ME01043B - Method for purifying fsh - Google Patents

Abstract

A method for the purification of recombinant FSH or some variant of FSH which involves subjecting a fluid containing FSH to affinity chromatography with staining; (1) hydrophobic reaction chromatography; and (2) chromatographic bulk phases that can be applied in any order. The patent contains another 23 claims.

Description

Field of technique

This invention relates to the field of follicle stimulating hormone (FSH) purification.

State of the art

Follicle-stimulating hormone (FSH) is an injectable protein that belongs to the class of gonadotropins. FSH is used to treat infertility and reproductive disorders in both male and female patients.

In nature, FSH is produced by the pituitary gland. For pharmaceutical use, FSH can be produced recombinantly (rFSH) or can be isolated from the urine of postmenopausal women (rFSH).

FSH is used in female patients to induce ovulation (01) as well as for controlled ovarian hyperstimulation (COH) in assisted reproductive technologies (ART). In a typical therapeutic regimen to induce ovulation, the patient receives daily injections of FSH or some variant (about 75 to 300 IJ FSH/day) over a period of about 6 to about 12 days. In a typical therapeutic regimen for controlled ovarian hyperstimulation, the patient receives daily injections of FSH or some variant (about 150-600 IU FSH/day) over a period of about 6 to about 12 days.

FSH is also used to induce spermatogenesis in men suffering from oligospermia. A regimen using 150 IJ FSH 3 times a week in combination with 2500 IJ hCG twice a week has been successful in improving sperm count in men suffering from hypogonadotropic hypogonadism [Burgues et al.; Subcutaneous self-administration of highly purified follicle-stimulating hormone and human chorionic gonadotropin for the treatment of male hypogonadotropic hypogonadism. Spanish Collaborative Group for Male Hypogonadotropic Hypogonadism; Hum. Reproduction; 1997,12, 980-6],

Due to the importance of FSH in the treatment of fertility disorders, it is desirable to provide FSH with high purity and high specific activity. FSH treatment requires repeated injections. Preparations with highly purified FSH can be administered subcutaneously, which enables self-administration by the patient and thereby increases patient suitability and cooperation.

Lynch et al. [Extraction and purification of human pituitary follicle-stimulating hormone and luteinizing hormone; Acta Endocrinologica, 1988, 288, 12-19] describe one method for

purification of human pituitary FSH. This method includes anion and cation exchange chromatography, immunoaffinity extraction and size exclusion chromatography. This method is said to result in pituitary FSH having a specific activity of 4,990 U (immunoassay)/mg, with 16 IJ/mg LH. Protein content was determined either on a dry weight basis or in solution by absorbance at 280 nm (assuming A280) cm /a 1 g/1 equals 1).

WO 98/20039 (IBSA Institut Biochimique SA) describes a process for the purification of human urinary FSH starting from urinary extracts called human menopausal gonadotropins (hMG). This process uses ion-exchange chromatography with an anionite resin on a weekly basis of the DEAE type followed by affinity chromatography on a resin having an anthraquinone derivative as a ligand. This process is reported to yield urinary FSH without LH and to have a specific activity of 6,870 IJ (immunoassay)/mg. The protein content was determined by assuming that an aqueous solution of 1 mg/ml protein has an optical density of 0.62 at 277 nm, in quartz cuvettes with a 1 cm path group.

WO 00/63248 (Institute Massone SA) describes a process for the purification of gonadotropins, including FSH, from human urine. This process involves the following steps: ion exchange chromatography with a strong cationic sulfopropyl resin, ion exchange chromatography with a strong anionic resin, and hydrophobic reaction chromatography (HIC). A preparation of FSH having a specific activity of 8,400 IJ/mg (Steelman-Pohley method: Follicle-stimulating hormone assay based on augmentation with human chorionic gonadotropin; Endocrinology, 1953, 53, 604-616) and less than 1 IJ LH (weight gain method rat seminal vesicles: Van Hell H, Matthijsen R & GA Overbeek; Ada Endocrinol, 1964, 47, 409) biological activity at 75 IJ FSH is reportedly obtained. Protein content was tested by Lowry's method [O.H. Lowry et al., J. Biol. Chem., 1951,795,265].

US 5,990,288 (Musick et al.) describes a method for the purification of FSH from biological samples, such as human pituitary glands or human postmenopausal urine. This process uses cation exchange chromatography on Fractogel EMD SO3-650M, followed by affinity chromatography with overstaining on Mimetic Orange 1 resin, followed by a hydrophobic reaction chromatography step on Bakerbond Wide Pore Hl-Propyl resin. This process is reported to yield human pituitary FSH having a specific activity of 7,066 IJ (immunoassay)/mg and less than 1 IJ (immunoassay)/mg LH, and urinary FSH having a specific activity of 6,298 IJ (immunoassay)/mg a less than 3 IJ (immunoassay)/mg of LH. Protein content was determined by absorbance at 280 nm (assuming A280!™ for 1 g/1 equals 1).

Chiba et al. [Isolation and partial characterization of LH, FSH and TSH from the pituitary gland of dogs; Endocrinol. J., 1997, 44, 205-218] describe a technique for the purification of canine pituitary gonadotropins, including FSH, using concanavalin (Con) affinity chromatography, hydrophobic reaction chromatography (HIC) and immobilized metal ion chromatography with Cu++. It was stated that the obtained FSH has a specific activity of 2.17 IJ/g of protein using a radioreceptor assay for FSH to measure the biological activity and with the help of a BioRad protein assay kit (BioRad Laboratories CA USA) to determine the protein content.

