WO2012168680A1

WO2012168680A1 - Pharmaceutical preparation comprising recombinant fsh

Info

Publication number: WO2012168680A1
Application number: PCT/GB2012/000491
Authority: WO
Inventors: Ian Cottingham; Daniel Plaksin; Bjorn Rikard Lorentz SANDSTROM
Original assignee: Ferring BV
Current assignee: Ferring BV
Priority date: 2011-06-06
Filing date: 2012-06-06
Publication date: 2012-12-13
Anticipated expiration: 2013-12-06
Also published as: EP2717904A1; WO2012168680A8; AR090095A1

Abstract

Preparations including FSH, for example recombinant FSH, wherein 17% to 23% of the sialyated glycan structures are tetra-sialylated glycan structures.

Description

PHARMACEUTICAL PREPARATION COMPRISING RECOMBINANT FSH

The present invention relates to gonadotrophins for use in the treatment of infertility. In particular it relates to follicle stimulating hormone (FSH).

The gonadotrophins are a group of heterodimeric glycoprotein hormones which regulate gonadal function in the male and female. They include follicle stimulating hormone (FSH), luteinising hormone (LH) and chorionic gonadotropin (CG).

FSH is naturally secreted by the anterior pituitary gland and functions to support follicular 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 sub-unit is post translationally modified by the addition of complex carbohydrate residues. Both subunits carry 2 sites for N-linked glycan attachment, the alpha sub-unit at amino acids 52 and 78 and the beta sub-unit at amino acid residues 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 infertility treatment; both to promote ovulation in natural reproduction and to provide oocytes for assisted reproduction technologies. Two recombinant versions of FSH, Gonal-F (Serono) and Puregon (Organon) became available in the mid-1990's. These are both expressed in Chinese hamster ovary (CHO) cells (Howies, 1996).

There is considerable heterogeneity associated with FSH preparations which relates to differences in the amounts of various isoforms present. 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 naturally derived pituitary FSH can contain a wide range of structures that can include combinations of mono-, bi-, tri- and tetra-antennary glycans (Pierce and Parsons, 1981. Ryan ef a/., 1987. Baenziger and Green, 1988). The glycans can carry further modifications: core fucosylation, bisecting glucosamine, chains extended with acetyl lactosamine, partial or complete sialylation, sialylation with a2,3 and a2,6 linkages, and sulphated galactosamine substituted for galactose (Dalpathado ef al., 2006). Furthermore, there are differences between the distributions of glycan structures at the individual 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. Commercially available rFSH products are derived from engineered Chinese hamster ovary cells (CHO cells). The range of glycan modifications in CHO cell derived rFSH are more limited than those found on the natural products. Examples of the reduced glycan heterogeneity found in CHO 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 a2,3 linkage (Kagawa et al, 1988, Takeuchi et al, 1988, Svensson et al., 1990); CHO cell derived rFSH only includes a2,3-linked sialic acid and does not include a2,6-linked sialic acid. This is different from naturally produced FSH (e.g. human Pituitary/ serum/ urinary FSH) which contains glycans with a mixture of a2,3 and a2,6- linked sialic acid, with a predominance of the former.

Further, it has also been demonstrated that a recombinant FSH preparation (Organon) differs in the amounts of FSH with an isoelectric point (pi) 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 recombinant products, Gonal-f (Serono) and Puregon (Organon) (Andersen et al. 2004). This must reflect a lower molar content 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 limitation in the manufacturing process (Bassett and Driebergen, 2005).

The circulatory life-time 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 pi, in which more acid equates to a higher negative charge. As previously stated the major contributor to overall molecular charge is the total sialic content 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 corresponding 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 pi fractions, the in vivo potency of the high pi (lower sialic acid content) fraction was decreased and it had a shorter plasma half-life (D' Antonio ef 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 a2,3 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 cr2,6 sialyl- transferase have not been reported.

Thus, as set out above, recombinant proteins expressed using the CHO system will differ from their natural counterparts in their type of terminal sialic acid linkages. This is an important consideration in the production of biologicals for pharmaceutical use since the carbohydrate moieties may contribute to the pharmacological attributes of the molecule. Thus, it is desirable to have a rFSH product that more closely replicates or mimics the physiochemical and pharmacokinetic profile of the product produced from human urine, it is also desirable to have an improved rFSH product.

The present applicants have developed a human derived recombinant FSH which is the subject of International Patent Application No. PCT/GB2009/000978, published as WO2009/127826A. Recombinant FSH with a mixture of both a2,3 and a2,6-linked sialic acid was made by engineering a human cell line to express both rFSH and a2,3 sialyltransferase. The expressed product is highly acidic and carries a mix of both a2,3- and a2,6-linked sialic acids; the latter provided by the endogenous sialyl transferase activity. It was found that the type of sialic acid linkage, a2,3- or σ2,6-, can have a dramatic influence on biological clearance of FSH, Recombinant FSH with a mixture of both a2,3 and a2,6-linked sialic acid has two advantages over rFSH expressed in conventional CHO cells: first the material is more highly sialylated due to the combined activities of the two sialyltransferases; and secondly the material more closely resembles the natural FSH. This is likely to be more biologically appropriate compared to CHO cell derived recombinant products that have produce only a2,3 linked sialic acid (Kagawa ef al, 1988, Takeuchi ef a/, 1988, Svensson ef al., 1990) and have decreased sialic acid content (Ulloa-Aguirre ef a/. 1995. , Andersen ef al. 2004) .

The rFSH product disclosed in International Patent Application No. PCT/GB2009/000978 contains branched glycan moieties. 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 residues or "antennae" 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. PCT/GB2009/000978 included mono-sialylated, di- sialylated, tri- sialylated and tetra- sialylated glycan structures with relative amounts as follows: 9-15% mono-sialylated; 27 - 30% di-sialylated; 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 structures on the FSH which are sialylated in any way (carry sialic acid). Thus, the phrase "20 - 36% tri-sialylated glycan structures" means that, of the total number of glycan structures on the FSH which carry sialic acid residues (that is, are sialylated), 20 to 36% of these glycan structures are tri sialylated (carry three sialic acid residues).

The applicants 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 PCT GB2009/000978 mentioned above) is markedly more potent then recombinant FSH products which are currently on the market.

According to the present invention there is provided follicle stimulating hormone including mono-, di-, tri- and tetra-sialylated glycan structures, wherein 15-24%, for example 17-23% of the sialylated glycan structures are tetrasialylated glycan structures (e.g. as shown by WAX analysis of charged glycans, as set out in the Examples below). Preferably the FSH includes cr2,3- and ct2,6- sialylation. The FSH (rFSH) according to the invention may have 1 % to 99% of the total sialylation being a2,3-sialylation. The FSH (rFSH) according to the invention may have 1% to 99% of the total sialylation being a2,6- sialylation. Preferably, 50 to 70%, for example 60 to 69%, for example about 65%, of the total sialylation is a2,3-sialylation. Preferably 25 to 50%, for example 30 to 50 %, for example 31 to 38%, for example about 35%, of the total sialylation is a2,6- sialylation.

