HK1071901A - Gonadotrophins for folliculogenesis - Google Patents

Description

Gonadotropins for folliculogenesis

Technical Field

The present invention relates to the field of gonadotropins, in particular their use in assisted reproductive technology, ovulation induction, intrauterine artificial insemination (IUI) and infertile male patients.

Background

Gonadotropins are a group of heterodimeric glycoproteins including Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) and Chorionic Gonadotropin (CG). These hormones regulate the reproductive function of both men and women.

Each of these hormones consists of two non-covalently linked subunits: one is an alpha-subunit common to FSH, LH and hCG, and the other is a beta-subunit unique to each hormone and conferring biospecificity to the individual hormones.

In all gonadotropins, each subunit has asparagine-linked (N-linked) oligosaccharide side chains. In the common alpha-subunit of the human hormone, they are linked at positions 52 and 78. In human FSH and CG, two N-linked oligosaccharide side chains are attached to the β -subunit, the attachment positions for FSH are 7 and 24, and the attachment positions for hCG are 13 and 30. In human LH, one oligosaccharide is attached to the β -subunit at position 30. hCG has four additional serine-linked (O-linked) oligosaccharide side chains located at the carboxy-terminal portion.

Like all glycoproteins, the oligosaccharide structure within gonadotropins changes, producing a number of isoforms found within the pituitary gland and in the circulation. In addition, the degree of "capping" of the terminal carbohydrates with sialic acid varies. Isoforms can be separated based on the charge they carry, which is determined primarily by the number and distribution of sialylated N-linked oligosaccharides. The highly sialylated form is more acidic than the average pl, referred to as "acidic". The lower sialylated form has a higher pl, termed "basic".

Due to their structural differences, gonadotropin isoforms also differ in their ability to bind to target cell receptors. The degree of sialylation affects their ability to survive in circulation. In the case of FSH, some groups have demonstrated that highly acidic/sialylated isoforms have a relatively long plasma half-life in animal models such as mouse and rat models⁽¹⁾。

It has been found that the patterns of endogenous FSH isoforms are different in humans. The acidic isoforms have a longer half-life in vivo and a lower in vitro bio-potency, and are mainly present in pre-pubertal children, hypogonadal patients and women in follicular phaseIn the serum of (1). In contrast, less sialylated, more basic isoforms have shorter half-lives in vivo and higher biological activities in vitro, and are found during adolescence, GnRH treatment, and around the mid-peak gonadotropin in women⁽²⁾。

FSH isoforms with higher sialic acid content have longer circulation time because the terminal sialic acid residues "cap" the galactose residues, thereby preventing interaction with the hepatic asialoglycoprotein receptor and preventing escape from the circulation⁽³⁾。

The oligosaccharide (glycan) moiety attached to the protein is branched, and each terminal sugar residue is called an antenna. The value of the parameter Z is a measure of how much of the carbohydrate moiety in the glycoprotein has charged residues in the antenna, e.g., sialic acid. The Z value of desialylated FSH is 0. The Z value of fully sialylated FSH is between 230 and 280.

Efficacy of FSH preparations was assessed in vitro using the Steelman-Pohley assay, which compares the ability of one preparation to increase ovarian mass in immature rats under specific conditions with an international standard/reference preparation, calibrated in International Units (IU)⁽⁴⁾。

The role of glycosylation and sialylation in influencing the FSH biological profile has been investigated by a number of groups.

D' Antonio et al assessed the metabolic clearance of acidic (pl < 4.8) and basic (pl > 4.8) rhFSH isoforms in female rats by chromatographic aggregation. As predicted, the basic isoform was found to clear faster than the acidic isoform (basic half-life 0.4 hours, acidic 0.9 hours). When comparing the acidic and basic forms in the Steelman-Pohley assay, it was found that the basic isoform is much less active than the acidic isoform (basic: ED)₅₀0.9 μ g/rat, acidic: ED (electronic device)₅₀0.3 μ g/rat). When these isoforms are compared on an international unit basis, there is no difference between them⁽⁵⁾。

An in vitro study was performed by Vitt et al, in which four were comparedThe ability of recombinant human FSH preparations of different pl to promote increased follicle size and estradiol production in isolated mice. As a result, basic FSH (pl ═ 5.0 to 5.6) was found to grow faster and to give maximum follicle size, and thus unfractionated recombinant FSH. Intermediate (pl ═ 4.5-5.0) and acidic (pl ═ 3.6-4.6) FSH preparations were inferior in both follicular growth rate and maximum size. Basic FSH induces estradiol secretion at a lower dose at an earlier stage than other isoforms. Follicles grown with acidic FSH, regardless of the concentration of acidic FSH, secrete measurable concentrations of estradiol only after a prolonged incubation period⁽⁶⁾。

Timossi et al used a chromatographic aggregation procedure to isolate 7 different components with different glycosylation/acidity from human pituitary FSH. These fractions were tested for their ability to cause upregulation of aromatase (required for estradiol production) and tissue plasminogen activator (tPA) expression in rat granulocytes in vitro. As a result, it was found that the ratio of biological activity to immunoreactivity (B/I) decreased with a decrease in pH of the model elution. The authors concluded that: the alkaline isoform has a higher ability to induce the expression of aromatase and tissue-type plasminogen activator mRNA's and proteins than the acidic variant⁽⁷⁾。

Zambrano et al used chromatographic aggregation to isolate 9 fractions with different pl from human pituitary FSH and tested the acidic and basic isoforms using three immunoassays and two in vitro assays: estradiol is produced by rat granulocytes and cAMP is produced by human fetal cell lines expressing the FSH receptor. The ratio of activity to immunoreactivity (B/I) in the bioassay was reduced in all bioassays with a decrease in type pl⁽⁸⁾。

Zambrrano et al, in another study, compared the binding affinities of 7 components of the acidic and basic isoforms of human pituitary FSH in the heterologous receptor system (rat granulocytes) and the homologous receptor system (recombinant human HEK-293 cells expressing the human FSH receptor). The binding affinity of the heterologous receptor increases with increasing type pl, which is not the case for the homologous receptor. cAMP production in HEK-293 cells also increases with increased isoform pI⁽⁹⁾。

Studies have shown that the more acidic FSH form is the most biologically active (by mass) in vivo when evaluated in a typical ovarian weight gain assay^(10，11). Timossi et al concluded that the basic form may be more active in vivo, but because of the shorter half-life, no effect on ovarian weight gain in rats was observed. They examined the effect of two formulations on a fast-reacting system: up-regulation of tissue plasminogen activator activity⁽¹²⁾. The authors concluded that: compared with the strong acid FSH preparation, the rhFSH with less acidic charge distribution has higher in vitro bioactivity and plasma clearance rate, and can more quickly induce the enzyme activity of the tissue plasminogen activator.

Gonadotropins play a key role in the reproductive cycle and their use is essential for assisted reproductive technologies such as In Vitro Fertilization (IVF) or IVF in combination with intracytoplasmic single sperm injection (IVF/ICSI) and Embryo Transfer (ET), as well as for inducing ovulation in anovulatory patients undergoing in vivo fertilization either naturally or by intrauterine artificial fertilization (IUI).

Assisted reproductive techniques typically use Controlled Ovarian Hyperstimulation (COH) to increase the number of gametes in women⁽¹³⁾. Standard protocol for COH⁽¹⁴⁾Comprising a down-regulation phase in which endogenous gonadotropins are suppressed by administration of a gonadotropin releasing hormone (GnRH) agonist, followed by a stimulation phase in which FSH is administered daily, typically about 150-225 IU/day, to induce follicular development (folliculogenesis). Another option is to start the stimulation after natural or induced menstruation, (usually starting at about day 6 of the stimulation phase), by administering a GnRH antagonist to prevent the appearance of LH surges at inappropriate times. When at least 3 follicles > 16 mm (one of which is 18 mm), a single bolus administration of hCG (5-10,000IU) mimics the natural LH surge and initiates ovulation. The time to recover oocytes was determined 36-38 hours after hCG injection.

In general, FSH is administered at a daily dose of about 75-150IU to induce ovulation. The down-regulation can be performed using GnRH agonists and antagonists, although less frequently than assisted reproductive technology indices. Administration of hCG prior to in vivo fertilization mimics a LH surge, which can be achieved by periodic intercourse or IUI.

The typical protocol described above for assisted reproductive techniques and ovulation induction requires daily injections of gonadotropin for a prolonged period of time, i.e. on average 10 days, and for some patients up to 20 days. Development of FSH preparations with higher efficacy may reduce the daily dose of FSH, and/or may shorten the treatment period (i.e. less volume injected), and/or may reduce the number of injections. Thereby facilitating the assisted reproduction technology and ovulation induction scheme and being more beneficial to patients.

