EP2717904A1 - Préparation pharmaceutique - Google Patents

Préparation pharmaceutique

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Publication number
EP2717904A1
EP2717904A1 EP12730594.4A EP12730594A EP2717904A1 EP 2717904 A1 EP2717904 A1 EP 2717904A1 EP 12730594 A EP12730594 A EP 12730594A EP 2717904 A1 EP2717904 A1 EP 2717904A1
Authority
EP
European Patent Office
Prior art keywords
fsh
stimulating hormone
follicle stimulating
sialylation
glycan structures
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP12730594.4A
Other languages
German (de)
English (en)
Inventor
Ian Cottingham
Daniel Plaksin
Bjorn Rikard Lorentz SANDSTROM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ferring BV
Original Assignee
Ferring BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ferring BV filed Critical Ferring BV
Priority to EP12730594.4A priority Critical patent/EP2717904A1/fr
Publication of EP2717904A1 publication Critical patent/EP2717904A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to gonadotrophins for use in the treatment of infertility.
  • gonadotrophins for use in the treatment of infertility.
  • FSH follicle stimulating hormone
  • 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 follicle stimulating hormone
  • LH luteinising hormone
  • CG chorionic gonadotropin
  • 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).
  • FSH FSH
  • 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).
  • glycan structures 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).
  • 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.
  • FSH e.g. human Pituitary/ serum/ urinary FSH
  • a recombinant FSH preparation 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).
  • 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.
  • the major contributor to overall molecular charge is the total sialic content of each FSH molecule.
  • rFSH Organon
  • urine-derived FSH has a higher sialic acid content
  • the corresponding plasma clearance rates in the rat are 0.34 and 0.14 ml/min (Ulloa-Aguirre et al. 2003).
  • 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.
  • 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.
  • 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.
  • branching 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.
  • 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.
  • X% mono-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).
  • 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).
  • 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).
  • 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.
  • 50 to 70% for example 60 to 69%, for example about 65%, of the total sialylation is a2,3-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.
  • 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).
  • 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).
  • 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.
  • FSH follicle stimulating hormone
  • 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.
  • 50 to 70% for example 60 to 69%, for example about 65%, of the total sialylation is a2,3- sialylation.
  • the term "recombinant FSH preparation" includes a preparation for e.g. pharmaceutical use which includes recombinant FSH.
  • the rFSH may be present as a single isoform or as a mixture of isoforms.
  • the FSH is a recombinant FSH ("rFSH" or "recFSH”).
  • 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).
  • 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).
  • 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.
  • 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).
  • % 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.
  • sialic acid content amount of sialylation per FSH molecule
  • 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).
  • 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).
  • 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.
  • 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.
  • recombinant FSH rFSH preparations; rFSH compositions
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ( ⁇ 2,6 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.
  • 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.
  • rFSH is produced using a2,3- sialyltransferase.
  • the rFSH may include Delinked sialic acids (a2,6 sialylation) provided by endogenous sialyl transferase activity.
  • a method of production of rFSH and/or an rFSH preparation as described herein 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.
  • 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.
  • 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).
  • the FSH includes ⁇ 2,3- 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.
  • 50 to 70% for example 60 to 69%, for example about 65%
  • of the total sialylation is a2,3- sialylation.
  • 25 to 50%, for example 30 to 50 %, for example 31 to 38%, for example about 35%, of the total sialylation is ⁇ 2,6- 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • recombinant FSH rFSH preparations; rFSH compositions
  • 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.
  • the rFSH is an FSH in which the 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) GalNAc.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • FSH follicle stimulating hormone
  • the glycans comprise (e.g. carry) bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc).
  • 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).
  • FSH follicle stimulating hormone
  • the glycans comprise (e.g. carry) bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc).
  • 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.
  • 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.
  • recombinant FSH rFSH preparations; rFSH compositions
  • 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.
  • the FSH is a recombinant FSH.
  • 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 ⁇ 2,3- sialylation.
  • the follicle stimulating hormone 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.
  • the FSH includes from 0.1 to 7% neutral glycan structures.
  • 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.
  • GalNAc N-Acetylgalactosamine
  • the rFSH is an FSH in which the 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) GalNAc.
  • the rFSH is an FSH in which the 40 to 55%, for example 42% to 52%, of the glycans comprise
  • GalNAc (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.
  • a follicle stimulating hormone wherein 20% or more of the glycans comprise (e.g. carry)
  • GalNAc a terminal GalNAc.
  • GalNAc Preferably 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc.
  • a pharmaceutical composition or a preparation comprising a follicle stimulating hormone (FSH) wherein 20% or more of the glycans comprise (e.g. carry) GalNAc.
  • FSH follicle stimulating hormone
  • FSH follicle stimulating hormone
  • glycans comprise (e.g. carry) GalNAc.
  • glycans Preferably 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc.
  • 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.
  • 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 FSH is a recombinant FSH.