WO 88/10270 (Institute di Ricerca Cesare Serono SPA) describes a method for the purification of human FSH from urine. This process involves immunochromatography with FSH-specific immobilized monoclonal antibodies bound to Sepharose 4B using divinyl sulfone, followed by reverse-phase HPLC. The resulting FSH does not contain LH and other urinary proteins and has a specific activity of 6,200 IJ/mg lyophilized powder (Steelman-Pohley method). This preparation was the first preparation of FSH that was suitable for subcutaneous administration due to its purity.

There remains a need for new methods for the purification of FSH and FSH variants. In particular, there is a need for purification methods that would avoid the use of expensive immunoaffinity chromatography steps.

Brief description of the invention

The aim of this invention is to provide a new method for the purification of recombinant FSH or some recombinant variant of FSH.

In a first aspect, the present invention provides a method for purifying recombinant human FSH or an FSH variant, starting from a liquid containing crude FSH, comprising the following steps:

(1) affinity chromatography with overstaining;

(2) chromatography with hydrophobic reaction; and

(3) reverse phase chromatography; which can be carried out in any order.

Brief description of the drawing

Figure 1 illustrates this invention, i.e. the purification process comprising the steps:

• afmite chromatography with overstaining,

• chromatography with hydrophobic reaction,

• reverse phase chromatography,

Figure 2 shows a flow diagram of one specific aspect of the present invention, ie. process

purification which includes steps:

• ultrafiltration/diafiltration,

• chromatography with anion exchange,

• affinity chromatography with overstaining,

• chromatography with hydrophobic reaction,

• reverse phase chromatography,

• chromatography with anion exchange,

• nanofiltration,

• ultrafiltration/diafiltration;

Abbreviations

The following abbreviations are used in the description of these inventions:

DF: diafiltration

FSH: follicle-stimulating hormone; r-FSH: recombinant FSH; hFSH: human FSH; r-hFSH: recombinant human FSH BV: substrate volume DEAE: diethylaminoethyl ELISA: enzyme-linked immunoassay DAC: cross-staining affinity chromatography IMAC: immobilized metal ion affinity chromatography OD: optical density

HIC: Hydrophobic Reaction Chromatography HPLC : High Performance Liquid Chromatography IRMA: Immunoradiometric Assay KD or kD: KiloDalton

HCP: host cell protein, proteins derived from the host cells used

the expression of FSH

IPC: hi-process controls

IEF: isoelectric focusing

PES: Polyethersulfone

RP-HPLC: reversed-phase high-performance liquid chromatography

Q FF: I'm not sure what to do with Q Sefarozi FF

RT: Room temperature

UF: ultrafiltration

WFI: water for injections

Detailed description of this invention

The present invention provides a method for purifying recombinant human FSH or a recombinant FSH variant starting from a liquid containing crude FSH, comprising the following steps:

(1) afmite chromatography with overstaining

(2) chromatography with hydrophobic reaction; and

(3) reverse phase chromatography; which can be applied in any order.

The purification method of this invention allows obtaining high-purity recombinant FSH mass, which can then be formulated into a ready-made medicine, e.g. Gonal-F (Serono). It has the advantage of providing a high degree of purity without the use of immunoaffinity chromatography. The crude FSH that forms the starting material for purification according to the present invention consists of harvested cell cultures containing recombinant FSH.

According to one preferred aspect, an antioxidant or some free amino acid or dipeptide with antioxidant or scavenger effects is included in some or all of the steps of the purification method according to the present invention. More precisely, this antioxidant is present in any buffer used to purify and/or concentrate and/or filter r-hFSH. This antioxidant prevents the oxidation of FSH during processing. The preferred antioxidant is L-methionine. Preferably, L-methionine is used at a concentration of at or about 10-100 mM. Further examples of antioxidants include t-butyl-4-methoxy-phenol, 2,6-bis(1,1-dimethylethyl)-4-methyl phenol; potassium or sodium bimeta-bisulfite, sodium bisulfite. Examples of free amino acids and dipeptides with antioxidant or scavenger effects are histidine, taurine, glycine, alanine, chamosine, anserine, 1-methylhistidine or combinations thereof.

Typically, the starting material is first purified and then optionally concentrated (eg, using ultrafiltration) and/or buffer exchanged (eg, via a diafiltration step) before being introduced into the first chromatographic step.

In the chromatography steps, polymer-based and agarose-based resins can be used. Membrane chromatography can also be used, where the resin is replaced by a functionalized membrane.

These 3 purification steps within the scope of this invention (ie, affinity chromatography with overstaining, chromatography with hydrophobic reaction, reverse phase chromatography) are described in more detail below.

Staining Affinity Chromatography Step (1)

This method of the present invention comprises the step of affinity chromatography with overstaining (1).