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 sialylated glycan structures are tetrasialylated glycan structures" refers to the number of glycan structures on the FSH which are tetra sialylated, i.e. carry four sialic acid residues, 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 "15-24% of the sialylated glycan structures are tetrasialylated glycan structures" means that, of the total number of glycan structures on FSH which carry sialic acid residues (that is, are sialylated), 15 to 24% of these glycan structures are tetra sialylated (carry four sialic acid residues).

According to the present invention there is also provided a follicle stimulating hormone preparation, for example a recombinant follicle stimulating hormone preparation, comprising follicle stimulating hormone (FSH) including mono-, di-, tri- and tetra-sialylated glycan structures, wherein 15-24%, for example 17-23% of the sialylated glycan structures are tetrasialylated glycan structures. Preferably the FSH includes a2,3- and a2,6- sialylation. The FSH (rFSH) preparation according to the invention may have 1% to 99% of the total sialylation being a2,3-sialylation. The FSH (rFSH) preparation according to the invention may have 1% to 99% of the total sialylation being a2,6-sialylation. Preferably, 50 to 70%, for example 60 to 69%, for example about 65%, of the total sialylation is a2,3- sialylation. Preferably, 25 to 50%, for example 30 to 50 %, for example 31 to 38%, for example about 35%, of the total sialylation is a2,6- sialylation. Herein, the term "recombinant FSH preparation" includes a preparation for e.g. pharmaceutical use which includes recombinant FSH. In embodiments of the invention, the rFSH may be present as a single isoform or as a mixture of isoforms. The applicants found the (e.g. recombinant) FSH and (e.g. recombinant) FSH preparations of the invention provide a higher response per dose in vivo compared to Gonal-F, a commercially available CHO cell derived product.

Preferably the FSH is a recombinant FSH ("rFSH" or "recFSH"). Preferably the FSH is a human cell line derived recombinant FSH.

The rFSH may preferably include 27 - 33%, for example 30 - 32%, tri-sialylated glycan structures. The rFSH may preferably include 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 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% 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 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 of glycan 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 (or rFSH preparation) according to the invention may have a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of 6 mol/mol or greater, for example between 6 mol/mol and 15 mo!/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 mol/mol and 13 mol/mol. The rFSH of the invention may be produced or expressed in a human cell line.

The rFSH (or rFSH preparation) according to the invention may have 10% or more of the total sialylation being a2,3-sia)ylation. For example, 20, 30, 40, 50, 60, 70, 80 or 90% or more of the total sialylation may be d2,3-sialylation. The rFSH (or rFSH preparation) may preferably include a2,3-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 rFSH (or rFSH preparation) of the invention may have 5% or more, for example 5% to 99%, of the total sialylation being a2,6- sialylation. The rFSH (or rFSH preparation) of the invention may have 50% or less of the total sialylation being a2,6-sialylation. The rFSH (or rFSH preparation) may preferably include a2,6-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 example around 35% of the total sialylation. By sialylation it is meant the amount of sialic residues present on the FSH carbohydrate structures. a2,3-sialylation means sialylation at the 2,3 position (as is well known in the art) and a2,6 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 sialyiated in the 2,3 position. The term "% of the total sialylation being a2,6-sialylation" refers to the % of the total number of sialic acid residues present in the FSH which are sialyiated in the 2,6 position.

The rFSH (or rFSH preparation) according to the invention may have a sialic acid content (amount 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. between 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 (or rFSH preparation) according to the invention may be FSH or a FSH preparation in which 16 % or fewer (e.g. 0.1 to 16%) of the glycans comprise (e.g. carry) bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc). Preferably the rFSH (or rFSH preparation) according to the invention is an FSH or FSH preparation in which 8 to 14.5% of the glycans comprise (e.g. carry) a bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc).

It will be understood that FSH comprises glycans attached to the FSH glycoproteins. It will also be understood that 00% 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 comprise (carry) bisecting N- acetylglucosamine" means that 16 % or fewer of the total number of glycans attached to the FSH glycoproteins include/carry bisecting N-acetylglucosamine, and so on.

The applicants have found that recombinant FSH (rFSH preparations; rFSH compositions) in which 16% or fewer (e.g. 8 to 14.5%) of the glycans comprised in the FSH glycoproteins carry bisecting GlcNac may have advantageous pharmacokinetic properties. It is believed the advantageous properties may arise because the amount of glycans which carry bisecting GlcNac is similar to that in the human urinary derived product Bravelle, which is rather less than that of other recombinant FSH preparations such as those disclosed in WO2012/017058.

The rFSH (or rFSH preparation) according to the invention may be an FSH or FSH preparation in which 20% or more of the glycans comprise (e.g. carry) N- Acety!galactosamine (GalNAc), for example in which 20% or more of the glycans comprise (e.g. carry) a terminal GalNAc. Preferably the rFSH (or rFSH preparation) according to the invention is an FSH or FSH preparation in which the 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) GalNAc. Preferably the rFSH (or rFSH preparation) according to the invention is an FSH or FSH preparation in which the 40 to 55%, for example 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 attached 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 a2,6- linkage increases the number of tetra sialylated structures, compared to CHO cell derived products which have only the a2,3- linkage available. The applicants have also found that their rFSH is distinguished over other approved products because of the sugar composition: it includes, or may include, a specific amount of GalNac. This may be linked to tetrasialylation and potency because the 2,6- sialylation is associated with GalNac. In other words, the present applicants have developed an rFSH product which includes specific characteristics (2,6- linker sites, GalNac) which provide rFSH with high degree of sialylation, which appears 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 of the invention 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 simplify (and render more efficient) 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 products; more acidic rFSH is produced and separation/removal of basic FSH is less problematic. The rFSH may be produced or expressed in a PER.C6® cell line, a PER.C6® derived cell line or a modified PER.C6® cell line. rFSH which is produced or expressed in a human cell line (e.g. PER.C6® cell line, HEK293 cell line, HT1080 cell line etc.) will include some a2,6-linked sialic acids (σ2,6 sialylation) provided by endogenous sialyl transferase activity [of the cell line] and will include some a2,3-linked sialic acids (a2,3 sialylation) provided by endogenous sialyl transferase activity. The cell line may be modified using a2,3-sialyltransferase. The cell line may be modified using a2,6-sialyltransferase. Alternatively or additionally, the rFSH may include a2,6-linked sialic acids (a2,6 sialylation) provided by endogenous sialyl transferase activity [of the cell line].

The rFSH may be produced using a2,3- and/or a2,6-sialyltransferase. In an example, rFSH is produced using a2,3- sialyltransferase. The rFSH may include Delinked sialic acids (a2,6 sialylation) provided by endogenous sialyl transferase activity. According to the present invention in a further aspect there is provided a method of production of rFSH and/or an rFSH preparation as described herein (according to aspects of the invention) comprising the step of producing or expressing the rFSH in a human cell line, for example a PER.C6® cell line, a PER.C6® derived cell line or a modified PER.C6® cell line, for example a cell line which has been modified using a2,3-sialyltransferase.

