RS50714B - FSH Purification Methods - Google Patents

Abstract

A method for purifying recombinant FSH or a variant of FSH comprising subjecting a liquid containing FSH to: (1) affinity chromatography with staining (2) chromatography with a hydrophobic reaction; and (3) reverse phase chromatography; which can be applied in any order. The application contains another 23 patent claims.

Description

Name. METHOD PURIFICATION

FSH

Field of technology

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) for 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) for 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 combined 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

Male Hypogonadotropic Hypogonadism Collaborative Group; Hum. Reprod. ; 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.

Lynchet al. [Extraction and purification of human pituitary follicle-stimulating hormone and luteinizing hormone; Acta Endocrinologica, 1988, 288, 12-19] describe a method for the 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 IJ (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 that A<280>^for 1 g/l is equal to 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 DEAE-type weekly base 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 includes 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-Pohlev method: Follicle-stimulating hormone assay based on augmentation with human chorionic gonadotropin; Endocrinology, 1953, 53,604-616) and less than 1 IJ LH (rat seminal vesicle weight gain method: Van Hell II, 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 (Musicket 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 of LH, and urinary FSH having a specific activity of 6.298 IJ (immunoassay)/mg and less than 3 IJ (immunoassay)/mg of LH. Protein content was determined by absorbance at 280 nm (assuming that A" ]Cm for 1 g/l is equal to 1).

Chibaet al. [Isolation and partial characterization of LH, FSH and TSH from the canine pituitary gland, Endocrinol../,1997,44,205-218] describe a technique for the purification of canine pituitary gonadotropins, including FSH, by concanavalin (Con) affinity chromatography, hydrophobic reaction chromatography (HIC) and immobilized metal ion chromatography with Cu<*>^. The resulting FSH was reported to have a specific activity of 2.17 IJ/g protein using a radioreceptor assay for FSH to measure biological activity and a BioRad protein assay kit (BioRad Laboratories CA USA) to determine 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 divinyl sulfone-linked Sepharose 4B, followed by reverse-phase HPLC. The obtained 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 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 the purification of recombinant human FSH or an FSH variant, starting from a liquid containing crude FSH, comprising the following steps: (1) affinity chromatography with staining;

(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:

• affinity chromatography with overstaining,

• chromatography with hydrophobic reaction,

• reverse phase chromatography,

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

purification which includes steps:

• ultrafiltration/diafiltration,

• anion exchange chromatography,

• affinity chromatography with overstaining,

• chromatography with hydrophobic reaction,

• reverse phase chromatography,

• anion exchange chromatography.

• nanofiltration,

• u ltrafil tracij e/dij afi ltrac ij e;

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: Volume of substrate

DEAE: diethylaminoethyl

ELISA: enzyme-linked immunoassay

DAC: affinity chromatography with overstaining

IMAC: immobilized metal ion affinity chromatography OD: optical density

HIC: Hydrophobic Reaction Chromatography

HPLC : high performance liquid chromatography

IRMA: an immunoradiometric assay

KD or kD: kiloDalton

HCP: host cell protein, proteins derived from host cells used to express f FSH

IPC: In-process controls

IEF: isoelectric focusing

PES: polyethersulfone

RP-HPLC: reversed phase high performance liquid chromatography Q FF: anion exchange on Q Sepharose FF

RT: Room temperature

UF: ultrafiltration

WFI: water for injection

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) affinity 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 finished drug, 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 steps of the purification method according to the present invention. More specifically, 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, carnosine, 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.

Flash chromatography step (1)

This method of the present invention comprises the step of flash chromatography with counting (l)

According to one 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 to mean that the ligand is derived in the sense that it is chemically bound to a 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: Tovopeari AF-blue-IIC-650M (Tosoh Bioscience), Tovopeari SuperButyl 550, Tovopeari Phenyl 650, Blue Cellthru BigBead (Sterogene), SwellGel Blue (Pierce), Cibachrome blue 3GA-agarose 100 (Sigma), Affi-Gel Blue (BioRad), Econo-Pac blue fill (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 particularly preferred 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 suitable 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 dye restaining (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-methyl phenol; potassium or sodium bimetabisulfite, sodium bisulfite.