According to a preferred aspect, the step of affinity chromatography with overstaining is carried out using a resin having as an immobilized ligand a dye compound well known to a person skilled in the art, i.e. Cibacron Blue F3G-A. The term "immobilized" is well understood by those skilled in the art and means that the ligand is derived in the sense that it is chemically bound to the given resin. A particularly preferred resin is Blue Sepharose FF (available from Amersham Biosciences Inc.). The technical characteristics of Blue Sepharose FF are as follows:

It is clear that this method can be applied with some alternative resins that have similar characteristics. Examples of alternative resins include: Toyopeari AF-blue-HC-650M (Tosoh Bioscience), Toyopeari SuperButyl 550, Toyopeari Phenyl 650, Blue Cellthru BigBead (Sterogene), SvvellGel Blue (Pierce), Cibachrome blue 3GA-agarose 100 (Sigma), Affi -Gel Blue (BioRad), Econo-Pac blue loading (Bio-Rad), Blue Sepharose HP (Amersham), Cibacron Blue 3GA (Sigma).

The elution step of immobilized affinity chromatography with overstaining is preferably carried out using a phosphate buffer, and it is especially preferable that it be sodium phosphate. The pH of the eluent should be about 6.0 or about 11.5, more preferably about 6.5 to about 8, and especially preferably about 7.0. Alternative buffers adequate for maintaining pH 7.0 include the following: MES, Bis-Tris, ADA, PIPES, ACES, BES, MOPS, TES, HEPES. The elution buffer for the flash-affinity chromatography step should preferably contain some salt to increase conductivity, preferably NaCl.

According to a particularly preferred aspect, the product containing the buffers for the step of affinity chromatography with redistribution (equilibration, washing and elution) contains an antioxidant, such as L-methionine. Further examples of antioxidants include t-butyl-4-methoxyphenol, 2,6-bis(1,1-dimethylethyl)-4-methylphenol; potassium or sodium bimetabisulfite, sodium bisulfite.

Chromatography step with hydrophobic interaction (2)

This method also includes a hydrophobic reaction chromatography step (2). According to a preferred aspect, hydrophobic reaction chromatography is performed with a resin such as Toyopearl Butyl 65OM (available from Tosoh Biosep In.).

It is clear that step (2) can be carried out using alternative resins, which have similar characteristics. Alternative resins that can be used are the following: Phenyl Sepharose 6 Fast flow (low sub); Phenyl Sepharose 6 Fast flow (high sub); Butyl Sepharose4 Fast Flow; Octyl Sepharose 4 Fast Flow; Phenyl Sepharose High efficiency; SOURCE 15ETH; SOURCE 15 ISO; SOURCE 15PHE all from Amersham Biosciences (800) 526-3593; (see www.amershambiosciences.com). Some additional resins are: Hydrocell C3 or C4; Hydrocell Fenil from BioChrom Labs Ina. (812) 234-2558; (see www.biochrom.com)

Bonding to the HIC resin is achieved in a high conductivity buffer obtained by adding salts (NaCl, (NH4)2SO4 or Na2SO4 for example). Elution in the hydrophobic reaction chromatography step is preferably carried out by reducing the conductivity of the mobile phase (reducing the salt concentration), using a buffer having a pH of about 6 to about 8, more preferably about 6.5 to about 7.5, most preferably about 7). One particularly preferred system contains sodium phosphate for buffering preferably at a pH of about 7 and ammonium sulfate. Alternative buffers are mentioned above.

According to a particularly preferred aspect, the buffers containing this product for step (2) for HIC (equilibration, washing, elution) contain an antioxidant, such as L-methionine. Alternative antioxidants are mentioned above.

Reverse phase chromatography step (31

This method of the present invention also includes a reverse phase chromatography (RPC) step (3). RPC is preferably carried out using a resin such as SOURCE 30 RPC (which can

obtain from Amersham Biosciences). It is clear that step (3) can be carried out using alternative resins which are well known to those skilled in the art and which have similar characteristics.

Chromatography is preferably carried out by buffering the mobile phase at a slightly basic pH, for example at about pH 7-8.5, more preferably at or around 7.5 or 7.6. According to one preferred aspect, the buffering species is ammonium acetate. Alternative buffers adequate for pH at or around 7.6 include: BES, MOPS, Phosphate, TES, HEPES. The buffer solutions used in this step may also contain some organic modifier whose concentration is modulated for the different phases of the step chromatography step (loading, washing, elution and regeneration). According to one preferred aspect, such organic modifier is some water-miscible organic solvent, preferably some alcohol (such as methanol, ethanol, etc.), most preferably 2-propanol (iso-propanol).

According to a particularly preferred aspect, the buffers containing this product for the RPC (equilibration, washing, elution) step contain an antioxidant, such as L-methionine. Alternative antioxidants are mentioned above.

Step 0 - optional further purification - ion exchange chromatography

In addition to the three main purification steps shown above, the present invention may include additional purification steps.

According to one aspect, the purification method of the present invention includes a preliminary step of ion exchange chromatography (0) preferably carried out with a strong anion exchange resin, particularly preferably with a quaternary ammonia resin such as Q Sepharose FF (available from from Amersham Biosciences), which has the following characteristics:

Alternatively, the ion exchange chromatography step (0) can be carried out using a resin

such as Fractogel EMD TMAE HICAP (available from Merck KGaA, Darmstadt Germany), or a resin with similar characteristics, see below:

The ion extraction chromatography step is preferably carried out using a buffer having a slightly alkaline pH (eg, from or about 7.2 to about 9.0, or about 8.0 to about 9.0, most preferably about 8.5). Suitable buffers include, for example, borate buffer, triethanolamine/iminodiacetic acid Tris, ammonium acetate, tricin, bicin, TES, HEPES, TAPS. The most preferred is a borate buffer, at a pH of around 8.5. Elution from the ion exchange resin is achieved by increasing the conductivity of the mobile phase by adding salt, preferably NaCl. According to one particularly preferred aspect, buffers containing this product for ion exchange chromatography (equilibration, washing, elution) contain an antioxidant, preferably L-methionine. Alternative antioxidants are mentioned above.