In addition, assisted reproductive techniques using in vitro fertilization may be accompanied by unfortunate events. For example, not every follicle can produce a viable oocyte, nor can every viable oocyte be successfully fertilized, and some embryos may not survive. In addition, transfer to the uterus and implantation may also be unsuccessful once viable embryos are selected. To maximize the chances of a safe birth of an infant, it is desirable to stimulate the growth and maturation of multiple follicles to ensure that multiple oocytes are harvested.

In contrast, the aim of ovulation induction is to obtain no more than 3, preferably 1, dominant follicles (to avoid multiple pregnancy).

For some patients receiving assisted reproductive techniques and ovulation induction, the number of follicles growing will be reduced when treated with conventional FSH preparations. This is a limiting factor in the success of assisted reproduction techniques, as it limits the number of embryos that can be transferred and/or cryopreserved. It is also a limiting factor in success for patients undergoing IUI (where access to more than one follicle is critical). Patients who develop this response include those over the age of about 33 to 35 years, those with an elevated baseline FSH, an elevated baseline estradiol or a reduced baseline inhibin b.

Spermatogenesis in males is dependent on stimulation of sertoli cells by FSH. Lack of FSH can cause oligospermia leading to infertility. Treatment of male infertility with conventional FSH preparations requires 3 injections of FSH a week for a maximum of 18 months.

The development of FSH preparations with a higher capacity to stimulate folliculogenesis has been sought. Furthermore, it is also a continuing desire to obtain new FSH preparations which attenuate the response to FSH when treating patients. It would also be desirable to produce FSH preparations with higher efficacy, thereby reducing the time of assisted reproductive technologies, ovulation induction and male infertility treatment regimens and/or reducing the cumulative dose and/or reducing the number of administrations.

Disclosure of Invention

It is an object of the present invention to provide a gonadotropin preparation for ovulation induction and COH, particularly in combination with assisted reproductive techniques.

In a first aspect of the invention there is provided an FSH preparation wherein the Z-value of the preparation is at least 200 or about 200.

A second aspect of the invention provides an FSH preparation, wherein the average pl of the preparation is less than 3.4 or 3.4.

In a third aspect the present invention provides a pharmaceutical composition comprising FSH wherein the Z-value of the FSH is at least 200 or about 200.

A fourth aspect of the invention provides the use of FSH for stimulating folliculogenesis, wherein the Z-value of FSH is at least 200 or about 200.

A fifth aspect of the invention provides the use of FSH for the preparation of a medicament for the stimulation of folliculogenesis, wherein the Z-number of FSH is at least 200 or about 200.

In a sixth aspect, the invention provides a method of inducing follicular development in a human patient, the method comprising administering FSH to the patient, wherein the Z-value of the FSH is at least 200 or about 200.

A seventh aspect of the invention provides a method of preparing an FSH preparation having a Z-value of at least 200 or about 200, the method including the steps of:

reacting FSH with a sialic acid donor in the presence of 2, 3-sialyltransferase;

selecting a suitable cell type for expression of recombinant FSH;

culturing cells, preferably recombinant cells, expressing FSH under conditions which favour high levels of sialylation;

separating the FSH isoforms with higher Z values by chromatographic techniques.

An eighth aspect of the invention provides the use of FSH for the treatment of male infertility, wherein the Z-number of FSH is at least 200 or about 200.

A ninth aspect of the invention provides the use of FSH for the preparation of a medicament for the treatment of male infertility, wherein the Z-number of the FSH is at least 200 or about 200.

A tenth aspect of the invention provides a method of treating male infertility in a human patient, the method comprising administering FSH to the patient, wherein the Z-number of the FSH is at least 200 or about 200.

Drawings

FIG. 1 is a chromatogram showing elution of glycans released from rFSH using a GlycoSep * C column; column 4.6X 100mM, packed with divinylbenzene resin (5 m) with polymer layer, mobile phase 20: 80 acetonitrile: water, linear gradient of ammonium acetate (500mM) per minute 0.25% during 5 min to 21 min and 0.525% during 21 min to 61 min. The X-axis represents retention time in minutes and the Y-axis represents signal intensity in millivolts (mV).

Figure 2 shows the number of follicles per size grade (Y-axis) on day 8 for patients receiving FSH acidic and basic isoforms up to day 7. The wavy line indicates the results for the acidic isoform and the diagonal line indicates the results for the basic isoform.

Figure 3 shows the number of follicles per size grade (Y-axis) on day 10 for patients receiving FSH acidic and basic isoforms up to day 7. The wavy line indicates the results for the acidic isoform and the diagonal line indicates the results for the basic isoform.

Figure 4 shows the mean FSH serum levels in patients after the last dose of FSH acidic and basic isoforms. The X-axis represents the time after the first FSH injection in hours, and the Y-axis represents the serum concentration of the immune response in IU/L. Square () is the serum concentration after injection of the acidic isoform; diamond () is the serum concentration after injection of the FSH basic isoform. Serum concentrations were determined by immunoassay, such as radioimmunoassay, using a kit provided by daiichi isotope Laboratory, japan.

Figure 5 shows the amino acid sequence of the alpha-subunit of mature human FSH.

Figure 6 shows the amino acid sequence of the β -subunit of mature human FSH.

Detailed Description

The present inventors have surprisingly found that the highly sialylated FSH isoforms have a better efficacy in inducing follicle production in human patients than the lower sialylated isoforms. The same or better clinical effect can be achieved with a lower cumulative dose of FSH using the FSH preparations of the present invention.

The present inventors have found that when a patient is treated with the same amount of acidic FSH and basic FSH (in IU), the number of growing follicles in a patient treated with acidic FSH is greatly increased.

As determined by routine experimentation, the number of growing follicles in patients treated with acidic FSH also increases significantly when the patients are treated with the same amount (by mass) of acidic FSH and basic FSH.

The number of growing follicles in some patients decreased when treated with conventional FSH preparations. This is the limiting factor in the success of assisted reproductive techniques. Patients exhibiting this response include those aged about 33 to 35 years old or older, those with elevated baseline FSH, elevated baseline estradiol or reduced baseline inhibin b. The FSH preparations of the present invention may be injected 1 time per day, or 1 time every other day, to elicit a better ovarian response than conventional preparations. Thus, the chances of these patients becoming pregnant are increased.

The inventors have also surprisingly found that such FSH preparations having better efficacy and reduced number of administrations may be prepared with FSH having a Z-value of at least 200 or about 200, preferably at least 210 or about 210, 220 or about 220, 230 or about 230, 240 or about 240, 250 or about 250, 260 or about 260, 270 or about 270, 280 or about 280 and 290 or about 290, in increasing order of preference (Z-values between these values are of course within the scope of the invention). To induce folliculogenesis, a conventional FSH preparation is generally administered at a daily dose of about 75-600 IU/day. For most patients, the same cumulative dose of conventional FSH preparations may be administered every other day in order to achieve the same clinical effect as daily injections⁽¹⁵⁾. The term "less frequent administration" means that the FSH preparation can be administered less frequently than every other day, and the same clinical effect as that obtained by daily or every other day administration of the conventional preparation in terms of total follicular volume is obtained.

The terms "acidic" and "basic" are commonly used to refer to FSH preparations having different degrees of sialylation. Since sialic acid is acidic, the higher the sialylation degree of the molecule, the smaller the pl. Using isoelectric focusing, chromatographic focusing, or other separation methods such as ion exchange chromatography, Fast Protein Liquid Chromatography (FPLC) and High Performance Liquid Chromatography (HPLC)⁽¹⁶⁾The mixture of isoforms may be separated into acidic or basic components, preferably based on the Z value.

The term "sialic acid" refers to any member of any family of nine-carbon carboxylated sugars. The most commonly used member of the sialic acid family is N-acetylneuraminic acid (2-keto-5-acetamido-3, 5-dideoxy-D-glyceryl-D-galactononopyranose-1-oic acid, (2-keto-5-acetamido-3, 5-dideoxy-D-glycerol-D-galactononopyranose-1-oic acid, commonly abbreviated as N-acetylneuraminic acidNeu5Ac, NeuAc, or NANA)). The second member of this family is N-glycolylneuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl group of NeuAc is hydroxylated. The third member of the sialic acid family is 2-keto-3-deoxy-nonanone sugar acid (KDN)⁽¹⁷⁾. In addition, some other 9-substituted sialic acids are included, such as 9-O-C, e.g. 9-O-lactoyl-Neu 5Ac or 9-O-acetyl-Neu 5Ac₁-C₆-acyl Neu5Ac, 9-deoxy-9-fluoro-Neu 5Ac and 9-azido-9-deoxy-Neu 5 Ac. For an understanding of the sialic acid family, see, e.g., Varki; glycobiology 21992; 25-40; sialic Acids: chemistry, Metabolism and Function, R.Schauer, Ed. (Springer-Verlag, New York (1992)).

