HK1108704B - Method for purifying fsh - Google Patents

Description

Method for purifying FSH

Technical Field

The present invention relates to the field of purification of Follicle Stimulating Hormone (FSH).

Technical Field

Follicle Stimulating Hormone (FSH) is an injectable protein belonging to the class of gonadotropins. FSH is used in the treatment of infertility and reproductive disorders in both female and male patients.

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

FSH is used in female patients to induce Ovulation (OI) and Controlled Ovarian Hyperstimulation (COH) in Assisted Reproductive Technologies (ART). In a typical ovulation induction treatment regimen, patients are given daily injections of FSH or a variant thereof (about 75 to 300IU FSH/day) over a period of about 6 to 12 days. In a typical course of controlled ovarian hyperstimulation therapy, patients are administered daily injections of FSH or a variant thereof (about 150-.

FSH is also used to induce spermatogenesis in male patients with oligospermia. In hypogonadal men with hypogonadotropic hormone secretion, a course of 3 times a week with 150IU FSH combined with 2,500IU hCG twice a week has successfully increased sperm count [ Burgues et al; (ii) a Subcutaneous self-administration of a high purity followified contamination hormone and a human chloroonicgoniophorbin for the treatment of the said reagent of a large hyperbolographic hyposona, a Spanish Collalibrative Group on a MaleHypolographic hyposona; hum. reprod.; 1997, 12, 980-6].

Because of the importance of FSH in the treatment of reproductive disorders, it is desirable to provide FSH of high purity and high specific activity. FSH treatment requires repeated injections. Highly purified FSH preparations can be administered subcutaneously or by the patient himself, thus providing great convenience and compliance to the patient.

Lynch et al describe a method for The purification of human pituitary FSH [ The extraction and purification of human pituitary follicle-stimulating hormone and stimulating hormone; acta Endocrinology, 1988, 288, 12-19]. The method comprises anion and cation exchange chromatography, immunoaffinity extraction and size exclusion chromatography. The specific activity of pituitary FSH obtained by this method was 4,990 (immunoassay) IU/mg, including 16 IU/mgLH. Protein content was determined by absorption at 280nm in dry weight or in solution (assuming A²⁸⁰ _1cmAt 1g/l equal to 1).

WO 98/20039(IBSA institute Biochimique SA) describes a human urinary FSH purification method with a urine extract as the starting material, also known as human menopausal gonadotropin (hMG). The method uses DEAE type weakly basic anion exchange resin to perform ion exchange chromatography, and then performs affinity chromatography on resin with anthraquinone derivative as ligand. The urinary FSH obtained by this method is said to be LH-free and to have a specific activity of 6,870 (immunoassay) IU/mg. The protein content was determined in a quartz cuvette with a path length of 1cm, assuming an optical density of 0.62 at 277nm for a 1mg/ml aqueous protein solution.

WO 00/63248(Instituto Massone SA) describes a method for purification of gonadotropins from FSH contained in human urine. The method comprises the following steps: ion exchange chromatography on a strong cationic hydrocarbyl sulphate type resin, ion exchange chromatography on a strong anionic resin and Hydrophobic Interaction Chromatography (HIC). The resulting FSH preparation was reported to have a specific activity of 8,400IU/mg (Steelman-Pohley method: Assay of the folloid cementing hormone basal on the amplification with human choricotino protein; Endocrinology; 1953, 53, 604-42) and less than 1IU LH bioactivity per 75IU FSH (rat mineral weight gain method: VanHell H, Matthijsen R & GA verbobek; Acta Endocrinol, 1964, 47, 409). Protein content was determined using the Lowry method [ o.h.lowry et al, j.biol.chem., 1951, 193, 265 ].

US 5,990,288(Musick et al) describes a method for purifying FSH from a biological sample, such as human pituitary gland or human post-menopausal urine. The method is used in Fractogel EMDSO₃Cation exchange chromatography on 650M, followed by dye affinity chromatography on a Mimetic orange 1 resin, and finally hydrophobic interaction chromatography on a Bakerbond Wide Pore HI-Propyl resin. The specific activity of human pituitary FSH obtained by this method is said to be 7,066IU (immunoassay)/mg and LH less than 1IU (immunoassay)/mg, and the specific activity of urinary FSH is said to be 6,298IU (immunoassay)/mg and LH less than 3IU (immunoassay)/mg. Protein content was determined by absorption spectroscopy at 280nm (assuming A²⁸⁰ _1cmAt 1g/l equal to 1).

