NZ620369B2 - Composition for controlled ovarian stimulation - Google Patents

Abstract

Discloses the use of follicle stimulating hormone (FSH) for the manufacture of a product for the treatment of infertility in a patient; wherein if the patient has serum AMH level of less than15 pmol/L, the product comprises a daily dose of, or daily dose equivalent to, 11 to 13 ?g recombinant FSH, the recombinant FSH includes ?2, 3- and ?2, 6- sialylation where 1% to 99% of the total sialylation is ?2, 6- sialyation, and 1% to 99% of the total sialylation is ? 2,3- sialyation; or wherein if the patient has serum AMH level of greater than 15 pmol/L, the product is formulated to be administered at a daily dose of, or daily dose equivalent to, 0.09 to 0.19 ?g recombinant FSH per kg bodyweight of the patient, the recombinant FSH including ?2, 3- and ?2, 6- sialylation wherein 1% to 99% of the total sialylation is ?2, 6- sialyation, and 1% to 99% of the total sialylation is ? 2,3- sialyation. the recombinant FSH includes ?2, 3- and ?2, 6- sialylation where 1% to 99% of the total sialylation is ?2, 6- sialyation, and 1% to 99% of the total sialylation is ? 2,3- sialyation; or wherein if the patient has serum AMH level of greater than 15 pmol/L, the product is formulated to be administered at a daily dose of, or daily dose equivalent to, 0.09 to 0.19 ?g recombinant FSH per kg bodyweight of the patient, the recombinant FSH including ?2, 3- and ?2, 6- sialylation wherein 1% to 99% of the total sialylation is ?2, 6- sialyation, and 1% to 99% of the total sialylation is ? 2,3- sialyation.

Description

Composition for controlled ovarian stimulation The present invention s to compositions and pharmaceutical products for the treatment of infertility. ed reproductive technology (ART) techniques such as in vitro fertilisation (IVF) are well known. These ART techniques generally require a step of controlled ovarian stimulation (COS), in which a cohort of follicles is stimulated to full maturity. rd COS regimens include stration of gonadotrophins, such as follicle stimulating hormone (FSH) alone or in combination with luteinising hormone (LH) activity to stimulate follicular development, normally with administration of a GnRH analogue prior to and/or during stimulation to prevent premature LH surge. The pharmaceutical itions generally used for COS include recombinant follicle stimulating hormone (rFSH), urinary derived FSH, inant FSH + LH preparations, urinary derived menotrophin [human menopausal gonadotrophin (hMG)] and highly purified human menopausal gonadotrophin (HP-hMG). IVF can be associated with a risk of ovarian hyperstimulation syndrome (OHSS), which can be life threatening in severe cases.

The ability to predict the response potential of women to controlled ovarian stimulation (COS) may allow the development of individualised COS ols. This could, for example, reduce the risk of OHSS in women predicted to have an excessive response to stimulation, and/or improve pregnancy outcomes in women classed as poor responders. The serum concentration of Ullerian hormone (AMH) is now established as a reliable marker of ovarian reserve. Decreasing levels of AMH are correlated with reduced n response to gonadotrophins during COS. r, high levels of AMH are a good predictor of excessive ovarian response, and an indicator of risk of OHSS.

In a preliminary study of women under 35 years old undergoing ART, the CONSORT dosing thm porating basal FSH, BMI, age and AFC) was used to predict the l FSH starting dose for COS in women at risk of developing OHSS (Olivennes et. al., 2009).

Individualising the dose did lead to adequate oocyte yield and good pregnancy rate. However, there were high rates of cancellations in the low dose group (75 IU FSH) due to inadequate response, and OHSS did occur in a significant proportion of the patients.

There is therefore a need for a composition for use in individualised COS protocols which provides adequate response to stimulation, and/or decreased risk of OHSS.

As indicated above, standard COS protocols may include administration of FSH. FSH is naturally secreted by the anterior pituitary gland and functions to support ular development and ovulation. FSH comprises a 92 amino acid alpha sub-unit, also common to the other glycoprotein hormones LH and CG, and a 111 amino acid beta sub-unit unique to FSH that confers the biological specificity of the hormone (Pierce and Parsons, 1981). Each it is post translationally modified by the addition of x ydrate residues. Both WO 20996 subunits carry 2 sites for N-linked glycan ment, the alpha sub-unit at amino acids 52 and 78 and the beta sub-unit at amino acid es 7 and 24 (Rathnam and Saxena, 1975, Saxena and Rathnam, 1976). FSH is thus glycosylated to about 30% by mass (Dias and Van Roey. 2001. Fox et al. 2001).

FSH purified from post-menopausal human urine has been used for many years in ility treatment; both to promote ovulation in natural reproduction and to provide oocytes for assisted reproduction technologies. The currently approved recombinant FSH (rFSH) products for ovarian stimulation, such as follitropin alfa -F, Merck Serono / EMD Serono) and follitropin beta (PUREGON / FOLLISTIM, MSD / Schering-Plough), are derived from a Chinese r Ovary (CHO) cell line. Currently, no rFSH products from a human cell line are commercially available.

There is considerable heterogeneity associated with FSH preparations which relates to differences in the amounts of s ms t. Individual FSH isoforms exhibit identical amino acid sequences but differ in the extent to which they are post-translationally modified; particular isoforms are characterised by heterogeneity of the carbohydrate branch structures and differing amounts of sialic acid (a terminal sugar) incorporation, both of which appear to influence the specific isoform bioactivity.

Glycosylation of natural FSH is highly complex. The glycans in lly derived pituitary FSH can contain a wide range of ures that can include combinations of mono-, bi- tri- and antennary glycans e and Parsons, 1981. Ryan et al., 1987. Baenziger and Green, 1988). The glycans can carry further modifications: core fucosylation, bisecting glucosamine, chains extended with acetyl lactosamine, partial or complete ation, sialylation with 02,3 and 02,6 linkages, and sulphated galactosamine substituted for galactose (Dalpathado et al., 2006). Furthermore, there are differences between the distributions of glycan structures at the dual glycosylation sites. A comparable level of glycan complexity has been found in FSH derived from the serum of individuals and from the urine of post- menopausal women (Wide et al., 2007).

The glycosylation of recombinant FSH products reflects the range of glycosyl- transferases present in the host cell line. The commercially available rFSH products are derived from engineered Chinese hamster ovary cells (CHO cells). The range of glycan cations in CHO cell derived rFSH are more limited than those found on the natural products. Examples of the reduced glycan heterogeneity found in CHO cell derived rFSH include a lack of bisecting glucosamine and a reduced content of core fucosylation and acetyl lactosamine extensions (Hard et al., 1990). In addition, CHO cells are only able to add sialic acid using the 02,3 linkage (Kagawa et al, 1988, Takeuchi et al, 1988, Svensson et al., 1990); CHO cell derived rFSH only includes 02,3-linked sialic acid and does not include inked sialic acid.

Thus CHO cell derived FSH is different from naturally produced FSH (e.g. human Pituitary/ serum/ urinary FSH) which contains glycans with a mixture of 02,3 and 02,6-linked sialic acid, with a predominance of the former.

Further, it has also been demonstrated that the commercially available recombinant FSH preparation differs in the amounts of FSH with an ctric point (pl) of below 4 (considered the acidic isoforms) when compared to pituitary, serum or post-menopausal urine FSH (Ulloa-Aguirre et al. 1995). The amount of acidic isoforms in the urinary preparations was much higher as compared to the CH0 cell d recombinant products, f (Merck Serono) and Puregon (Schering Plough) sen et al. 2004). This must reflect a lower molar t of sialic acid in the recombinant FSH since the content of negatively-charged glycan modified with sulphate is low in recombinant FSH. The lower sialic acid content, compared to natural FSH, is a feature of both commercially available recombinant FSH products and may reflect a tion in the manufacturing process.

The circulatory ime of FSH has been documented for materials from a variety of sources. Some of these materials have been fractionated on the basis of overall molecular charge, as characterised by their pl, in which more acid equates to a higher negative .

As previously stated the major contributor to overall molecular charge is the total sialic t of each FSH molecule. For instance, rFSH (Organon) has a sialic acid content of around 8 mol/mol, whereas urine-derived FSH has a higher sialic acid content (de Leeuw et al. 1996).

The ponding plasma clearance rates in the rat are 0.34 and 0.14 ml/min (Ulloa-Aguirre et al. 2003). In another example where a sample of recombinant FSH was split into high and low pl fractions, the in vivo potency of the high pl (lower sialic acid content) fraction was decreased and it had a shorter plasma half-life onio et al. 1999). It has also been reported that the more basic FSH circulating during the later stages of the ovulation cycle is due to the down- regulation of 02,3 sialyl-transferase in the anterior pituitary which is caused by increasing levels of estradiol (Damian-Matsumara et al. 1999. Ulloa-Aguirre et al. 2001). Results for the 02,6 sialyl-transferase have not been reported.

Thus, as set out above, recombinant proteins expressed using the CH0 system will differ from their natural counterparts in their type of terminal sialic acid linkages. This is an important consideration in the tion of icals for pharmaceutical use since the carbohydrate moieties may contribute to the pharmacological attributes of the molecule.

The present applicants have developed a human d recombinant FSH which is the subject of International Patent Application No. , hed as WO2009/127826A. Recombinant FSH with a mixture of both 02,3 and 02,6—linked sialic acid was made by engineering a human cell line to express both rFSH and 02,3 transferase.

The expressed product is highly acidic and carries a mix of both 02,3- and 02,6-linked sialic acids; the latter provided by the endogenous sialyl transferase activity. It was found that the type of sialic acid linkage, 02,3- or 02,6-, can have a dramatic influence on biological clearance of FSH. Recombinant FSH with a mixture of both 02,3 and 02,6-linked sialic acid has two advantages over rFSH expressed in conventional CHO cells: first the material is more highly WO 20996 sialylated due to the combined activities of the two sialyltransferases; and secondly the material more closely les the natural FSH. This is likely to be more biologically appropriate compared to CH0 cell derived recombinant products that have produce only 02,3 linked sialic acid (Kagawa et al, 1988, Takeuchi et al, 1988, Svensson et al., 1990) and have decreased sialic acid content (Ulloa-Aguirre et al. 1995., Andersen et al. 2004).

The rFSH product disclosed in International Patent Application No. contains branched glycan es. FSH comprises glycans (attached to the FSH glycoproteins) and these glycans may contain a wide variety of structures. As is well known in the art, branching (of a glycan) can occur with the result that the glycan may have 1, 2, 3, 4 or more terminal sugar residues or “antennae”; glycans with 1, 2, 3 or 4 terminal sugar es or nae” are referred to respectively as mono-antennary, di-antennary, tri- antennary or tetra-antennary structures. Glycans may have sialylation presence on mono- antennary and/or di-antennary and/or tri-antennary and/or tetra-antennary structures. An example rFSH disclosed in International Patent Application No. included ialylated, di- ated, tri- sialylated and tetra- sialylated glycan structures with relative amounts as s: 9-15% ialylated; 27 — 30% a|y|ated; 30 — 36% tri--sialylated and 25 — 29 % tetra--sialylated. As is well known, a mono-sialylated glycan structure carries one sialic acid residue; a di-sialylated glycan structure carries two sialic acid residues; a tri- sialylated glycan structure carries three sialic acid residues; and a tetra-sialylated glycan structure carries four sialic acid residues. Herein, terminology such as “X% mono-sialylated”, “X% di-sialylated”, “X% tri-sialylated” or “X% tetra-sialylated” refers to the number of glycan structures on FSH which are mono-, di, tri or tetra sialylated (respectively), expressed as a percentage (X%) of the total number of glycan ures on the FSH which are sialylated in any way (carry sialic acid). Thus, the phrase “30 — 36% alylated glycan ures” means that, of the total number ofglycan structures on the FSH which carry sialic acid residues (that is, are sialylated), 30 to 36% of these glycan structures are tri sialylated (carry three sialic acid residues). The applicants have surprisingly found that FSH having a specific amount of tetra- sialylated glycan structures (which is different to that of the example rFSH product disclosed in mentioned above) is markedly more potent then inant FSH products which are currently on the market. The amino acid sequence of the applicant’s products is the native sequence and is cal to natural human FSH and ng CHO-derived rFSH products. However, the present applicants have found that human derived recombinant FSH products (i.e. recombinant FSH produced or expressed in a human cell line e.g. made by engineering a human cell line) which have a mixture of both 02,3 and d2,6-linked sialic acid and/or a specific amount of tetra-sialylated glycan structures may be particularly effective when utilised in (e.g. individualised) COS protocols.