Chromatography step with hydrophobic interaction (2)

This method also includes a hydrophobic reaction chromatography step (2). In a preferred embodiment, hydrophobic reaction chromatography is performed with a resin such as Tovopeary Butyl 650M (available from Tosoh Biosep Inc.).

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 15ISO; SOURCE 15PHE all from Amersham Biosciences (800) 526-3593; (see www.amershambiosciences.com). Some additional resins are: Hvdrocell C3 or C4; Hvdrocell Phenyl by BioChrom Labs Inc. (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 (3)

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 (available 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 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 having similar characteristics, see below:

The ion exchange 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, tricine, bicine, TES, HEPES, TAPS. The most preferred is a borate buffer, at a pH of about 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, the 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 one 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 Tovopeari Butyl 650M];

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

Optional further purification step (- 1) - ultrafiltration/ diafiltration

Before the ion exchange chromatography step (0), it may be desirable to perform an ultrafiltration step to concentrate the crude FSH. Ultrafiltration (or diafiltration) is preferably carried out using a membrane whose value is about 3-10 kD, most preferably about 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 preferred to follow the order below:

(0) anion-exchange chromatography, (1) affinity chromatography with overstaining, (2)

hydrophobic reaction chromatography (HIC), (3) RPC reverse phase 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 a 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 carried out 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 column

FF);

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

Sepharose FF);

(2) hydrophobic reaction chromatography (HIC) (preferably using a column with

Butyl 650M c);

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

RPC); (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 about 5 kD).

It may be desirable to subject the FSFI sample to a nanofiltration step, particularly in the form of 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 performed at any stage of the purification process, however, it is particularly preferred that nanofiltration be performed after the second ion exchange chromatography step, or after reverse phase chromatography, or after hydrophobic reaction chromatography. 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 jc 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

(preferably TMAE hiccup resin);

(4') Nanofiltration,

(5) Ultrafiltration (preferably with a 5 kD membrane).

The advantage of this invention is that the purification method is free from the expensive step of immunoaffinity 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 provides (eg, immunoglobulins washed from the resin).

Storage/Lyophilization

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 may be formulated for injection, intramuscular or subcutaneous, preferably subcutaneous. 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 Pluronic 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.

Sequences:

SEQ ID 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 for example WO 85/01958). One example of a method of FSH expression using recombinant technology is the transfection of cukaryotic 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 may be cDNA or it may contain introns. Another example of the use of recombinant technology in the production of FSH is the application of homologous recombination to insert a heterologous regulatory segment into an operative link with the 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 with a homologous gene. This method of the present invention can be used to purify expressed FSH 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 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 the wild-type α-subunit and some hybrid (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 LaPoltet al; Endocrinologv; 1992,131,2514-2520 or Klein et al.; Development and characterization of recombinant FSH agonists Long-acting hFSH; Human Reprod. 2003, 50-56] Also included is CTP-FSH, a single-chain molecule consisting of the following sequences (N-terminal to C-terminal):

indicated that pFSH denotes the β-subunit of FSH, phCG CTP (113-145) denotes the carboxy terminal peptide of hCG while ocFSH denotes the α-subunit of FSH, as described by Kleinet al. [ Pharmacokinetics and pharmacodynamics of single-chain recombinant human

follicle-stimulating hormone containing human chorionic carboxyterminal peptide

gonadotropin in rhesus monkeys, Fertility & Slerility, 2002, 77.1248-1255]. Other examples of FSH variants include molecules having additional glycosylation sites incorporated into the ua- and/or P-subunit, as disclosed in WO 01/58493 (Maxygen) and FSH molecules with S-S bonds between subunits, as disclosed in WO 98/58957. Further examples of FSH variants include chimeric molecules containing sequences from FSH and sequences from hCG or LH, such as

which are 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 ID 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 a preferred embodiment, FSH is produced recombinantly in CHO cells, either in serum or in a 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 enables self-administration by the patient.

