombinant human FSH (FSH (invention); preparation 1: open squares, preparation 2: closed triangles) or FSH obtained from CHO cells (Gonal F; closed diamonds).

[0215] FIG. 15 shows the progesterone synthesis of isolated granulosa cells stimulated with different concentrations of the improved recombinant human FSH (FSH (invention); preparation 1: open squares, preparation 2: closed triangles) or FSH obtained from CHO cells (Gonal F; closed diamonds).

[0216] FIG. 16 shows the cAMP release of isolated granulosa cells stimulated with different concentrations of the improved recombinant human FSH (FSH (invention); open squares) or urinary FSH (Fostimon; closed diamonds).

[0217] FIG. 17 shows the estradiol synthesis of isolated granulosa cells stimulated with different concentrations of the improved recombinant human FSH (FSH (invention); open squares) or urinary FSH (Fostimon; closed diamonds).

[0218] FIG. 18 shows the progesterone synthesis of isolated granulosa cells stimulated with different concentrations of the improved recombinant human FSH (FSH (invention); open squares) or urinary FSH (Fostimon; closed diamonds).

[0219] FIG. 19 shows the results of the Steelman-Pohley assay using the improved recombinant human FSH in comparison to standard urinary FSH and standard recombinant FSH obtained from CHO cells. The ovarian weight gain in immature female rats after daily administration for three days is plotted against the used FSH concentration.

[0220] FIG. 20 shows schematic drawings of complex-type glycan structures which may be attached to the FSH glycosylation sites. Shown are (a) biantennary, (b) triantennary and (c) tetraantennary structures. One or more of the sialic acid and galactose residues may also be absent in these structures and the structures may further comprise, for example, a bisecting GlcNAc residue, a fucose residue and/or sulfate groups. Sia: sialic acid; Gal: galactose, also referred to herein as terminal galactose; GlcNAc: N-acetylglucosamine; Man: mannose.

EXAMPLES

Example 1

Preparation of FSH (Invention)

[0221] FSH is produced by cultivation of GT-5s cells stably transfected with two expression constructs harbouring the alpha and beta chain of human FSH (alpha chain accession number NT_007299.13; beta chain accession number NT.sub.1'009237.18). The plasmid for the expression of the FSH alpha chain is carrying the gene of a mutated version of the murine dihydrofolate reductase (dhfr) with higher resistance to the enzyme inhibitor methotrexate than the native form and the second plasmid for the expression of the FSH alpha chain is carrying the puromycin resistance gene.

[0222] Transfection of the cell line for FSH (invention) expression was performed by nucleofection using the two expression plasmids described above. For selection and amplification of stable antibody producing cell clones puromycin and methotrexate were added at increasing concentrations. Amplified cell pools were seeded in a semi-solid matrix for single cell cloning by the Clone PixFL technology or single cell cloning by limited dilution. The clones were screened for high secretion of intact FSH molecules.

[0223] FSH is produced by fermentation of the final FSH producing GT-5s clone in batch, fed-batch or perfusion process under serum free conditions. The fermentation is usually run for 2-3 weeks.

[0224] After fermentation the supernatant is filtered through 2 m filters to eliminate cells and cell debris prior to a sterile filtration step using 0.2 m filters. The purification process utilizes a reverse phase chromatography (RPC) as capture step followed by a concentration step and a subsequent size exclusion chromatography (SEC). Optionally, the eluate is then applied to an anion exchange chromatography (AEC) to eliminate the less acidic FSH contents. This is done by washing the bound FSH with washing buffer at pH 5.0 (enrichment at pH 5.0) or pH 4.5 (enrichment at pH 4.5) to elute less acidic FSH isoforms prior to elution of the desired FSH fraction. As a polishing step a hydrophobic interaction chromatography (HIC) is used to gain FSH at high purity.

Example 2

Phase II Clinical Studies with FSH (Invention)

[0225] A phase II clinical study with FSH (invention) and a comparator agent (Gonal-F) was performed to investigate the therapeutic efficacy and safety of various dosages of the FSH preparations.

[0226] 240 randomized female human patients with indication for intracytoplasmatic sperm each comprising 40 patients, the patients were treated with 52.5 IU, 75 IU, 112.5 IU or 150 IU FSH (invention) per day, 150 IU FSH (invention) every second day or 150 IU Gonal-f per day.