Thus, according to a preferred aspect, this method of the present invention comprises the following steps:

(0) anion-exchange chromatography, preferably on a strong ion-exchange resin [preferably a quaternary ammonia resin, such as Q Sepharose FF or Fractogel EMD TMAE];

(1) affinity chromatography with overstaining [preferably on Blue Sepharose FF];

(2) chromatography with a hydrophobic reaction [preferably on Toyopearl Butyl 650M];

(3) reverse phase chromatography [preferably on Source 30 RPC].

Optional further purification step M) - ultrafiltration/dialysis

Before the ion exchange chromatography step (0), it may be desirable to carry out an ultrafiltration step, in order to concentrate the crude FSH. Ultrafiltration (or diafiltration) is preferably carried out using a membrane whose value is around 3-10 kD, most preferably around 8 kD.

Optional further purification step (4) - anion exchange chromatography

According to an additional preferred aspect, this method of the present invention also comprises a second step of anion exchange chromatography (4). A preferred resin is Fractogel EMD TMAE HICAP (available from Merck KGaA, Darmstadt Germany), or resins having similar characteristics as mentioned above. Alternatively, the second step of anion-exchange chromatography can be performed on Q Sepharose FF, or other resins having similar characteristics, as mentioned above.

The steps of anion exchange chromatography, cross-color affinity chromatography, hydrophobic reaction chromatography (HIC), reverse phase chromatography, and the second anion exchange chromatography step can be performed in any order, although it is preferred that the anion exchange chromatography step be performed first. The remaining steps of flash-affinity chromatography, hydrophobic reaction chromatography (HIC), and optional other anion-exchange chromatography steps can be performed in any order, although it is preferable to follow the order below:

(0) anion-exchange chromatography, (1) affinity chromatography with overstaining, (2) hydrophobic reaction chromatography (HIC), (3) reversed-phase RPC chromatography; and

(4) second anion exchange chromatography.

Optional further purification step (5) - ultrafiltration/diafiltration

According to an additional preferred aspect, after any chromatography step (especially after the reverse phase chromatography step), the FSH sample is subjected to a concentration step. Preferably, this step is carried out using ultrafiltration in combination with diafiltration to obtain a mass of the desired composition. Ultrafiltration (or diafiltration) is preferably carried out with the help of a membrane whose value is around 3-10 kD, most preferably around 5 kD.

According to a particularly preferred aspect, the following steps are performed in the order shown below:

(-1) Ultrafiltration (preferably with a membrane whose limit value is around 8 kD),

(0) Anion exchange chromatography (preferably using a Q Sepharose FF column);

(1) affinity chromatography with overstaining (preferably using a column with Blue Sepharose FF);

(2) chromatography with a hydrophobic reaction (H1C) (preferably using a Butyl 650M c column);

(3) reverse phase chromatography (RPC) (preferably using a Source 30 RFC column);

(4) anion exchange chromatography on a strongly basic anion exchange resin preferably using TMAE hiccup resin); and

(5) ultrafiltration (preferably with a membrane whose value is around 5 kD).

It may be desirable to subject the FSH sample to a nanofiltration step, particularly as a virus purification step, ie. to reduce the risk of contamination of the FSH preparation with viruses or virus-like particles from cell culture. Nanofiltration can be done at any stage of the purification process, however, it is particularly preferred that nanofiltration be performed after the second step of ion-exchange chromatography, or after reversed-phase chromatography, or after chromatography with a hydrophobic reaction. Nanofiltration can be performed multiple times, for example it can be performed twice.

According to a particularly preferred aspect, this method of the present invention comprises the following steps:

(-1) Ultrafiltration (preferably with a membrane whose value is around 8 kD),

(0) Anion exchange chromatography (preferably with Q Sepharose FF),

(1) Affinity chromatography with overstaining (preferably with Blue Sepharose FF),

(2) Hydrophobic Reaction Chromatography (HIC) (preferably with Butyl

650M),

(3) Reverse phase chromatography (RPC) (preferably with Source 30 RPC),

(4) Anion-exchange chromatography on a strongly basic anion-exchange resin

(preferentno TMAE hicap smola);

(4') Nanofiltration,

(5) Ultrafiltration (preferably with a membrane whose value is 5 kD).

The advantage of this invention is that the purification method is free from the expensive step of immuno-affinity chromatography and that, regardless of this, it gives FSH of high purity and specific bioactivity. Also, the purified FSH of the present invention does not contain unwanted impurities that immunoaffinity chromatography gives (e.g., immunoglobulins washed from the resin).

Storage/Lyophilisation

The liquid composition obtained from the purification process as described above and containing the purified FSH can be frozen for storage as is, or after purification the eluate can be subjected to freeze-drying to eliminate the solvent. The resulting liquid or lyophilized product is called "bulk FSH".

