HUT76660A - New composition of glycoprotein isoforms having follicle stimulating activity - Google Patents

Description

The present invention relates to a follicular stimulant composition comprising a mixture of glycoprotein isoforms; and perfection of follicle growth and maturation.

The follicle-stimulating hormone (FSH) produced in the anterior pituitary gland plays an important role in female and male reproduction by stimulating the differentiation and maturation of the gonadal glands, the testes, the Sertoli cells, and the ovarian granulosa. FSH is produced and secreted by the pituitary gland in various molecular forms (in the form of isohormones or isoforms), all of which have their charge, receptor binding affinity, biological activity.

84065-760C-WORD-tree and residence time in plasma vary. This microheterogeneity is due to differences in the amount and / or composition of carbohydrate residues, particularly sialic acid. Various types of gonadotropin have been isolated from the anterior lobe, serum, and urine of several non-mammalian and mammalian species, including humans. FSH isoforms of relatively acidic nature, which are multiple sialylated, have less receptor affinity and in vitro biological activity than the more basic isoforms. In contrast, due to their longer plasma residence time, the in vivo biological effects of these acidic forms are stronger [Ulloa-Aguirre et al. Reprod. 3: 491-501 (1988)].

FSH is used during in vitro fertilization (IVF for short) to induce ovulation and to stimulate the ovaries in a controlled manner. The purpose of controlled superovulation is to increase the number of mature oocytes (ova) and subsequent embryo transfer (ET) for in vitro fertilization. Usually up to three embryos are transferred per transfer. Because more than one treatment is usually required, most infertility clinics freeze the spare embryos or fertilized oocytes and transfer them to subsequent cycles. Assuming normal fertilization, the more oocytes there are, the greater the number of possible transmissions, and thus the chances of a woman becoming pregnant after a single cycle of treatment. In the case of male infertility, the chance of fertilization and thus of pregnancy increases with the number of oocytes obtained.

Previously it has been shown that the pl-value (isoelectric point) of the majority of circulating FSH isoforms during the follicular and luteal phases of the normal menstrual cycle is less than 4.8; whereas significantly more alkaline FSH isoforms are produced in the middle of the cycle or after treatment with estrogens [Padmanabhan et al., J. Clin. Endocrinol. Metab. 67: 465-473 (1988)].

This means that more acidic FSH isoforms play a role in the growth and enhancement of ovarian follicles during the follicular phase.

So far, FSH preparations isolated from natural sources have been used for clinical purposes. Isohormone distribution profiles of commercially available formulations have been reported [e.g., Harlin et al., Fert. Faster. 46, 10551061 (1986). These FSH formulations appear to consist of relatively acidic isohormone fractions.

Surprisingly, it has been found that a mixture of isoforms of glycoproteins having isoelectric points in the range of 4.8 to 4.2, and under the same clinical conditions, perform better than known glycoprotein formulations, and with follicular stimulating activity greater than 15% derived from isoforms with an isoelectric point greater than 4.8 and less than 30% derived from isoforms with an isoelectric point less than 4.2.

Clinical trials were carried out with the composition of the invention containing the glycoprotein isohormone prepared by recombinant DNA techniques. Isolation of FSH iso-hormones by their isoelectric point (p1) has shown that the composition of the invention contains relatively basic isoforms. The isoelectric points reported herein were determined by chromatofocusing (see Example 1).

The profile of commonly known glycoprotein isohormone formulations (one example of which is Metrodin® used in our clinical trial) has a more acidic profile.

The fact that a more basic isohormone profile leads to better results in superovulation is surprising, since it is believed in the literature that bioavailability in vivo is the most important factor in the follicle stimulating effect of glycoproteins (see EP 388 223). application). The basic isoforms have a shorter half-life than the more acidic isoforms, which would render the basic isoforms less suitable for use in in vitro fertilization procedures; this is a teaching other than the present invention.

Preferably, the glycoprotein isohormone mixture of the present invention contains a relatively high proportion of relatively basic isoforms. This may be based on the preference for formulations containing glycoprotein isoforms which contain more than 15% of isoforms with an isoelectric point greater than 4.8 and less than 30% with an isoelectric point of less than 4.2; and mixtures of isoforms where the isoforms are more than 15% has an isoelectric point above 4.8 and less than 25% with an isoelectric point below 4.2; further preferred are compositions wherein more than 25% of the isoforms have an isoelectric point of greater than 4.8 and less than 25% of the isoforms have an isoelectric point of less than 4.2; more preferably, more than 25% of the isoforms have an isoelectric point greater than 4.8 and less than 20% have an isoelectric point below 4.2.

The percentage of isoforms as used herein is defined as the relative amount of immunoreactive isoforms obtained after chromatofocusing. Alternatively, protein content may be determined by colorimetry.