The carbohydrate (also called "glycan") moiety is attached to the peptide backbone through a monosaccharide, or through an O-or N-linked glycosidic linkage. When the carbohydrate moiety is processed, branching occurs, resulting in 1, 2, 3, or 4 (and sometimes more) terminal sugar residues or "antennae" in the carbohydrate moiety. These carbohydrate moieties are designated as mono-, di-, tri-or tetra-chain. The parameter Antennal Index (AI) is a measure of the degree of branching of the carbohydrate residues and is also an index of the three-dimensional size of the carbohydrate moiety. To determine this parameter, a glycoprotein is chemically treated to release all carbohydrate residues, for example by heating with hydrazine, or the carbohydrate can be enzymatically cleaved, for example with an endoglycosidase (N-glycanase)⁽¹⁸⁾. The carbohydrate mixture is isolated. If desired, the carbohydrate mixture is reacted with a label, such as a radioactive label, a chromogenic label (i.e., a UV-visible active), a fluorescent label, an immunoreactive label, or the like. The labeled carbohydrate mixture is then subjected to a desialylation reaction with a sialidase to produce a labeled neutral carbohydrate mixture (alternatively the order of labeling and desialyzing steps may be reversed). The fractions are separated from the labeled neutral carbohydrate mixture by chromatography which distinguishes the different species (mono-, di-, tri-or tetra-chain). Chromatography (normal or reverse phase) can be carried out using essentially any method, including, for example, thick or thin layer chromatography, or HPLC. In addition, the first and second substrates are,the separated neutral carbohydrate mixture may be reacted with a reagent to render the components volatile, and the mixture may be subjected to Gas Chromatography (GC). Visualization can be achieved by methods appropriate to the labels and chromatography used. For example, if a fluorescent label is used, it can be detected by a fluorometer; if a chromogenic label is used, it can be detected with an ultraviolet-visible spectrophotometer. If not labeled, mass spectrometry can be used to measure the peak and residence time. Peaks of different species of mono-, di-, tri-or tetra-branched chains can be identified by mass spectrometry or by comparison with known standards.

The peaks associated with the two, three, and four branched carbohydrates in the chromatogram were then combined for analysis. The AI is calculated by substituting the percentage of each species in total carbohydrate into the following equation:

AI＝2P_di+3P_tri+4P_tetra

wherein AI is the antenna index, P_di、P_triAnd P_tetraThe percentages of di-, tri-, and tetra-branched carbohydrates in total carbohydrate are expressed, respectively. The remaining amount of other components (e.g., an antenna) may be present but will not have a significant effect on the AI value.

A high antennal index indicates a high degree of branching of the carbohydrate moiety, with many antennals. The AI value of recombinant human FSH is typically about 220-280, or on average about 255.

The value of the parameter Z measures how many antennae in the carbohydrate moiety of a glycoprotein have charged residues, such as sialic acid. To determine the Z value, the carbohydrate moiety is released from the peptide and, if desired, labeled, as described above. The mixture was subjected to ion exchange chromatography to separate the various species based on charge. The eluted peaks are visualized by means of the above-mentioned markers, or by some other method such as mass spectrometry. The peaks associated with the one-, two-, three-, and four-charged carbohydrate species in the chromatogram were then combined for analysis. The Z value was calculated by substituting the percentage of each species in total carbohydrate into the following equation:

Z＝P’_mono+2P’_di+3P’_tri+4P’_tetra

wherein Z is a Z number, P'_mono、P’_di、P’_triAnd P'_tetraRepresenting the percentage of carbohydrates with one, two, three, and four charges, respectively, to the total carbohydrate.

A high value of Z indicates that many antennae have charged residues, and thus the glycoprotein is highly charged and acidic in the case of sialic acid residues. The Z value of recombinant human FSH is generally from about 150 to about 190, or on average about 184.

The present inventors have surprisingly found that FSH isoforms with Z-values above 200 or about 200 are more effective in terms of number of follicles than FSH isoforms with a "same dose" (in IU) of Z-values below 200. By "same dose" is meant that the IU dose is the same when measuring the amount of FSH of different isoforms in a conventional in vivo assay (by comparing the ability to increase ovarian mass in rats in vivo under specific conditions). In other words, there are different clinical effects when different isotypes, determined as the same IU dose in vivo in rats, are administered to humans.

FSH preparations with increasing Z values may be isolated by any means. For example, a batch of recombinant FSH may be subjected to isoelectric focusing, or to chromatographic focusing, e.g., Mulders et al⁽¹⁹⁾Zambrano et al⁽²⁰⁾And Timossi et al⁽²¹⁾The method as set forth in one of them. Fractions with different average pl values can also be isolated. Preferred FSH preparations of the invention have a pl average of less than 3.4 or about 3.4, more preferably less than 3.3 or about 3.3, particularly preferably less than 3.2 or about 3.2, with the preferred degree increasing with decreasing pl average.

The Z value parameter reflects the average degree of sialylation of a batch of FSH. There may be cases where: FSH preparations with high Z values may still have a significant proportion of basic (less sialylated) species. Such basic species may act as antagonists of the FSH receptor and are therefore undesirable. The "spreading" of an included species can be determined by isoelectric or chromatographic aggregation. The Z value is analyzed to know the extension of each kind. The formulation suitably contains less than 4% or about 4% neutral carbohydrate species (i.e. the glycan moiety is uncharged), and less than 16% or about 16% mono-sialylated species, whereas the formulation preferably contains less than 3% or about 3%, less than 2% or about 2%, or less than 1% or about 3% neutral carbohydrate species, and less than 15% or about 15%, less than 12% or about 12%, less than 10% or about 10%, less than 8% or about 8%, or less than 5% or about 5% mono-sialylated species, preferably the degree increases with decreasing percentage.

FSH preparations with increased folliculogenesis efficacy due to increased sialylation of one or more additional glycosylation sites on the protein are within the scope of the present invention. Substitution of residues in the FSH protein backbone by serine, threonine, lysine or asparagine residues, for example, by mutagenesis techniques, may be introduced at such sites. Example 7 gives an example of the method that can be used to generate these mutant forms of FSH. For in vivo glycosylation, the site introduced should be an "N-glycosylation site" that forms the following sequence: N-X ' -S/T/C-X ", wherein X ' is any amino acid residue other than proline, X" is any amino acid residue which may be the same or different from X ', preferably not proline, N is asparagine, and S/T/C denotes a residue which may be serine, threonine or cysteine, preferably serine or threonine, most preferably threonine. The acidic isoform (pl ≦ 3.4) of these FSH molecules is within the scope of the present invention. These modified FSH molecules with additional glycosylation sites are described, for example, in WO 01/58493 (Maxygen). The following mutations are particularly preferred:

in the β -subunit: E4N, A70N, L73N, V78N, G100N, Y103N, F19N/121T, L37N/Y39T, D41N/A43T, E55N/A43T, E59N/V61T and R97N/L99T;

in the α -subunit: E9N, F17T, F17N, R67N, V68T, E56N, H83N, and F33N/R35T;

wherein A is alanine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is glycine, H is histidine, I is isoleucine, L is leucine, N is asparagine, R is arginine, T is threonine, V is valine, Y is tyrosine, and the symbol "E4N" indicates the substitution of glutamic acid (E) at position 4 with asparagine (N). For sequence numbering, the amino acid sequence of human FSH α is as shown in fig. 5 or SEQ ID NO: 1, mature sequence number. The amino acid sequence of human FSH β is as shown in fig. 6 or SEQ ID NO: 2, mature sequence number shown in seq id no.