According to the present invention in a first aspect there is provided a product (e.g. a pharmaceutical composition) comprising follicle stimulating hormone (FSH) for use in the treatment of infertility in a t (e.g. a patient having serum AMH level of 0.05 pmol/L or above, for example 0.5 pmol/L or above), wherein the product comprises a dose of, or a dose equivalent to, 1-24 pg, for e 2-24 pg, for example 2 to 15 pg, human derived recombinant FSH. Preferably the product comprises a dose of, or a dose equivalent to, 4.5 to 12.5 pg, for e 5 to 12.5 pg, for example 6 to 12.5 pg, for example 6.3 to 10.5 pg, human derived recombinant FSH.

According to the invention there is provided a product (e.g. a pharmaceutical composition) comprising follicle stimulating hormone (FSH) for use in the treatment of infertility in a patient having serum AMH level of <15 pmol/L (e.g. 0.05 pmol/L to 14.9 pmol/L), wherein the product comprises a (e.g. daily) dose of, or dose lent to, 9 to 14 pg, for example 11 to 13 pg, for example 12 pg human derived recombinant FSH. Preferably the FSH is a recombinant FSH (“rFSH” or “recFSH”). ably the FSH is a human cell line derived recombinant FSH. The dose es an ive response while minimising risk of OHSS.

Preferably the treatment of infertility comprising a step of determining (e.g. measuring) the serum AMH level of the patient, and administering the dose to a patient having serum AMH level of<15 pmol/L (e.g. 0.05 pmol/L to 14.9 pmol/L).

According to the invention in a further aspect there is provided a product (e.g. a pharmaceutical composition) comprising follicle stimulating hormone (FSH) for use in the treatment of infertility in a patient having serum AMH level of 215 pmol/L, wherein the product comprises a (e.g. daily) dose of, or dose equivalent to, 5 to 12.5 pg, for example 6 to 10.5 pg human derived recombinant FSH. Preferably the FSH is a recombinant FSH (“rFSH” or “recFSH”). ably the FSH is a human cell line derived recombinant FSH. The dose provides an ive response while minimising risk of OHSS. ably the treatment of infertility comprising a step of determining (e.g. measuring) the serum AMH level of the patient, and administering the dose to a patient having serum AMH level of 215 pmol/L. In one embodiment, the product is for use in the treatment of ility in a patient having serum AMH level of 15 to 24.9 , and the product is for administration at a (e.g. daily) dose of, or dose equivalent to, 5 to 12 pg, for example 7 to 12 pg, for example 8.7 to 10 pg, human derived recombinant FSH rably 9 to 10 pg human derived recombinant FSH) In this embodiment, the ent of infertility may comprise a step of determining (e.g. measuring) the serum AMH level of the patient, and administering the dose to a patient having serum AMH level of 15 to 24.9 pmol/L. In another embodiment, the product is for use in the treatment of infertility in a patient having serum AMH level of 25 to 34.9 pmol/L, and the product is for administration at a (e.g. daily) dose of, or dose equivalent to, 5 to 12 pg, for example 6 to 9 pg, for example 7 to 8 pghuman derived recombinant FSH (preferably 7.3 to 8 pg human derived recombinant FSH).

In this embodiment, the treatment of infertility may comprise a step of determining (e.g. measuring) the serum AMH level of the patient, and administering the dose to a patient having WO 20996 serum AMH level of 25 to 34.9 pmol/L. In another embodiment, the product is for use in the treatment of infertility in a patient having serum AMH level of 2 35 pmol/L, and the t is for stration at a (e.g. daily) dose of, or dose equivalent to, 5 to 11 pg, for example 6.3 to 7 pg, human derived recombinant FSH (preferably 6 to 7 pg human derived recombinant FSH).

In this embodiment, the treatment of infertility may comprise a step of determining (e.g. measuring) the serum AMH level of the t, and administering the dose to a patient having serum AMH level of 2 35 pmoI/L.

The doses above may be for treatment of infertility in the patient’s (subject’s) first stimulation protocol. It will be appreciated that for further stimulation cycles, the doses may be adjusted according to actual ovarian response in the first cycle.

The applicants have found that it is generally necessary to retrieve in the region of nine oocytes in order to enable selection of two high quality oocytes for er.

The applicants have found that for subjects having low AMH (AMH < 15 pmol/L per litre) a reasonably high dose of recombinant FSH is required (for example 12 pg) to achieve this. At this dose, 8 to 14 oocytes will be retrieved from 60% of subjects with low AMH. This is an unexpected and significant improvement over treatment of subjects with low AMH treated with 150 IU GonaI-f, where 8 to 14 oocytes are retrieved from only 33% of subjects. The applicants have found that there is no need to adjust this dose according to the bodyweight of the t.

However, 60 % of the population (and 80% of women under 30 d for infertility) have high AMH (that is, AMH of 215 pmol/L). For these subjects it is lly fairly straightfonNard to retrieve a mean of 9 to 11 s; the problem with ation protocols is the risk of OHSS. The ants have found that in patients dosed at low doses of human recombinant FSH that there is a relationship between oocytes retrieved and body weight of the subject. This means that there may be a risk associated with treatment with a fixed dose of FSH (which is usual in the art). The present ants have established a relationship between dose of FSH and AMH level and weight of the t which provides an improved safety profile (reduced risk of OHSS) with acceptable or improved oocyte retrieval compared to the known treatment protocols (see example 10).

According to the invention in a further aspect there is provided a product (e.g. a pharmaceutical composition) comprising follicle ating hormone (FSH) for use in the treatment of infertility in a patient having serum AMH level of 215 pmol/L, wherein the product is for administration at a (e.g. daily) dose of, or dose equivalent to, 0.09 to 0.19 pg (for example 0.09 to 0.17 pg) human derived recombinant FSH per kg bodyweight of the patient. Preferably the treatment of ility comprises a step of determining (e.g. measuring) the serum AMH level of the patient, and administering the dose to a patient having serum AMH level of 215 pmol/L. In one embodiment, the product is for use in the treatment of infertility in a patient having serum AMH level of 15 to 24.9 pmol/L, and the product is for administration at a (e.g. daily) dose of, or dose equivalent to, 0.14 to 0.19 pg human derived recombinant FSH WO 20996 (preferably 0.15 to 0.16 pg human derived inant FSH) per kg bodyweight of the patient.

In this embodiment, the treatment of infertility may comprise a step of determining (e.g. measuring) the serum AMH level of the t, and administering the dose to a patient having serum AMH level of 15 to 24.9 . In another ment, the t is for use in the treatment of infertility in a patient having serum AMH level of 25 to 34.9 pmo|/L, and the product is for administration at a (e.g. daily) dose of, or dose equivalent to, 0.11 to 0.14 pg human derived recombinant FSH rably 0.12 to 0.13 pg human d inant FSH) per kg bodyweight of the patient. In this embodiment, the treatment of infertility may comprise a step of determining (e.g. measuring) the serum AMH level of the t, and administering the dose to a patient having serum AMH level of 25 to 34.9 pmo|/L. In a still further embodiment, the product is for use in the treatment of infertility in a patient having serum AMH level of 2 35 pmo|/L, and the product is for administration at a (e.g. daily) dose of, or dose equivalent to, 0.10 to 0.11 pg human derived recombinant FSH per kg bodyweight of the patient. In this ment, the treatment of ility may comprise a step of determining (e.g. measuring) the serum AMH level of the patient, and stering the dose to a patient having serum AMH level of 2 35 pmo|/L. ably the FSH is a recombinant FSH (“rFSH” or “recFSH”).

Preferably the FSH is a human cell line derived recombinant FSH. The doses provide an effective response while minimising risk of OHSS.

The doses above may be for treatment of infertility in the patient’s (subject’s) first stimulation protocol. It will be appreciated that for further stimulation cycles, the doses may be adjusted according to actual ovarian response in the first cycle.

According to the invention in a still further aspect there is provided a product (e.g. a pharmaceutical composition) comprising follicle stimulating hormone (FSH) for use in the treatment of infertility in a patient having serum AMH level of <15 pmo|/L, wherein the product is for administration at a (e.g. daily) dose of, or dose equivalent to, 0.15 to 0.21 pg, (for example 0.19 to 0.21 pg) human derived recombinant FSH per kg bodyweight of the patient. Preferably the treatment of infertility comprises a step of ining (e.g. measuring) the serum AMH level of the patient, and administering the dose to a patient having serum AMH level of <15 pmo|/L. However, it is not ed that patients having serum AMH level of <15 pmo|/L are dosed by body weight. It will be appreciated that these doses may be readily converted to treat patients with dosing according to their BMI, using conversions well known in the art.

The product (e.g. pharmaceutical compostion) may be for use in the treatment of infertility in a patient having serum AMH of 50-149 pmo|/L, wherein the t comprises a dose of, or dose equivalent to, 6 to 18 pg, for example 8 to 11 pg, for example 8.5 to 10.2 pg human derived recombinant FSH. The product may be for use in the treatment of infertility in a patient having serum AMH 15.0-29.9 pmo|/L, wherein the product comprises a dose of, or a dose equivalent to, 4.8 to 15 pg, for example 6 to 9 pg, for example 6.8 to 8.5 pg human derived recombinant FSH. The product may be for use in the treatment of infertility in a patient having serum AMH 30-44.9 pmol/L, wherein the product comprises a dose of, or a dose equivalent to, 3.6 to 12 pg, for example 4 to 7 pg, for e 5.1 to 6.8 pg human derived recombinant FSH. The product may be for use in the treatment of infertility in a patient having serum AMH 45 pmol/L or greater, wherein the product comprises a dose of, or a dose equivalent to, 2 to 9 pg, for example 2.4 to 9 pg (for example 3.4 to 5.1 pg) or 2 to 5 pg human derived recombinant FSH. The t may comprise follicle ating hormone (FSH) for use in the treatment of ility in a patient having serum AMH of 5 pmo|/L or less, wherein the product comprises a dose of, or a dose equivalent to 7.2 to 24 pg, for example 10 to 15 pg for example 10.2 to 13.6 pg, human derived recombinant FSH. The product may be for use in the ent of infertility in a patient n the product ses a dose of, or dose equivalent to, 4.8 to 18 pg, for example 6 to 11 pg, for example 6.8 to 10.2 pg human derived recombinant FSH. Preferably the FSH is a recombinant FSH (“rFSH” or “recFSH”). Preferably the FSH is a human cell line derived recombinant FSH.

Preferably the rFSH (e.g. human cell line derived recombinant FSH) es d2,3- and 02,6- sialylation. The FSH (rFSH) for use according to the invention may have 1% to 99% of the total sialylation being d2,3-sialylation. The FSH (rFSH) according to the invention may have 1% to 99% of the total sialylation being d2,6-sialylation. Preferably, 50 to 70%, for example 60 to 69%, for example about 65%, of the total sialylation is d2,3-sialylation. Preferably 25 to 50%, for example 30 to 50 %, for example 31 to 38%, for example about 35%, of the total ation is 02,6- sialylation.

Preferably the rFSH (e.g. human cell line derived recombinant FSH) includes mono-, di- tri- and tetra-sia|y|ated glycan structures, wherein 15-24%, for example 17-23% of the sia|y|ated glycan structures are tetrasialylated glycan structures (e.g. as shown by WAX analysis of charged glycans, as set out in the Examples below). The FSH comprises glycans (attached to the FSH glycoproteins). It is well known that glycans in FSH may contain a wide variety of structures. These may include combinations of mono, bi, tri and tetra-antennary glycans. Herein, terminology such as “X% of the sia|y|ated glycan structures are ialylated glycan structures” refers to the number of glycan structures on the FSH which are tetra sia|y|ated, i.e. carry four sia|ic acid residues, expressed as a percentage (X%) of the total number of glycan structures on the FSH which are sia|y|ated in any way (carry sia|ic acid).

Thus, the phrase “15-24% of the sia|y|ated glycan structures are tetrasialylated glycan structures” means that, of the total number ofg|ycan structures on FSH which carry sia|ic acid residues (that is, are sia|y|ated), 15 to 24% of these glycan structures are tetra sia|y|ated (carry four sia|ic acid residues).

The rFSH may be present as a single isoform or as a mixture of isoforms.

The applicants have devised “individualised” COS protocols n specific doses of recombinant FSH having ic teristics are used to treat patients based on their ic AMH , thereby increasing the likelihood of adequate response to stimulation (e.g. in patients having a low response potential), and/or decreased risk of OHSS (e.g. in patients classed as high or excessive responders).