According to the present invention in a further aspect there is provided rFSH produced (e.g. expressed) in a human cell line, the rFSH including including mono-, di-, tri- and tetra-sialylated glycan structures, wherein 15-24%, for example 17-23% of the sialylated glycan structures are tetrasialylated glycan structures (e.g. as shown by WAX analysis of charged glycans, as set out in the Examples below). Preferably the FSH includes σ2,3- and a2,6- sialylation. The FSH (rFSH) according to the invention may have 1% to 99% of the total sialylation being a2,3-sialylation. The FSH (rFSH) according to the invention may have 1% to 99% of the total sialylation being a2,6-sia!ylation. Preferably, 50 to 70%, for example 60 to 69%, for example about 65%, of the total sialylation is a2,3- sialylation. Preferably 25 to 50%, for example 30 to 50 %, for example 31 to 38%, for example about 35%, of the total sialylation is σ2,6- sialylation.

The rFSH may be produced or expressed in a PER.C6® cell line, a PER.C6® derived cell line or a modified PER.C6® cell line. The cell line may be modified using a2,3- sialyltransferase. The cell line may be modified using a2,6-sialyltransferase. Alternatively or additionally, the rFSH may include a2,6-linked sialic acids (a2,6 sialylation) provided by endogenous sialyl transferase activity [of the cell line]. The rFSH (or rFSH preparation) may have 40% or more of the total sialylation being a2,3-sia)ylation, for example 50-70%, for example 60 to 69%, for example about 65%, of the total sialylation may be a2,3- sialylation. The rFSH of the invention may have 5% or more, for example 5% to 99%, of the total sialylation being d2,6-sialylation. The rFSH (or rFSH preparation) of the invention may have 50% or less of the total sialylation being a2,6-sialylation, for example 30 to 50 %, for example 31 to 38%, for example about 35%,of the total sialylation may be a2,6- sialylation. The rFSH may have a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of 6 mol/mol or greater, for example between 6mol/mo! and 15 mol/mol.

The rFSH (produced in the human cell line) may be an FSH comprising glycans wherein 16 % or fewer (e.g. 0.1 to 16%) of the glycans comprise (e.g. carry) bisecting N- acetylglucosamine (bisecting GlcNAc or bisGlcNAc). Preferably the rFSH is an FSH comprising glycans wherein 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 glycans attached 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 comprise (carry) bisecting N- acetylglucosamine" means that 16 % or fewer of the total number of glycans attached to the FSH glycoproteins include/carry bisecting N-acetylglucosamine, and so on.

The applicants have found that recombinant FSH (rFSH preparations; rFSH compositions) in which 16% or fewer (e.g. 8 to 14.5%) of the glycans comprised in the FSH glycoproteins carry bisecting GlcNac may have advantageous pharmacokinetic properties.

The rFSH (produced in the human cell line) may be an FSH 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. Preferably the rFSH is an FSH in which the 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) GalNAc. Preferably the rFSH is an FSH in which the 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc.

It appears that the availability of the a2,6- linkage increases the number of tetra sialylated structures, compared to CHO cell derived products which have only the ct2,3- linkage available. The applicants also found that their rFSH is distinguished over other approved products because of the sugar composition: it includes, or may include, a specific amount of GalNac. This may be linked to tetrasialylation and potency because the 2,6- sialylation is associated with GalNac. In other words, the present applicants have developed an rFSH product which includes specific characteristics (2,6- linker sites, GalNac) which provide rFSH with high degree of sialylation, which appears to lead to improved potency in vivo.

The rFSH (produced in a human cell line) 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 (produced in a human cell line) 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.

According to the present invention in a still further aspect there is provided a (recombinant) follicle stimulating hormone (FSH) wherein 16 % or fewer (e.g. 0.1 to 16%) of the glycans comprise (e.g. carry) bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc). Preferably 8 to 14.5% of the glycans comprise (e.g. carry) a bisecting N- acetylglucosamine (bisecting GlcNAc or bisGlcNAc). According to the present invention in a further aspect there is provided a pharmaceutical composition or a preparation comprising a (recombinant) follicle stimulating hormone (FSH) wherein 16 % or fewer (e.g. 0.1 to 16%) of the glycans comprise (e.g. carry) bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc). Preferably 8 to 14.5% of the glycans comprise (e.g. carry) a bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc).

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 attached to the FSH glycoproteins. Thus, herein, the terminology "8 to 1 .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 comprise (carry) bisecting N- acetylglucosamine" means that 16 % or fewer of the total number of glycans attached to the FSH glycoproteins include/carry bisecting N-acetylglucosamine, and so on.

The applicants have found that recombinant FSH (rFSH preparations; rFSH compositions) in which 6% or fewer (e.g. 8 to 14.5%) of the glycans comprised in the FSH glycoproteins carry bisecting GlcNac may have advantageous pharmacokinetic properties. It is believed the advantageous properties may arise because the amount of glycans which carry bisecting GlcNac is similar to that in the human urinary derived product Bravelle, and is rather less than that of other recombinant FSH preparations such as those disclosed in WO2012/017058.

Preferably the FSH is a recombinant FSH. Preferably the recombinant FSH is produced or expressed in a human cell line, for example a PER.C6® cell line, a PER.C6® derived cell line or a modified PER.C6® cell line.

The follicle stimulating hormone (FSH) may include a2,3- sialylation and a2,6- sialylation. The FSH (rFSH) may have 1% to 99% of the total sialylation being a2,3- sialylation. The FSH (rFSH) may have 1% to 99% of the total sialylation being a2,6- sialylation. Preferably 25 to 50%, for example 30 to 50%, of the total sialylation is a2, 6- sialylation. Preferably 50 to 70% of the total sialylation is σ 2,3- sialylation.

The follicle stimulating hormone (FSH) may include mono-, di-, tri- and tetra- sialylated glycan structures, wherein 15-24%, for example 17-23% of the sialylated glycan structures are tetrasialylated glycan structures. Preferably 27 to 33% of the sialyated glycan structures are tri-sialylated glycan structures. Preferably, 24 to 33% of the sialyated glycan structures are di-sialylated glycan structures. Preferably, 12 to 21% of the sialyated glycan structures are mono-sialylated glycan structures. Preferably the FSH includes from 0.1 to 7% neutral glycan structures.

Preferably the FSH has a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of 6 mol/mol or greater, for example between 6mol/mol and 15 mol/mol.

The rFSH may be an FSH 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. Preferably the rFSH is an FSH in which the 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) GalNAc. Preferably the rFSH is an FSH in which the 40 to 55%, for example 42% to 52%, of the glycans comprise

(e.g. carry) terminal GalNAc.

The rFSH may have 6 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 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.