FSH preparations with increased folliculogenesis efficacy due to increased sialylation of one or more additional glycosylation sites on the accessory peptide are also within the scope of the present invention. By "accessory peptide" is meant any peptide that includes a glycosylation site and is linked to the amino and/or carboxy terminus of the α -and/or β -subunit of FSH without adversely affecting the FSH activity of the resulting molecule. For example, the β -subunit of hCG is much larger than the β -subunits of other gonadotropins, since the approximately 34 additional amino acids on the C-terminus referred to herein constitute the Carboxy Terminal Portion (CTP). In urinary hCG, CTP contains 4 mucin-like O-linked oligosaccharides. The CTP may be linked to the β -subunit of FSH, preferably at the carboxy terminus of the β -subunit of FSH, resulting in a molecule with FSH activity and four additional glycosylation sites. The acidic isoforms of these FSH molecules (pl ≦ 4.4) are within the scope of the present invention. WO 93/06844(Washington University) and Boime et al⁽²²⁾Such molecules are also disclosed. WO 90/09800(Washington University) discloses other FSH molecules with modified glycosylation sites.

In the present context, FSH preparations with additional glycosylation sites will be referred to as FSH^gly+To indicate. When additional glycosylation sites are added, the value of the parameter Z, which is the sum of the percentages, is normalized, so that this parameter can no longer be compared with "normal" FSH preparations (i.e. those with four glycosylation sites). When it is against FSH^gly+When the preparation is used for analyzing the glycan species, the parameter Z can be calculated by referring to the calculation method of the Z value⁺And (4) counting. FSH of the invention^gly+Z of the preparation⁺A number greater than 200 or about 200, preferably greater than 210 or about 210, greater than 220 or about 220, greater than 230 or about230. Greater than 240 or about 240, greater than 250 or about 250, greater than 260 or about 260, greater than 270 or about 270, with degrees of preference depending on Z⁺The number increases and increases.

FSH of the invention^gly+The pl value of the formulation is much lower than normal FSH. Particularly preferred FSH^gly+Formulations are those with improved efficacy and have a pl average of less than 4.4 or about 4.4, more preferably a pl average of less than 4.2 or about 4.2, less than 4.0 or about 4.0, less than 3.8 or about 3.8, less than 3.6 or about 3.6, less than 3.4 or about 3.4, less than 3.3 or about 3.3, and less than 3.2 or about 3.2, with the preferred levels increasing with decreasing pl average.

All embodiments of the invention preferably use recombinant FSH. Preferably, human recombinant FSH is used for treatment of human patients. The formulations of the invention may be isolated from conventional recombinant FSH, or they may be isolated from FSH^gly+Separating the preparation.

The present invention also provides a method for increasing sialic acid content using what is termed a "sialic acid boosting" method. Treatment of recombinant FSH (preferred) or recombinant FSH with an enzyme (e.g. a glycosyltransferase, especially a sialyltransferase) in the presence of a sialic acid donor (e.g. CMP-sialic acid) as described in WO 98/31826(Cytel Corporation)^gly+Preparations (also preferred) or urinary FSH, may be used to fortify sialic acid. For example, U.S. Pat. No. 5,541,083(University of California; Amgen) describes examples of recombinant sialyltransferases and methods of producing recombinant sialic acid. At least 15 different mammalian sialyltransferases have been described to date, of which 13 cDNAs have been cloned. These cDNAs can be used to recombinantly produce sialic acid, which can be reused in the methods of the invention.

The sialyltransferases used are capable of transferring sialic acid to the sequence Gal β 1, 4GlcNAc, which are the most common penultimate moieties on which terminal sialic acids on sialylated glycoproteins are based. A sialyltransferase which can be used is ST3Gal III, which is also known as (2, 3) -sialyltransferase (EC 2.4.99.6). This enzyme catalyzes the transfer of sialic acid to Gal-beta-1, 3-glycosylOn Gal of NAc or Gal-beta-1, 4-glycosylNAc glycosides⁽²³⁾. Sialic acid is linked to a galactose (Gal) residue, forming an alpha linkage between the two sugars. The intersugar linkage is between position 2 of NeuAc and position 3 of Gal. The specific enzyme can be isolated from rat liver⁽²⁴⁾(ii) a Human cDNA⁽²⁵⁾And genome⁽²⁶⁾DNA sequences are well known and thus facilitate the production of such enzymes by recombinant expression. In a preferred embodiment, the sialylation process uses ST3Gal III (preferably from rat), ST3Gal IV, ST3Gal I, ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAc I, or ST6GalNAc II, more preferably ST3Gal III, ST6Gal I, ST3GalIV, ST6Gal II, or ST3Gal V, and especially preferably ST3Gal III from rat.

The amount of sialyltransferase is preferably in the range of 50 or about 50mU or less per mg FSH, more preferably in the range of 5-25 or about 5-25mU per mg FSH. Preferably, the concentration of sialyltransferase is from 10 to 50 or about 10 to 50mU/ml and the concentration of FSH is 2 or about 2 mg/ml.

Transfection of recombinant cells or other cells expressing FSH with a gene encoding sialyltransferase, in which the gene is expressed, may result in FSH enriched in the acidic isoform. The gene may contain genomic coding sequences (i.e., introns) or it may contain cDNA coding sequences. Alternatively, a construct capable of causing expression of FSH may be inserted into the genome of a cell if the genome of the cell contains endogenous sequences encoding sialyltransferases. Expression of sialyltransferases may be increased by insertion of a non-natural regulatory sequence active in the cell and operably linked to an endogenous sequence encoding the sialyltransferase. An amplification gene operably linked to a sequence encoding sialyltransferase can also be inserted to effect amplification of the genomic sialyltransferase coding sequence. These manipulations can be carried out by homologous recombination methods, see for example EP 0505500(Applied Research Systems ARSHOLDING N.V.).

The degree of sialylation of the FSH preparation may also be increased by selecting cells expressing recombinant FSH which are known to favour sialylation. These cells include selected pituitary cells and Chinese Hamster Ovary (CHO) cells that express high levels of sialyltransferase. The FSH preparation prepared in such a cell may also be used to isolate the highly sialylated isoforms using the separation method described above.

Culturing cells expressing FSH, preferably recombinant FSH, under conditions conducive to high levels of sialylation may also increase the degree of sialylation of the FSH preparation. Sialylation may be facilitated by the addition of neuraminidase and/or a direct intracellular precursor for the synthesis of sialic acid, such as acetylmannosamine, to the culture broth. Preparation of FSH prepared under these culture conditions the highly sialylated isoforms may also be isolated using the isolation methods described above.

If a sialic acid potentiating approach is used, it is desirable that the FSH preparation has a higher AI prior to enzymatic sialylation, thereby providing a number of antennae for the attachment of sialic acid residues. The AI of the FSH should preferably be greater than 220 or about 220, more preferably the AI is greater than 240 or about 240, and particularly preferably the AI is greater than about 270. For example, FSH with higher AI can be isolated by affinity chromatography on concanavalin-A (Con-A) derivatized agarose (methyl glucose gradient as eluent), or by preparative HPLC.

The method for enhancing sialic acid according to the present invention is suitably applied to FSH preparations which have been modified to introduce one or more additional glycosylation sites (FSH)^gly+Formulation). These FSHs^gly+The preparation may also be used to isolate a component with a higher AI prior to the sialic acid fortification.

The present invention includes FSH preparations prepared by expressing FSH in cells that are unable to sialylate and then subjecting the FSH to sialic acid fortification. For example, WO 99/13081(Akzo Nobel n.v.) describes the expression of wild type FSH and muteins in unicellular eukaryotic dictyostelium, particularly muteins with additional glycosylation sites. Dictyostelium does not sialylate glycans. The present invention includes FSH preparations prepared by subjecting wild-type FSH or a mutein expressed in dictyostelium to sialic acid fortification.

After sialic acid fortification, FSH preparations with the desired degree of sialylation may be isolated by ion exchange chromatography, isoelectric aggregation, chromatographic aggregation, or concanavalin-A (Con-A) chromatography.

The Z value of the FSH of the invention is at least 200 or about 200, more preferably at least 210 or about 210, particularly preferably at least about 220, most preferably at least about 230, 240, 250, 260 or 270, with increasing Z values being preferred. The Z value of fully sialylated FSH is 230 or about 230 to 280 or about 280, depending on the antennal index. The Z value of the very preferred FSH preparations of the present invention is 230 or about 230 to 280 or about 280.

The FSH preparations of the present invention are formulated to consistently have a Z value of at least 200 or about 200, or the preferred Z values as described above. The FSH of the present invention may be isolated from a mixture of isoforms by a number of methods well known to those skilled in the art. For example, isotypes can be separated on the basis of pl using isoelectric focusing, chromatographic focusing, or ion exchange chromatography. The different fractions are analyzed for sialic acid content and the desired fraction is selected. Examples of suitable conditions for ion exchange chromatography are given in the examples. These separation methods may be used to isolate the FSH of the invention from conventionally produced rFSH or urinary FSH (ufsh), or the desired isoform may be isolated from FSH by sialyltransferase treatment or other recombinant techniques as mentioned above.