In the clinical study reported herein, it has been found that the number of oocytes obtained in the controlled superovulation of the two FSH formulations, i.e., the composition of the invention and Metrodin ^R , is different. Several oocytes were obtained from the group treated with the composition of the invention. In addition, the treatment required significantly lower doses of FSH and a shorter duration of treatment.

The glycoprotein mixture of the invention may be derived from urine. In the case of naturally occurring FSH formulations, the number and relative amounts of all isoforms depend on the source (pituitary, serum, or urine); donor age and endocrine status; and the isolation method used for purification [Wide: J. Clin. Endocrinol. Metab. 55: 682-688 (1981); Wide and Hobson:

• · · · · · · · ·

J. Clin. Endocrinol. Metab. 56: 371-375 (1983)].

Alternatively, the glycoprotein may be a recombinant glycoprotein.

In the case of recombinant FSH (in short: recFSH), heterogeneity is determined by the host cell strain selected for production and cell culture conditions. Obviously, in the production of glycoproteins such as FSH, the Chinese hamster ovary cell strain (in short, the CHO cell strain) is chosen because these cells are known to produce glycoproteins identical or closely related to the oligosaccharides of human glycoproteins [Sasaki et al. . Chem. 262: 12095-12076 (1987). Other host cell strains may be used.

The recombinant FSH composition of the invention is produced by a CHO (Chinese Hamster Ovary) cell strain transfected with a plasmid containing two gene subunits encoding human FSH (human FSH, in short, hFSH).

This CHO cell strain produces intact, glycosylated hFSH, which is secreted into the culture medium and a source of material for further purification. Similar batches can be prepared as described (van Wezenbeck et al., From Clone to Clinic, Kluwer Academic Publishers, 1990, 245-251). Preliminary preclinical studies have shown that the binding affinity of the recFSH receptor and its in vitro and in vivo efficacy are compatible with those of FSH isolated from natural sources (Mannaerts et al., Endocrinology 129: 2623-2630 (1991)).

The FSH compositions of the present invention may be selected for their chromatofocusing profile. In addition, the profile can be influenced by selecting a particular host cell strain or by modifying the culture conditions.

Alternatively, mixtures of glycoprotein isoforms of the present invention can be isolated from cell strains that stimulate the expression (expression) of a glycoprotein gene by introducing a regulatory sequence for site-specific targeting of the glycoprotein gene (see WO 92/19255).

In addition to wild-type cell strains, basic isohormones are expressed by expression of recombinant FSH in cell lines lacking glycosylation. Such a cell strain may be, for example, a cell strain lacking the enzyme N-acetylglucosamine transferase or lacking the transport of sialic acid to the Golgi (Galway et al., 1990, Endocrinology 127: 93-100).

Other embodiments of the invention are mixtures of basic glycoprotein isoforms obtainable by enzymatic or chemical modification. This treatment removes portions of the carbohydrate chains without touching the amino acid sequence. Glycoprotein batches can be treated, for example, with HF [Chen et al., J. Biol. Chem. 257: 14446-14452 (1982). Partial desialylation can be accomplished by enzymatic hydrolysis with neuraminidase [Vaitukaitis and Ross, J. Clin. Endocrinol. Metab. 33: 308-311 (1971)].

The invention also relates to basic glycoprotein hormone mixtures which have a biological, i. E., Follicular stimulating effect, and which are prepared by removing one or more nitrogen bound oligosaccharide chains from one of the subunits. These chains can be removed by site-directed mutagenesis of the gene encoding the glycoprotein. In this way, a single amino acid can be substituted so that the carbohydrate binding site is no longer available. As a result, one or more carbohydrate chains can no longer be attached to the α or B subunit [Matzuk and Boimé, J. Cell Biol. 106: 1049-1059 (1988)].

According to the present invention, a mixture of relatively basic isohormone forms can be used in the manufacture of a medicament for use in inducing ovulation or controlled ovarian stimulation.

Accordingly, the present invention also provides pharmaceutical compositions comprising such glycoprotein isoforms in admixture with pharmaceutically acceptable excipients. Methods for preparing formulations and formulations can be found in Remington's Pharmaceutical Sciences 1463-1497. pages [16. edition, Mack Publ. Co. of Easton, Pa., USA (1980)]. Ampoules containing the pharmaceutical composition of the invention may contain, for example, from 1 to 1000 µg of glycoprotein mixture (for example, 75 IU (International Units) are considered therapeutically effective amount).

The present invention also relates to an improved method of inducing follicular growth and maturation comprising the use of a pharmaceutical composition as described above comprising a mixture of basic glycoprotein isoforms. According to the invention, it is not necessary to include a mixture of several acidic isoforms in the treatment regimen.

A further improvement of this method consists in the repeated administration of pharmaceutical compositions containing a mixture of glycoprotein isoforms having a thymostimulant activity, wherein the profile of each composition, i.e. the isoform profile, is the same. Accordingly, the administration of only relatively basic isoforms per treatment is sufficient.