The term "crude recombinant FSH" refers to the cell culture supernatant from recombinant FSH-expressing cells, before it is subjected to any chromatographic steps. This term includes the crude form of the supernatant (as isolated from the cells) as well as the 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 [a follicle-stimulating hormone assay based on augmentation with human chorionic gonadotropin; Endocrinology; 1953, 53,604-616], or follicular growth in female patients. Follicular growth in female patients can be assessed 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 against an 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 Steelman Pohley's bioassay [divided by the amount of protein, which is determined by some assay for total protein content, such as Lowry's assay [O.H. Lowry. N.J. Rosebrough, A.L. Farr and R.J. Randall

(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 as indicated by the biological activity being measured by Steelman-Pohley bioassay and the protein content being measured by means of SE-HPLC

FSH samples can be analyzed for their purity at different stages of this 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 a and a p subunit. Some dissociation of subunits may occur, and this can be monitored by observing the amount of free α-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 a specific immunoassay

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: IEF

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 pH). 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 diagrams 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 (1): Affinity chromatography with overstaining on Blue Sepharose

The initial FSH material for purification was prepared from harvested cells from culture containing recombinant FSH, i.e. FSH produced recombinantly with CHO cells in either serum- or serum-free medium. 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 Tovopeare Butyl 650M

The Blue Sepharose FF eluate from step (I) was applied to a Tovopeary 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 on 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, 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-mentioned 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

Clarified crude material was first concentrated by ultrafiltration using a 10KD polyether sulfonic 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 the 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.

Step (0): Anion exchange on Q Sepharose 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 Tovopeare Butyl 650M

The Blue Sepharose FF eluate was loaded onto a Tovopeary 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 on 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).

Step (4) chromatography with modification on Fractogel EMD TMAE hiccup resin

The column with Fractogel EMD TMAE hicap 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 (5) Ultrafiltration of the bulk substance

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 with buffer by diafiltration with WFI.

Sample purity

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

Claims (24)

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 the final FSH in bulk obtained using the methods from Example 2 are given in Table 1. Requirements1. A method for purifying 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, characterized in that the affinity chromatography with color transfer from step (1) is carried out with a resin having immobilized Cibacron Blue F3G-A. 3. The method of claim 1 or 2, characterized in that the resin used for affinity chromatography with overstaining from step (1) is Blue Sepharose FF. 4. The method of any one of claims 1 to 3, characterized in that the flash-over affinity chromatography of step (1) is carried out using a sodium phosphate buffer at a pH of about 6.5 to 11.5 as 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 Tovopeary Butyl 650M, or some resin having similar characteristics. 6. The method of any one of claims 1 to 5, characterized in that the hydrophobic reaction chromatography of step (2) is carried out using sodium phosphate / ammonium sulfate as an 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) affinity chromatography with overstaining; (2) chromatography with hydrophobic reaction; and then (3) reverse 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, characterized in that 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 hydrophobic reaction; and (3) reverse phase chromatography. 13. The method of claim 10, 11 or 12, characterized in that the anion exchange chromatography of step (0) is carried out with Q Sepharose FF resin or Fractogel EMD TMAE HiCap resin. 14. The method of any one of claims 10 to 13, characterized in that 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 step of anion exchange chromatography from step (4) is carried out 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 overstaining; (2) Chromatography with hydrophobic reaction; (3) Reverse phase chromatography; and (4) Anion exchange chromatography 19. The method of any one of claims 16 to 18, characterized in that the second anion exchange chromatography step of step (4) is carried out using Fractogel EMD TMAE HiCap resin or Q SepharoseFF resin. 20. The method from claims 16 to 19, which is indicated by the fact that the second step of anion-exchange chromatography from step (4) is carried out with the use of 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'), followed by a second anion exchange chromatography step (4). 22. The method of claim 21, comprising an ultrafiltration step (5), after a nanofiltration step (4'). 23. The method of any one of claims 1 to 22, which is indicated by the fact that any eluent and/or buffer may contain some 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 on Q Sepharose FF with borate / NaCl, and L-methionine at a pH of about 8.5 as eluent; (1) Subjecting the eluate from step (0) to an affinity 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 hydrophobic reaction chromatography step on Tovopeary 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.