In one aspect the invention provides a pharmaceutical composition comprising FSH of the invention (i.e. a Z value of at least 200 or about 200, preferably the minimum Z value is as set out above). Such pharmaceutical compositions may be used in conjunction with techniques such as ovulation induction or assisted reproduction to stimulate folliculogenesis. Because the FSH of the present invention is particularly effective in inducing multiple follicular development and maturation, it is particularly useful in assisted reproductive technologies where it is desirable to harvest multiple oocytes.

Alternatively, by careful selection of appropriate doses, the FSH of the invention may be used to induce the production of a single follicle in ovulation induction, or a small number of follicles (up to about 3 follicles) in IUI for in vivo fertilization. The production of a single follicle can be achieved by either decreasing the dose of FSH or decreasing the number of doses administered compared to conventional FSH preparations. For example, in ovulation induction, the FSH preparation of the invention may be administered 225-400IU every 3 days, or less, depending on the patient response. The patient's response can be determined by ultrasonography.

The FSH of the present invention is typically formulated as a pharmaceutical composition which further comprises a diluent or excipient. Those skilled in the art are aware of the various diluents or excipients that are suitable for use in formulating pharmaceutical compositions.

The FSH of the present invention is generally formulated in unit doses in solid form which are readily dissolved to form sterile injectable solutions suitable for intramuscular or subcutaneous use. The solid is typically prepared by lyophilization. Typical excipients and carriers include sucrose, lactose, sodium chloride, buffers such as sodium dihydrogen phosphate and sodium hydrogen phosphate. Injectable solutions can be prepared by dilution with water just prior to use.

The FSH of the present invention may also be formulated as an injectable solution, which includes any of the excipients and buffers listed above, as well as other excipients and buffers known to those skilled in the art.

The FSH of the present invention may be used in Controlled Ovarian Hyperstimulation (COH). Standard protocol for COH⁽²⁷⁾Comprising a down-regulation phase in which endogenous luteinising hormone is down-regulated by administration of a gonadotropin releasing hormone (GnRH) agonist, followed by a stimulation phase in which Follicle Stimulating Hormone (FSH) is administered daily, typically at 75-600 or about 75-600 IU/day, preferably 150-225 IU/day or about 150-225 IU/day, to induce follicular development (folliculogenesis). Another option is to start stimulation with FSH after natural or induced menstruation and then administer the GnRH antagonist (usually starting at about day 6 of the stimulation phase). When at least 3 follicles > 16 mm (one of which is 18 mm), a single bolus administration of hCG (5-10,000IU) mimics the natural LH surge and induces ovulation. In general, hCG is injected on any of days 10 to 14, but hCG can also be injected at a later time, depending on the time that the above parameters are met. The time for recovering the oocytes was determined 36-38 hours after the hCG injection。

The FSH of the invention may also be used in ovulation induction and IUI. For example, priming with the FSH preparation of the invention is initiated after natural or induced menstruation at a daily dose of 75-150 IU. When 1 or 3 follicles reach a diameter of at least 16 mm, one bolus administration of hCG induces ovulation. In vivo fertilization was performed by periodic sexual intercourse or IUI.

Because the FSH of the invention has a better efficacy compared to known FSH preparations, such regimens as described above can be modified with lower IU doses of FSH, and/or with shorter FSH priming cycles, to achieve the same or better response in terms of number of follicles and viability of follicles. For example, sufficient folliculogenesis may be achieved with an FSH preparation of the invention at 50-150 or about 50-150IU FSH, preferably 50-100 or about 50-100IU FSH, more preferably 50-75 or about 50-75IU FSH. Typically, FSH is administered daily or semi-daily. The administration period may be less than 14 days or about 14 days, preferably less than 12 days or about 12 days, more preferably less than 11 days or about 11 days or 10 or about 10 days.

For ovulation induction, the FSH preparation of the invention may be administered at a dose of 25-150IU FSH/day, preferably 50-125IU FSH/day.

To treat male infertility, the FSH formulation of the invention may be administered at 150-300IU weekly until sperm production by periodic sexual intercourse or assisted reproductive techniques reaches a level sufficient for fertilization.

The inventors have also found that because of the superior efficacy of FSH preparations having a Z value of at least 200 or about 200, these preparations may be administered less frequently than FSH preparations having a Z value of less than 200. (for clarity of description, the term FSH is used⁺²⁰⁰、FSH⁺²¹⁰、FSH⁺²²⁰And the like, which means that the Z value is in the range of about 200-210, or about 200-210, 211-220, or about 211-220, 221-230, or about 221-230, and the like. ) This means that for patients who normally require daily administration of e.g. 150IU of conventional FSH to achieve sufficient folliculogenesis, only 225IU of FSH need be administered e.g. every 3 days⁺²⁰⁰Or 300IU FSH every 4 days⁺²⁰⁰The same effect can be obtained. Because FSH is a relatively common FSH preparation⁺²⁰⁰The efficacy of (a) is higher, so the above cited doses can be reduced in those patients presenting a good response. For FSH preparations of the invention having a Z value of not less than 230 or about 230, injections may be given every 5, 6 or 7 days, depending on the patient's response. The response can be assessed by ultrasonography and/or by measuring serum estradiol levels. Other suitable schemes are as follows: 100IUFSH every 2 days⁺²¹⁰(ii) a 200IU FSH every 3 days⁺²¹⁰(ii) a 275 or 300IU FSH every 4 days⁺²¹⁰(ii) a 80-100IU FSH every 2 days⁺²²⁰(ii) a 180-IU FSH every 3 days⁺²²⁰(ii) a 260 IU FSH every 4 days⁺²²⁰(ii) a 75-100IU FSH every 2 days⁺²³⁰(ii) a 170 IU FSH once every 3 days⁺²³⁰(ii) a 250-300IU FSH every 4 days⁺²³⁰(ii) a 275-IU FSH every 5 days⁺²⁵⁰(ii) a 375-450IU FSH every 6 days⁺²⁵⁰(ii) a 450-⁺²⁵⁰。

The term "higher efficacy" as used herein in relation to the effect of folliculogenesis includes any measurable improvement or increase in the number of follicles and/or the viability of follicles in an individual, e.g. when compared to the number of follicles and/or the viability of follicles in one or more patients treated at the same dose (IU/IU), as determined in conventional assays for ovarian weight gain in rats, or as determined for FSH preparations having a Z-value of less than 200. The improvement or increase is preferably statistically significant, and the p-value is preferably less than 0.05. Methods for determining statistically significant results are well known and documented in the art, and any suitable method may be used.

The invention will now be further described by way of the following non-limiting examples.

Examples

EXAMPLE 1 determination of Z value

The Z value of the glycoprotein can be determined from the glycan profile.

Recombinant human FSH was reacted with hydrazine at 100 ℃ for 5 hours, and the glycan moiety could be released using an Oxford Glycosciences GlycoPrep * 1000 full-automatic analyzer or the like.

The polysaccharides were separated from unreacted hydrazine and amino acid hydrazide by using a column coated with glass beads. The polysaccharides were eluted with sodium acetate reagent.

Acetylation of glycans with acetic anhydride. Excess reagent was removed using a mixed bed ion exchange column. Any unreduced glycans were collected in a dilute acetate buffer solution.

The accumulated sugars were collected on a 0.5 meter filter (Oxford GlycoSecenes) and lyophilized. The dried polysaccharide is labeled by reacting the dried polysaccharide with a reducing agent having a fluorophore (e.g., 2-aminobenzamide) at 65 ℃ for 120 minutes under acidic conditions.

The labeled glycans are separated from the excess reagent by a hydrophilic absorbent membrane, and the glycans remain on the absorbent membrane. Recovering polysaccharide with water, and freezing for chromatographic separation.

The labeled polysaccharides are separated by anion exchange chromatography. The chromatography procedure was performed as follows:

the column was a GlycoSep * C column, 4.6X 100mm, packed with divinylbenzene resin (5 m) with a polymer layer;

the flow rate of the mobile phase was 0.4 ml/min;

mobile phase A: acetonitrile (chromatography gradient)

Mobile phase B: ammonium acetate 500mM pH4.5

Mobile phase C: ultrapure water

Detection with a fluorometer, setting λ_Excitation：330nm，λ_Launching：420nm；

Elution under the following elution conditions:

initial conditions: 20% mobile phase A, 80% mobile phase C

The mobile phase B was a linear gradient (0.25% per minute) from 5 to 21 minutes, and the mobile phase A was constantly maintained at 20%

From 21 to 61 minutes the mobile phase B was a linear gradient (0.525% per minute) and the mobile phase A was kept constant at 20%

The temperature of the column was maintained at 30. + -. 2 ℃.