Such mixtures can be prepared only by isolation of basic isoforms, for example by preparative chromatofocusing. The isoelectric point of such isoforms is preferably above 4.2. It will be appreciated that fractionation of isoform mixtures on glycoprotein batches of various origins, such as urine or recombinant DNA cell strains, may be accomplished with or without chemical or enzymatic modification.

The pharmaceutical compositions of the present invention may be combined, for example, with GnRH antagonists or agonists and / or LH activity such as HCG or LH to induce superovulation in clinical treatments.

The invention is further illustrated by the following non-limiting examples.

• ··· ·· · ·

Example 1

Recombinant human FSH (Org. 32489) and was obtained from urine

Comparison of the immunoreactive isohormone profile of FSH (Metrodin ^R ) FSH

In this study, the isohormone profile of several batches of Org 32489 (recombinant human FSH, NV Organon) from Chinese hamster ovary cells transfected with genes encoding hFSH was compared and compared with commercial batches of hFSH, i.e. Metrodin ^{R, from} urine.

Purified (at least 99%) Org 32489 starting material and final formulation product were purchased from Diosynth and Organon (The Netherlands). The urinary hFSH preparation, Metrodin ^R (from Serono, Rome, Italy; reported as 70/45 in vivo bioactivity of 75 IU / ampoule compared to the International Standard (IS)) was used as reference. A total of 12 Org 32489 feedstock batches, 5 Org 32489 clinical formulations (CPs) and 12 Metrodin ^R batches were used to compare isohormone formulations. Batches of Metrodin ^R labeled in-house clinical preparation code were used in an experiment with clinical preparation batches of Org 32489 to compare clinical efficacy (see Example 2).

During chromatofocusing, the different hFSH isoforms were separated by their isoelectric point. The distribution of hFSH was determined enzymatically by quantitative immunoassay using antibodies with comparable activity to the isoforms.

···· · «· ··

- 11 Chromatofocusing was performed at pH 6 and pH 3 with protein liquid chromatography (fast procedure, FPLC, for short) on an HR 5/20 column (Pharmacia, Woerden, The Netherlands) equipped with the Polypuff Buffer # 94 (PBE-94). Pharmacia), equilibrated with 0.034 M L-histidine solution (Aldrich Chemie, Steinheim, Germany) and adjusted to pH 6.2 with hydrochloric acid. Prior to use, each column was eluted with 53 ml polypeptide 73 (Pharmacia) diluted 1:11 (v / v) in distilled water and adjusted to pH 3.0 with hydrochloric acid (eluting buffer) until pH with a constant slope (slope) of 6-3 pH range. After each experiment, the column was eluted with elution buffer and 2 mL of 0.034 M L-histidine solution containing 100 μg / mL human serum albumin (HSA; Behring, Marburg, Germany).

Approximately 225 IU of FSH from each hormone formulation, as defined above for in vivo bioactivity, was dissolved in 3 ml equilibration buffer (stock solution) and 2 ml (150 IU) of this solution was applied to the column. The column was then eluted with the eluting buffer; At a flow rate of 1 ml / min, 1 ml fractions were collected. After 53 fractions, 2 mL of 2M saline was applied to the column and another 7 fractions were collected. Fractions 1, 2 and 3 and 4, 5 and 6 were combined for quantitative measurement of FSH. All other fractions were supplemented with 1 g / L bovine serum albumin (BSA; Sigma, St. Louis, MO, · · · · · · · · · · · · · · · · · · · · · · · · · ·

- 12 US) (medium ⁺ ) supplemented with a 1: 1 mixture of HAM F12 and DMEM (Gibco, Grands Islands, NY, USA) to 2.5 ml. In addition, 0.3 mL of FSH stock solution was supplemented with 2.2 mL of medium ^{+ to} 2.5 mL. All FSH fractions were desalted by loading each fraction onto a 12-ml medium ⁺ equilibrated PD-10 column (Pharmacia). After elution with 3.5 ml medium ⁺ , the collected samples were stored at -20 ° C until FSH immunoreactivity was determined.

Immunoreactivity of FSH was measured by a two-site sandwich ^, 'enzyme immunoassay, using a β-directed capture antibody (48 monoclonal antibodies) and an α-directed HRP-labeled detection antibody (monoclonal antibody 116B) according to a previous publication [Mannaerts Endocrinology 129: 2623-2630 (1991). This definition only recognizes intact dimers and has been found to be equally suitable for the detection of all FSH isoforms. The sensitivity of the method, expressed in International Standard 70/45, is 0.4 IU / liter; the coefficients of variation within and between measurements are 7% and 8% respectively. The degree of cross-reactivity with hLH and hCG is less than 0.01% and 0.01%, respectively.