FSH formulations

FSH or an FSH variant of the present invention or purified according to the method of the present invention can be formulated for injection, intramuscularly or subcutaneously, preferably subcutaneously. This formulation of FSH can be freeze-dried, when it is then dissolved in water for injection immediately before injection. The FSH formulation can also be a liquid formulation, in which case it is directly injected, without prior dilution.

The formulation of FSH can be in the form of a single dose or multiple doses. If it is a multiple dose, it should preferably contain some bacteriostatic agent, such as, for example, benzyl alcohol, meta-cresol, thymol or phenol, preferably benzyl alcohol or meta-cresol. Single dose formulations may also contain some bacteriostatic agent.

The FSH of the present invention can be formulated with known excipients and stabilizers, for example with sucrose and mannitol. It may also contain antioxidants, such as methionine. It can further contain a surfactant, such as TWEEN (preferably TWEEN 20), or Pluronic (preferably Pfuronic F68).

In a particularly preferred multi-dose formulation, the FSH produced by the method of the present invention is formulated by dissolving in water for injection with sucrose, phosphate buffer (pH 7), Pluronic F68, methionine and meta-cresol or benzyl alcohol.

One particularly preferred multi-dose liquid formulation of recombinant FSH for subcutaneous or intramuscular injection is as follows:

Indications

The FSH of the present invention is suitable for use in all treatments where FSH is indicated. It is particularly suitable for subcutaneous administration to induce ovulation, controlled hyperstimulation of the ovaries in assisted reproductive technologies as well as in the treatment of oligospermia. It can be used together with other gonadotropins such as LH and hCG. It can also be used with other compounds that enhance the FSH response, such as clomiphene citrate, aromatase inhibitors, such as Anastrozole, Letrozole, Fadrozole, and YM-511.

Sequence:

SEQID NO. 1: human glycoprotein, α-subunit;

SEQ ID NO. 2: hFSH P-subunit SEQ ID NO. 3: hFSH P-subunit variant 1 SEQ ID NO. 4: hFSH p-subunit variant 2 SEQ ID NO. 5: hFSH p-subunit variant 3

Follicle-stimulating hormone, or FSH, as used herein refers to human FSH (hFSH) produced as a full-length mature protein. FSH is a dimer consisting of the alpha-subunit of human glycoprotein and the beta-subunit of human FSH. The protein sequence of the alpha subunit of human glycoprotein is given in SEQ ID NO: 1 while the protein sequence of the beta subunit of human FSH is given in SEQ ID NO: 2. The use of the term "recombinant" refers to preparations of FSH that are produced using recombinant DNA technology (see example WO 85/01958). One example of a method of FSH expression using recombinant technology is the transfection of eukaryotic cells with DNA sequences encoding an alpha and beta subunit of FSH, regardless of whether it is provided on one vector or two vectors where each subunit has a separate promoter, as described in European patents no. EP 0 211 894 and EP 0 487 512. The DNA encoding FSH can be cDNA or it can contain introns. Another example of the use of recombinant technology in the production of FSH is the application of homologous recombination for the insertion of a heterologous regulatory segment in operative connection with endogenous sequences encoding one or both subunits of FSH, as described in European patent no. EP 0 505 500 (Applied Research Systems ARS Holding NV). Also contemplated are methods such as those disclosed in WO 99/57263 (transkaryotic therapies), where one of the subunits is inserted heterologously into the cell and the other subunit is expressed by activating the genomic sequences of the heterologous regulatory segment by homologous recombination. This method of the present invention can be used to purify expressed FSII using these and other methods.

The term "recombinant cell" refers to a cell produced by the insertion of heterologous DNA, including any of the aforementioned methods of genetic manipulation.

Preferably, FSH is produced in Chinese Hamster Ovary (CHO) cells transfected with a vector or vectors comprising DNA encoding the alpha-subunit of human glycoprotein and the beta-subunit of FSH. The DNA encoding the alpha and beta subunits can be present on the same or on different vectors.

The term "FSH variant" is intended to encompass those molecules that differ in amino acid sequence, glycosylation pattern, or subunit linkages from human FSH but exhibit FSH activity. Examples include CTP-FSH, a modified extended-acting recombinant FSH consisting of a wild-type α-subunit and some hybrid (a β-subunit in which the carboxy-terminal hCG peptide is fused to the C-terminal part of the β-subunit of FSH, as described by LaPolt et al.; Endocrinology; 1992, 131, 2514-2520 or Klein et al.; Development and characterization of a long-acting recombinant hFSH agonist; Human Reprod. 2003,18, 50-56]. Also included is CTP-FSH with a single chain, a single-chain molecule consisting of the following sequences (from N-terminal to C-terminal end):

indicated that PFSH denotes the β-subunit of FSH, phCG CTP (113-145) denotes the carboxy terminal peptide of hCG while aFSH denotes the α-subunit of FSH, as described by Klein et al. [Pharmacokinetics and pharmacodynamics of a single-chain recombinant human follicle-stimulating hormone containing the carboxy-terminal peptide of human chorionic gonadotropin in rhesus monkeys, Fertility & Sterility, 2002, 77,1248-1255]. Other examples of FSH variants include molecules having additional glycosylation sites incorporated into the α- and/or β-subunit, as disclosed in WO 01/58493 (Maxygen) and FSH molecules with SS bonds between subunits, as disclosed in WO 98/ 58957. Further examples of FSH variants include chimeric molecules comprising sequences from FSH and sequences from hCG or LH, such as those described in WO 91/16922 and WO 92/22568.