According to the present invention in a still further aspect there is provided a follicle stimulating hormone (FSH) wherein 20% or more of the glycans comprise (e.g. carry)

GalNAc. Preferably 20% or more of the glycans comprise (e.g. carry) a terminal GalNAc. Preferably 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry)

GalNAc. Preferably 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc. According to the present invention in a further aspect there is provided a pharmaceutical composition or a preparation comprising a follicle stimulating hormone (FSH) wherein 20% or more of the glycans comprise (e.g. carry) GalNAc. Preferably 20% or more of the glycans comprise (e.g. carry) a terminal GalNAc. Preferably

40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) GalNAc.

Preferably 40 to 55%, for example 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 attached 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 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.

The follicle stimulating hormone (FSH) may include a2,3- sialylation and a2,6- sialylation. The FSH (rFSH) may have 1% to 99% of the total sialylation being a2,3- sialylation. The FSH (rFSH) may have 1% to 99% of the total sialylation being a2,6- sialylation. Preferably 25 to 50%, for example 30 to 50%, of the total sialylation is a2, 6- sialylation. Preferably 50 to 70% of the total sialylation is a 2,3- sialylation.

As discussed above, it appears that the availability of the σ2,6- linkage increases the number of tetra sialylated structures, compared to CHO cell derived products which have only the a2,3- linkage available. Ferring have also found that their rFSH is distinguished over other approved products because of the sugar composition: it includes, or may include, a specific amount of GalNac. This may be linked to tetrasialylation and potency because the 2,6- sialylation is associated with GalNac. In other words, the present applicants have developed an rFSH product which includes specific characteristics (2,6- linker sites, GalNac) which provide rFSH with high degree of sialylation, which appears to lead to improved potency in vivo.

The rFSH (produced in the human cell line) may be an FSH comprising glycans wherein 16 % or fewer (e.g. 0.1 to 16%) of the glycans comprise (e.g. carry) bisecting N- acetylglucosamine (bisecting GlcNAc or bisGlcNAc). Preferably the rFSH is an FSH comprising glycans wherein 8 to 14.5% of the glycans comprise (e.g. carry) a bisecting N- acetylglucosamine (bisecting GlcNAc or bisGlcNAc).

The rFSH 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 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.

According to the present invention in a further aspect there is provided a recombinant follicle stimulating hormone (FSH) wherein 16 to 24% of the glycans comprise (e.g. terminal) 1 fucose-lewis. The rFSH may have 15 to 23% 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. According to the present invention in a further aspect there is provided a recombinant follicle stimulating hormone (FSH) wherein 1.5 to 4.5% of the glycans comprise (e.g. terminal) 2 fucose-lewis. The rFSH 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. According to the present invention in a further aspect there is provided a pharmaceutical composition comprising a recombinant follicle stimulating hormone (FSH) wherein 16 to 24% of the glycans comprise (e.g. terminal) 1 fucose-lewis. The rFSH may have 15 to 23% 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. According to the present invention in a further aspect there is provided a pharmaceutical composition comprising a recombinant follicle stimulating hormone (FSH) wherein 1.5 to 4.5% of the glycans comprise (e.g. terminal) 2 fucose-lewis. The rFSH 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.

According to the present invention in a further aspect there is provided a pharmaceutical composition comprising (e.g. recombinant) follicle stimulating hormone (rFSH) including including mono-, di-, tri- and tetra-sialylated glycan structures, wherein 15- 24%, for example 17-23% of the sialylated glycan structures are tetrasialylated glycan structures (e.g. as shown by WAX analysis of charged glycans, as set out in the Examples below).

The pharmaceutical composition may further comprise hCG and/or LH and/or LH activity.

The rFSH may include a2,3- and a2,6-sialylation. The FSH (rFSH) may have 1% to 99% of the total sialylation being a2,3-sialylation. The FSH (rFSH) may have 1% to 99% of the total sialylation being a2,6-sialylation. The rFSH may be produced or expressed in a PER.C6® cell line, a PER.C6® derived cell line or a modified PER.C6® cell line. The cell line may be modified using a2,3-sialyltransf erase. The cell line may be modified using a2,6-sialyltransferase. Alternatively or additionally, the rFSH may include a2,6-linked sialic acids (a2,6 sialylation) provided by endogenous sialyl transferase activity [of the cell line]. The rFSH (or rFSH preparation) may have 40% or more of the total sialylation being a2,3- sialylation, for example 50-70%, for example 60 to 69%, for example about 65%, of the total sialylation may be a2,3-sialylation. The rFSH of the invention may have 5% or more, for example 5% to 99%, of the total sialylation being a2,6-sialylation. The rFSH (or rFSH preparation) of the invention may have 50% or less of the total sialylation being a2,6- sialylation, for example 25-50%, for example 30 to 50 %, for example 31 to 38%, for example about 35%, of the total sialylation may be a2,6- sialylation. The rFSH may have a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of 6 mol/mol or greater, for example between 6mol/mol and 15 mol/mol. Preferably, 50 to 70% of the total sialylation is a2, 3-sialylation. Preferably 25 to 50 %, e.g. 30 to 50%, of the total sialylation is a2, 6- sialylation.

The rFSH may be an FSH comprising glycans wherein 16 % or fewer (e.g. 0.1 to 16%) of the glycans comprise (e.g. carry) bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc). Preferably the rFSH is an FSH comprising glycans wherein 8 to 14.5% of the glycans comprise (e.g. carry) a bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc).

The rFSH may be an FSH 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. Preferably the rFSH is an FSH in which the 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) GalNAc. Preferably the rFSH is an FSH in which the 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc.

According to the present invention in a further aspect there is provided a pharmaceutical composition comprising (e.g. recombinant) follicle stimulating hormone (rFSH) wherein 16 % or fewer (e.g. 0.1 to 16%) of the glycans comprise (e.g. carry) bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc). Preferably 8 to 14.5% of the glycans comprise (e.g. carry) a bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc). The pharmaceutical composition may further comprise hCG and/or LH and/or LH activity.

According to the present invention in a further aspect there is provided a pharmaceutical composition (e.g. recombinant) follicle stimulating hormone (rFSH) wherein 20% or more of the glycans comprise (e.g. carry) GalNAc. Preferably 20% or more of the glycans comprise (e.g. carry) a terminal GalNAc. Preferably 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) GalNAc. Preferably 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc. The pharmaceutical composition may further comprise hCG and/or LH and/or LH activity. hCG can be obtained by any means known in the art. hCG as used herein includes humah-derived and recombinant hCG. Human-derived hCG can be purified from any appropriate source (e.g. urine, and placenta) by any method known in the art. Methods of expressing and purifying recombinant hCG are well known in the art.

LH can be obtained by any means known in the art. LH, as used herein, includes human-derived and recombinant LH. Human-derived LH can be purified from any appropriate source (e.g. urine) by any method known in the art. Methods of expressing and purifying recombinant LH are known in the art.