The polysaccharides elute according to their charge, being neutral, mono-, di-, tri-and tetra-sialylated. A typical chromatogram is shown in FIG. 1.

The peaks on the resulting chromatogram were grouped by the range of residence times corresponding to the sialylation degree listed in table 1.

TABLE 1 residence time and number of charges of glycans released from rFSH
			Dwell time (minutes)	Polysaccharides	Number of charges
2 to 4	Neutral property	0
			15 to 21	A sialic acid group	Pmono
21 to 35	Two sialic acids	Pdi
			35 to 45	Three sialic acids groups	Ptri
45 to 52	Four sialic acids groups	Ptetra

The results for each group of saccharides are expressed as the percentage of the total area occupied by the different groups of glycans (neutral, mono-, di-, tri-and tetra-sialyl), and the Z values are given by the different saccharides (P)_glycan) The proportion calculation of (2):

Z＝P’_mono+2P’_di+3P’_tri+4P’_tetra

example 2 determination of the Antenna Index (AI)

The glycans were released from the peptide backbone by hydrazinolysis as described in example 1, and these carbohydrates were then fluorescently labeled with 2-aminobenzamide.

The 2-aminobenzamide-labeled glycans were enzymatically desialylated with sialidase (Vibrio cholerae) in 250mM ammonium acetate pH5.5 containing 20mM calcium chloride at 37 ℃ for 18 hours. Approximately 0.05U sialidase was used for the initial amount of 100 micrograms of rhFSH glycans.

The desialylated glycans were dried in vacuo and stored at-20 ℃ until separation by preparative reverse phase HPLC, under the following conditions:

the column is a GlycoSep * R column;

the flow rate of the mobile phase was 0.7 ml/min;

eluent A: ammonium acetate 50mM pH6.0

Eluent B: ammonium acetate 50mM pH6.0 containing 8% acetonitrile

Detection with a fluorometer, setting λ_Excitation：330nm，λ_Launching：420nm；

Temperature of the column: at 30 ℃.

The dried sample was reconstituted with eluent a (200 μ l) before loading into the column: 50 microliters of this solution was added.

The following gradient was used:

t ═ 0 (min) 55% a; 45% B

t 15 (min) 55% a; 45% B

t ═ 70 (min) 0% a; 100% B

t-75 (min) 0% a; 100% B

t 76 (min) 55% a; 45% B

Two, three and four tentacle peaks were confirmed by electrospray mass spectrometry (ESMS) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

The results are expressed as relative percentages of two antennas, three antennas and four antennas, 100% being the sum of all glycans. AI is then calculated according to the following formula:

AI＝2P_di+3P_tri+4P_tetra

wherein AI is the antenna index, P_di、P_triAnd P_tetraThe percentages of di-, tri-, and tetra-branched carbohydrates in total carbohydrate are expressed, respectively.

Example 3 separation of different fractions of FSH based on the degree of sialylation

The recombinant FSH was separated into acidic and basic components by anion exchange chromatography on DEAE sepharose FF.

The column used was packed with DEAE sepharose FF resin: for experimental grade purity (about 60 mg whole protein), the column is Φ 1.6 × 20 cm (XK Pharmacia or equivalent), for larger grade purity, the column is Φ 3.4 × 40 cm (Vantadge Amicon or □ □ material);

the flow rate of the mobile phase was 150-;

equilibration buffer 1: 2M Tris-HCl pH7.0 + -0.1;

equilibration buffer 2: 25mM Tris-HCl pH7.0 + -0.1, conductivity is 2.15 + -1.5 mS/cm;

elution buffer 1: 25mM Tris pH 7.0. + -. 0.1, 35mM sodium chloride, conductivity 5.8. + -. 0.4mS/cm (this buffer elutes the more basic isoform);

elution buffer 2: 25mM Tris pH 7.0. + -. 0.1, 150mM sodium chloride, conductivity 18.3. + -. 0.5mS/cm (this buffer elutes the more acidic isoform);

regeneration solution: 0.5M sodium hydroxide, 1M sodium chloride;

storing the solution: 10mM sodium hydroxide

The temperature of the column is maintained at 23. + -. 3 ℃ or 5. + -. 3 ℃.

FSH loaded into the column was prepared as follows:

whole frozen rhFSH was thawed at 5 ± 3 ℃. After thawing was complete, the solution was diluted with 2M Tris-HCl pH 7.0. + -. 0.1 (3-4 mg rhFSH per ml resin, estimated as optical density at 276.4nm) at the following ratio: 1 part buffer to 79 parts bulk rhFSH. The final concentration of Tris-HCl was 25 mM. The pH was adjusted to 7.0. + -. 0.1 with 1M HCl.

The column was prepared by washing with 3 Bed Volumes (BV) of 0.5M sodium hydroxide followed by 6BV water. Equilibration was performed by rinsing with 4-5BV equilibration buffer 1 until a pH of about 7 was measured. The washing was continued with 7-8BV of equilibration buffer 2.

A sample of rhFSH prepared as described above was loaded into the column. After completion of the column packing, the column was washed with 3BV of equilibration buffer 1.

Then, elution with elution buffer 1 was started, and as the absorbance (276.4nm) started to rise, collection of the basic component was started, continuing until 20. + -.1 bed volume. The eluate was then exchanged for elution buffer 2 and collection of the acidic component started as the absorbance (276.4nm) started to rise, continuing until 3. + -.1 bed volume.

Next, the fractions were concentrated by ultrafiltration using 8400(Amicon or equivalent) ultrafiltration cell type equipped with YM3 membrane (for alkaline fraction) and YM10 membrane (for acidic fraction). All manipulations were carried out at 5. + -. 3 ℃.

Example 4 clinical study comparing FSH isoforms

Two experimental rhFSH batches were evaluated for comparative efficacy in volunteers.

Two FSH preparations were obtained by separating two components of rhFSH by ion exchange chromatography as described in example 3 above. Run A was designated as "acidic" and the Z number was 220 (i.e., the acidic component of example 3), while run B was designated as "basic" and the Z number was 160 (i.e., the basic component of example 3).

FSH was added to ampoules of lots a and B to contain approximately 150IU FSH each, using a routine trial of rat ovarian weight gain.

The characteristics of the two batches are listed in table 2. It should be noted that because these bottles add FSH in IU units, the actual amount of FSH in the basic lot B ampoules is about 250% of that of the acidic lot a (about 24 micrograms versus about 9 micrograms).

TABLE 2 characterization of the batch FSH used in the clinical study
				Batch number A "acidic"	Batch number B "basic"
FSH content per ampoule	8.7 microgram/ampoule	23.8 microgram/ampoule
			Specific biological activity	19,753 IU/mg	7,386 IU/mg
Z number	220	160
			Feeler index (AI)	274	237

Specific biological activity was calculated by dividing the activity (expressed in IU) by the weight of protein.

A group of 32 pre-menopausal female volunteers was formed. These patients received daily injections of dabigatran (0.1 mg) to down-regulate the pituitary gland. After 14 days, ultrasonography was carried out and stimulation was started with rFSH (150 IU/day) of lot A or lot B in the absence of cysts. Follicular growth was assessed daily by ultrasonography and serum estradiol concentrations.

During the FSH priming phase, the follicles develop and grow in diameter. The number of follicles was measured for each patient on days 8 and 10 of stimulation and counted, and the number of follicles belonging to the size scale of 0-10mm, 11-15mm and 16-25mm was recorded. Figure 2 shows the average number of follicles per size scale per patient on day 8 for patients receiving treatment with both acidic and basic isoforms. Figure 3 shows the same pattern made on day 10.

The results of the study show that the size of the follicles in the alkaline group grows regularly over time, while the follicles in the acidic group produce a second group of follicles, slightly later than the first group, with the result that follicular formation increases very rapidly, approximately 2-fold compared to the alkaline group. The size of the second group of follicles increased from day 8 to day 10. The results were: patients treated with "acidic" FSH all had a total of 18 follicles greater than 11mm at day 10, whereas patients treated with the "basic" isotype had on average only 11 follicles greater than 11mm on day 10.

The average total number of follicles in the "acidic" group was 28, while the "basic" group was 19.

The mean Total Follicle Volume (TFV) was determined for each patient by ultrasonography. The TFV of the group receiving "acidic" FSH was 30% higher than the group receiving "basic" FSH.