The FSH variants referred to herein also include carboxy-terminal deletions of beta subunits that are shorter than the full-length mature protein of SEQ ID NO:2. Carboxy-terminal deletions of human beta subunits are provided in SEQ 1DS NOS: 3,4, and 5. It is clear that the carboxy-terminal variants of the beta chain form a complex with some known alpha subunit to form some variant FSH heterodimer.

According to one preferred aspect, FSH is produced recombinantly in CHO cells, either in serum or in serum-free medium.

According to one preferred aspect, the purified FSH produced according to the method of the present invention is suitable for subcutaneous administration, which allows self-administration by the patient.

The term "crude recombinant FSH" refers to cell culture supernatant from recombinant FSH-expressing cells, before it is subjected to any chromatographic steps. This term includes crude supernatant (as isolated from cells) as well as concentrated and/or filtered and/or ultrafiltered supernatant.

The term "biological activity" related to FSH activity refers to the ability of an FSH formulation to induce biological responses associated with FSH, such as an increase in ovarian weight in the Steelman-Pohley assay [Follicle-stimulating hormone assay based on augmentation with human chorionic gonadotropin ; Endocrinology; 1953, 53,604-616], or follicular growth in female patients. The growth of follicles in female patients can be evaluated with the help of ultrasound, for example, in terms of the number of follicles that have a mean diameter of about 16 mm on the 8th day of stimulation. Biological activity is evaluated in relation to the accepted standard for FSH.

The LH content of an FSH preparation can be measured, for example, using an LH-specific immunoassay, such as Delfia hLH Spec (Wallac Oy, Turku, Finland).

The term "specific activity" in connection with FSH, means the biological activity in IJ of this preparation in some recognized bioassay for FSH, such as the Steelman Pohley bioassay [divided by the amount of protein, which is determined by some assay for total protein content, such as is Lowry's essay [O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randjall (1951) J. Biol. Chem. 193: 265; Hartree E.E. (1972). Anal. Biochem. 48: 422; J.R. Dulley and P.A. Grieve (1975) Anal. Biochem. 64:136], Bradford essay [Bradford, M. M. (1976) Anal. Biochem. 72, 248] or absorbance at 280 nm.

Preferably, the FSH obtained by the present invention has a specific activity greater than about 8000 IJ/mg, more preferably greater than about 9000 IJ/mg, even more preferably greater than about 10000 IJ/mg, even more preferably greater than about 14000 IJ/mg which is indicated by biological activity is measured by Steelman-Pohley bioassay and protein content is measured by SE-HPLC

FSH samples can be analyzed for purity at various stages of the procedure, using, for example, techniques such as those listed below:

r-hFSH quantification/free alpha subunit/purity/oxidized forms: RP-HPLC

As mentioned above, FSH is a heterodimeric glycoprotein, consisting of an α and β subunit. Some dissociation of subunits may occur, and this can be monitored by observing the amount of free ot-subunits present in a sample. In addition, FSH subunits can undergo oxidation. Oxidized contaminants can be quantified using RP-HPLC, while free subunits can be assessed using SDS-PAGE.

Quantification of r-hFSH: Immunoassay

FSH content can be determined in a sample using an immunoassay specific for FSH, such as the DELFIA FSH immunoassay.

Total protein: Bradford assay, Lowry assay, Absorbance at 280 nm

Like any other protein preparation, total protein content can be determined using techniques such as Bradford assay, Lowry assay or absorbance at 280 nm. Isoform pattern: 1EF

As mentioned above, FSH is a glycoprotein that has multiple oligosaccharide residues joined at different sites on both subunits. These oligosaccharide residues can be branched to varying degrees and can be capped with sialic acid residues. Sialic acid residues are negatively charged (at neutral pll). Differences in capping lead to heterogeneity, with a mixture of species having different isoelectric points (pl). This can be assessed using a charge-separating technique, such as isoelectric focusing (IEF).

Host Cell Protein (HCP)

The host cell protein can be analyzed using an ELISA assay. For example, antibodies can be raised in a "mock culture", which is a culture of host cells without the FSH gene.

EXAMPLES

This invention will now be illustrated by two examples.

Corresponding flow charts illustrating the aforementioned two examples are given in Figures 1 and 2. The resulting purified r-hFSH is called "bulk r-hFSH".

EXAMPLE 1 (cf Figure 1)

Step CO: Affinity chromatography with overstaining on Blue Sepharose

The initial FSH material for purification was prepared from harvested cells from the culture containing recombinant FSH, i.e. FSH produced recombinantly with CHO cells in either serum- or serum-free media. Affinity chromatography with a dye column (Blue Sepharose FF resin) was first equilibrated with a low conductivity buffer at pH 8.5 containing L-methionine. The liquid containing FSH was then applied directly to the resin. After loading, unbound material was washed away using equilibration buffer. FSH was finally eluted by washing the column with sodium phosphate buffer at pH 7.0, containing NaCl and L-methionine. The elution pool was processed directly to the next step. This step was carried out at 2-8°C.