The pharmaceutical composition (according to any aspect of the invention) may be for the treatment of infertility, e.g. for use in e.g. assisted reproductive technologies (ART), ovulation induction or intrauterine insemination (IUI). The pharmaceutical composition may be used, for example, in medical indications where known FSH preparations are used. The present invention also provides the use of rFSH and/or an rFSH preparation described herein (according to aspects of the invent/on) for, or in the manufacture of a medicament for, the treatment of infertility. The pharmaceutical compositions of the present invention can be formulated into well-known compositions for any route of drug administration, e.g. oral, rectal, parenteral, transdermal (e.g. patch technology), intravenous, intramuscular, subcutaneous, intrasusternal, intravaginal, intraperitoneal, local (powders, 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, buffers and the like, as described in Remington's Pharmaceutical Sciences fifteenth edition (Matt Publishing Company, 1975), at pages 1405 to 1412 and 1461 - 87, and the national formulary XIV fourteenth edition (American Pharmaceutical 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 thereof, vegetable oils (such as olive oil), and injectible organic esters such as ethyl oleate.

The compositions of the present invention also can contain additives such as but not limited to preservatives, wetting agents, emulsifying agents, and dispersing agents. Antibacterial and antifungal agents can be included to prevent growth of microbes and includes, for example, m-cresol, benzyl alcohol, paraben, chlorobutanol, phenol, sorbic acid, and the like. Furthermore, it may be desirable to include isotonic agents such as sugars, sodium chloride, and the like.

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 dissolution which, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenteraliy administered FSH combination form is accomplished by dissolving 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 polymer 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.

Injectable 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 supplied in any suitable container, e.g. vial, pre-filled syringe, injection cartridges, and the like.

Formulations (e.g. injectable formulations) may be supplied as a product having pharmaceutical compositions containing FSH (optionally with hCG, LH etc.) 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 together. If administered separately, administration can be sequential. The product can be supplied in any appropriate package. For example, a product can contain a number of pre-filled syringes or vials containing either FSH, hCG, or a combination of both FSH and hCG. The syringes or vials may be packaged in a blister package or other means to maintain sterility. A product can optionally contain instructions for using the FSH and hCG 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, 7^th ed. In a preferred embodiment, the compositions of the invention are supplied as compositions for parenteral administration. General methods for the preparation of the parenteral formulations are known in the art and are described in REMINGTON; THE SCIENCE AND PRACTICE OF PHARMACY, supra, at pages 780-820. The parenteral compositions can be supplied in liquid formulation or as a solid which will be mixed with a sterile injectable medium just prior to administration. In an especially preferred embodiment, the parenteral compositions are supplied in dosage unit form for ease of administration and uniformity of dosage.

Detailed description of the invention

The present invention will now be described in more detail with reference to the attached drawings in which:

Figure 1 shows a plasmid map of the pFSHalpha/beta expression vector;

Figure 2 shows the a2,3-sialyltransf erase (ST3GAL4) expression vector;

Figure 3 shows the a2,6-sialyltransferase (ST6GAL1) expression vector;

Figure 4 shows % abundance sialic acid distribution of examples of recombinant FSH produced by PER.C6® cells stably expressing FSH after engineering with 02,3- 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 a2,3- sialyltransferase;

and Figure 6 shows a comparison of concentration of inhibin-B following administration of 225IU Gonal f (bottom line, dotted line) and 225 IU of the Example (top line, full line) of Invention.

Sequence Selection

Human FSH

The coding region of the gene for the FSH alpha polypeptide was used to according to Fiddes and Goodman. (1981). The sequence is banked as AH007338 and at the time of construction there were no other variants of this protein sequence. The sequence is referred herein as SEQ ID NO:1.

The coding region of the gene for FSH beta polypeptide was used according to Keene et al (1989). The sequence is banked as N _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.

Sialyltransferase

a2,3-Sialyltransferase - The coding region of the gene for beta-galactoside alpha-

2, 3-sialy (transferase 4 (a2,3-sialyltransferase, ST3GAL4) was used according to Kitagawa and Paulson (1994). The sequence is banked as L23767 and referred herein as SEQ ID

NO:3.

a2,6-Sialyltransferase - The coding region of the gene for beta-galactosamide alpha-2,6-sialyltransferase 1 (a2,6-sialyltransferase, ST6GAL1) was used according to Grundmann et al. (1990). The sequence is banked as N _003032 and referred herein as SEQ ID NO:4.

EXAMPLES

Example 1 Construction of the FSH expression vector

The coding sequence of FSH alpha 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 S'-CCGGGTTAACTTATTATTCTTTCATTTCACCAAAGG-S' (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 alpha DNA was digested with BamHI and Nhe\ and inserted into the sites BamYW and Nhe) on the expression vector already containing the FSH beta polypeptide DNA. The vector DNA was used to transform the DH5a strain of E.coli. Colonies were picked for amplification. Colonies containing the vector containing both FSH alpha and beta were selected 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).

Example 2 Construction of the ST3 expression vector

The coding sequence of beta-galactoside alpha-2,3-sialyltransf erase 4 (ST3, L23767, SEQ ID NO:3) was amplified by PCR using the primer combination 2,3STfw and 2,3STrev.

2,3STfw 5'-CCAGGATCCGCCACCATGTGTCCTGCAGGCTGGAAGC-3' (SEQ ID NO:13) 2,3STrev 5'-TTTTTTTCTTAAGTCAGAAGGACGTGAGGTTCTTG-3' (SEQ ID NO:14)

The resulting amplified ST3 DNA was digested with the restriction enzymes BamHl and AM and inserted into the SamHI and Afl \ 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 (Figure 2) contained the correct sequence according SEQ ID NO:3 and was selected for transfection. Example 3 Construction of the ST6 expression vector

The coding sequence of beta-galactosamide alpha-2,6-sialyltransferase 1 (ST6, NM_003032, SEQ ID NO:4) was amplified by PCR using the primer combination 2,6STfw and 2,6STrev. 2,6STfw 5'-CCAGGATCCGCCACCATGATTCACACCAACCTGAAG-3' (SEQ ID NO: 15) 2,6STrev 5'-TTTTTTTCTTAAGTTAGCAGTGAATGGTCCGG-3' (SEQ ID NO: 16) The resulting amplified ST6 DNA was digested with the restriction enzymes SamHI and AfHl and inserted into the SamHI and Affll sites on the CMV driven mammalian expression vector carrying a hygromycin resistance marker. The vector was amplified as previously described and sequenced. Clone pST6#11 (Figure 3) contained the correct sequence according SEQ ID N0.4 and was selected for transfection.

Example 4 Stable expression of pFSH α+β in PER.C6® cells. Transfection isolation and screening of clones.

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

To obtain stable clones a liposome based transfection agent with the pFSH α+β construct. Stable clones were selected in VPRO supplemented with 10% FCS and containing G418. Three weeks after transfection G418 resistant clones grew out. Clones were selected 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 determine 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 sufficient productivity and quality were selected for sialyltransferase engineering.