[0227] The treatment cycle for each patient included down-regulation of the endogenous hormone level using a GnRH agonist protocol, stimulation of follicle growth by administration of FSH, retrieval of the oocytes, ICSI and embryo transfer. Stimulation with FSH was done for up to 18 days until at least one follicle reached a diameter of at least 20 mm. Mean duration of FSH treatment was about 9 to 10 days. Then final maturation of the oocytes was induced by administration of a single dose of hCG about 1 day after the last FSH dose. 32 to 36 hours after hCG administration, all follicles having a size of at least 12 mm were punctured. 2 to 3 days after oocyte retrieval, selected oocytes were fertilized by ICSI and a maximum of two embryos per patient were transferred.

[0228] As result, stimulation of follicle growth with FSH (invention) led to a higher number of follicles and retrieved oocytes than with Gonal-f at half or three quarter the dose of Gonal-f:

TABLE-US-00002 TABLE 2 Comparison of FSH (invention) and Gonal-f FSH (invention) 150 IU every Gonal-F Mean number of 75 IU 2.sup.nd day 112.5 IU 150 IU follicles 12 mm 13.0 12.8 13.9 12.4 (follicles large enough (+5%) (+3.2%) .sup.(+12%) for puncture) retrieved COCs 12.6 13.4 14.4 11.1 (obtained oocyte (+13.5%) (+20.7%) (+29.5%) complexes) retrieved metaphase II 9.4 10.1 10.7 8.6 oocytes (+9.5%) (+17.4%) (+24.5%) (obtained mature oocytes) 2PN oocytes (one day 7.3 7.5 7.5 6.2 after puncture) (+17.5%) .sup.(+21%) .sup.(+21%) (oocytes fertilized by ICSI (two core stadium))

[0229] The results show that FSH (invention), in comparison to Gonal-f, induces the development of a significantly increased number of large follicles even at lower doses (down to only 50% of the dose of Gonal-f). Likewise, also the number of retrieved cumulus-oocyte complexes (COCs), the number of metaphase II oocytes and the number of successfully fertilized oocytes are significantly higher for FSH according to the present invention when compared to Gonal-f at a up to 2-fold higher dose. This impressively demonstrates the superior activity of FSH (invention).

[0230] Furthermore, also the relative number of successful fertilizations based on the identified developed follicles is increased for FSH (invention). For example, in the patients treated with 150 IU FSH (invention) every second day, 59% of all follicles with a diameter of at least 12 mm which were identified in the treated patients were successfully fertilized and formed a two core oocyte (2PN). In contrast, only 50% of the follicles 12 mm identified in the patients treated with Gonal-f were successfully fertilized. Hence, the follicles induced by FSH (invention) demonstrated a higher quality than those induced by Gonal-f.

Example 3

Phase I Clinical Studies with FSH (Invention)

[0231] A phase I clinical study with FSH (invention) and comparator agents (Gonal-F and Bravelle) was performed to determine the therapeutic efficacy of the FSH preparations.

[0232] FSH was administered to female volunteers and the pharmacokinetic and pharmacodynamic parameters were determined. In a first study, the healthy female volunteers received 25 IU, 75 IU, 150 IU or 300 IU FSH (invention) in a single subcutaneous dose and the mean follicle size in relation to the pre-dose size was determined by daily measurements from day 4 post administration. As control, volunteers received placebo or 100 IU Bravelle or Gonal-F. As shown in FIGS. 1, 2 and 3, the mean follicle size significantly increased after a single dose of FSH (invention). Increase in follicle size was dose dependent. The increase in follicle size was significantly greater when compared to placebo or the reference FSH preparations (Bravelle and Gonal-F). Hence, it was shown that FSH (invention) has a much higher potency of inducing follicular growth than the commonly used FSH preparations and is capable of inducing significant follicular growth even after a single dose.