The serum level of AMH may be determined (e.g. measured) by any method known in the art. Preferably the serum AMH level is measured using the AMH Gen-ll enzyme linked immunosorbent assay, a kit (Beckman Coulter, |nc., r, Texas). This assay can detect can detect AMH trations r than 0.57 pmol/L with a minimum limit of quantitation of 1.1 pmol/L. Other assays may be used.

Herein, serum AMH values are generally recited in terms of pmol/L. This may be ted to ng/mL using the conversion equation 1ng/ml AMH = 7.1 pmol/L AMH.

Herein the terms “patient” and “subject” are used interchangeably.

The product (e.g. pharmaceutical composition) ably ses a daily dose of, or a daily dose equivalent to, the amounts of human derived rFSH defined above, herein, and in the claims. The ) dose may be an initial dose (i.e. it may be reduced, increased, or maintained during the treatment).

The product (e.g. pharmaceutical composition) may be for (daily) administration of FSH starting on day one of treatment and continuing for seven to thirteen days, for example nine to thirteen days, for example 10 to 13 days, for example 10 to 11 days. The product (e.g. pharmaceutical composition) may be for administration 12 to 16, e.g. 13 to 15, e.g. 14 days after administration of (e.g. after initiation of administration of, e.g. after initiation of daily administration of) a GnRH agonist (e.g. Synarel, Lupron, Decapeptyl). The product (e.g. pharmaceutical composition) may be for administration with a GnRH agonist. The t (e.g. pharmaceutical composition) may be for administration prior to administration of a GnRH nist (e.g. ganirelix, cetrorelix), for example for administration five or six days prior to administration of a GnRH antagonist. The product (e.g. pharmaceutical composition) may be for administration with a GnRH antagonist. Preferably the product (e.g. pharmaceutical composition) is for administration prior to administration of a high (ovulatory) dose of hCG (for example 4,000 to 11,000 IU hCG, e.g. 5,000 IU hCG, 10,000 IU hCG etc.; or 150 to 350 microgram inant hCG, for example 250 microgram inant hCG) to induce final follicular maturation.

It will be appreciated that the product may be for dosing at frequencies more (or less) than daily, in which case the relevant doses will be equivalent to the (daily) doses specified herein.

Herein the term “treatment of infertility” includes treatment of infertility by lled ovarian stimulation (COS) or methods which include a step or stage of controlled ovarian stimulation (COS), for example lntra Uterine Insemination (lUl), in vitro fertilisation (IVF), or intracytoplasmic sperm injection (ICSI). The term “treatment of infertility” includes treatment of infertility by ovulation induction (OI) or by methods which e a step or stage of ovulation induction (OI). The term “treatment of infertility” includes treatment of infertility in a subject having tubal or unexplained ility, including treatment of infertility in a subject having endometriosis, for e stage I or stage II endometriosis, and/or in a subject having anovulatory infertility, for example WHO type II anovulatory ility, and/or in a subject with a partner with male factor infertility. The t (or ition) may be for (use in) the treatment of infertility (and/or for controlled ovarian ation) in a subject having triosis, for e in a subject having stage I or stage II endometriosis, as defined by The American Society for Reproductive ne (ASRM) classification system for the various stages of endometriosis, (stage IV most severe; stage I least severe) [American Society for Reproductive Medicine. Revised American Society for Reproductive Medicine fication of endometriosis: 1996. Fertil Steril 1997; 67,817 821.].

The product (composition) may be for (use in) the ent of infertility (and/or for controlled ovarian stimulation) in a subject having normal serum FSH level of 1 to 16 lU/L, for example 1 to 12 lU/L, in the early follicular phase.

The product (composition) may be for (use in) the treatment of infertility (and/or for controlled ovarian stimulation) in a subject aged 18 to 42 years, for example 25 to 37 years.

The product may be for (use in) the treatment of infertility r for controlled ovarian stimulation) in a subject having BMI >1 and BMI < 35 kg/m2, for example a subject having BMI >18 and BMI < 25 kg/m2, for example a t having BMI >20 and BMI < 25 kg/m2.

The rFSH may preferably include 27 — 33%, for example 30 — 32%, tri-sialylated glycan structures. The rFSH may preferably e 24 — 33%, for example 26 — 30%, di-sialylated glycan structures. The rFSH may preferably include 12 — 21%, for example 15 — 17%, mono- sialylated glycan structures. The rFSH preferably includes mono-sialylated, di- sialylated, tri- sialylated and tetra- sialylated glycan structures with relative amounts as follows: 15 to 17% mono-sialylated; 26 — 30% di--sialylated; 27 — 33% (e.g. 29 to 32%, e.g 30-32%, e.g 30 to 31%) tri--sialylated and 17 — 23 % tetra--sialylated (e.g. as shown by WAX analysis of charged glycans, as set out in the Examples). The rFSH may include from 0 to 7%, for example 0.1 to 7%, for example 3 to 6%, for example 5 to 6%, neutral sialylated structures. The FSH comprises glycans (attached to the FSH glycoproteins). Herein, terminology such as “X% mono-sialylated”, “X% lylated”, “X% tri-sialylated” or “X% tetra-sialylated” refers to the number of glycan structures on FSH which are mono-, di, tri or tetra sialylated (respectively), expressed as a percentage (X%) of the total number of glycan structures on the FSH which are sialylated in any way (carry sialic acid). Thus, the phrase “27 — 33% tri-sialylated glycan structures” means that, of the total number ofglycan structures on FSH which carry sialic acid residues (that is, are sialylated), 27 to 33% of these glycan structures are tri sialylated (carry three sialic acid residues).

The rFSH may have a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of n] of 6 mol/mol or greater, for example between 6 mol/mol and 15 mol/mol, e.g between 8 mol/mol and 14 mol/mol, for example between 10 mol/mol and 14 mol/mol, e.g between 11 mol/mol and 14 mol/mol, e.g between 12 mol/mol and 14 mol/mol, e.g. between 12 moI/mol and 13 moI/mol. The rFSH may be produced or expressed in a human cell line.

The FSH (rFSH) for use according to the invention may have 1% to 99% of the total sialylation being 02,3-sialylation. The rFSH may have 10% or more of the total sialylation being d2,3-sia|y|ation. For example, 20, 30, 40, 50, 60, 70, 80 or 90% or more of the total sialylation may be 02,3-sialylation. The rFSH may preferably include 02,3-sialylation in an amount which is from 50 to 70% of the total sialylation, for example from 60 to 69% of the total sialylation, for example from 63 to 67%, for example around 65% of the total sialylation. The FSH (rFSH) for use according to the invention may have 1% to 99% of the total sialylation being 012,6- ation. The rFSH (or rFSH preparation) of the invention may have 5% or more, for e 5% to 99%, of the total sialylation being ia|y|ation. The rFSH may have 50% or less of the total sialylation being 02,6-sialylation. The rFSH may ably include 02,6- sialylation in an amount which is from 25 to 50% of the total sialylation, for example from 30 to 50% of the total sialylation, for example from 31 to 38%, for e around 35% of the total sialylation. By sialylation it is meant the amount of sialic residues present on the FSH carbohydrate structures. d2,3-sia|y|ation means sialylation at the 2,3 position (as is well known in the art) and 02,6 sialylation at the 2,6 position (also well known in the art). Thus “% of the total sialylation may be a 2,3 sialylation” refers to the % of the total number of sialic acid residues present in the FSH which are sialylated in the 2,3 position. The term “% of the total sialylation being ia|y|ation” refers to the % of the total number of sialic acid residues present in the FSH which are sialylated in the 2,6 on.

The rFSH may have a sialic acid content t of sialylation per FSH molecule) of (based on the mass of protein, rather than the mass of protein plus carbohydrate) of 6% or greater (e.g. between 6% and 15%, e.g. n 7% and 13%, e.g. between 8% and 12%, e.g. between 11% and 15%, e.g. between 12% and 14%) by mass.

The rFSH may be rFSH or a rFSH preparation in which 16 % or fewer (e.g. 0.1 to 16%) of the glycans comprise (e.g. carry) ing N-acetylglucosamine (bisecting GIcNAc or bisGlcNAc). Preferably the rFSH (or rFSH preparation) is an rFSH or rFSH preparation in which 8 to 14.5% of the glycans comprise (e.g. carry) a bisecting N-acetylglucosamine (bisecting GIcNAc or bisGlcNAc).

It will be understood that FSH comprises s ed to the FSH glycoproteins. It will also be understood that 100% of the glycans refers to or means all of the glycans attached to the FSH glycoproteins. Thus, herein, the terminology “8 to 14.5% of the glycans comprise (carry) bisecting N-acetylglucosamine” means that 8 to 14.5% of the total number of glycans attached to the FSH glycoproteins include/carry bisecting N-acetylglucosamine; “16% or fewer of the glycans se (carry) bisecting N-acetylglucosamine” means that 16 % or fewer of the total number of s attached to the FSH glycoproteins include/carry bisecting N- acetylglucosamine, and so on.

The applicants have found that recombinant FSH (rFSH preparations; rFSH itions) in which 16% or fewer (e.g. 8 to 14.5%) of the glycans comprised in the FSH glycoproteins carry ing GIcNac may have advantageous pharmacokinetic properties. It is believed the advantageous properties may arise because the amount of glycans which carry bisecting GIcNac is similar to that in the human urinary derived product Bravelle, which is rather less than that of other inant FSH preparations such as those disclosed in W02012/017058.

The rFSH (or rFSH ation) may be an rFSH or rFSH preparation in which 20% or more of the glycans comprise (e.g. carry) N-Acetylgalactosamine (GalNAc), for example in which 20% or more of the glycans comprise (e.g. carry) a terminal GalNAc. ably the rFSH (or rFSH preparation) is an FSH or FSH preparation in which the 40 to 55%, for example 42% to 52%, of the s comprise (e.g. carry) GalNAc. ably the rFSH (or rFSH preparation) is an FSH or FSH preparation in which the 40 to 55%, for e 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc.

It will be understood that FSH comprises glycans attached to the FSH glycoproteins. It will also be understood that 100% of the glycans refers to or means all of the glycans ed to the FSH glycoproteins. Thus, herein, the terminology “wherein 20% or more of the glycans comprise (e.g. carry) GalNAc” means that 20% or more of the total number of glycans attached to the FSH glycoproteins include/carry N-Acetylgalactosamine (GalNAc); “40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc” means that 40 to 55 %, for example 42% to 52%, of the total number of glycans attached to the FSH glycoproteins include/carry terminal GalNAc, and so on.

It appears that the availability of the 02,6- linkage increases the number of tetra sia|y|ated structures, compared to CH0 cell derived products which have only the d2,3- linkage ble. The applicants have also found that their rFSH is distinguished over other approved products because of the sugar composition: it includes, or may e, a specific amount of GalNac. This may be linked to tetrasialylation and potency because the 2,6- sia|y|ation is associated with GalNac. In other words, the present applicants have developed an rFSH product which includes specific characteristics (26- linker sites, GalNac) which provide rFSH with high degree of ation, which s to lead to improved potency in vivo.

The rFSH (or rFSH preparation) may have 16 to 24% of the glycans comprising (e.g. terminal) 1 fucose-lewis, for example 16.5 to 18% of the glycans comprising (e.g. terminal) 1 fucose-lewis. The rFSH (or rFSH preparation) may have 1.5 to 4.5%, for example 2 to 4%, for example 3.7%, of the glycans comprising (e.g. terminal) 2 fucose -lewis. The content of fucose-lewis may have an effect on potency.

The rFSH may be produced or expressed in a human cell line, for example a Per.C6 cell line, a HEK293 cell line, a HT1080 cell line etc.. This may fy (and render more ent) the production method because manipulation and control of e.g. the cell growth medium to retain sialylation may be less critical than with known processes. The method may also be more efficient because there is little basic rFSH produced compared to production of known rFSH ts; more acidic rFSH is produced and separation/removal of basic FSH is less problematic. The rFSH may be produced or expressed in a PER.CS® cell line, a PER.CG® derived cell line or a modified PER.CG® cell line. rFSH which is produced or expressed in a human cell line (e.g. PER.CG® cell line, HEK293 cell line, HT1080 cell line etc.) will include some d2,6-linked sialic acids (012,6 sialylation) provided by endogenous sialyl erase activity [of the cell line] and will include some d2,3-linked sialic acids (012,3 sialylation) provided by endogenous sialyl transferase activity. The cell line may be modified using (12,3- sialyltransferase. The cell line may be modified using d2,6-sialyltransferase. Alternatively or additionally, the rFSH may e d2,6-linked sialic acids (02,6 sialylation) provided by endogenous sialyl transferase activity [of the cell line]. Herein, the term “human derived recombinant FSH” means recombinant FSH which is produced or sed in a human cell line (e.g. recombinant FSH made by engineering a human cell line).