Higher FSH serum levels in patients in the "basic" group as predicted by the injected protein mass □ by radioimmunoassay; however, this difference is only about 30% from the 250% administration (see fig. 4), which is consistent with higher metabolic tolerance of the acidic isoform.

Example 5 separation of FSH from different fractions based on the Antenna Index (AI)

HPLC or affinity chromatography on concanavalin-A (Con-A) derived agarose may isolate FSH preparations with higher than normal antennary indices.

Example 6 "fortification of sialic acid" with sialyltransferase "

Recombinant human FSH ("starting material"; 10 mg) was dissolved in buffer (0.1M HEPES, pH 7.5) at a concentration of 4.3 mg/ml. To this solution was added 100mU/ml of recombinant rat sialyltransferase (ST3Gal III) and 20mM cytidine-5' -monophosphate-N-acetylneuraminic acid (CMP-NeuAc) as sialic acid donor. Another option is that sialic acid donors can be generated in situ with 20mM NeuAc and 2mM CMP in the presence of CMP-sialic acid synthetase. The reaction was incubated at 37 ℃ for 24 hours. The sialic acid enriched fraction was isolated using the technique described in example 3.

Sialic acid fortification may also be carried out using starting material consisting of FSH with a higher antennal index as prepared in example 5.

Alternatively, sialic acid fortification may be carried out using FSH starting material which already has a higher Z value relative to conventional recombinant FSH. These starting materials can be isolated using the technique of example 3.

Example 7 Generation of FSH mutants

cDNAs for the alpha-and beta-subunits of human FSH were subcloned into the pDONR vector (Invitrogen). Using QuikChange^TMThe site-directed mutagenesis kit (Stratagene) introduces N-linked glycosylation sites in the alpha-and beta-subunits of FSH. QuikChange^TMThe system uses two synthetic oligonucleotide primers containing the desired mutation. The following oligonucleotide pairs were used to introduce N-linked glycosylation sites: V78N is CC TTGTAT ACA TAC CCA AAC GCC ACC CAG TGT CAC and GTG ACA CTG GGTGGC GTT TGG GTA TGT ATA CAA GG, A70N is GC TGT GCT CAC CATAAC GAT TCC TTG TAT ACA TAC C and GGT ATG TAT ACA AGG AAT CGTTAT GGT GAG CAC AGC, D41N/A43T is GAT CTG GTG TAT AAG AACCCA ACT AGG CCC AAA ATC CA and TGG ATT TTG GGC CTA GTT GGGTTC TTA TAC ACC AGA TC, G100N is TGT ACT GTG CGA GGC CTG AACCCC AGC TAC TGC TCC and GGA GCA GTA GCT GGG GTT CAG GCC TCGCAC AGT ACA, E56N is G AAC GTC ACC TCA AAC TCC ACT TGC TG and CA GCA AGT GGA GTT TGA GGT GAC GTT C, and F17T is CAG GAAAAC CCA ACC TTC TCC CAG CC and GG CTG GGA GAA GGT TGG GTTTTC CTG. Use of ABI PRISM BigDye^TMThe Terminator v3.0 Ready Reaction Cycle sequencing kit confirmed the DNA sequence of the mutant cDNA, and then analyzed with ABI PRISM 310 gene analyzer.

The pCI mammalian expression vector (Promega) was converted to a GATEWAY destination vector using the GATEWAY vector conversion System (Invitrogen). By GATEWAY^TMCloning techniques (Invitrogen) the alpha-and beta-mutants were subcloned into the pCI expression vector along with the wild type subunit. The pCI expression vector contains the human cytomegalovirus immediate-early enhancer/promoter (used to regulate the inserted gene)Expression) □ gene upstream (to initiate expression) and monkey virus 40 late polyadenylation signal downstream of the inserted gene (to terminate transcription). The E56N and F17T α -mutants in pCI were co-transfected with wild type FSH β in pCI, whereas the A70N, G100N, V78N and D41N/A43T β -mutants in pCI were co-transfected with wild type α -subunit in pCI. As a control, the wild-type β -subunit of FSH in pCI was co-transfected with the α -subunit in pCI. Plasmids were transiently transfected into HEK293 cells (ATTC, CRL-10852) using the calcium phosphate method (see, e.g., WO 96/07750). In addition, a pCI plasmid containing either the wild-type β -subunit or the V78N β -mutant was co-transfected with the wild-type α -subunit in pCI. The plasmid may also be transiently or stably transfected into CHO cells. The day after transfection the medium was changed to DMEM/F12(Invitrogen, 11320-033) containing 1. mu.g/ml insulin (Invitrogen, 18140-020), 6.8 ng/ml sodium selenite (Sigma, S5261) and 12.2 ng/ml ferric citrate. One day after the media was switched, conditioned media were collected and centrifuged at about 800Xg for 5 minutes at 4 ℃ to remove any cell residue. The supernatant was removed and centrifuged at 16,000Xg for 5 minutes in a Biofuge fresco (Heraeus Instruments) and the broth was further clarified by filtration through a 0.45m Acrodisc filter (Gelman Sciences, 4184). To the clarified cell extract, 1M Tris pH7.4 was added to a final concentration of 50mM Tris, and Tween20 was added to a final concentration of 0.1% Tween 20.

The FSH mutants were purified from cell extracts by immunoaffinity chromatography using agarose derived from anti-FSH mabs immobilized with divinyl sulfone (immune resin anti-FSH-mab-divinyl sulfone-agarose). These resins can be prepared by methods known to the person skilled in the art, for example as described in WO 88/10270.

The resin was equilibrated at 4 ℃ with an equilibration buffer consisting of 0.1M Tris-HCl, 0.3M pH7.5 sodium chloride buffer. The column is loaded with a large amount of IU FSH (by radioimmunoassay, (RIA), corresponding to 80-90% of the total FSH binding capacity of the column.

Non-retained protein was eluted with equilibration buffer (same above) until OD of the eluate₂₈₀Less than 0.02.

The absorbed mutant FSH was eluted from the immune resin with 1M aqueous ammonia solution at 4 ℃. Combining at 4 deg.C to obtain eluate with volume about 4 times of the volume of the immune resin, adding glacial acetic acid to adjust pH to 9.0, collecting, ultrafiltering in Amicon device (membrane molecular weight cut-off of 10,000Da), and concentrating to small volume.

The concentrated mutant FSH solution was then subjected to a reverse phase HPLC procedure using a Waters Prep LC 500A liquid chromatograph equipped with a uv detector and a preparative gradient generator. The pH of the solution was adjusted to about 5.6 prior to loading into the column. Charging the solution into C₁₈Reversed phase column (Prepak 500C)₁₈cartidges Waters) which had been equilibrated at room temperature with 0.05M ammonium acetate buffer ph 5.6. The flow rate was 100 ml/min and the eluent was monitored at 280 nm.

Mutant FSH is eluted with a gradient of up to 50% isopropanol in the mobile phase. The fractions were examined by analytical gas chromatography (GPC) and Radioimmunoassay (RIA). The organic solvent was distilled off under vacuum at a temperature below 40 ℃, the solution was stored cold and lyophilized.

The mutant FSH preparations expressed in CHO cells were subjected to ion exchange chromatography to isolate Z as described in example 3⁺A number greater than 180, 190, 200, 210, 220, 230, 240, 250 and higher.

Mutant FSH preparations expressed in CHO cells or HEK293 cells were subjected to sialic acid fortification as described in example 6. After sialic acid fortification, the mutant FSH was subjected to ion exchange chromatography to isolate Z⁺A number greater than 180, 190, 200, 210, 220, 230, 240, 250 and higher.

Reference to the literature

1.Wide，L；The regulation of metabolic clearance of human FSH in mice by variation ofthe molecular structure of the hormone；Acta Endocrinologica 112 1986；336-344.

2.Chappel et al.；Endocr.Rev.4 1983；179-211；Padmanabhan et al.；J.Clin.Endocrinol.Met.67 1988；465-473；Wide et al.；J.Clin.Endocrinol.Met.70 1990；271-276；Anobile et al.Glycoform composition of serum gonadotropins through the normalmenstrual cycle and in the post-menopausal state；Mol.Human Reproduct.4 1998；631-639.

3.Morell et al.；J.Biol.Chem.246 1971；1461-1467；Ashwell et al.；Annu.Rev.Biochem.51 1982；531-554.

4.Steelman & Pohley；Assay of the follicle stimulating hormone based on theaugmentation with human chorionic gonadotropin；Endocrinology 53 1953；604-616.