[0233] Furthermore, a multiple dose clinical study was performed. FSH (invention), Gonal-F and Bravelle were administered with daily doses of 150 IU for seven days. In a further cohort, FSH (invention) was given at daily doses of 75 IU for seven days. In another cohort, FSH (invention) was administered every second day in a dose of 150 IU. FSH was administered after down-regulation of the menstruation cycle, resulting in the stimulation of follicle growth. Serum levels of FSH, inhibin-b and estradiol were monitored and the number and size of the follicles were determined. As shown in FIG. 4, the serum levels of FSH were comparable for the different FSH preparations administered at equal doses. FSH (invention) given at half dose or every second day resulted in halving of the FSH serum level, with the administration every second day showing the expected fluctuation (see FIG. 4). However, as shown in FIGS. 5 to 7, the administration of FSH (invention) results in a markedly increased number and size of induced follicles compared to Gonal-F and Bravelle. Administered at half dose, FSH (invention) results in a follicular growth comparable to that of Gonal-F (see FIG. 8). Furthermore, the administration of FSH (invention) every second day results in a comparable number, but markedly increased size of the induced follicles when compared to Gonal-F administered with the same dose, but every day instead of every second day (see FIG. 9). A similar increase in follicle size can also be seen when comparing the administration of 150 IU FSH (invention) every second day and 75 IU FSH (invention) every day, which results in the same amount of FSH administered. Hence, the same amount of FSH (invention) can result in a much increased follicular size when given every second day rather than every day. This difference is also observed in inhibin-b and estradiol levels in the patient serum, wherein FSH (invention) given every second day at 150 IU shows a significantly increased level compared to FSH (invention) administered every day at 75 IU or Gonal-F administered every day at 150 IU (see FIG. 10).

[0234] In addition, the high number of follicles induced with FHS (invention) is observed for several days in the patients. In particular The number of follicles having a size of at least 10 mm is maintained in the patient treated with FSH (invention) on average for about 5 days (see FIG. 11). For example, the number of large follicles is essentially constant throughout days 8/9 to 14/15, i.e. after termination of the FSH administration. In contrast, Gonal-f shows a peak in the number of large oocytes on days 9 and 10, which thereafter rapidly declines. Hence, FSH (invention) shows a trailing effect, maintaining the developmental status of the follicles for several days after termination of the FSH application. This effect is important for infertility treatment as it broadens the window for a successful induction of the final oocyte maturation. In common treatments, hCG or other ovulation inducers have to be administered to the patient about 1 day after termination of the FSH administration. Else, the induced large follicles will regress and final maturation of the oocytes is no longer possible. With FSH (invention), the oocytes stay significantly longer, i.e. about 5 to 6 days, at the achieved size and maturation status after termination of the FSH administration. Hence, stimulation of the final oocyte maturation and induction of ovulation, using, e.g. hCG, is possible for a much longer time interval when using FSH (invention) for stimulation of follicle growth compared to the use of Gonal-f.

[0235] Furthermore, the pharmacokinetic of FSH was analyzed after the above-described multiple dosage regimen. For this, the serum level of FSH was monitored following the multiple dose administration. FIG. 12 shows the mean concentration versus time curves for plasma FSH after the last injection of multiple subcutaneous injections of 75 or 150 IU FSH (invention) administered daily and 150 IU FSH (invention) administered every second day, 150 IU Bravelle and 150 IU QD Gonal-f administered daily on a linear scale. The plots show an increase in the plasma FSH concentration after the last subcutaneous injection of multiple injections. After the peak plasma FSH concentration (C.sub.max) the plasma FSH concentration decreased to baseline level. The C.sub.max of plasma FSH increased with an increasing dose level of FSH (invention). C.sub.max decreased when 150 IU FSH (invention) was administered once every two days instead of once daily. When comparing the concentration versus time plots (FIG. 12) of 150 IU FSH (invention administered daily with the same dose levels of the comparators Bravelle and Gonal-f, it can be seen that the curves are highly similar. The C.sub.max after 150 IU FSH (invention) (12.989 mIU/mL), Bravelle (13.370 mIU/mL), and Gonal-f (12.281 mIU/mL) were comparable. The AUC.sub.0-last of Bravelle (1172.066 h*mIU/mL) was higher than the AUC.sub.0-last of FSH (invention) (824.897 h*mIU/mL) and Gonal-f (917.400 h*mIU/mL). Also the circulation half-life t.sub.112 of the different FSH preparations was comparable, with that of Bravelle being slightly higher (FSH (invention): 33 h; Gonal-f: 36 h; Bravelle: 54 h). This is remarkable as Bravelle showed a significantly lower pharmaceutical efficacy (see above).

[0236] In conclusion, it was demonstrated in the phase I clinical study that FSH (invention) has a much higher therapeutic efficacy in terms of follicular growth compared to the same amount of Gonal-F and Bravelle. Furthermore, a dosage regimen wherein FSH (invention) is administered every second day results in markedly increased follicular size compared to administration every day.