  • 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 a 2,3- sialylation.
  • 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.
  • 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 follicle stimulating hormone 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.
  • the FSH includes from 0.1 to 7% neutral glycan structures.
  • 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 (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).
  • 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.
  • a recombinant follicle stimulating hormone 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.
  • a recombinant follicle stimulating hormone 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.
  • a pharmaceutical composition comprising a recombinant follicle stimulating hormone (FSH) wherein 16 to 24% of the glycans comprise (e.g. terminal) 1 fucose-lewis.
  • FSH recombinant follicle stimulating hormone
  • 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.
  • 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.
  • FSH follicle stimulating hormone
  • 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.
  • 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).
  • rFSH follicle stimulating hormone
  • 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.
  • 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.
  • 50 to 70% of the total sialylation is a2, 3-sialylation.
  • 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).
  • 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.
  • GalNAc N-Acetylgalactosamine
  • the rFSH is an FSH in which the 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) GalNAc.
  • 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.
  • 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.
  • a pharmaceutical composition e.g. recombinant follicle stimulating hormone (rFSH) wherein 20% or more of the glycans comprise (e.g. carry) GalNAc.
  • 20% or more of the glycans comprise (e.g. carry) a terminal GalNAc.
  • 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) GalNAc.
  • 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 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.
  • 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.
  • 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.
  • aqueous and non-aqueous pharmaceutical carriers, diluents, solvents or vehicles examples 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.
  • 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.
  • isotonic agents such as sugars, sodium chloride, and the like.
  • FSH FSH
  • other active ingredients if present
  • the rate of absorption of FSH then depends upon its rate of dissolution which, in turn, can depend upon crystal size and crystalline form.
  • 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.
  • the rate of FSH release can be controlled.
  • 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 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.
  • 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.
  • 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.
  • the parenteral compositions are supplied in dosage unit form for ease of administration and uniformity of dosage.
  • 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;
  • 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.
  • 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.
  • 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.
  • FSH alpha polypeptide AH007338, SEQ ID NO:1
  • FSH beta polypeptide NM_003032, SEQ ID NO:2
  • FSHa-fw and FSHa-rev and FSHb-fw and FSHb-rec 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' FSHa-rev 5'-GGATGGCTAGCTTAAGATTTGTGATAATAAC-3' (SEQ ID NO.10)
  • FSHb-fw 5'-CCAGGCGCGCCACCATGAAGACACTCCAGTTTTTC-3' 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.
  • 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).
  • 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.
  • the coding sequence of beta-galactosamide alpha-2,6-sialyltransferase 1 was amplified by PCR using the primer combination 2,6STfw and 2,6STrev.
  • Example 4 Stable expression of pFSH ⁇ + ⁇ 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).
  • a liposome based transfection agent with the pFSH ⁇ + ⁇ 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.
  • 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.
  • FSH from a2,3- clone (Example 5) was prepared using a using a modification of the method described by Lowry ef al. (1976).
  • PER.C6®-FSH 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 0.3x10 6 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% C0 2 .
  • Excell 525 JRH Biosciences
  • the cells were sub- cultured to a cell density of 0.2 or 0.3x10 6 cells/ml and further cultured in shaker flasks at 37°C, 5% C0 2 and 00 rpm.
  • VPRO serum-free production medium
  • VPRO JRH Biosciences
  • PER.C6®cells very high cell densities (usually > 10 7 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 1x10 6 cells/ml. The cells were then cultured in a shaker flask for 7-10 days at 37°C, 5% C0 2 and 100 rpm.
  • the cells grew to a density of > 10 7 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.
  • 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.
  • WAX Weak Anion Exchange
  • 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 ⁇ 2,6 sialylation.
  • 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).
  • WAX Weak Anion Exchange
  • 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.
  • 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.
  • WAX Weak Anion Exchange
  • 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 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).
  • Gonal F CHO cell derived recombinant FSH
  • Bravelle human urinary FSH
  • the amount of bisecting GlcNac in the FSH of the invention varies between 8.7 and 13.9%, averaging approximately at 10.9%.
  • the amount of 1 Fucose Lewis in the FSH of the invention varies between 16.1 and 23.3%, averaging approximately at 19%.
  • Example 9 A multiple dose study investigating the safety, tolerability, pharmacokinetics, pharmacodynamics, and immunogenicity of FE 999049 in comparison to GONAL-F. Study population
  • 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%.
  • FE 999049 and GONAL-F were 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.
  • AEs Adverse Events
  • vital signs vital signs
  • ECG ECG
  • clinical laboratory measurements ECG
  • physical examination No serious adverse event or death occurred during the study.
  • 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.
  • Timossi CM Barrios-de-Tomasi J, GonzaJez-Suarez R, Arranz MC, Padmanabhan V, Conn PM, and Ulloa-Aguirre A. (2000). Differential effects of the charge variants of human follicle-stimulating hormone. J Endocrinol. 165(2), 93-205.
  • Protein sequence of FSH alpha (SEQ ID NO:5) 1 MDYYRKYAAI FLVTLSVFLH VLHSAPDVQD CPECTLQENP FFSQPGAPIL QC GCCFSRA
  • Beta-galactosamide alpha-2,6-sialyltransferase 1 Accession number NM 003032 Nucleotide sequence of ST6GAL1