The rFSH may be produced using d2,3- and/or 02,6-sialyltransferase. In an e, rFSH is produced using 012,3- sialyltransferase. The rFSH may include d2,6-linked sialic acids (012,6 ation) provided by endogenous sialyl transferase activity.

The product may be a pharmaceutical composition. The pharmaceutical composition is for the treatment of infertility. The treatment of infertility may comprise ed reproductive technologies (ART), ovulation induction or intrauterine insemination (lUl). The pharmaceutical composition may be used, for example, in medical indications where known FSH preparations are used.

The product or composition can be formulated into well-known itions for any route of drug administration, e.g. oral, rectal, parenteral, transdermal (e.g. patch technology), intravenous, uscular, subcutaneous, intrasusternal, intravaginal, intraperitoneal, local rs, ointments or drops) or as a buccal or nasal spray. A typical composition comprises a pharmaceutically acceptable carrier, such as aqueous solution, non toxic excipients, including salts and preservatives, s and the like, as described in Remington’s Pharmaceutical Sciences fifteenth edition (Matt Publishing y, 1975), at pages 1405 to 1412 and 1461 — 87, and the national formulary XIV fourteenth edition (American ceutical Association, 1975), among others.

Examples of suitable aqueous and non-aqueous pharmaceutical carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures f, vegetable oils (such as olive oil), and injectible organic esters such as ethyl oleate. The compositions of the t invention also can contain additives such as but not limited to preservatives, wetting agents, emulsifying , surfactants and sing agents.

Antibacterial and ngal agents can be included to prevent growth of es and includes, for example, m-cresol, benzyl alcohol, n, chlorobutanol, phenol, sorbic acid, and the like.

If a preservative is included, benzyl alcohol, phenol and/or m-cresol are preferred; however, the preservative is by no means limited to these es. Furthermore, it may be ble to include isotonic agents such as sugars, sodium de, and the like. The product or composition may further comprise a salt comprising a pharmaceutically acceptable alkali metal cation selected from the group consisting of Na+- or K+- salts, or a combination thereof.

Preferably the salt is a Na+- salt, for example NaCl or NaZSO4.

Preferably the product or composition comprises recombinant FSH and one or more of Polysorbate 20, L-methionine, phenol, disodium sulphate and sodium phosphate buffer.

In some cases, to effect prolonged action it is desirable to slow the absorption of FSH (and other active ingredients, if present) from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of FSH then depends upon its rate of ution which, in turn, can depend upon crystal size and crystalline form. atively, delayed absorption of a parenterally administered FSH combination form is accomplished by ving or suspending the FSH combination in an oil vehicle. Injectable depot forms can be made by forming microencapsule matrices of the FSH (and other agents, if present) in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of FSH to polymer and the nature of the particular r employed, the rate of FSH release can be controlled.

Examples of other biodegradable polymers include polyvinylpyrrolidone, poly(orthoesters), poly(anhydrides) etc. Depot injectable formulations are also prepared by entrapping the FSH in liposomes or microemulsions which are compatible with body tissues. able formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use. Injectable formulations can be ed in any suitable container, e.g. vial, pre-filled syringe, injection cartridges, and the like.

The t or composition may be formulated for single use or for multiple use (multiple dose). If the product or composition is ated for multiple use, it is preferred that a preservative is included. If a preservative is included, benzyl alcohol, phenol and/or ol are preferred; however, the preservative is by no means limited to these examples. The single use or multiple use formulated product or composition may further comprise a salt comprising a pharmaceutically acceptable alkali metal cation selected from the group consisting of Na+- or K+- salts, or a combination thereof. Preferably the salt is a Na+- salt, for example NaCl or NaZSO4. 2012/065507 The product or composition may be included in a container such as a vial, prefilled cartridge (e.g. for single administration or multiple use) or an injection device such as a “pen” for e.g. administration of multiple doses .

The product or composition may be a formulation (e.g. injectable formulation) including FSH (optionally with hCG, LH, LH activity etc.) The LH activity, if present, may originate from LH or human chorionic gonadotropin, hCG. If there is more than one active ingredient (i.e.

FSH and e.g. hCG or LH) these may be suitable for administration separately or er. If administered separately, administration can be sequential. The product can be supplied in any appropriate package. For example, a product can include a number of containers (e.g. pre- filled syringes or vials) containing either FSH or hCG, or a combination (or combination) of both FSH and hCG. The hCG may be recombinant hCG or urinary hCG. If the product includes a number of containers (e.g. pre-filled syringes or vials) containing FSH, e.g. recombinant FSH, each container may include the same amount of FSH. One or more containers may e different amounts of FSH. The es or vials may be packaged in a blister e or other means to maintain sterility. Any product can optionally contain instructions for using the FSH (and e.g. hCG if t) formulations. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted in accordance with routine practice in this field. See GOODMAN and GILMAN’s THE PHARMACOLOGICAL BASIS FOR THERAPEUTICES, 7th ed. In a preferred embodiment, the compositions of the invention are supplied as itions for parenteral administration. l methods for the preparation of the parenteral formulations are known in the art and are described in REMINGTON; THE SCIENCE AND PRACTICE OF CY, supra, at pages 780-820. The parenteral itions can be supplied in liquid formulation or as a solid which will be mixed with a sterile injectable medium just prior to stration. In an ally preferred embodiment, the parenteral compositions are supplied in dosage unit form for ease of stration and uniformity of dosage.

According to the present invention in a further aspect there is ed a method of treatment of infertility comprising: (a) measuring the serum AMH level of a subject; and (b) stration to the subject a dose of, or a dose equivalent to, 1-24 pg, for example 2-24 pg, for example 2 to 15 pg, human derived recombinant FSH. Preferably the dose is, or is equivalent to, 4.5 to 12.5 pg, for e 5 to 12.5 pg, for example 6 to 12.5 pg, for example 6.3 to 12 pg, human derived recombinant FSH.

According to the present invention in a further aspect there is provided a method of treatment of infertility comprising: (a) determining (e.g. measuring) the serum AMH level of a subject; and (b) administering a (e.g. daily) dose of, or dose equivalent to, 9 to 14 pg, for example 11 to 13 pg, for example 12 pg human derived recombinant follicle stimulating hormone (FSH) to a (the) subject having serum AMH level of <15 pmol/L (e.g. 0.05 pmol/L to 14.9 pmol/L). ably the FSH is a recombinant FSH (“rFSH” or “recFSH”). Preferably the FSH is a human cell line derived recombinant FSH. The dose provides an effective se while minimising risk of OHSS.

According to the present invention in a further aspect there is provided a method of treatment of infertility comprising: (a) determining (e.g. measuring) the serum AMH level of a t; and (b) administering a (e.g. daily) dose of, or dose equivalent to, 5 to 12.5 pg human derived recombinant follicle stimulating hormone (FSH) to a (the) subject having serum AMH level of 215 pmol/L. The (e.g. daily) dose may be, or be equivalent to, 6 to 10 pg human derived recombinant follicle ating hormone (FSH). Preferably the FSH is a inant FSH (“rFSH” or “recFSH”). Preferably the FSH is a human cell line derived recombinant FSH.

The dose es an effective response while minimising risk of OHSS.

In one ment, the method includes a step of administering a (e.g. daily) dose of, or dose equivalent to, 5 to 12 pg, for e 7 to 12 pg, for example 8.7 to 10 pg, human derived recombinant FSH (preferably 9 to 10 pg human derived recombinant FSH) to a (the) subject having serum AMH level of 15 to 24.9 pmol/L In another embodiment, the method includes a step of administering a (e.g. daily) dose of, or dose equivalent to, 5 to 12 pg human derived recombinant FSH (for example 7 to 12 pg, for example 6 to 9 pg, for example 7 to 8 pg, for example 7.3 to 8 pg human d recombinant FSH) to a (the) subject having serum AMH level of 25 to 34.9 pmol/L. In another embodiment, the method includes a step of administering a (e.g. daily) dose of, or dose equivalent to, 5 to 11 pg human derived recombinant FSH (for example 6 to 7 pg, for example 6.3 to 7 pg, human derived inant FSH) to a (the) subject having serum AMH level of 2 35 pmol/L.

According to the present invention in a further aspect there is provided a method of treatment of infertility comprising: (a) determining (e.g. ing) the serum AMH level of a subject; and (b) administering a (e.g. daily) dose of, or dose lent to, 0.09 to 0.19 pg (for example 0.09 to 0.17 pg) human derived recombinant FSH per kg bodyweight of the subject, wherein the subject has serum AMH level of 215 pmol/L. Preferably the FSH is a recombinant FSH (“rFSH” or “recFSH”). ably the FSH is a human cell line derived recombinant FSH.

The dose provides an effective response while minimising risk of OHSS.

In one ment, the method includes a step of administering a (e.g. daily) dose of, or dose equivalent to, 0.14 to 0.19 pg human derived recombinant FSH (preferably 0.15 to 0.16 pg human derived recombinant FSH) per kg bodyweight of the subject, the subject having serum AMH level of 15 to 24.9 pmol/L . In another embodiment, the method includes a step of administering a (e.g. daily) dose of, or dose equivalent to, 0.11 to 0.14 pg human derived recombinant FSH (preferably 0.12 to 0.13 pg human derived recombinant FSH) per kg bodyweight of the subject, the subject having serum AMH level of 25 to 34.9 pmol/L. In r ment, the method includes a step of administering a (e.g. daily) dose of, or dose equivalent to, 0.10 to 0.11 pg human derived recombinant FSH per kg bodyweight of the subject, the subject having serum AMH level of 235 pmol/L. Preferably the FSH is a recombinant FSH (“rFSH” or “recFSH”). Preferably the FSH is a human cell line derived recombinant FSH. These doses provide an effective response while minimising risk of OHSS.

According to the t invention in a r aspect there is provided a method of treatment of infertility comprising: (a) determining (e.g. measuring) the serum AMH level of a subject; and (b) administering a (e.g. daily) dose of, or dose equivalent to, 0.15 to 0.21 pg (for example 0.19 to 0.21 pg) human derived recombinant FSH per kg bodyweight of the subject, wherein the subject has serum AMH level of <15 pmol/L.

The administration preferably comprises a daily dose of, or a daily dose equivalent to, the amount of FSH defined above and in the claims. The (daily) dose may be an initial dose (it may be d, increased, or maintained during the treatment).

The method may be a method of treatment of infertility in the patient’s (subject’s) first stimulation protocol. It will be appreciated that for further stimulation cycles, the doses may be ed according to actual ovarian response in the first cycle.

According to the present invention in a further aspect there is provided a method of treatment of infertility comprising: (a) determining (e.g. measuring) the serum AMH level of a subject; and (b) if the subject has serum AMH level of <15 pmol/L (e.g. 0.05 pmol/L to 14.9 pmol/L), administering to the subject a dose of, or dose lent to, 10 to 14 pg, for e 11 to 13 pg, for example 12 pg, human derived recombinant follicle stimulating hormone (FSH); or if the subject has serum AMH level of 15 to 24.9 , administering to the subject a dose of, or dose equivalent to, 0.14 to 0.19 pg human derived recombinant FSH (preferably 0.15 to 0.16 pg human derived inant FSH) per kg bodyweight of the subject; or if the subject has serum AMH level of 25 to 34.9 pmol/L pmol/L, administering to the subject a dose of, or dose equivalent to, 0.11 to 0.14 pg human derived recombinant FSH (preferably 0.12 to 0.13 pg human derived recombinant FSH) per kg bodyweight of the subject; if the subject has serum AMH level of 235 pmol/L pmol/L, administering to the subject a dose of, or dose lent to, 0.10 to 0.11 pg human d recombinant FSH per kg bodyweight of the t.

For a patient (subject) having serum AMH of 50-149 pmol/L, a dose of, or dose equivalent to, 6 to 18 pg, for example 8 to 11 pg, for example 8.5 to 10.2 pg human derived recombinant FSH may be administered. For a patient (subject) having serum AMH 15.0-29.9 pmol/L, a dose of, or a dose equivalent to, 4.8 to 15 pg, for example 6 to 9 pg, for e 6.8 to 8.5 pg human derived recombinant FSH may be administered. For a patient (subject) having serum AMH 30-44.9 pmol/L, a dose of, or a dose equivalent to, 3.6 to 12 pg, for example 4 to 7 pg, for example 5.1 to 6.8 pg human derived recombinant FSH may be administered. For a patient (subject) having serum AMH 45 pmol/L or greater, a dose of, or a dose equivalent to, 2 to 9 pg, for example 2.4 to 9 pg (for example 3.4 to 5.1 pg) or 2 to 5 pg human derived recombinant FSH may be administered. For a patient (subject) having serum AMH of 5 pmol/L or less, a dose of, or a dose lent to 7.2 to 24 pg, for example 10 to 15 pg for example .2 to 13.6 pg, human derived recombinant FSH may be administered. In some es, a dose of, or dose equivalent to, 4.8 to 18 pg, for example 6 to 11 pg, for e 6.8 to 10.2 pg human derived recombinant FSH is administered. Preferably the FSH is a recombinant FSH (“rFSH” or “recFSH”). Preferably the FSH is a human cell line d recombinant FSH. The administration preferably comprises a daily dose of, or a daily dose equivalent to, the amount of FSH defined above and in the claims. The (daily) dose may be an initial dose (it may be reduced, increased, or maintained during the ent.