Example 4

Granulosa Cell Assay

[0237] In order to perform a granulosa cell assay primary cells are isolated from the follicular fluid of IVF patients during the collection of the oocytes. After a Ficoll gradient centrifugation which eliminates other cell types as e.g. red blood cells the granulosa cells are seeded in 24 to 96 well plate format for 5-7 days in culture medium containing androstendione or testosterone. After that period, the cells (2 to 4*10.sup.4 cells per well) are stimulated with FSH ranging between 1 pg/ml to 2 g/ml in the steps shown in the diagram (400 l medium per well). After three to four hours incubation half of the supernatant is collected for performing the cAMP assay. Another 24 h later the cells are lysed by freeze thaw in the remaining supernatant. The lysate is applied in the progesterone and estradiol assays.

[0238] Comparison of FSH (invention) and Gonal F

[0239] In the first set of experiments FSH (invention) is compared to Gonal F (Merck Serono SA). Gonal F is FSH recombinantly produced in CHO cells. The results are shown in FIGS. 13 to 15. While the second messenger cAMP is produced at comparable FSH concentrations of Gonal F and FSH (invention) products in comparable amounts, the steroids progesterone and estradiol are released at much lower FSH concentrations in the case of FSH (invention) products compared to FSH recombinantly produced in CHO cells (Gonal F).

[0240] Comparison of FSH (invention) and Fostimon

[0241] In another set of experiments the FSH (invention) was compared against Fostimon (IBSA Institut Biochimique SA), the FSH product isolated out of human urine. The results are shown in FIGS. 16 to 18. While the cAMP level rises similarly at comparable dose ranges of FSH for both products, the sex steroids are produced at a significantly lower concentration of FSH (invention) compared to Fostimon.

[0242] Note: Since the assays are performed using different donors, differences in the stimulation profile may account to the donors used in each assay.

Example 5

Steelman-Pohley Assay

[0243] The activity of FSH was also determined by the Steelman-Pohley assay. The assay was performed according to the pharmacopeia. In particular, the ovarian weight gain in immature female rats was measured after administration of three different FSH concentrations each given daily for three days. The potency is calculated using the parallel line evaluation. The Steelman-Pohley assay was used to determine the standard international units (IU) of the FSH preparations according to the invention.

[0244] As demonstrated by the Steelman-Pohley assay, the in vivo activities of the FSH (invention) and of the urinary and recombinant standard FSH are similar in rat (see FIG. 19).

Example 6

Glycoprofiling

[0245] The glycoprofiles of the different FSH preparations were determined by structural analysis of the glycosylation. Glycoprofiling generates information on the complex glycan structure of the glycosylation sites. For glycoprofiling, the intact N-glycans were released from the protein core and the reducing ends of N-glycans were labeled with a fluorescence marker. The purified sample of the labeled N-glycans was separated by UPLC. Peak areas based on fluorometric detection were employed for calculation of the relative molar abundances of the N-glycan structures. Estimated data for the FSH are summarized in Table 3. The values represent the relative molar contents of N-glycans containing the interesting type of monosaccharide (e.g. fucose).

TABLE-US-00003 TABLE 3 Relative amounts of the different glycosylation properties Sample F S S0 S1 S2 S3 S4 G B Sulf FSH (invention) 38% 97% 2% 21% 43% 19% 14% 99% 34% 9% Fostimon 48% 83% 91% 28% Puregon.sup.1 29% 91% 91% 0% F: glycans containing fucose; S: glycans containing at least one sialic acid; S0: glycans containing no sialic acid; S1: glycans containing one sialic acid; S2: glycans containing two sialic acids; S3: glycans containing three sialic acids; S4: glycans containing four sialic acids; G: glycans containing galactose; B: glycans containing bisecting N-acetylgalactosamine; Sulf: sulfated N-glycans .sup.1literature values (Hrd, K. et al. (1990) European Journal of Biochemistry 193, 263-271)

[0246] Shown are the relative amounts of N-glycans on the FSH which carry the indicated units. Puregon is another recombinant human FSH produced in CHO cells.

[0247] Furthermore, the ratio of 2,3-coupled and 2,6-coupled sialic acids in the glycans of the FSH was analyzed by comparing the amount of sialic acid released by sialidase A (cleaving off 2,3- and 2,6-coupled sialic acids) and sialidase S (cleaving off only 2,3-coupled sialic acids).