For quantitative characterization of Org 32489 and Methrodinic isohormone formulations, the distribution of immunoreactivity of FSH after chromatofocusing was divided into the following six pH ranges:

> 5.3;

5.30 to 4.81;

• ·

- 13 • ·· ······· · · · ·

4.80 to 4.20;

4.19 to 3.60;

3.59 to 3.00; and <3.0 (salt fraction).

These pH ranges were chosen to symmetrically divide the bell-shaped distribution of the immunoreactivity of FSH after chromatofocusing Org 32489. The amount of FSH immunoreactivity eluted within each pH range was expressed as a percentage of the total eluted FSH immunoreactivity. In addition, the yield, i.e., the percentage of total eluted FSH immunoreactivity expressed as a percentage of the column loaded FSH immunoreactivity, pH range and pH peak was determined. Yields less than 75% and greater than 125% were not considered acceptable and therefore the results of these chromatofocusings were not used.

The yield, pH range, pH peak, and distribution of FSH immunoreactivity after chromatofocusing the Org 32489 stock and Metrodin ^R batches are shown in Table 1. Representative chromatofocusing profiles of Org 32489 are shown in Figure 1. The yield of FSH immunoreactivity obtained after chromatofocusing Org 32489 and Metrodin ^R was similar: 93.7 (SD 8.9) percent and 89.1 (SD 6.1) percent, respectively. (SD stands for standard deviation). Org 32489 showed a bell-shaped distribution of FSH immunoreactivity at 5.68 (SD 0.11) and 3.18 (SD at 0.09 pH range, with a 4.53 peak (SD 0.08)) (n = 12).

- 14 The immunoreactive profile of Metrodin ^R ranges from 5.58 (SD 0.18) to 3.07 (SD 0.05), peak at pH 4.28 (SD 0.13) (n = 12).

The distribution of FSH immunoreactivity in a pH range above six showed that compared with the product Metrodin ^R Org 32489 contained a relatively basic isoforms is approximately two times higher percentages of which the isoelectric point (pI) higher than 4.8 (20.9 % versus 12.7%), and approximately twice the percentage of relatively acidic isoforms with an isoelectric point lower than 4.2 (21.1% versus 41.7%) (see Figure 1). and Tables 2). Furthermore, all Metrodin ^R batches contained more FSH immunoreactivity in their salt fraction (0.8% for Org 32489 and 3.3% for Metrodin ^R batches); this refers to FSH isoforms with an isoelectric point of less than 3.0.

In addition to the batches of Org 32489 starting material described above, the chromatographic profile of five clinical preparations was examined. The mean FSH distribution of these clinical preparations was very similar to that of the parent batches (Table 1). Similar to the twelve batches of Org 32489 and Metrodin ^R used to compare isohormone, clinical preparations of Org 32489 and Metrodin ^{R were} used to compare clinical efficacy (see Example 2); the difference between the relative amounts of the basic and acidic isoforms in the two formulations is the same, ie the clinical • · ···

The formulations contained more basic isoforms than the Metrodin ^R clinical formulations (17.02% versus 12.7% if the isoelectric point is greater than 4.8).

This comparative study, which covers the chromatofocusing profile of Org 32489 and urinary hFSH, i.e. Metrodin ^R , clearly showed that Org 32489 has more basic isoforms with an isoelectric point greater than 4.8 and less isoelectric with less than 4.2 containing an acidic isoform such as Metrodin ^R.

Example 2

Comparative study of the efficacy of Org 32489 in in vitro fertilization (IVF) patients

Study design

This study was designed in a multicentre, randomized, evaluator-blind (blinded) group comparative study comparing the safety and efficacy of Org 32489 with Metrodin ^R in infertile, defective pituitary-function subjects with IVF and embryo transfer. (ET). Approximately one thousand subjects were enrolled in this study, with a ratio of 3: 2 to Org 32489 treated subjects compared to individuals treated with Metrodin ^R. The duration of the study did not exceed three treatment cycles. Perform efficacy analysis only during first treatment cycles * ··· · «

- 16 • · »·» · • * - - · · · · · · «« «··· · * · ·· r ·

Individuals were enrolled according to the following entry criteria:

Criteria for participating in the study

- Ages 18 and over at the time of screening.

- The cause of an individual's infertility is presumably resolved by IVF.

- Up to three previous attempts have been made on IVF, gamete intrauterine transfer (GIFT) or zygote intrauterine transfer (ZIFT), with at least one oocyte harvesting.

- Ovarian cycles are normal, with an average duration of 24 to 35 days, with an individual variation of + 3 days (but not exceeding 24-35 days).

- Healthy physically and intellectually.

- Your weight is between 80% and 130% of your ideal weight; and

- gives its written consent after being informed.

Exclusion Criteria

- Infertility caused by endocrine abnormalities such as hyperprolactinemia, polycystic ovarian syndrome, ovarian failure.