Detailed description of the invention The present invention will now be described in more detail with nce to the attached drawings in which: Figure 1 shows a plasmid map of the pFSHalpha/beta expression vector; Figure 2 shows the q2,3-sialyltransferase (ST3GAL4) expression vector; Figure 3 shows the q2,6-sialyltransferase (ST6GAL1) expression ; Figure 4 shows % abundance sialic acid bution of examples of recombinant FSH produced by PER.C6® cells stably expressing FSH after engineering with 023- sialyltransferase; Figure 5 shows % abundance of glycan charge distribution of examples of recombinant FSH produced by PER.C6® cells stably expressing FSH after engineering with (12,3- sialyltransferase; Figure 6 shows a comparison of concentration of inhibin-B following administration of 225|U Gonal f (bottom line, dotted line) and 225 IU of the Example (top line, full line) of Invention; Fig 7 shows the effect of body weight on oocytes retrieved in the low AMH treatment group (Example 10, 10A); and Fig 8 shows the effect of Body weight on oocytes retrieved in the high AMH treatment group ce Selection Human FSH The coding region of the gene for the FSH alpha polypeptide was used to according to Fiddes and Goodman. (1981). The ce is banked as AH007338 and at the time of construction there were no other variants of this protein sequence. The ce is referred herein as SEQ ID NO:1.

The coding region of the gene for FSH beta polypeptide was used according to Keene et a/ (1989). The ce is banked as NM_000510 and at the time of construction there were no other variants of this protein sequence. The sequence is referred herein as SEQ ID NO: 2 SiaIyItransferase d2,3-Sialyltransferase - The coding region of the gene for alactoside alpha-2,3- siaIyItransferase 4 (d2,3-sialyltransferase, ST3GAL4) was used ing to Kitagawa and Paulson (1994). The sequence is banked as L23767 and referred herein as SEQ ID NO: 3. ialyltransferase - The coding region of the gene for beta-gaIactosamide aIpha- 2,6—sialyltransferase 1 (02,6-sialyltransferase, ST6GAL1) was used according to Grundmann et al. (1990). The sequence is banked as 032 and referred herein as SEQ ID NO: 4.

EXAMPLES Example 1 Construction of the FSH expression vector The coding sequence of FSH aIpha polypeptide (AH007338, SEQ ID NO: 1) and FSH beta polypeptide (NM_003032, SEQ ID NO: 2) were amplified by PCR using the primer combinations FSHa-fw and FSHa-rev and FSHb-fw and FSHb-rec respectively.

FSHa-fw 5'-CCAGGATCCGCCACCATGGATTACTACAGAAAAATATGC-3' (SEQ ID NO:9) FSHa-rev 5'-GGATGGCTAGCTTAAGATTTGTGATAATAAC-3' (SEQ ID NO:10) FSHb-fw 5'-CCAGGCGCGCCACCATGAAGACACTCCAGTTTTTC-3' (SEQ ID NO: 11) FSHb-rev GGTTAACTTATTATTCTTTCATTTCACCAAAGG-3' (SEQ ID NO: 12) The resulting amplified FSH beta DNA was digested with the restriction enzymes Ascl and Hpal and inserted into the Ascl and Hpal sites on the CMV driven mammalian expression vector carrying a neomycin selection marker. Similarly the FSH aIpha DNA was digested with BamHI and Nhel and inserted into the sites BamHI and Nhel on the expression vector aIready containing the FSH beta polypeptide DNA.

The vector DNA was used to transform the DH5d strain of E.coli. Colonies were picked for ication. Colonies containing the vector containing both FSH alpha and beta were ed for sequencing and all contained the correct sequences according to SEQ ID NO: 1 and SEQ ID NO: 2. Plasmid pFSH A+B#17 was selected for transfection (Figure 1). e 2 Construction of the ST3 expression vector The coding sequence of beta-gaIactoside aIpha-2,3-siaIyItransferase 4 (ST3, L23767, SEQ ID NO: 3) was amplified by PCR using the primer combination 2,3Swa and 2,3STrev. 2,38wa 5'-CCAGGATCCGCCACCATGTGTCCTGCAGGCTGGAAGC-3' (SEQ ID NO: 13) 2,3STreV 5'-TTTTTTTCTTAAGTCAGAAGGACGTGAGGTTCTTG-3' (SEQ ID NO: 14) The resulting amplified ST3 DNA was ed with the restriction enzymes BamHI and Aflll and inserted into the BamHI and Aflll sites on the CMV driven mammalian expression vector carrying a hygromycin resistance marker. The vector was amplified as previously described and sequenced. Clone pST3#1 e 2) contained the correct sequence according SEQ ID NO: 3 and was selected for transfection. e 3 Construction of the ST6 expression vector The coding sequence of beta-galactosamide aIpha-2,6-sialyltransferase 1 (ST6, NM_003032, SEQ ID NO: 4) was amplified by PCR using the primer combination 2,GSwa and 2,GSTrev. 2,GSTfW 5'-CCAGGATCCGCCACCATGATTCACACCAACCTGAAG-3' (SEQ ID NO: 15) 2,GSTrev 5'-TTTTTTTCTTAAGTTAGCAGTGAATGGTCCGG-3' (SEQ ID NO: 16) The ing amplified ST6 DNA was digested with the restriction enzymes BamHI and Aflll and inserted into the BamHI and Aflll sites on the CMV driven mammalian expression vector carrying a hygromycin resistance . The vector was amplified as previously described and sequenced. Clone pST6#11 (Figure 3) contained the correct sequence according SEQ ID NO: 4 and was selected for transfection.

Example 4 Stable expression of pFSH a+l3 in PER.C6® cells. Transfection isolation and screening of .

PER.C6®cIones producing FSH were generated by expressing both polypeptide chains of FSH from a single plasmid (see Example 1).

To obtain stable clones a Iiposome based ection agent with the pFSH 0L+B construct. Stable clones were ed in VPRO mented with 10% FCS and containing G418. Three weeks after transfection G418 resistant clones grew out. Clones were ed for isolation. The isolated clones were cultured in selection medium until 70-80% confluent. Supernatants were assayed for FSH protein content using an FSH selective ELISA and pharmacological activity at the FSH receptor in cloned cell line, using a cAMP accumulation assay. Clones expressing functional protein were progressed for culture expansion to 24 well, 6 well and T80 flasks.

Studies to ine productivity and quality of the material from seven clones were initiated in T80 flasks to generate sufficient material. Cells were cultured in supplemented media as previously described for 7 days and the supernatant harvested. Productivity was determined using the FSH selective ELISA. The isoelectric profile of the material was determined by Isoelectric focusing (IEF), by methods known in the art. Clones with ient productivity and quality were selected for sialyltransferase engineering. e 5 Level of sialylation is increased in cells that over s (12,3- sialyltransferase. Stable expression of pST3 in FSH expressing ® cells; Transfection isolation and ing of clones.

PER.C6® clones producing highly sialylated FSH were generated by expressing 02,3 sialyltransferase from separate plasmids (Example 2) in PER.C6® cells already expressing both ptide chains of FSH (from Example 4). Clones produced from PER.C6® cells as set out in Example 4 were selected for their characteristics including productivity, good growth profile, production of functional protein, and produced FSH which included some sialylation.

Stable clones were generated as previously described in Example 4. Clones were isolated, ed and assayed. The d2,3-sialyltransferase clones were adapted to serum free media and suspension conditions.

As before, clones were assayed using a FSH selective ELISA, functional response in an FSH or cell line, IEF, metabolic nce rate and Steelman Pohley analysis. Results were compared to a commercially available recombinant FSH (Gonal-f, Serono) and the parental FSH PER.C6®cell lines. FSH produced by most of the clones has significantly improved sialylation (i.e. on average more FSH isoforms with high numbers of sialic acids) compared to FSH expressed without d2,3- sialyltransferase. In conclusion expression of FSH together with sialyltransferase in PER.C6®cells resulted in increased levels of sialylated FSH compared to cells expressing FSH only.

Example 6 Production and purification overview A procedure was developed to produce FSH in PER.C6®cells that were cultured in suspension in serum free medium. The procedure is described below and was applied to several FSH-producing PER.C6®cell lines.

FSH from d2,3- clone (Example 5) was prepared using a using a modification of the method described by Lowry et al. (1976).

For the production of PER.C6®-FSH, the cell lines were adapted to a serum- free medium, i.e., Excell 525 (JRH ences). The cells were first cultured to form a 70%-90% confluent yer in a T80 e flask. On e the cells were re-suspended in the serum free medium, Excell 525 + 4 mM L-Glutamine, to a cell density of 0.3X106 cells/ml. A 25 ml cell suspension was put in a 250 ml shaker flask and shaken at 100 rpm at 37°C at 5% C02.

After reaching a cell density of > lxl06 cells/ml, the cells were sub-cultured to a cell density of 0.2 or 6 cells/ml and further cultured in shaker flasks at 37°C, 5% C02 and 100 rpm. 2012/065507 For the production of FSH, the cells were transferred to a serum- free production medium, i.e., VPRO (JRH Biosciences), which supports the growth of PER.C6®cells to very high cell densities (usually > 107 cells/ml in a batch e). The cells were first cultured to > 1xl06 cells/ml in Excell 525, then spun down for 5 min at 1000 rpm and subsequently suspended in VPRO medium + 6 mM L-glutamine to a density of 1x106 cells/ml. The cells were then cultured in a shaker flask for 7-10 days at 37°C, 5% C02 and 100 rpm. During this period, the cells grew to a density of > 107 cells/ml. The culture medium was harvested after the cell viability d to decline. The cells were spun down for 5 min at 1000 rpm and the atant was used for the quantification and purification of FSH. The concentration of FSH was determined using ELISA (DRG ElA 1288).

Thereafter, purification of FSH was carried out using a modification of the method described by Lowry et al. (1976). cation using charge selective chromatography was carried out to enrich the highly ated forms by methods well known in the art.

During all chromatographic procedures, enrichment of the sialylated forms of FSH as claimed herein was confirmed by RIA (DRG ElA 1288) and/or lEF.

Example 7 Quantification of relative amounts of 02,3 and 02,6 sialic acid The relative tage amounts of 02,3 and 02,6 sialic acid on purified rFSH (Example 6) were measured using known techniques.

N-Glycans were released from the samples using PNGase F under denaturative conditions and then labelled with 2-aminobenzamide. Released glycan forms were then separated and analysed by Weak Anion Exchange (WAX) column for determination of charge distribution. Labelled glycans treated with 2,3,6,8 sialidase for determination of total sialic acid and 2,3 sialidase for determination of 2,3 sialic acid, were further analyzed by wax column.

The relative percentages of the charged glycans were calculated from structures t in the undigested and digested glycan pools and are shown in Figure 4 (for 8 samples).

These were found to be in the ranges 50% - 70% (e.g. about 60% or 65%) for 02,3 sialylation and 28 to 50%, generally 30 to 35% (e.g. about 31% or 35%), for 02,6 ation.

Example 8 fication of relative amounts mono, di, tri and tetra ated glycan structures The relative percentage amounts of mono, di, tri and tetra sialylated structures on glycans extracted from purified rFSH (Example 6) were measured using known techniques.

N Glycans were released from the s using PNGase F under denaturative conditions and then were labeled with 2-aminobenzamide. Glycans were released from the samples using PNGase F under denaturative conditions and then d with 2- aminobenzamide. Released glycan forms were then separated and analysed by Weak Anion ge (WAX) column for determination of sialylation distribution. The relative amounts of neutral, mono-sialylated, di-sialylated, tri-sialylated and tetra-sialylated structures are shown in Figure 5 (for the 8 samples shown in Fig 4).

The rFSH includes neutral di- tri- tetra- , mono-sialylated, sialylated, sialylated and sialylated glycan structures with relative s as follows: neutral 5-6 %; 15-17% mono- sialylated; 26-30% alylated; 30-32% tri--sialylated and 17-23 % tetra—sialylated.