TABLE-US-00004 TABLE 4 Relative amounts of the sialic acid linkage Sample 2,3-linked sialic acid 2,6-linked sialic acid FSH (invention) 43% 57% Bravelle 75% 25% Gonal F/Puregon 100% 0%

[0248] In FSH (invention), the sialic acid residues are coupled to the glycans by 2,3- as well as 2,6-bonds in a ratio of about 1:1, comprising even more 2,6-coupled sialic acids than 2,3-coupled sialic acids, while in the urinary FSH Bravelle (Ferring Pharmaceuticals Inc.) the ratio is about 3:1 in favor of 2,3-linked sialic acid. Due to their recombinant production in CHO cells, Puregon (Organon/EssexPharma) and Gonal F (Merck Serono) do not have any bisecting N-acetylgalactosamines and only comprises 2,3-coupled sialic acids.

[0249] Antennarity, terminal galactose units and Z-number were calculated from the above measurements and by determination of the charge distribution of the glycans after release from the FSH.

TABLE-US-00005 TABLE 5 Antennarity of the glycosylation of the different FSH Sample Bi Tri Tetra FSH (invention) 51% 26% 21% Fostimon 39% 45% 16% GonalF.sup.1 ~65% ~25% ~10% Puregon.sup.2 53% 26% 12% Bi: biantennary N-glycans; Tri: triantennary N-glycans; Tetra: tetraantennary N-glycans .sup.1literature values (Gervais, A. et al. (2003) Glycobiology 13(3), 179-189) .sup.2literature values (Hrd, K. et al. (1990) European Journal of Biochemistry 193, 263-271)

[0250] Shown are the relative amounts of bi-, tri- and tetraantennary N-glycans on the FSH.

TABLE-US-00006 TABLE 6 Z-number of different FSH Sample Z-number FSH (invention) 220 Gonal F (rFSH) 218 Puregon (rFSH) 204 Fostimon (uFSH) 212 Bravelle (uFSH) 244

[0251] Shown is the Z-number, i.e. the relative acidity, of the FSH preparations. A higher Z-number indicates a more acidic FSH preparation.

[0252] In conclusion, the FSH according to the present invention (FSH (invention)) has a high degree of bisecting N-acetlyglucosamine, a high antennarity, a high degree of sialylation and a high sulfation degree. It is assumed that because of one or more of these three glycosylation parameters, the FSH (invention) has a superior activity compared to the common recombinant or urinary FSH preparations.

[0253] Furthermore, the FSH (invention) has a ratio of 2,3- to 2,6-sialylation of about 1:1 or even a higher amount of 2,6-sialylation.

[0254] Furthermore, the glycan structures of the FSH preparations were also analyzed by mass spectroscopy of the released glycans. The following results were obtained:

TABLE-US-00007 TABLE 7 Relative amounts of different glycosylation properties Sample F S0 S1 S2 S3 S4 S > 0 G0 G1 G2 G3 G4 G > 0 B Gonal F 55 1 16 45 28 9 98 0 1 55 30 14 100 0 Bravelle 43 1 11 45 34 9 99 0 7 39 39 14 99 14 FSH (inv.) 43 1 18 35 31 15 99 0 7 45 30 20 102 28 shown are the relative amounts of glycans having the following property: F: fucose; S0: no sialic acid; S1: one sialic acid; S2: two sialic acids; S3: three sialic acids; S4: four sialic acids; S > 0: at least one sialic acid; G0: no galactose; G1: one galactose; G2: two galactoses; G3: three galactoses; G4: four galactoses; G > 0: at least one galactose; B: bisecting GlcNAc

TABLE-US-00008 TABLE 8 Antennarity of the glycosylation of the different FSH Sample Bi Tri Tetra FSH (invention) 48% 31% 21% Bravelle 45% 43% 12% Gonal-f 56% 30% 14% Bi: biantennary N-glycans; Tri: triantennary N-glycans; Tetra: tetraantennary N-glycans

TABLE-US-00009 TABLE 9 Relative amount of sulfated glycans Sample Sulfation FSH (invention) 15% Bravelle 2% Gonal F 0%

[0255] Shown are the relative amounts of N-glycans on the FSH which carry a sulfate group.