- Male infertility as defined by the following criteria: sperm count less than 10x10 ⁶ per ml; and / or how much sperm cells with normal morphology 17 -

less than 40%; and / or the number of sperm cells with normal motility is less than 40%.

- GnRH analogs, FSH, hMG and / or hCG are contraindicated.

- Any ovarian and / or abdominal abnormality which may interfere with proper ultrasound examination.

- Hypertension (diastolic blood pressure greater than 90 mmHg in sitting position and / or systolic blood pressure greater than 150 mmHg in sitting position).

- Chronic cardiovascular, hepatic, renal or pulmonary disease.

- History of continuous alcohol or drug use (within 12 months); and

- administration of test drugs within three months prior to screening.

The treatment regime

On the first day of the menstrual cycle, buserelin treatment was started. The first day of the menstrual cycle is defined as the first day a person awakens with menstrual bleeding. Treatment with Org 32489 (75 IU / ampoule) or Metrodin ^R (75 IU / ampoule) was initiated 14-18 days after the onset of buszerelin, when this treatment induced a hypogonadotrophic state (i.e., a serum E2 value of 50 pg / g). less than ml). If this condition was not achieved, Org 32489 or Metrodin ^R treatment was postponed and the dose of buserelin increased to 4 x 300 μg / day. This increased dose of buserelin was maintained for an additional complete treatment period until the FSH treatment was discontinued. Buserelin pre-treatment was not continued for more than five weeks. If, after complete reduction, a follicular cyst of at least 20 mm was formed on the first day of FSH treatment, this cyst was punctured before the first FSH injection.

Dosing was performed according to the following schedule in each treatment cycle.

buserelin Dose; 4 x 150 pq daily the route of administration through the nasal mucosa Org 32489 / dose Treatment 1-4. day: / ^ Metrodin 150-225 IU daily; from day 5 on dose for each individual set it up the route of administration intramuscular (i.m.)

HCG (10,000 IU) was administered when at least three follicles of 17 mm or larger were present. Up to three embryos were returned and frozen embryos were returned to natural cycles or stimulated cycles. If no embryo transfer was made, the cycle was considered null and void. The luteal phase was monitored by evaluation of progesterone. After injection of 10,000 IU hCG, the luteal phase was supported for at least two weeks (e.g., three injections of 1,500 IU hCG or at least 50 mg progesterone daily by intramuscular injection or 400 mg progesterone daily by vaginal injection).

Statistical evaluation

The primary effect variable is the total number of oocytes obtained during the first treatment cycle. Other variables analyzed during the first treatment cycle were the number of FSH ampoules administered to stimulate the ovaries and the duration of FSH treatment. The results of these parameters were analyzed using a combination of Cochran's unique central results. Furthermore, to confirm the results of the Cochran method, we used Wilcoxon's sum (rank sum test and variance analysis (ANOVA)).

Output of the first treatment cycles of the study

FSH treatment was initiated in a total of 981 individuals in 18 study centers. A total of 907 individuals were punctured, of which 546 belonged to the Org 32489 group and 361 to the Metrodin group. With this number of punctured individuals, it was possible to detect a difference of at least 1.2 oocytes between the two treatment groups with a probability of 0.8 (the so-called performance of the test).

The number of oocytes obtained per center in each treatment group is shown in Table 3. In all centers, the average number of oocytes obtained in the Org 32489 group was higher than in the Metrodin group. The total mean number of oocytes (measured per center) in the Org 32489 group was 10.84 oocytes in the Metrodin group

It was found to be 8.95 oocytes, resulting in a treatment difference of 1.89 oocytes in favor of Org 32489. The difference of 1.89 oocytes is more than five times the standard error and is very significant (P <0.0001). The resulting 95% confidence interval indicates that patients treated with Org 32489 receive at least 1.2 and up to 2.6 oocytes more than those treated with Metrodin. The exclusion of immature oocytes from the total number of oocytes obtained did not affect the effect of the treatment (see Table 4).

The total number of ampoules administered to the pungent subjects per center is shown in Table 5. The total volume of Org 32489 administered in 14 centers out of 18 was less than the total amount of Metrodin administered. The mean total FSH administered (measured per center) was 82.5 ampoules in the Org 32489 group and 31.8 ampoules in the Metrodin group, resulting in a treatment difference of 3.3 ampoules, which is statistically very significant (P <0.0001). The resulting 95% confidence interval indicates that subjects treated with Org 32489 received, on average, at least 2.1 and at most 4.5 ampoules less than subjects treated with Metrodin.

The duration of FSH treatment in pungous individuals by center is shown in Table 6. Duration of treatment was shorter in 13 out of 18 centers in the Org 32489 group than in the Metrodin group. The average duration of treatment (measured per center) was 10.7 days in the Org 32489 group and 11.3 days in the Metrodin tube21; as a result, the treatment difference was 0.6 days, which is statistically very significant (P <0.0001). The resulting 95% confidence limit indicates that subjects treated with Org 32489 have a treatment duration of at least 0.3 days and at most 0.9 days shorter than those treated with Metrodin.