Step (2): Chromatography with hydrophobic reaction (HIC) on Tovopearl Butyl 650M

The Blue Sepharose FF eluate from step (1) was applied to a Toyopearl Butyl 650M column equilibrated with sodium phosphate buffer, pH 7.0, containing ammonium sulfate and L-methionine. Unbound material was washed with equilibration buffer. FSH eluted with the same buffer but with a reduced concentration of ammonium sulfate. This eluate was processed in the next step. This step was performed at room temperature (RT)

Step (3): Reverse phase to Source 30 RPC

The HIC eluate (from step (2)) was first conditioned by adding IPA (isopropanol). The Source 30RPC column was equilibrated with ammonium acetate buffer, pFI 7.6, containing L-methionine, and 2-propanol at a concentration equivalent to that of the conditioned input material. After washing the unbound material with equilibration buffer, the resin was washed with ammonium acetate buffer, pH 7.6, containing L-methionine, and an increased concentration of 2-propanol. FSH was finally eluted by further increasing the concentration of 2-propanol. The elution pool was finally diluted by mixing with water containing L-methionine. This diluted pool was processed to the next step. This step is performed at room temperature (RT).

If the above procedure is followed, the purification factor - i.e. the ratio of the purity of FSH in the purified sample in relation to the purity of FSH in the starting material (raw FSH) - is about 40,000.

EXAMPLE 2 (cf Figure 2)

Step (-1): Ultrafiltration/Diafiltration of concentrated r-hFSH

All operations were performed under refrigerated conditions (2-8°C). The crude FSH that forms the starting material for purification is derived from an urban cell culture containing recombinant FSH.

Clarification

Crude r-hFSH was first filtered through a 0.5 µm filter (such as Pali Profile II filters or equivalent).

Ultrafiltration

The clarified crude material was first concentrated by ultrafiltration using a 10KD polyether sulfome membrane. The concentrated retentate was then diafiltered through at least 5 diavolumes of borate buffer, pFI 8.5 containing L-methionine as an antioxidant. The conductivity and pH of this retentate were measured to monitor the progress of diafiltration. The retentate is then further concentrated before draining the system. The ultra-filtration apparatus was finally washed with diafiltration buffer and the liquid remaining after washing was mixed with the obtained retentate. This pool is transferred to the next step.

Filtration

The concentrated product was filtered through a 0.2 µm (or equivalent) polyether sulfone filter.

Korak (0); Izmena aniona na O Sefarozi FF

The filtered material was then applied to a strong anion exchange resin (Q-sepharose FF) equilibrated with sodium borate buffer, pH 8.5, containing L-methionine. After loading, the column was washed with equilibration buffer to wash away any unbound material. The column was then eluted with sodium borate buffer pH 8.5, containing NaCl (to increase conductivity) and L-methionine (as an antioxidant). The elution of the collected pool was processed by affinity chromatography with overstaining.

Step (1): Two affinity chromatography on Blue Sepharose

Affinity flash column chromatography (Blue Sepharose FF resin) was first equilibrated with the elution buffer from the Q-Sepharose FF step. The captured eluate was then applied directly to this resin. After loading, unbound material was washed away using equilibration buffer. FSH was finally eluted by washing the column with sodium phosphate buffer at pH 7.0 containing NaCl and L-methionine. The elution pool was processed directly into the next coral. This step was carried out at 2-8°C.

Step (2): Chromatography with hydrophobic interaction on Tovopearl Butyl 650M

The Blue Sepharose FF eluate was loaded onto a Toyopearl Butyl 650M column equilibrated with sodium phosphate buffer, pH 7.0, containing ammonium and L-methionine. Unbound material was washed with equilibration buffer. FSH was eluted with the same buffer, but with a reduced concentration of ammonium sulfate. This eluate was processed in the next step. This step was carried out at room temperature (RT).

Step (3): Reverse phase to Source 30 RPC

The HIC eluate (from step (2)) was first conditioned by adding IPA (isopropanol). The Source 30RPC column is equilibrated with ammonium acetate buffer, pH 7.6, containing L-methionine and 2-propanol at a concentration equivalent to that of the conditioned input material. After washing the unbound material with equilibration buffer, this resin was washed with ammonium acetate buffer, pH 7.6, containing L-methionine and an increased concentration of 2-propanol. FSH was finally eluted by further increasing the concentration of 2-propanol. This elution pool was finally diluted with stirring, with water containing L-methionine. The diluted pool was processed to the next step. This step was carried out at room temperature (RT).

Korak (4) hromatografiia sa izmcnom koia na Fractogel EMP TMAE hicap smoli

The Fractogel EMD TMAE hicap column was first equilibrated with sodium borate buffer, pH 8.5, containing L-methionine. Diluted post-RPC material (from step (3) was applied to the column. Unbound material was washed away using equilibration buffer. FSH eluted from the column increased the salt concentration in a linear fashion. This step was carried out at 2-8°C.

Step (4*) Nanofiltration

This eluate from the Fractogel EMD-TMAE step (4) was applied directly to a 20nm nanofiltration device under 3 bar pressure under nitrogen conditions. This filtrate was processed to the next step. The operation was performed at 2-8°C.

Step (51) Ultrafiltration of bulk substances

Nanofiltered FSH material was concentrated by tangential flow filtration on a 5KD polyether sulfone membrane. When this retentate reached about one-half of the initial volume, the material was replaced by buffer by diafiltration with WFI.