5.D’Antonio et al.；Biological characterisation of recombinant human folliclestimulating hormone isoforms；Human Reproduction 14 1999；1160-1167.

6.Vitt et al.；Isoforms of human recombinant follicle-stimulating hormone：comparisonof effects on murine follicle development In Vitro；Biol.Reproduct.59 1998；854-861

7.Timossi et al.；Differential effects of the charge variants of human follicle-stimulatinghormone；J.Endocrinol.165 2000；193-205.

8.Zambrano et al.；Studies on the relative in-vitro biological potency of the naturally-occurring isoforms of intrapituitary follicle stimulating hormone；Mol.Hum.Reprod.2 1996；563-71.

9.Zambrano et al.；Receptor binding activity and in vitro biological activity of thehuman FSH charge isoforms as disclosed by heterologous and homologous assaysystems：implications for the structure-function relationship of the FSH variants；Endocrine 10 1999；113-121.

10.Wide et al.；Influence of the assay method used on the selection of the most activeforms of FSH from the human pituitary；Acta Endocrinol.(Copenhagen)113 1986；17-22.

11.Mulders et al.；Prediction of the in vivo biological activity of human recombinantfollicle stimulating hormone using quantitative isoelectric focussing；Biologicals 251997；269-281.

12.Timossi et al.；A less acidic human follicle-stimulating hormone preparation inducestissue-type plasminogen activator enzyme activity earlier than a predominantly acidicanalogue in Phenobarbital-blocked pro-oestrous rats；Mol.Human Reproduct.4 1998；1032-1038.

13.Healy et al.；Lancet 343 1994；1539-1544.

14.for example，a technique is described in EP 0 170 502(Serono Laboratories，Inc.).

15.Buckler et al.；Ovulation induction with low dose alternate day recombinant folliclestimulating hormone；Hum.Reprod.14 1999；2969-73.

16.H*rd et al.；Isolation and structure determination of the intact sialylation N-linkedcarbohydrate chains of recombinant human follitropin expressed in Chinese hamseterovary cells，Eur.J.Biochem.193 1990；263-271.

17.Nadano et al.；J.Biol.Chem.261 1986；11550-11557；Kanamori et al.；J.Biol.Chem.265 1990；21811-21819.

18.Swedlow at al.；Deglycosylation of gonadotropins with an endoglycosidase；Proc.Soc.Experiment.Biol.& Med.181 1986；432-437.

19.Mulders et al.；Biologicals 25 1997；269-281.

20.Zambrano et al.；Mol.Hum.Reprod.2 1996；563-571.

21.Timossi et al.；Neuroendocrinology 67 1998；153-163.

22.Boime et al.；Glycoprotein hormone structure-function and analog design；RecentProg.Horm.Res.54 1999；271-88.

23.Wen et al.；J.Biol.Chem.267 1992；21011；Van den Eijnden et al.Enzymaticamplification involving glycosyltransferases forms the basis for the increased size ofasparagine-linked glycans at the surface of NIH 3T3 cells expressing the N-ras proto-oncogene；J.Biol.Chem.266 1991；21674.

24.Weinstein et al.J.Biol.Chem.257 1982；13845.

25.Sasaki et al.J.Biol.Chem.268 1993；22782-22787；Kitagawa & Paulson；J.Biol.Chem.269 1994；1394-1401.

26.Kitagawa et al.；J.Biol.Chem.271 1996；931-938.

27.for example，a conventional technique is described in EP 0 170 502(SeronoLaboratories，Inc.).

Sequence listing

<120> gonadotropin for folliculogenesis

<130>

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<170>PatentIn version 3.0

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<213> Intelligent (Homo sapiens)

<400>1

Ala Pro Asp Val Gln Asp Cys Pro Glu Cys Thr Leu Gln Glu Asn Pro

1 5 10 15

Phe Phe Ser Gln Pro Gly Ala Pro Ile Leu Gln Cys Met Gly Cys Cys

20 25 30

Phe Ser Arg Ala Tyr Pro Thr Pro Leu Arg Ser Lys Lys Thr Met Leu

35 40 45

Val Gln Lys Asn Val Thr Ser Glu Ser Thr Cys Cys Val Ala Lys Ser

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Lys Val Glu Asn His Thr

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Ala Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

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Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys Glu Glu

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Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly Tyr Cys

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Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys Ile Gln

35 40 45

Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Arg Val Pro

50 55 60

Gly Cys Ala His His Ala Asp Ser Leu Tyr Thr Tyr Pro Val Ala Thr

65 70 75 80

Gln Cys His Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val

85 90 95

Arg Gly Leu Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met Lys Glu

100 105 110

Claims (39)

1. An FSH preparation characterized by: the Z value of the formulation is at least 200 or about 200.

2. The FSH preparation according to claim 1, wherein: the Z value of the formulation is at least 210 or about 210.

3. The FSH preparation according to claim 1, wherein: the Z value of the formulation is at least 220 or about 220.

4. The FSH preparation according to claim 1, wherein: the Z value of the formulation is at least 230 or about 230.

5. The FSH preparation according to claim 1, wherein: the Z value of the formulation is at least 240 or about 240.

6. The FSH preparation according to claim 1, wherein: the Z value of the formulation is at least 250 or about 250.

7. The FSH preparation according to claim 1, wherein: the Z value of the formulation is at least 260 or about 260.

8. A pharmaceutical composition comprising FSH, wherein: the FSH has a Z value of at least 200 or about 200.

9. The pharmaceutical composition of claim 8, wherein: the FSH has a Z value of at least 210 or about 210.

10. The pharmaceutical composition of claim 8, wherein: the FSH has a Z value of at least 220 or about 220.

11. The pharmaceutical composition of claim 8, wherein: the FSH has a Z value of at least 230 or about 230.

12. The pharmaceutical composition of claim 8, wherein: the FSH has a Z value of at least 240 or about 240.

13. The pharmaceutical composition of claim 8, wherein: the FSH has a Z value of at least 250 or about 250.

14. The pharmaceutical composition of claim 8, wherein: the FSH has a Z value of at least 260 or about 260.

15. The pharmaceutical composition according to any one of claims 8 to 14, wherein: the pharmaceutical composition is used in controlled ovarian hyperstimulation.

Use of an FSH preparation for folliculogenesis, said FSH preparation comprising: the FSH has a Z value of at least 200 or about 200.

17. The use of claim 16, wherein: the FSH has a Z value of at least 210 or about 210.

18. The use of claim 16, wherein: the FSH has a Z value of at least 220 or about 220.

19. The use of claim 16, wherein: the FSH has a Z value of at least 230 or about 230.

20. The use of claim 16, wherein: the FSH has a Z value of at least 240 or about 240.

21. The use of claim 16, wherein: the FSH has a Z value of at least 250 or about 250.

22. The use of claim 16, wherein: the FSH has a Z value of at least 260 or about 260.

Use of FSH for the preparation of a medicament for folliculogenesis, said medicament comprising: the FSH has a Z value of at least 200 or about 200.

24. The use of claim 23, wherein: the FSH has a Z value of at least 210 or about 210.

25. The use of claim 23, wherein: the FSH has a Z value of at least 220 or about 220.

26. The use of claim 23, wherein: the FSH has a Z value of at least 230 or about 230.

27. The use of claim 23, wherein: the FSH has a Z value of at least 240 or about 240.

28. The use of claim 23, wherein: the FSH has a Z value of at least 250 or about 250.

29. The use of claim 23, wherein: the FSH has a Z value of at least 260 or about 260.

30. A method of preparing an FSH preparation having a Z-value of at least 200 or about 200, comprising:

the method comprises the step of reacting FSH with a sialic acid donor in the presence of 2, 3-sialyltransferase.

31. The method of claim 30, wherein: the FSH has a Z value of at least 210 or about 210.

32. The method of claim 30, wherein: the FSH has a Z value of at least 220 or about 220.

33. The method of claim 30, wherein: the FSH has a Z value of at least 230 or about 230.

34. The method of claim 30, wherein: the FSH has a Z value of at least 240 or about 240.

35. The method of claim 30, wherein: the FSH has a Z value of at least 250 or about 250.

36. The method of claim 30, wherein: the FSH has a Z value of at least 260 or about 260.

37. A method according to any one of claims 30 to 36, wherein: the sialic acid donor is cytidine-5' -monophosphate-sialic acid (CMP-sialic acid).

38. A method according to any one of claims 30 to 37, wherein: the sialyltransferase is rat ST3Gal III.

39. A method of preparing an FSH preparation having a Z-value of at least 200 or about 200, comprising: the method comprises an ion exchange chromatography step.