Wilcoxon's sum test and variance analysis confirmed all of our observations.

Based on the foregoing, it can be concluded that in this study, superovulation controlled by Org 32489 treatment was superior to Metrodin ^R treatment in terms of both the number of oocytes obtained and the significantly lower FSH dose and shorter duration of treatment.

Text of the figures

Figure 1:

Figure 1 shows the distribution of FSH immunoreactivity obtained after chromatofocusing a specific batch of Org 32489. An example is the profile of a batch. The upper and lower parts of the figure represent separate preparation (sf = salt fraction). Total yields were 91.9% and 91.3%, respectively.

- 22 Table 1

Average yield, pH range, pH peak, and distribution of FSH immunoreactivity based on chromatofocusing of twelve Org 32489 batches, five Org 32489 end products (ie clinical product) and twelve Metrodin ^R batches

Org 32489 MetrodinR commodity clinical products n 12 5 12 Yield 93.7 ± 8.9% 84.2 ± 3.6% 89.1 ± 6.1% pH range 5.68-3.18 5.60-3.13 5.58 - 3.07 pH tip 4:53 4:52 4:28 Distribution of FSH: pH range> 5.3 2.0% 2.2% 1.3% 5.30-4.81 18.9% 18.0% 11.4% 4.80-4.20 58.2% 55.5% 45.6% 4.19-3.60 18.6% 21.7% 33.8% 3.59 - 3.00 1.7% 2.0% 4.6% <3.0 0.8% 0.8% 3.3%

Table 2

Percentage of basic isoforms of Org 32489 and Metrodin® clinical preparations used to compare clinical efficacy at iso-electric points greater than 4.8 expressed in FSH immunoreactivity; and the percentage of acidic isoforms at an isoelectric point below 4.2 expressed as FSH immunoreactivity (see Example 2)

preparation Izohormon-distribution basic% (pl> 4.8) own% (pl <4.2) Org 32489: number of raw material batches (n = 12) 20.9 21.1 The efficacy of clinical preparations comparative study (n = 2) 17.1 23.9 Metrodin ^R s number of batches (n = 12) 12.7 41.7 The efficacy of clinical preparations comparative study (n = 3) 11.9 44.4

- 24 Table 3

Statistical analysis of the total number of oocytes recovered using Cochran's approximation)

Total number of oocytes recovered

Group

Org 32489 minus Metrodin

First iklus I T Org 32489! I I I I - I 1 Metrodin The treatment difference estimated is [The estimated | value standard failure 95% reliability interval (P-value) í centers n ! S46 J 361 J altogether average 10.84 8.95 1.89 ! 0:37 1.2 - 2.6 set to center I I << 0.0001) I I interaction test · j (0.88) Center 1 1 First n ! 67 J 41! average 12:54 11.80 0.73 ! 1:33 2 n ¹² ! 7 1 1 average 1 12-67 8.71 3.95 ι ^3.64 ! 3 n ⁴ S! 1 1 average 1 ⁹ · 50 4:00 5:50 j ^1-74 ! 4 n ! 11! 7! 1 1 average 1- ^7-91 S.43 j 2:48 {1.89 J 5 n ! 13 | ⁸ 1 1 1 average 15:46 10:38 S .09 ! ³²⁰ ! 6 n ! ³⁰ ! 17! 1 1 · average 12.60 9:53 3:07 J 2.04 j Ί n ! 44! 24 * I | average 7:23 5:21 2:02 í ^x · ⁰¹ ! 8 n ! ^{19 15} ! 1 1 average 1 ⁷ · ⁶⁸ 6.60 1:08 J 1.92 J 9 n ! 27 · 19! 1 1 average 10.78 9.79 0.99 ! ¹⁵⁸ ! 10 n ! 41 ' 25 j 1 1 t average 1 ni ² 9:08 2:04 9 ° ^9β 11 n ! ² 4: 14! Ι | average i 9-50 J 8.71 0.79 ! ¹⁵⁶ ! 12 n ! 16 ' 13! 1 1 1 average ι ⁹³⁸ 1 8.62 0.76 ! ¹⁸⁵ i 1 j 13 n 1 33 j 22 J 1 1 1 average 1 ¹¹⁷⁰ 1 10:45 1:24 í ³ · ⁴⁷ 1 1 14 n j 64 | 46! 1 1 1 average 12.80 12:17 0.62 ! ¹ · ²⁹ ! 1 | 15 n ! ⁸³ 1 ⁵⁹ ! 1 1 1 average 12:48 9.81 2.67 1 ¹¹³ ! 1 1 16 n 1 ²⁷ ! 17! 1 1 1 1 average ; 13.70 12.78 0.94 ! ² · ⁴⁰ 1 17 n '17' 1 13 ' ! 1 1 average 13.29 i 11.23, 2:06 ^! 1.93: í 18 n '14 9 1 1 average , 16.4 3! 11.67 4.76 l 3 77 * 1 ^J * ·