Example 5 Level of sialylation is increased in cells that over express a2,3- sialyltransferase. Stable expression of pST3 in FSH expressing PER.C6® cells; Transfection isolation and screening of clones.

PER.C6® clones producing highly sialylated FSH were generated by expressing a2,3 sialyltransferase from separate plasmids (Example 2) in PER.C6® cells already expressing both polypeptide 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, expanded and assayed. The a2,3-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 receptor cell line, IEF, metabolic clearance 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 a2,3- 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 a2,3- clone (Example 5) was prepared using a using a modification of the method described by Lowry ef al. (1976).

For the production of PER.C6®-FSH, the cell lines were adapted to a serum- free medium, i.e., Excell 525 (JRH Biosciences). The cells were first cultured to form a 70%- 90% confluent monolayer in a T80 culture flask. On passage the cells were re-suspended in the serum free medium, Excell 525 + 4 m 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 > IxlO⁶ cells/ml, the cells were sub- cultured to a cell density of 0.2 or 0.3x10⁶ cells/ml and further cultured in shaker flasks at 37°C, 5% C0₂ and 00 rpm.

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 culture). The cells were first cultured to > 1x10^s 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 started to decline. The cells were spun down for 5 min at 1000 rpm and the supernatant was used for the quantification and purification of FSH. The concentration of FSH was determined using ELISA (DRG EIA 1288). Thereafter, purification of FSH was carried out using a modification of the method described by Lowry et al. (1976). Purification using charge selective chromatography was carried out to enrich the highly sialylated 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 EIA 1288) and/or IEF.

Example 7 Quantification of relative amounts of a2,3 and σ2,6 sialic acid

The relative percentage amounts of a2,3 and a2,6 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 present 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 a2,3 sialylation and 28 to 50%, generally 30 to 35% (e.g. about 31% or 35%), for σ2,6 sialylation.

Example 8 Quantification of relative amounts mono, di, tri and tetra sialylated 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 samples 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 labeled with 2- aminobenzamide. Released glycan forms were then separated and analysed by Weak Anion Exchange (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 , mono-sialylated, di- sialylated, tri- sialylated and tetra- sialylated glycan structures with relative amounts as follows: neutral 5-6 %; 5- 7% mono- sialylated; 26-30% di-sialylated; 30-32% tri-sialylated and 17-23 % tetra-sialylated. Example 8a

The relative percentage amounts of a2,6 sialic acid on purified rFSH extracted from nine samples of purified rFSH (produced by the methods of 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 (see Example 8). The analysis allows calculation of a2,6 sialic acid.

The relative percentages 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 a2,6 sialylation.

The relative percentage amounts of bisecting GlcNac, 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 released 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 MALDI-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 recombinant FSH) and Bravelle (human urinary FSH).

Value of 3.1 is total ¼ Fucose Lewis. ² 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 GlcNac 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, produced according to Example 6) or 16.5 g of Gonal-F for seven days. The composition has relative percentage amounts of mono, di, tri and tetra sialylated structures as set out in Example 8 [neutral 5-6 %; 15-17% mono-sialylated; 26-30% di-sialylated; 30-32% tri-sialylated and 17-23 % tetra— sialylated.] The amount of GalNAc is approximately 50%, the amount of bisGlcNac about 9%, the amount of 1 Fucose Lewis is about 20 % and the amount of 2 Fucose Lewis about 3%.

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, clinical laboratory measurements, and physical examination. 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 immediately 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 administration of FE 999049 and GONAL-F, however to a greater extent following administration of FE 999049 compared to GONAL-F. Both number and size distribution 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 amounts of a2,3 sialylation and a2,6 sialylation is markedly more potent then recombinant FSH products which are currently on the market. References

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SEQ ID NO:1

Follicle stimulating hormone alpha polypeptide

Accession number AH007338

Nucleotide sequence of FSH alpha

1 ATGGATTACT ACAGAAAATA TGCAGCTATC TTTCTGGTCA CATTGTCGGT GTTTCTGCAT

61 GTTCTCCATT CCGCTCCTGA TGTGCAGGAT TGCCCAGAAT GCACGCTACA GGAAAACCCA

121 TTCTTCTCCC AGCCGGGTGC CCCAATACTT CAGTGCATGG GCTGCTGCTT CTCTAGAGCA

181 TATCCCACTC CACTAAGGTC CAAGAAGACG ATGTTGGTCC AAAAGAACGT CACCTCAGAG 241 TCCACTTGCT GTGTAGCTAA ATCATATAAC AGGGTCACAG TAATGGGGGG TTTCAAAGTG

301 GAGAACCACA CGGCGTGCCA CTGCAGTACT TGTTATTATC ACAAATCTTA A

Protein sequence of FSH alpha (SEQ ID NO:5) 1 MDYYRKYAAI FLVTLSVFLH VLHSAPDVQD CPECTLQENP FFSQPGAPIL QC GCCFSRA

61 YPTPLRSK T MLVQKNVTSE STCCVAKSYN RVTVMGGFKV ENHTACHCST CYYHKS

SEQ ID NO:2

Follicle stimulating hormone beta polypeptide

Accession number NM_0D0510

Nucleotide sequence of FSH beta

1 ATGAAGACAC TCCAGTTTTT CTTCCTTTTC TGTTGCTGGA AAGCAATCTG CTGCAATAGC 61 TGTGAGCTGA CCAACATCAC CATTGCAATA GAGAAAGAAG AATGTCGTTT CTGCATAAGC 121 ATCAACACCA CTTGGTGTGC TGGCTACTGC TACACCAGGG ATCTGGTGTA TAAGGACCCA 181 GCCAGGCCCA AAATCCAGAA AACATGTACC TTCAAGGAAC TGGTATATGA AACAGTGAGA 241 GTGCCCGGCT GTGCTCACCA TGCAGATTCC TTGTATACAT ACCCAGTGGC CACCCAGTGT 301 CACTGTGGCA AGTGTGACAG CGACAGCACT GATTGTACTG TGCGAGGCCT GGGGCCCAGC 361 TACTGCTCCT TTGGTGAAAT GAAAGAATAA

Protein sequence of FSH beta (SEQ ID NO:6)

1 M TLQFFFLF CCWKAICCNS CELTN1TIAI EKEECRFCIS INTT CAGYC YTRDLVYKDP 61 ARPKIQ TCT FKELVYETVR VPGCAHHADS LYTYPVATQC HCGKCDSDST DCTVRGLGPS 121 YCSFGEMKE SEQ ID NO:3