Example 8a The relative tage amounts of 02,6 sialic acid on purified rFSH extracted from nine samples of purified rFSH (produced by the methods of Example 6) were measured using knowntechmques N-Glycans were released from the samples using PNGase F under denaturative conditions and then labelled with obenzamide. Released glycan forms were then ted and analysed by Weak Anion Exchange (WAX) column for determination of charge distribution. Labelled glycans treated with 2,3,6,8 sialidase for determination of total sialic acid and 2,3 sialidase for determination of 2,3 sialic acid, were further analyzed by wax column (see Example 8). The analysis allows calculation of 02,6 sialic acid.

The relative tages of the charged glycans were calculated from structures present in the undigested and digested glycan pools and are shown in the following Table.

These were found to be in the ranges 25 to 50%, generally 30 to 35% for 02,6 sialylation.

The relative tage amounts of bisecting , GalNac and 1-Fucose Lewis on glycans extracted from the nine samples of purified rFSH (produced by the methods of Example 6) were measured using known techniques. N-Glycans were ed from the glycoprotrein using PNGase F and labeled with 2-aminobenzamide (2AB). The analysis was done by two dimensional (2D) HPLC analysis in combination with enzymatic degradation of the glycans. For verification, the glycans were analyzed by MS The relative amounts of alpha 2,6-sialic acid and the terminal residues are shown in the following table, together with those for Gonal F (CHO cell derived inant FSH) and Bravelle (human urinary FSH).

Ref. Aver Gona Brav Sample mm.--O E; I: 5 III-1 .=N Z a:(Qm 'n elle % aouepunqe % aouepunqe % aouepunqe % aouepunqe % aouepunqe % nqe % aouepunqe % aouepunqe % aouepunqe % aouepunqe % aouepunqe % aouepunqe sialic 27-7 acid 34.9 26.2 30.1 31.1 28.3 30.4 (.0 01 (.0 (.0 30.7 0 55.4 1GalNA 0 11.3 51 44.6 50.7 44.7 49 47.6 45.3 46.4 44.9 47.1 Bisectin Glcimc 10 12.4 10.2 8.9 8.7 11.8 11.4 10.8 13.9 10.9 55 14 Fucose 21.1 18.7 23.3 18.1 20.3 18.1 17.9 18.7 19.0 19.0 3.11 2.2 Lewis Fucose 4 4.1 4.3 1.9 3.1 4.2 3.8 3.9 4.4 3.7 — n.d.2 Lewis 1 2 Value of 3.1 is total 1/2 Fucose Lewis. Not determined.

It can be seen that the amount of GalNac in the FSH of the invention varies between about 44.9 and 51%, averaging about 47.1%.

It can be seen that the amount of bisecting GIcNac in the FSH of the invention varies between 8.7 and 13.9%, averaging approximately at 10.9%.

It can be seen that the amount of 1 Fucose Lewis in the FSH of the invention varies between 16.1 and 23.3%, averaging approximately at 19%.

It can be seen that the amount of 2 Fucose Lewis in the FSH of the invention varies between 1.9 and 4.4%, averaging approximately at 3.7%.

Example 9 — A multiple dose study investigating the safety, tolerability, pharmacokinetics, pharmacodynamics, and immunogenicity of FE 999049 in comparison to GONAL-F.

Study population A total of 48 (24 on each drug) healthy women received daily doses of 14.6 pg of FE 999049 (a composition according to the invention, ed according to Example 6) or 16.5 pg of Gonal- F for seven days.

Safety results Multiple dose administration of FE 999049 and GONAL-F was safe and generally well tolerated as assessed by Adverse Events (AEs), vital signs, ECG, al laboratory ements, and physical ation. No serious adverse event or death occurred during the study.

Pharmacokinetic results Following the administration of FE 999049 and GONAL-F over 7 days, the FSH concentration values as assessed ately prior to the next injection increased and seemed to reach a steady state level after 6—7 days. However the exposure (AUC and Cmax) of FE 999049 was 60% higher in comparison to Gonal-F.

Pharmacodynamic results The concentrations of inhibin-B (see figure 6), oestradiol, and progesterone all increased subsequent to stration of FE 999049 and GONAL-F, r to a greater extent following stration of FE 999049 compared to GONAL-F. Both number and size bution of follicles showed a greater response to FE 999049 compared to GONAL-F.

Example 9 demonstrates that FSH having a specific amount (17-23%) of tetra-sialylated glycan structures and e.g. specific s of 02,3 sialylation and (12,6 sialylation is markedly more potent then recombinant FSH products which are currently on the market.

Example 10 — A multiple dose study investigating FE 999049 in comparison to GONAL-F.

The following describes a ised, controlled, assessor-blind, parallel groups, multinational, multicentre trial assessing the dose-response relationship of FE 999049 in patients undergoing controlled ovarian stimulation for in vitro fertilisation (lVF) / intracytoplasmic sperm ion (ICSI). The patient population was 265 lVF patients aged between 18 to 37 years, with BMI 18.5 to 32.0 kg/m2.

The trial was designed as a dose-response trial with number of s retrieved as the primary endpoint. Secondary endpoints will explore the qualitative and quantitative impact of different doses of FE 999049 with regard to endocrine profile, follicular development, oocyte fertilisation, embryo quality and treatment efficiency (i.e. total tropin ption and duration of stimulation). The trial is designed to evaluate the efficacy of FE 999049 to establish pregnancy when used in controlled ovarian stimulation for lVF/ICSI cycles.

Subjects were assessed within 3 months prior to randomisation for compliance with the inclusion and exclusion criteria, including an anti-Miillerian hormone (AMH) assessment to se homogeneity of the trial population in on to ovarian response and minimise the number of potential poor and hyper-responders to the FE 999049 doses and F dose used in the trial. The AMH assessment was measured using the AMH Gen-ll enzyme linked immunosorbent assay kit (Beckman Coulter, lnc., Webster, Texas). This assay can detect AMH concentrations greater than 0.57 pmol/L with a minimum limit of tation of 1.1 .

On day 2-3 of their menstrual cycle, subjects were randomised in a 1:1:1:1:1:1 fashion to treatment with either 90 lU, 120 lU, 150 lU, 180 IU or 210 IU FE 999049 or 150 IU GONAL-F, and n stimulation initiated. Randomisation was stratified according to AMH level at screening [5.0-14.9 pmol/L (low AMH) and 15.0 to 44.9 pmol/L (high AMH)).

Gonal-F is filled by mass (FbM) at FDA request; referring to pg dose is therefore appropriate. The Gonal —F label indicates 600 lU/44 pg, which indicates that 150 IU is 11 pg.

However, there is some variation and the batch certificate for this trial indicated that 11.3 pg Gonal-F was equivalent to 150 IU. The FE999049 doses are presented by protein content (pg) rather than biological activity. Thus the doses of FE999049 were 5.2119 (90 IU), 6.9119 (120 IU), 8.6119 (150 IU), 10.3pg (180 IU) or 121119 (210 IU).

The subject and dose distribution is set out as follows (data are number of subjects): Table 1 FE 999049 GONAL-F .2pg 6.9pg 8.6ug 10.3pg 12.1pg 11.3(11)pg Total Screened 334 Randomised 42 45 44 45 46 43 265 and exposed thAMH strata 23 26 24 24 26 25 148 (56%) (15.0 - 44.9 pmol/L) Low AMH (5:31am 9 19 19 20 20 21 18 117 (44%) pmol/L) Per-protocol 40 42 42 44 44 43 255 The daily dose level of FE 999049 or GONAL-F is fixed throughout the entire stimulation . During stimulation, subjects are monitored on stimulation day 1, 4 and 6 and hereafter at least every second day. When 3 follicles of 215 mm are observed, visits are performed daily. Subjects are d with FE 999049 or GONAL-F for a maximum of 16 days.

To t a premature LH surge, a GnRH antagonist elix e, ORGALUTRAN, MSD / Schering-Plough) may be initiated on stimulation day 6 at a daily dose of 0.25 mg and continued throughout the stimulation . Triggering of final follicular maturation is done on the day when 23 follicles with a diameter 217 mm are observed. if there are <25 follicles with a diameter 212 mm, 250 pg recombinant hCG (choriogonadotropin alfa, LLE, Merck Serono / EMD Serono) is administered. If there are 25-35 follicles with a diameter 212 mm, 0.2 mg GnRH agonist (triptorelin acetate, DECAPEPTYL / GONAPEPTYL, SUBSTITUTE SHEET (RULE 26) Ferring Pharmaceuticals) is administered. In case of excessive ovarian response, d as >35 follicles with a er 212 mm, the treatment is cancelled. In case of poor ovarian response, defined as <3 follicles with a diameter 210 mm observed on ation day 10, the cycle could be cancelled.

Oocyte retrieval takes place 36h (i 2h) after triggering of final follicular maturation and the oocytes inseminated by lVF and/or ICSI. Fertilisation and embryo development are assessed from oocyte retrieval to the day of transfer. For ts who unden/vent triggering of final follicular maturation with hCG, one blastocyst of the best quality available is transferred on day 5 after oocyte retrieval while remaining blastocysts are frozen. For subjects who undergo triggering of final follicular maturation with GnRH agonist, no embryo transfer takes place in the fresh cycle and cysts are instead frozen on day 5. Vaginal progesterone tablets (LUTINUS, Ferring Pharmaceuticals) 100 mg 3 times daily are provided for luteal phase support from the day after oocyte retrieval until the day of the clinical ncy visit. A BhCG test is performed 13-15 days after embryo transfer and clinical pregnancy will be confirmed by transvaginal ultrasound (TVU) 5-6 weeks after embryo transfer.

Results The number of oocytes retrieved (primary nt) is shown in the following Table.

Table 2 FE 999049 GONAL—F .2 pg 6.9 pg 8.6 pg 10.3 pg 12.1 pg 11.3 (11)pg Oocytes retrieved All 5.2 (3.3) 7.9 (5.9) 9.2 (4.6) 10.6 (7.0) 12.2 (5.9) 10.4 (5.2) HighAMH 9) 9.1 (6.4) 10.6(4.8) 13.6(7.8) 14.4(5.8) 12.4(5.4) Low AMH 4.5 (2.2) 6.3 (4.9) 7.4 (3.8) 6.9 (3.6) 9.4 (4.9) 7.8 (3.4) Data are mean (SD) The primary obiective was met: a significant dose-response relationship was established for FE 999049 with respect to number of oocytes retrieved. This finding was observed not only for the l trial population, but also for each of the two AMH strata used at randomisation.

A significant dose-response for FE 999049 was demonstrated for all key objective pharmacodynamic parameters, e.g. estradiol. inhibin B and inhibin A. At a r microgram dose level, the pharmacodynamic responses with FE 999049 were larger than with GONAL-F (these results not shown).

The serum FSH concentrations after exposure to FE 999049 were significantly higher than for GONAL-F. The s confirm that the PK profile of FE 999049 differs from that of GONAL-F.

Fertilisation rates, blastocyst development and pregnancy rates in lVF/lCSl patients treated with FE 999049 were within expectations.

There were no safety ns with the use of FE 999049. A good local tolerability was 1O nted.

Further Analysis The applicants have further ed the data to fy the FE 999049 dose(s) that fulfil the following criteria with respect to number of oocfles ved: . Oocytes retrieved in the range 8-14 . Minimise proportion of patients with <8 oocytes - Minimise proportion of ts with .220 oocytes The applicants also investigated the impact of body weight. If relevant, the dose is converted into pg/kg for an average subject. This value of ug/kg and $0.01 ug/kg are evaluated in a model with respect to distribution of oocytes retrieved as well as safety profile, and the optimal dose is identified.

Low AMH strata As seen in Table 2, the dose of FE999049 which fulfilled the first criterion (Oocytes retrieved in the range 8-14) was 12.1 ug (mean 9.4 oocytes retrieved). The distribution of oocytes is shown in Table 3 below.

Table 3 FE 999049 GONAL-F .2pg 6.909 8.6119 10.3ug 12.109 11.3 (1 1)pg Oocytes retrieved <4 32% 24% 15% 10% 10% 6% SUBSTITUTE SHEET (RULE 26) 2012/065507 4-7 63% 42% 45% 60% 20% 56% 8-14 5% 24% 35% 30% -19 0% 5% 5% 0% 5% 6% 220 0% 5% 0% 0% 5% 0% Data are % of subjects As shown by the box and arrow, a dose of 12.1 pg FE999049 provides retrieval of the most ble number of oocytes in 60% of subjects in the low AMH group. This is a marked improvement on GonaI-F (most desirable number of oocytes in only 33% of subjects).