Table 4

Statistical analysis of the number of oocytes recovered (using Cochran's approximation method) considering all pungous individuals

Number of mature oocytes recovered

Group

Org 32489 minus Metrodin

1st cycle 1 I [org 32489! 1 1 1 1 .. .... 1 Metrodin The treatment difference estimated is jI appreciates | value standarc | failure τ · t | d 1 95 micron MEG ^- reliability interval (P-value) centers 1 ⁿ ! ! 361! J- - 1 1 altogether average 8th SS | 6.76 1.79 0:33 1.1 - 2.4 Set to center I 1 1 1 (<0.00011 1 interaction test j I 1 1 (0.89) Center 1 i i -1- | 1 n ! 67 J 41! 1 1 average ^{12 48} 11:29 1:18 1:31 1 I 2 n ! I · ² ! 7 J 1 | average j 10.92 7:43 3:49 3:10 1 1 3 1 ⁿ 4! S | 1 1 average 1 ⁵ .75 1.60 4:15 1:24 1 1 4 i n ! 1 °! 7 J 1 1 average 7:50 5:43 2:07 2:02 1 1 s 1 ! 13! 8! 1 1 atlag 1 13.23 8:25 4.98 3:20 1 | 6 1 ⁿ 30 | 17 I 1 i average 1 10.53 8:35 2:18 1.81 1 | 7 i? 44 [ 24 J 1 ; average 1 ⁷ · ²³ 5:21 2:02 1:01 1 I 8 J n 19 j 15 j 1 1 atlag ¹⁴³⁷ 4:27 0:10 1:30 1 1 9 1 ⁿ 27 I 19! 1 ! average | 10:59 9.74 0.86 1:59 1 | 10 i ⁿ ! ⁴¹ ! 25! 1  average 1 ^6-93 i 5:40 1:53 0.99 1 I 11 i? ! 24! 1 «! 1 . average ¹⁸¹⁷ 7:21 0.9S 1:27 1 | 12 i n ! i ^fi ! 13! 1 average 1 ⁶⁸¹ i 7.15 -0.34 1.98 1 | 13 • n ^{3 3} ! ²² 1 average [10.94 9.82 1:12 1:48 1 | 14  n 64! 46! 1 'average 12:59 11:35 1:25 1:33 1 I IS 1 > n í 83 | 59! 1  average 7:39 4.85 2.S4 0.79 1 1 Youth : n ! 27 17 j 1 _: average 1 12.56 11:24 1:32 2:33 1 | 17  n ! I ⁷ I 13 ¹ 1 average 13.29; 11.23 i 2:06 1.93 1 i 18  n i ¹³ i ⁹ ! 1 i average 1 ¹⁰ - ³¹ ! 1 1 8.11 J 2:20 2.78 1 1 , 1

- 26 Table 5

Statistical analysis of total doses of FSH administered (using the Cochran Approach), including all pungous individuals

The total dosed FSH _T ...., Group Org 32489 minus Metrodin number of vials / ampoules 1 1 | | The treatment iA is estimated! 95 X- 1st cycle Org 32489 Metrodin difference value | reliability 1 I estimated standard | interval 1 1 is an error of j 1 1 (P value) . | __ centers j n j S46 i j average 28.49 31.82 -3.33 0.62 -4.5 - 2.1 altogether t 1 j (<0.0001) l set to control panel 1 j 1 J (0.06) J interaction test! . Center 1 i i I X í ⁿ ! «»! ⁴¹ ! 1 1 average j 29.29} 38.13 -8.84 3:44 1 2 í ! ¹² ! 7 1 1 average 28.08 27.43 0.65 2.80 I 3 í ⁿ 4! S! 1 | average 39.S0 42.60 -3.10 8:10 1 l 4 ! ⁿ 11 j 7 J 1 1 average 36.36 37.86 -1.49 5:49 1 1 5 ! ⁿ ! ¹³ ! ⁸ ! 1 j atlag 30.54 42.00 -11.46 4.86 I 1 £ ! ⁿ ! ³⁰ ! ¹⁷ 1 1 average J 26.87 2B.76 -1.90 2.95 1 1 7 ! « 44! ²⁴ ! 1 j atlag ^{July 27} 33.83 -6.77 2:21 1 1 8 ! ⁿ ! ³⁹ ! ³⁹ ! 1 j atlag 49.26 48.77 o.so S.66 1 1 9 ! ⁿ ! ²⁷ ! ³⁹ ! 1 1 average 1 ^3S · ⁰⁴ 32.79 2:25 3:09 1 1 10 ! ⁿ 41! 25! 1 1 average 1 ³⁵ · ⁷⁰ 37.88 -2.18 3:59 1 I 11 J n ! 24! ³⁴ ! 1 1 average ¹³ ° ^-7S 38.07 -7.32 2.78 1 1 12 ! ! ³⁶ ! ³³ ! 1 i average 32.38 32.00 0:38 6:01 1 13 ! n ! ³³ ! ²² ! 1 ί average 27.61 29.00 -1.39 2:15 1 14 i ⁿ ! ! ⁴⁶ ! 1 1 average 30.59 31.22 -0. 6 1:29 1 IS t ⁸³ ! ⁵⁹ ! l | average ^{23 67} 28.19 -4.52 1:44 1 16 1 ⁿ ! ²⁷ ! ³⁷ ! 1 1 average 1 ²³²⁶ 28.47 -5.21 2.65 1 17 ! n 17! ³³ ! I 'average 25.79 j 33.23 -7.44 3.64 i IS ! » i 14! ⁹ ! 1 | average ! ²⁶⁹³ ! 3S.S6 1 -8.63 3:33 » 1 1