Beta-galactoside alpha-2,3-sialyltransferase 4

Accession Number L23767

Nucleotide sequence of ST3GAL4

1 ATGTGTCCTG CAGGCTGGAA GCTCCTGGCC ATGTTGGCTC TGGTCCTGGT CGTCATGGTG

61 TGGTATTCCA TCTCCCGGGA AGACAGGTAC ATCGAGCTTT TTTATTTTCC CATCCCAGAG

121 AAGAAGGAGC CGTGCCTCCA GGGTGAGGCA GAGAGCAAGG CCTCTAAGCT CTTTGGCAAC

181 TACTCCCGGG ATCAGCCCAT CTTCCTGCGG CTTGAGGATT ATTTCTGGGT CAAGACGCCA

241 TCTGCTTACG AGCTGCCCTA TGGGACCAAG GGGAGTGAGG ATCTGCTCCT CCGGGTGCTA

301 GCCATCACCA GCTCCTCC T CCCCAAGAAC ATCCAGAGCC TCAGGTGCCG CCGCTGTGTG

361 GTCGTGGGGA ACGGGCACCG GCTGCGGAAC AGCTCACTGG GAGATGCCAT CAACAAGTAC

421 GATGTGGTCA TCAGATTGAA CAATGCCCCA GTGGCTGGCT ATGAGGGTGA CGTGGGCTCC

4 8 1 AAGACCACCA TGCGTCTCTT CTACCCTGAA TCTGCCCACT TCGACCCCAA AGTAGAAAAC

541 AACCCAGACA CACTCCTCGT CCTGGTAGCT TTCAAGGCAA TGGACTTCCA CTGGATTGAG

601 ACCATCCTGA GTGATAAGAA GCGGGTGCGA AAGGGTTTCT GGAAACAGCC TCCCCTCATC

661 TGGGATGTCA ATCCTAAACA GATTCGGATT CTCAACCCCT TCTTCATGGA GATTGCAGCT

721 GACAAACTGC TGAGCCTGCC AATGCAACAG CCACGGAAGA TTAAGCAGAA GCCCACCACG

781 GGCCTGTTGG CCATCACGCT GGCCCTCCAC CTCTGTGACT TGGTGCACAT TGCCGGCTTT

841 GGGTACCCAG ACGCCTACAA CAAGAAGCAG ACCATTCACT ACTATGAGCA GATCACGCTC

901 AAGTCCATGG CGGGGTCAGG CCATAATGTC TCCCAAGAGG CCCTGGCCAT TAAGCGGATG

961 CTGGAGATGG GAGCTATCAA GAACCTCACG TCCTTCTGA

Protein Sequence of ST3GAL4 (SEQ ID NO:7)

1 MCPAGWKLLA MLALVLWMV WYSISREDRY I ELFYFPI PE KKEPCLQGEA ESKASKLFGN

61 YSRDQPIFLR LEDYF VKTP SAYELPYGTK GSEDLLLRVL AITSSSIPKN IQSLRCRRCV

121 WGNGHRLRN SSLGDAINKY DWIRLNNAP VAGYEGDVGS KTTMRLFYPE SAHFDPKVEN

181 NPDTLLVLVA FKAMDFHWIE TILSDKKRVR KGFWKQPPLI WDVNPKQIRI LNPFFMEIAA

241 DKLLSLPMQQ PRKIKQKPTT GLLAITLALH LCDLVHIAGF GYPDAYNKKQ TIHYYEQITL

301 SMAGSGHNV SQEALAIKRM LEMGAIKNLT SF

SEQ ID NO:4

Beta-galactosamide alpha-2,6-sialyltransferase 1 Accession number NM 003032 Nucleotide sequence of ST6GAL1

1 ATGATTCACA CCAACCTGAA GAAAAAGTTC AGCTGCTGCG TCCTGGTCTT TCTTCTGTTT

61 GCAGTCATCT GTGTGTGGAA GGAAAAGAAG AAAGGGAGTT ACTATGATTC CTTTAAATTG

12 1 CAAACCAAGG AATTCCAGGT GTTAAAGAGT CTGGGGAAAT TGGCCATGGG GTCTGATTCC

18 1 CAGTCTGTAT CCTCAAGCAG CACCCAGGAC CCCCACAGGG GCCGCCAGAC CCTCGGCAGT

24 1 CTCAGAGGCC TAGCCAAGGC CAAACCAGAG GCCTCCTTCC AGGTGTGGAA CAAGGACAGC

301 TCTTCCAAAA ACCTTATCCC TAGGCTGCAA AAGATCTGGA AGAATTACCT AAGCATGAAC

361 AAGTACAAAG TGTCCTACAA GGGGCCAGGA CCAGGCATCA AGTTCAGTGC AGAGGCCCTG

421 CGCTGCCACC TCCGGGACCA TGTGAATGTA TCCATGGTAG AGGTCACAGA TTTTCCCTTC

48 1 AATACCTCTG AATGGGAGGG TTATCTGCCC AAGGAGAGCA TTAGGACCAA GGCTGGGCCT

54 1 TGGGGCAGGT GTGCTGTTGT GTCGTCAGCG GGATCTCTGA AGTCCTCCCA ACTAGGCAGA

601 GAAATCGATG ATCATGACGC AGTCCTGAGG TTTAATGGGG CACCCACAGC CAACTTCCAA

661 CAAGATGTGG GCACAAAAAC TACCATTCGC CTGATGAACT CTCAGTTGGT TACCACAGAG

721 AAGCGCTTCC TCAAAGACAG TTTGTACAAT GAAGGAATCC TAATTGTATG GGACCCATCT

78 1 GTATACCACT CAGATATCCC AAAGTGGTAC CAGAATCCGG ATTATAATTT CTTTAACAAC

84 1 TAGAAGACTT ATCGTAAGCT GCACCCCAAT CAGCCCTTTT ACATCCTCAA GCCCCAGATG

901 CCTTGGGAGC TATGGGACAT TCTTCAAGAA ATCTCCCCAG AAGAGATTCA GCCAAACCCC

961 CCATCCTCTG GGATGCTTGG TATCATCATC ATGATGACGC TGTGTGACCA GGTGGATATT

1021 TATGAGTTCC TCCCATCCAA GCGCAAGACT GACGTGTGCT ACTACTACCA GAAGTTCTTC

1081 GATAGTGCCT GCACGATGGG TGCCTACCAC CCGCTGCTCT ATGAGAAGAA TTTGGTGAAG

114 1 CATCTCAACC AGGGCACAGA TGAGGACATC TACCTGCTTG GAAAAGCCAC ACTGCCTGGC

1201 TTCCGGACCA TTCACTGCTA A

Op-

Protein Sequence of ST6GAL1 (SEQ ID N0.8)

1 MIHTNLKKKF SCCVLVFLLF AVICVWKEKK KGSYYDS FKL QTKEFQVLKS LGKLAMGSDS 61 QSVSSSSTQD PHRGRQTLGS LRGLAKAKPE ASFQVWNKDS SSKNLI PRLQ KIWKNYLSMN 12 1 KYKVSYKGPG PGI KFSAEAL RCHLRDHVNV SMVEVTDFPF NTSEWEGYLP KES IRTKAGP 18 1 WGRCAWSSA GSLKSSQLGR EI DDHDAVLR FNGAPTANFQ QDVGTKTTIR LMNSQLVTTE 24 1 KRFLKDSLYN EGILIVWDPS VYHSDI PKWY QNPDYNFFNN YKTYRKLHPN QPFYILKPQM 30 1 PWELWDILQE I SPEEIQPNP PSSGMLGI I I MMTLCDQVDI YEFLPSKRKT DVCYYYQKFF 3 61 DSACTMGAYH PLLYEKNLVK HLNQGT DEDI Y.LLGKATLPG FR I HC

Claims

1. A follicle stimulating hormone (FSH) including mono-, d/-, tri- and tetra-sialylated glycan structures, wherein 15-24%, for example 17-23% of the sialylated glycan structures are tetrasialylated glycan structures.