Purity of samples

The purity of the FSH samples was determined after the purification step:

Biological activity of samples

The biological activity of purified r-hFSH was measured using the Steelman-Pohley method of ovarian weight increase. Specific activity was calculated using the biological activity divided by the FSH content determined using the SE-HPLC method, as described below.

The specific activity of the final bulk product obtained is typically between 10,000 and 17,000 IJ/mg. Exemplary values of 2 samples of final FSH in bulk obtained using the method from Example 2 are given in Table 1.

Table 2. Specific activity of bulk purified rhFSH of the present invention

Claims (24)

1. A method for the purification of recombinant FSH or an FSH variant comprising subjecting a liquid containing FSH to: (1) affinity chromatography with staining; (2) chromatography with hydrophobic reaction; and (3) reverse phase chromatography; which can be applied in any order. 2. The method of claim 1, wherein the affinity chromatography with overstaining of step (1) is carried out with a resin having immobilized Cibacron Blue F3G-A. 3. The method of claim 1 or 2, wherein the resin used for the affinity chromatography with overstaining of step (1) is Blue Sepharose FF. 4. The method of any one of claims 1 to 3, characterized in that the flash-affinity chromatography of step (1) is carried out using a sodium phosphate buffer at a pH of about 6.5 to 11.5 as an eluent. 5. The method of any one of claims 1 to 4, characterized in that the hydrophobic reaction chromatography (HIC) of step (2) is carried out using Toyopearl Butyl 650M, or a resin having similar characteristics. 6. The method of any one of claims 1 to 5, characterized in that the chromatography with the hydrophobic reaction of step (2) is carried out using sodium phosphate / ammonium sulfate as eluent. 7. The method of any one of claims 1 to 6, wherein the reverse phase chromatography step of step (3) is carried out using Source 30 RPC as the resin. 8. The method of any one of claims 1 to 7, characterized in that the reverse phase chromatography step of step (3) is carried out using ammonium acetate with 2-propanol as eluent. 9. The method of any one of claims 1 to 8, wherein the steps are carried out in the following order: (1) flash-affinity chromatography; (2)    chromatography with a hydrophobic reaction; and then (3)    reversed phase chromatography. 10. The method of any one of claims 1 to 9, including the step of anion exchange chromatography (0). 11. The method of claim 10, wherein the step of anion exchange chromatography is carried out before steps (1), (2) and (3). 12. The method of claim 11, wherein the steps are carried out in the following order: (0) anion exchange chromatography; (1)    affinity chromatography with overstaining; (2)    chromatography with a hydrophobic reaction; and (3)    reversed phase chromatography. 13. The method of claim 10, 11 or 12, wherein the anion exchange chromatography of step (0) is performed with Q Sepharose FF resin or Fractogel EMD TMAE HiCap resin. 14. The method of any one of claims 10 to 13, wherein the ion exchange chromatography of step (0) is carried out using borate buffer as eluent. 15. The method of claim 14, wherein the borate buffer has a pH of about 8.5. 16. The method of any one of claims 10 to 15, further comprising a second step of anion exchange chromatography (4). 17. The method of claim 16, characterized in that the second anion exchange chromatography step of step (4) is performed after steps (1), (2) and (3). 18. The method of claim 17, wherein the steps are carried out in the following order: (0) anion exchange chromatography; (1)    Affinity Chromatography with Staining; (2)    Hydrophobic reaction chromatography; (3)    Reversed phase chromatography; and (4)    Anion exchange chromatography 19. The method of any one of claims 16 to 18, wherein the second step of the anion exchange chromatography of step (4) is carried out using Fractogel EMD TMAE HiCap resin or Q SepharoseFF resin. 20. The method of claims 16 to 19, characterized in that the second step of anion-exchange chromatography from step (4) is carried out using a borate buffer and a gradient of increasing NaCl concentration. 21. The method of any one of claims 16 to 20, comprising a nanofiltration step (4'), a second anion exchange chromatography step (4) is performed. 22. The method of claim 21, comprising the step of ultrafiltration (5), after the step of nanofiltration (4'). 23. The method of any of claims 1 to 22, wherein any eluent and/or buffer may contain an antioxidant, especially L-methionine. 24. A method for purifying human recombinant FSH comprising the steps of subjecting the FSH to: (-1) ultrafiltration; (0) anion exchange chromatography Q Sepharose FF with borate /NaCl, and L-methionine with a pll of about 8.5 as eluent; (1) Subjecting the eluate from step (0) to a flash chromatography step with overstaining on Blue Sepharose FF and with phosphate, NaCl, L-methionine, at a pH of about 7 as eluents; (2) subjecting the eluate from step (l) to a step of hydrophobic reaction chromatography on Toyopearl Butyl 650M, with phosphate, ammonium sulfate, L-methionine at a pH of about 7 as eluent; (3) subjecting the eluate from step (2) to a reverse phase chromatography step on a Source 30 RPC with ammonium acetate, L-methionine, with 2-propanol at pH about 7.6 as eluent; (5) subjecting the eluate from step (3) to an anion exchange chromatography step on Fractogel (6) EMD TMAE hicap resin, with borate, L-methionine, at a pH of or about 8.5 and NaCl; (4') subjecting the eluate from step (4) to a nanofiltration step and (7) subjecting the permeate from step (4*) to an ultrafiltration step.