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Abstract

L'invention concerne des préparations incluant de la FSH (hormone folliculostimulante), par exemple de la FSH recombinante, 17% à 23% de structures de type glycane sialylé étant des structures de type glycane tétrasialylé.
EP12730594.4A 2011-06-06 2012-06-06 Préparation pharmaceutique Withdrawn EP2717904A1 (fr)

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EP12730594.4A EP2717904A1 (fr) 2011-06-06 2012-06-06 Préparation pharmaceutique

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EP11168816 2011-06-06
EP12730594.4A EP2717904A1 (fr) 2011-06-06 2012-06-06 Préparation pharmaceutique
PCT/GB2012/000491 WO2012168680A1 (fr) 2011-06-06 2012-06-06 Préparation pharmaceutique

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TWI488640B (zh) 2008-04-16 2015-06-21 Ferring Int Ct Sa 藥學製劑
US20180264086A1 (en) * 2015-09-17 2018-09-20 Glycotope Gmbh Mammalian follicle-stimulating hormone composition with increased stability
IL272763B2 (en) 2017-09-01 2024-02-01 Ferring Bv The composition for controlled stimulation of the ovary

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MEP38608A (en) * 2001-10-22 2011-02-10 Merck Serono Sa Gonadotrophins for folliculogenesis
US8609370B2 (en) * 2004-02-13 2013-12-17 Glycotope Gmbh Highly active glycoproteins-process conditions and an efficient method for their production
TWI488640B (zh) 2008-04-16 2015-06-21 Ferring Int Ct Sa 藥學製劑
TWI532495B (zh) * 2009-10-05 2016-05-11 菲瑞茵國際中心股份有限公司 藥學製劑
WO2012016576A1 (fr) 2010-08-04 2012-02-09 Glycotope Gmbh Hormone folliculostimulante humaine recombinante améliorée

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