2. A follicle stimulating hormone (FSH) according to claim 1 including a2,6- sialylation.

3. A follicle stimulating hormone (FSH) according to claim 2 wherein 25 to 50%, for example 30 to 50%, of the total sialylation is a2, 6- sialylation.

4. A follicle stimulating hormone (FSH) according to any preceding claim including a2,3- sialylation.

5. A follicle stimulating hormone (FSH) according to claim 4 wherein 50 to 70% of the total sialylation is a 2,3- sialylation.

6. A follicle stimulating hormone (FSH) according to any preceding claim which is a recombinant FSH.

7. A recombinant follicle stimulating hormone according to any preceding claim including a2,3- and a2,6- sialylation, wherein 50 to 70% of the total sialylation is a2, 3-sialylation and 25 to 50 % of the total sialylation is a2,6- sialylation.

8. A follicle stimulating hormone (FSH) according to any preceding claim wherein 27 to 33% of the sialyated glycan structures are tri-sialylated glycan structures.

9. A follicle stimulating hormone (FSH) according to any preceding claim wherein 24 to 33% of the sialyated glycan structures are di-sialylated glycan structures.

10. A follicle stimulating hormone (FSH) according to any preceding claim wherein 2 to 21 % of the sialyated glycan structures are mono-sialylated glycan structures.

1 1. A follicle stimulating hormone (FSH) according to any preceding claim including from 0.1 to 7% neutral glycan structures.

12. A follicle stimulating hormone (FSH) according to any preceding claim having a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of 6 mol/mol or greater, for example between 6mol/mol and 15 mol/mol.

13. A recombinant follicle stimulating hormone (FSH) according to any preceding claim produced or expressed in a human cell line, for example a PER.C6® cell line, a PER.C6® derived cell line or a modified PER.C6® cell line.

14. A recombinant follicle stimulating hormone (FSH) according to any preceding claim in which 16 % or fewer of the glycans comprise bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc).

15. A recombinant follicle stimulating hormone (FSH) according to any preceding claim in which 8 to 14.5% of the glycans comprise a bisecting N-acetylglucosamine.

16. A recombinant follicle stimulating hormone (FSH) according to any preceding claim in which 20% or more of the glycans comprise N-Acetylgalactosamine (GalNAc).

17. A recombinant follicle stimulating hormone (FSH) according to any preceding claim in which 40 to 55%, for example 42% to 52%, of the glycans comprise a terminal GalNAc.

18. A recombinant follicle stimulating hormone (FSH) according to any preceding claim in which 6 to 24%, for example 8 to 20%, of the glycans comprise a (e.g.

terminal) 1 fucose lewis.

19. A recombinant follicle stimulating hormone (FSH) according to any preceding claim in which 1.5 to 5%, for example 3.7%, of the glycans comprise a (e.g. terminal) 2 fucose lewis.

20. A recombinant follicle stimulating hormone (FSH) wherein 16 % or fewer of the glycans comprise bisecting N-acetylglucosamine.

21. A recombinant follicle stimulating hormone (FSH) according to claim 20 wherein 8 to 14.5% of the glycans comprise a bisecting N-acetylglucosamine.

22. A follicle stimulating hormone (FSH) wherein 20% or more of the glycans comprise GalNAc.

23. A follicle stimulating hormone (FSH) according to claim 22 wherein 20% or more of the glycans, for example 42% to 52% of the glycans, comprise a terminal GalNAc.

24. A follicle stimulating hormone (FSH) according to claim 22 or 23 which is a

recombinant FSH.

25. A recombinant follicle stimulating hormone (FSH) wherein 16 to 24% of the glycans comprise (e.g. terminal) 1 fucose-lewis.

26. A recombinant follicle stimulating hormone (FSH) wherein 1.5 to 4.5%, for example 3.7%, of the glycans comprise (e.g. terminal) 2 fucose-lewis.

27. A recombinant follicle stimulating hormone (FSH) according to any of claims 20 to

26 which includes a2,3- sialylation and a2,6- sialylation.

28. A recombinant follicle stimulating hormone (FSH) according to any of claims 20 to

27 which includes mono-, di-, tri- and tetra-sialylated glycan structures, wherein 15- 24%, for example 17-23% of the sialylated glycan structures are tetrasialylated glycan structures.

29. A follicle stimulating hormone (FSH) preparation comprising a follicle stimulating hormone (FSH) according to any preceding claim.

30. A pharmaceutical composition comprising a follicle stimulating hormone (FSH) according to any preceding claim.

31. A pharmaceutical com osition for use in the treatment of infertility comprising a follicle stimulating hormone (FSH) including mono-, di-, tri- and tetra-sialylated glycan structures, wherein 15-24%, for example 17-23% of the sialylated glycan structures are tetrasialylated glycan structures.

32. A pharmaceutical composition comprising a follicle recombinant stimulating hormone (rFSH) wherein 16 % or fewer of the glycans comprise bisecting N- acetylglucosamine.

33. A pharmaceutical composition comprising a recombinant follicle stimulating

hormone (rFSH) wherein 20% or more of the glycans comprise GalNAc.

34. A method of treatment of infertility comprising a step of administration to a subject a follicle stimulating hormone (FSH) including mono-, di-, tri- and tetra-sialylated glycan structures, wherein 15-24%, for example 17-23% of the sialylated glycan structures are tetrasialylated glycan structures.

35. A method of treatment of infertility comprising a step of administration to a subject a recombinant follicle stimulating hormone (rFSH) wherein 16 % or fewer (e.g. 0.1 to 16%) of the glycans comprise (e.g. carry) bisecting N-acetylglucosamine;

and/orwherein 20% or more of the glycans comprise (e.g. carry) GalNAc.

36. Use, in the manufacture of a medicament for the treatment of infertility, of a follicle stimulating hormone (FSH) including mono-, di-, tri- and tetra-sialylated glycan structures, wherein 15-24%, for example 17-23% of the sialylated glycan structures are tetrasialylated glycan structures.

37. Use, in the manufacture of a medicament for the treatment of infertility, of a follicle stimulating hormone (FSH) wherein 16 % or fewer (e.g. 0.1 to 16%) of the glycans comprise (e.g. carry) bisecting N-acetylglucosamine; and/or wherein 20% or more of the glycans comprise (e.g. carry) GalNAc.