«· · ·

- 27 Table 6

Statistical analysis of FSH treatment duration (using the Cochran Approach), including all pungous individuals

Duration of FSH treatment (days) First cycle centers altogether ι n | average i set to control panel 1 interaction test Center 1 n average ⁿ average n average average n average < n average n average β n average 9 n average 10 n average n average 1 1 1 12 n average i 1 i 13 n average t t 1 i 14 n average i 15 . n * average ΐ 1 1 ' ! n • average 1 1 i 17 ·. n  average i ! i 18 • n ι average i 1 1

Org 32489

Group

54 «

10. ««

Metrodin

3S1

11:27

Org 32489 minus Metrodin

Estimated treatment difference | Estimated value I standard error I -% confidence interval (P-value)

-0. (0 sec

0:13

"7 11.99 41 13.73 -1.75 I 0.65 ¹² ! 7 j 1 I 9:08 9:00 0:08 0.C6 4! 5 J 1 1 13.75 14:00 -0.25 1:41 ¹¹ 1 7 [ 1 i 11 '4 11:57 0:06 1:15 ²² ! 8 1 | 9.92 11.38 J -1.45 0.69 30! ¹⁷ ! 1 I 10.83 11.24 [ -0.40 0:50 44 | 24! 1 I 12:41 13:46 -1.05 O.SS 19! 15! 1 1 13:11 13:53 -0.43 0.96 27 ' ¹⁵ ! 1 1 11.IS 10.2 ' 0.88 0:59 ⁴¹ ! 25 j 1 12:24 12.72 -0.48! 0 .43 24! 14 J 1 1 11:33 12.93 -1.S0 0:57 i «! 13! 1 1 12:25 11:21 1:02 1:08 33! ²² ! 1 1 8.55 J 8:45 0.09 j 0:35 «4! 4 «! 1 10:09 10.3 3 -0.23 0:35 83 ' 59! 1 1 10.81 12:02 -1.21 0:37 ²⁷ ! ¹⁷ ! 1 I 10.9 ' 11.82 j -0.86 0:48 ¹⁷ ! 13! 1 1 10.0 ' 11.77 j -1.71 0.67 14 9 j 1 t 9.79 11.33} -1.S5 0.84

-0.9 - -0.3 («0.0001) (0.02) ♦ ·

Claims (9)

A pharmaceutical composition comprising a mixture of follicular stimulating isoforms of a glycoprotein hormone with a range of 4.8 to 4.2 and having isoelectric points, in admixture with pharmaceutically acceptable excipients, characterized in that the isoforms are more than less than 30% has an isoelectric point below 4.8 and less than 30% has an isoelectric point below 4.2. The composition of claim 1, wherein more than 15% of the isoforms have an isoelectric point greater than 4.8 and less than 25% have an isoelectric point Below 4.2. The composition of claim 1, wherein more than 25% of the isoforms have an isoelectric point greater than 4.8 and less than 25% have an isoelectric point Below 4.2. The composition of claim 1, wherein more than 25% of the isoforms have an isoelectric point above 4.8 and less than 20% have an isoelectric point Below 4.2. 5. A composition according to any one of claims 1 to 6, wherein the glycoprotein is a recombinant follicle stimulating hormone. 6. A composition according to any one of claims 1 to 6, wherein the glycoprotein is modified by enzymatic or chemical treatment. The composition of claim 5, wherein the recombinant follicle stimulating hormone is modified by removing at least one of the nitrogen-bound oligosaccharide chains. 8. A method for inducing follicular growth and maturation in a female subject, characterized in that the female subject has a method according to claims 1-7. A composition according to any one of claims 1 to 6. 9. A method of inducing follicular growth and maturation by administering identical pharmaceutical compositions containing a mixture of isoforms of glycoprotein having follicular stimulating activity during the ovulation cycle, wherein the isoforms have an isoelectric point greater than 4.2.