Table 4 below shows the analysis of signs of excessive response in the low AMH strata (data are number of subjects). It can be seen that there were no indications of early OHSS of a moderate or severe nature and there were no incidences of preventative action being required; there are no concerns associated with the dose of 12.1 pg FE999049 in a patient having low AMH.

Table 4 FE 999049 GONAL-F .2ug 6.9ug 8.6ug 10.3ug 12.1ug 11.3(11)pg All ts 19 19 20 20 21 18 Early OHSS, mod/sev 0 0 0 0 O 0 GnRH agonist 0 0 0 0 0 0 triggering Preventive action“ 0 0 O 0 0 O 215 oocytes 0 2 1 0 2 1 Any of the above 0 2 1 0 2 1 SUBSTITUTE SHEET (RULE 26) WO 20996 Fig 7 shows the effect of body weight on oocytes retrieved (for the low AMH strata), for the various doses. The arrows indicate the number of oocytes retrieved from subjects of ight between 45kg and 90kg treated at the 12.1 pg dose. As can be seen (text box) the variation between patients of bodyweight 45kg and those of 90kg is less than around 0.5 oocytes; in other words dosing by body weight is not required in patients with low AMH when dose of FE999049 is at least 12 pg, because there is not a significant variation in oocytes retrieved with body weight, at this dose.

Thus the applicants have found that a dose of, or dose equivalent to, 6 to 18 pg, for example 9 to 14 pg, for example 12 pg, human d recombinant FSH is suitable for use in the treatment of infertility in a patient having serum AMH <15 pmol/L, for example 0.05-14.9 pmol/L for example 5.0-14.9 pmol/L. The dose es an ive response while minimising risk of OHSS.

High AMH strata As seen in Table 2, three doses of FE999049 fulfilled the first criterion (oocytes retrieved in the range 8—14): 6.9 pg (mean 9.1 oocytes retrieved), 8.6 pg (mean 10.6 oocytes retrieved), and .3 pg (mean 13.6 oocytes retrieved).

Fig 8 shows the effect of body weight on oocytes retrieved (for the high AMH strata), for the various doses. The arrows indicate the number of oocytes retrieved from subjects of body weight between 45kg and 90kg treated at the 6.9 pg, 8.6 pg and 10.3 pg doses. As can be seen (text boxes) the variation is significant: for the 6.9 pg dose 6 onal oocytes will be retrieved from a 45 kg t compared to a 90 kg patient; for the 8.6 pg dose 4 additional oocytes will be retrieved from a 45 kg patient compared to a 90 kg patient; and for the 10.1 pg dose 2.5 additional s will be retrieved from a 45 kg patient compared to a 90 kg patient.

In other words dosing by body weight has an impact in ts with high AMH when the dose of 49 is less than 12 pg, because there is a significant variation in oocytes retrieved with body weight, at these doses.

Table 5a below shows a further breakdown of oocytes retrieved (from Table 2) by AMH. This shows the doses which fulfilled the first criterion (oocytes retrieved in the range 8—14) for each sub strata of AMH (boxes).

Table 5a FE 999049 .2pg 6.9 pg 8.6pg 10.3pg 12.1pg Oocytes retrieved moI/L 4.9(3.8) 7.3(3.6) 10.6(5.1) 11.5(6.7) 12.3 (5.9) 2534 pmoI/L 8) 9.1 (6.8) 7) 15.5(6.4) 16.7(4.9) -45pmol/L 21.0(1.4) 18.0 15.7(6.5) (12.2) Table 5 b below shows the analysis of patients where treatment was cancelled due to either excessive response or agonist triggering, for these subgroups. For example, one patient in the 25-34 pmol/L AMH strata led due to excessive response following the dose of 10.3 pg and one patient in the 25-34 pmol/L AMH strata cancelled due to excessive response following the dose of 12.1 pg; one patient in the 35-45 pmol/L AMH strata cancelled following agonist triggering following dose of 10.3 pg; and one patient in the 35-45 pmoI/L AMH strata cancelled following agonist ring following dose of 6.9 pg.

Table 5b FE 999049 .2pg 6.9pg 8.6pg 10.3pg 12.1pg _———————_—______ OHSS***, cancellation due to excessive response or agonist triggering**** -24 pmol/L 0 0 0 o 0 -34 pmol/L o o 0 1m 1*“ -45 pmol/L 0 1**** 0 1am 0 SUBSTITUTE SHEET (RULE 26) It can be seen therefore that tailoring of dose by bodyweight (Fig 8) and AMH level would be useful in the high AMH strata, to minimise cancellations and maximise oocyte retrieval.

The applicants have found that the ing doses provide an effective response while minimising risk of OHSS (kg is kg body weight of patient). -—_15-24 pmol/L 0.14 0.19 pg/kg, for example 0.15-0.16 (12 pg) -34 pmol/L 0.11-0.14 pg/kg; for example 0.12-0.13 (12 pg) pg/kg, preferably 0.13 pglkg - 0.10-0.11 pg/kg, preferably 0.11 pg/kg (12 pg) The following are appropriate if dosing by bodyweight is not desired. 2 35 pmol/L (12 Hg) The following are riate if fewer ries of AMH are required. l4AMH categories 3 AMH categories 2 AMH categories One dose I {AMH Dose AMH Dose AMH Dose AMH Dose <15 12 pg <15 12 pg <15 12 pg — 0.16 HQ/kg -24 0.15-0.16 pg/kg 15-24 0.15-0.16 215 0.14 pg/kg ug/kg -34 0.12-0.13 pg/kg 225 0.12 pg/kg 235 0.10-0.11 pg/kg SUBSTITUTE SHEET (RULE 26) The following are appropriate if dosing by bodyweight is not desired.

I , i4 AMH ries 3 AMH categories 2 AMH ries One dose iAMH ' Dose AMH Dose AMH Dose AMH Dose g <15 12 pg <15 12 pg <15 12 pg - 9.3 pg or10 pg -24 9.3-10pg 15-24 9.3—10pg 215 8.7pg —34 7.3-8 pg 225 7.3 pg 235 6.3-7 pg Thus the applicants have found that a dose of, or dose equivalent to, 9 to 14 pg, for example 12 pg, human derived inant FSH is suitable for use in the treatment of infertility in a t having serum AMH <15 pmollL, for example 0.05-149 pmol/L for example 5.0-14.9 pmol/L.

The dose provides an effective se while minimising risk of OHSS.

The applicants have found that a dose of, or dose equivalent to, 5 to 12.5 pg, for example 6 to .5 pg, human derived recombinant FSH is suitable for use in the treatment of ility in a patient having serum AMH 215 pmol/L. The dose provides an effective response while minimising risk of OHSS.

The applicants have found that a (e.g. daily) dose of, or dose equivalent to, 0.09 to 0.19 pg human d recombinant FSH per kg bodyweight of the patient is suitable for use in the treatment of infertility in a patient having serum AMH level of 215 pmol/L. The applicants have found that a (e.g. daily) dose of, or dose equivalent to, 0.14 to 0.19 pg human derived recombinant FSH (preferably 0.15 to 0.16 pg human derived recombinant FSH) per kg bodyweight of the patient is suitable for use in the treatment of infertility in a patient having serum AMH level of 15 to 24.9 pmol/L. The applicants have found that a (e.g. daily) dose of, or dose equivalent to, 0.11 to 0.14 pg human derived recombinant FSH (preferably 0.12 to 0.13 SUBSTITUTE SHEET (RULE 26) human derived inant FSH) per kg bodyweight of the patient is suitable for use in the treatment of infertility in a t having serum AMH level of 25 to 34.9 pmol/L. The applicants have found that a (e.g. daily) dose of, or dose equivalent to, 0.10 to 0.11 pg human derived recombinant FSH per kg bodyweight of the patient is suitable for use in the treatment of infertility in a patient having serum AMH level of 2 35 pmol/L. These doses provide an effective response while minimising risk of OHSS.

The ants have found that a (e.g. daily) dose of, or dose equivalent to, 0.15 to 0.21 pg (e.g. 0.16 pg) human derived recombinant FSH per kg bodyweight of the patient is suitable for use in the treatment of infertility in a patient having serum AMH level of <15 pmol/L, for example for the first stimulation cycle with human derived recombinant FSH. . However, it is not required that patients are dosed by body weight at this level of AMH.

Example 10 A - Individualised COS protocol (low AMH) The selected patients are about to undergo COS for in vitro fertilisation (IVF) / intracytoplasmic sperm injection (ICSI) by methods known in the art. The pre-treatment protocol includes assessment/screening of the patient’s serum AMH using the AMH Gen-ll enzyme linked sorbent assay kit (Beckman Coulter, lnc., Webster, . This assay can detect AMH concentrations greater than 0.57 pmol/L with a minimum limit of quantitation of 1.1 pmol/L. AMH may be measured using other Assay kits (e.g. available from Roche).

The COS protocol proceeds in the usual manner apart from administration of the initial dose of FE 999049 ing to AMH level at ing. A patient with an AMH level of <14.9 pmol/L would be administered an initial daily dose of imately 12 pg FE 999049, a human derived inant FSH product manufactured according to the method of Example 6. A patient with an AMH level of 15 to 24.9 pmol/L would receive an l daily dose of 0.15 to 0.19 pg of the human derived recombinant FSH per kg bodyweight of the patient. A patient with an AMH level of 25 to 34.9 pmol/L would receive an initial daily dose of 0.11 to 0.13 pg of the human derived recombinant FSH per kg bodyweight of the patient. A patient with an AMH level of 2 35 pmol/L would receive an initial daily dose of 0.10 to 0.11 pg of the human derived recombinant FSH per kg ight of the patient.

Example 11 — Individualised COS protocols.

The doses in this protocol are less preferred that Example 10A.

The selected patients are about to undergo COS for in vitro fertilisation (IVF) / intracytoplasmic sperm injection (ICSI) by methods known in the art. The pre-treatment ol includes assessment/screening of the patient’s serum AMH using the AMH Gen-ll enzyme linked immunosorbent assay kit an Coulter, lnc., Webster, Texas). This assay can detect AMH concentrations greater than 0.57 pmol/L with a minimum limit of quantitation of 1.1 pmol/L.

The COS protocol proceeds in the usual manner apart from stration of the l dose of FE 999049 according to AMH level at screening in line with the following table. Thus a patient with an AMH level of 5—14.8 pmol/L would be administered 180 IU FSH in the form of approximately 8-11 pg FE 999049, a human derived recombinant FSH product manufactured according to the method of Example 6. A patient with an AMH level of 30-449 pmol/L would be administered 120 IU FSH in the form of approximately 4-7 pg FE 999049, a human d recombinant FSH product manufactured according to the method of Example 6. If the AMH level is not available, the patient recombinant would be administered 120-180 lU FSH in the form of approximately 6-11 pg FE 999049, a human derived inant FSH product manufactured according to the method of Example 6.

AMH Level Starting Approx Dose FSH lent in <5 pmolll 210 IU 10-15 pg -14.9 pmolll 180 IU 8-11 pg >15-29.9 pmolll 150 IU 6-9 pg >30-44.9 pmolll 120 IU 4-7 pg >45 pmolll 90 IU 2-5 pg Not Available 120-180 lU 6-11 pg References Andersen CY, Westergaard LG, and van Wely M. (2004). FSH isoform composition of commercial gonadotrophin preparations: a neglected aspect? Reprod Biomed Online. 9(2), 231-236.

Arey BJ, Stevis PE, Deecher DC, Shen ES, Frail DE, Negro-Vilar A, and Lopez FJ. (1997) Induction of promiscuous G protein coupling of the follicle-stimulating hormone (FSH) receptor: a novel mechanism for ucing pleiotropic actions of FSH isoforms. Mol Endocrinol. 11(5), 517-526.

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Figs 1, 2 and 3: Plasmid maps of the pFSHalpha/beta ,pST3 and pST6 expression vectors.

CMV = Cytomegalovirus er, BGHp(A) = Bovine Growth Hormone poly-adenylation sequence, fl ori = fl origin of replication, SV40 = Simian Virus 40 promoter, Neo = Neomycin resistance marker, Hyg = ycin resistance marker, SV40 p(A) = Simian Virus 40 poly-adenylation sequence, FSH A = Follicle ating hormone alpha polypeptide, FSH B = Follicle ating hormone beta polypeptide, ST3GAL4 = 02,3- sialyltransferase, ST6GAL1 - 02,6-sialyltransferase, CoIEI = ColEI origin of replication, Amp = ampicillin resistance marker.

SUBSTITUTE SHEET (RULE 26) SEQ ID NO: 1 Follicle stimulating hormone alpha polypeptide Accession number AH007338 Nucleotide sequence of FSH alpha 1 ATGGATTAC':_‘ ACAGAAAATA TGCAGCTATC TTTCTGGTCA CATTGTCGGT GTTTCTGCAT 61 GTTCTCCAT':_‘ CTGA TGTGCAGGAT TGCCCAGAAT GCACGCTACA GGAAAACCCA 121 TCCC AGCCGGGTGC CCCAATACTT CAGTGCATGG GCTGCTGCTT CTCTAGAGCA 181 AC':1C CACTAAGGTC CAAGAAGACG ATGTTGGTCC AAAAGAACGT CACCTCAGAG 241 TCCACT':1GCT GTGTAGCTAA ATCATATAAC ACAG TAATGGGGGG AGTG 3 0 1 GAGAACCACA CGGCGTGCCA CTGCAGTACT TGTTATTATC ACAAATCTTA A Protein sequence of FSH alpha (SEQ ID NO: 5) 1 MDYYRKYAAI F.4VTLSVFL-I VLHSAPDVQD CP *.CTT.Q nNP l: l: SQPGAP I L QCMGCCFSRA 61 YPTPLRSKKT MJVQKNVTSIZ1 KSYN RVTVMGGFKV ENHTACHCST CYYHKS SEQ ID NO: 2 Follicle stimulating hormone beta polypeptide Accession number 510 Nucleotide sequence of FSH beta ATGAAGACAC TCCAGTTT T T CTTCCTTTTC TGT TGCTGGA TCTG CTGCAATAGC 61 TGTGAGCTGA CCAACATCAC CATTGCAATA GAGAAAGAAG AATGTCGTTT CTGCATAAGC 121 ATCAACACCA CTTGGTGTGC TGGCTACTGC TACACCAGGG ATCTGGTGTA TAAGGACCCA 181 GCCAGGCCCA AAATCCAGAA AACATGTACC T TCAAGGAAC TGGTATATGA AACAGTGAGA 241 GTGCCCGGCC_‘ GTGCTCACCA TTCC TTGTATACAT ACCCAGTGGC CACCCAGTGT 301 CACC1GTGGCA AGTGTGACAG CGACAGCACT GATTGTACTG TGCGAGGCCT GGGGCCCAGC 361 TAC':1GCTCCC_‘ TTGGTGAAAT GAAAGAATAA 40 Protein sequence of FSH beta (SEQ ID NO: 6) 1 MKTLQFFF.4F CCWKAICCNS TIAI “.K‘. “.CRJ: CI S INTTWCAGYC YTRDLVYKDP 61 ARPKIQKTCT l: K<.T.VY “.TVR V HADS .4YTYPVATQC HCGKCDSDST DCTVRGLGPS 12 1 YCSJ: G*-MK~.

SEQ ID NO: 3 alactoside aIpha-2,3-sialyltransferase 4 Accession Number L23767 Nucleotide sequence of STBGAL4 ; ATGTGTCCTG CAGGCTGGAA GCTCCTGGCC ATGTTGGCTC TGGTCCTGGT CGTCATGGTG 6; TGGTATTCCA TCTCCCGGGA AGACAGGTAC ATCGAGCTTT TTTATTTTCC AGAG 12L AAGAAGGAGC CGTGCCTCCA GGGTGAGGCA GAGAGCAAGG CCTCTAAGCT CTTTGGCAAC 18L TACTCCCGGG ATCAGCCCAT CTTCCTGCGG CTTGAGGATT ATTTCTGGGT CAAGACGCCA 24; TCTGCTTACG AGCTGCCCTA CAAG GGGAGTGAGG ATCTGCTCCT GCTA 30; GCCATCACCA GCTCCTCCAT CCCCAAGAAC ATCCAGAGCC TCAGGTGCCG CCGCTGTGTG 36L GTCGTGGGGA ACGGGCACCG GCTGCGGAAC AGCTCACTGG GAGATGCCAT CAACAAGTAC 42; GATGTGGTCA TCAGATTGAA CAATGCCCCA GTGGCTGGCT ATGAGGGTGA CGTGGGCTCC 48L AAGACCACCA TGCGTCTCTT CTACCCTGAA TCTGCCCACT TCGACCCCAA AGTAGAAAAC 54L AACCCAGACA CACTCCTCGT CCTGGTAGCT TTCAAGGCAA TGGACTTCCA CTGGATTGAG 60; ACCATCCTGA GTGATAAGAA GCGGGTGCGA AAGGGTTTCT AGCC TCCCCTCATC 66; TGGGATGTCA ATCCTAAACA GATTCGGATT CCCT TCTTCATGGA GATTGCAGCT 72; GACAAACTGC TGAGCCTGCC AATGCAACAG CCACGGAAGA AGAA CACG 78L GGCCTGTTGG CCATCACGCT CCAC CTCTGTGACT TGGTGCACAT TGCCGGCTTT 84L GGCTACCCAG ACGCCTACAA CAAGAAGCAG ACCATTCACT ACTATGAGCA GATCACGCTC 90; AAGTCCATGG CGGGGTCAGG CCATAATGTC TCCCAAGAGG CCCTGGCCAT TAAGCGGATG 96L CTGGAGATGG GAGCTATCAA GAACCTCACG TCCTTCTGA Protein Sequence of STBGAL4 (SEQ ID NO: 7) 1 MCPAGWK 1 . .A M .WMV WYSISRnDRY In .tYJ: PIP . KKfiPCIQGfiA nSKAS<IFGN 61 YSRDQ?IF.R L?DYFWVKT? SAY L‘J LPYGTK GS?D.LLRV. AITSSSIP<W IQSLRCRRCV 121 VVGNGiRIRN INKY DVVIRLWNAP VAGYEGDVGS KTTMRItY?n SAHtD?<V BZ 181 NPDTL.V.VA F<AMDFHWI'_‘J TIISDK<RVR KGFW<QPPLI WDVNP<QIRI LNPFF EIAA 241 DKLLSJPMQQ PR<IKQKPTT G..AIT.A.H LCDLVHIAGF NK<Q TIHYYEQITL 301 GiNV SQEALAIKR LEMGAI<NJT SF SEQ ID NO: 4 Beta-galactosamide aIpha-2,6-sialyltransferase 1 Accession number NM_003032 Nucleotide sequence of ST6GAL1 I ATGA”TCACA CCAACCTGAA GAAAAAGTTC AGCTGCTGCG G"C"T TC""CTG""T 61 A"Cr1 GTG"GTGGAA GGAAAAGAAG AAAGGGAGTr1 ACTATGAT"C A""G 12L CAAACCAAGG AA""CCAGGT GTTAAAGAGT CTGGGGAAAr1 r1GGCCA”GGG G"C"GAT"CC 18L CAG"CTG"Ar1 CC"CAAGCAG GGAC CCCCACAGGG GCCGCCAGAC CC"CGGCAGT 24L CTCAGAGGCC r1AGCCAAGGC AGAG GCCTCC""CC AGG"G"GGAA CAAGGACAGC ; TCT"CCAAAA ACCTTATCCC r1AGGCTGCAA AAGA"C"GGA AGAAT"ACCT AAGCATGAAC 36; AAG"ACAAAG r1G"CCTACAA GGGGCCAGGA CCAGGCA"CA AGT"CAGTGC AGAGGCCC"G 421 CGC"GCCACC r1CCGGGACCA r1GTGAATGTA TCCA"GG"AG AGG"CACAGA TT""CCCT"C 48; AATACCTC”G AA”GGGAGGG r"'"ATC"(3CCC AAGGAGAGCA TTAGGACCAA GGC"GGGCCT 54L AGGT G"GC"GTTGT AGCG GGATC"C"GA AGTCC"CCCA AC"AGGCAGA 601 GAAATCGA"G A"CA"GACGC AG"CC"GAGG GGGG CACCCACAGC CAACTTCCAA 66L CAAGATG"GG GCACAAAAAC r1ACCA"TCGC C"GATGAACT C"CAG""GGr1 TACCACAGAG 721 AAGCGCT"CC TCAAAGACAG r1T"G"ACAAr1 GAAGGAA"CC TAA""G"A"G GGACCCATCT 781 GTA"ACCACr1 CAGA"ATCCC AAAG"GGTAC CAGAATCCGG A"TA"AA""" CTTTAACAAC 84; TACAAGAC"" ATCG"AAGCr1 CAAr1 CAGCCCT"TT ACA"CC"CAA GCCCCAGA”G 901 CC""GGGAGC r1ATGGGACAr1 r1C"TCAAGAA A"CTCCCCAG ""CA GCCAAACCCC 961 CCA"CC"C"G GGATGC"TGG r1A"CATCATC A"GA"GACGC TG"GTGACCA GG”GGATA”T 102; TA"GAG"TCC r1CCCATCCAA GCGCAAGACT GACG"GTGCr1 AC"ACTACCA GAAGT"CT"C 1081 GA"AGTGCCT GCACGA”GGG r1GCCTACCAC CCGC"GC"Cr1 ATGAGAAGAA TT"GG"GAAG 114; CA"CTCAACC AGGGCACAGA r1GAGGACATC C"TG GAAAAGCCAC AC”GCCTGGC 120:. ACCA TTCACTGCTA A Protein ce of ST6GAL1 (SEQ ID NO: 8) 1 MIiTNLKKKF SCCVLVFJLF AVICVWKEK< KGSYYDSFKL QTK?FQVL<S .GKLAMGSDS 61 QSVSSSSTQD ?H¥GRQTIGS .RGuAKA<P? ASFQVWNKDS SSKVLIPRJQ (IWKNYLSMW 121 KY<VSYKGPG ?GI<FSA?AL HVNV SMVLVTDEPE NTSfiWfiGY.P KAG? 181 WGRCAVVSSA QIGR :IDDHDAVLR FNGAPTANFQ Q DVGT<TTIR . NSQIVTT_‘J 241 KRFIKDS.YW ?GI.IVWDPS VYHSDIP<WY QNPDYNFFNN Y<TYR<LH?N Q?FYIJKPQ 301 PW?.WDI.Qfi IS?fifiIQ?NP ?SSGMLGIII MMTJCDQVDI YEFLPSKR<T DVCYYYQKFF 361 DSACTMGAYi ?L.Y?KN.VK {LNQGTD?DI YLLGKATLPG F

Claims (8)

1. The use of le stimulating hormone (FSH) for the manufacture of a product for the treatment of ility in a patient having serum AMH level of <15 pmol/L, wherein the product comprises a daily dose of, or daily dose equivalent to, 11 to 13 µg recombinant FSH, the 5 recombinant FSH including α2, 3- and α2, 6- ation wherein 1% to 99% of the total sialylation is α2, 6- sialyation, and 1% to 99% of the total sialylation is α 2,3- sialyation.

2. A use according to claim 1 wherein the product comprises a daily dose of, or daily dose equivalent to 12 µg human derived recombinant FSH.

3. A use according to claim 1 or claim 2 wherein the treatment of ility comprises a 10 step of determining the serum AMH level of the patient, and a step of administering the dose to a patient having serum AMH level of <15 pmol/L.

4. The use of follicle stimulating hormone (FSH) for the cture of a product for the treatment of ility in a patient having serum AMH level of ≥15 , wherein the t is formulated to be administered at a daily dose of, or daily dose equivalent to, 0.09 to 0.19 µg 15 recombinant FSH per kg bodyweight of the patient, the recombinant FSH including α2, 3- and α2, 6- sialylation wherein 1% to 99% of the total sialylation is α2, 6- sialyation, and 1% to 99% of the total sialylation is α 2,3- tion.

5. A use according to claim 4 in the treatment of a patient having serum AMH level of 15 to 24.9 pmol/L, wherein the product is formulated to be administered at a daily dose of, or daily 20 dose equivalent to, 0.14 to 0.19 µg recombinant FSH per kg bodyweight of the patient.

6. A use according to claim 4 in the treatment of a patient having serum AMH level of 25 to 34.9 pmol/L, wherein the product is formulated to be administered at a daily dose of, or daily dose equivalent to, 0.11 to 0.14 µg recombinant FSH per kg bodyweight of the patient.

7. A use according to claim 4 in the treatment of a patient having serum AMH level of ≥ 35 25 pmol/L, wherein the product is formulated to be stered at a daily dose of, or daily dose equivalent to, 0.10 to 0.11 µg human derived recombinant FSH per kg bodyweight of the patient.

8. A use according to any preceding claim wherein the FSH includes mono-, di-, tri- and tetra-sialylated glycan structures, wherein 15-24%, for example 17-23% of the ated glycan 30 structures are tetrasialylated glycan structures.