Chiba et al [ Isolation and partial characterization of LH, FShand TSH from saline pipeline; Endocrinol.J., 1997, 44, 205-]A technique for purifying canine pituitary gonadotropins including FSH using concanavalin (Con) A affinity chromatography, Hydrophobic Interaction Chromatography (HIC) and Cu is described⁺⁺And (4) fixed metal ion chromatography. It was reported that the bioactivity was measured by FSH radioreceptor assay and the protein content was measured by BioRad protein kit (BioRad Laboratories CA USA), and the specific activity of the obtained FSH was 2.17IU/g protein.

WO 88/10270(Instituto di Ricecrca Cesare Serono SPA) describes a method for purification of FSH from human urine. The method involves immunochromatography using FSH-specific immobilized monoclonal antibodies conjugated with bis-vinylsulfone to Sepharose 4B, followed by reverse phase HPLC. The obtained FSH was free of LH and other urinary proteins and had a specific activity of 6,200IU/mg (lyophilized powder) (Steelman-Pohley method). The formulation is the first FSH formulation and is suitable for subcutaneous injection due to its high purity.

There is therefore a need to develop new methods for purification of FSH or FSH variants. In particular, there is a need for purification methods that avoid the use of costly immunoaffinity chromatography steps.

Brief description of the invention

The object of the present invention is to provide a novel method for purification of recombinant FSH or recombinant FSH variants.

The present invention firstly provides a method for purifying recombinant human FSH or FSH variants starting from a liquid containing raw FSH, comprising the steps of:

(1) dye affinity chromatography;

(2) hydrophobic interaction chromatography, and;

(3) reversed phase chromatography;

may be performed in any order.

Brief Description of Drawings

FIG. 1 shows the present invention, namely a purification process comprising the following steps:

dye affinity chromatography, followed by a chromatographic analysis,

the hydrophobic interaction chromatography is carried out in the presence of a hydrophobic interaction agent,

(ii) reverse-phase chromatography (reversed-phase chromatography),

FIG. 2 shows a flow chart of a specific embodiment of the present invention, namely a purification method comprising the following steps:

-an ultrafiltration/diafiltration step, in which the ultrafiltration step is carried out,

(ii) anion-exchange chromatography, wherein,

dye affinity chromatography, followed by a chromatographic analysis,

the hydrophobic interaction chromatography is carried out in the presence of a hydrophobic interaction agent,

(ii) reverse-phase chromatography (reversed-phase chromatography),

(ii) anion-exchange chromatography, wherein,

the nano-filtration is carried out in a reactor,

ultrafiltration/diafiltration;

abbreviations;

the following abbreviations are used in the description of the present invention:

DF: diafiltration (Perfect Inflation)

FSH: follicle stimulating hormone;

r-FSH: recombinant follicle stimulating hormone;

hFSH: human follicle stimulating hormone;

r-hFSH: recombinant human follicle stimulating hormone

BV: volume of the column bed

DEAE: diethylaminoethyl

ELISA: enzyme linked immunoassay

DAC: dye affinity chromatography

IMAC: stationary metal ion chromatography

OD: optical density

HIC: hydrophobic interaction chromatography

HPLC: high performance liquid chromatography

IRMA: immunoradiometric assay

KD or kD: kilodalton (kilodalton)

HCP: host cell protein for expressing FSH

IPC: in the process control

IEF: isoelectric focusing

PES: polyether sulfone

RP-HPLC: reversed phase high performance liquid chromatography

QFF: anion exchange on Q Sepharose FF

RT: at room temperature

UF: ultrafiltration

WFI: water for injection

Detailed Description

The present invention provides a method for purifying recombinant human FSH or FSH variants starting from a liquid containing raw FSH, said method comprising the steps of:

(1) dye affinity chromatography;

(2) hydrophobic interaction chromatography; and

(3) reversed phase chromatography;

the above steps may be performed in any order.

The purification method of the present invention allows to obtain a high purity recombinant FSH material which can be formulated as a final drug, such as Gonal-F (Serono). It has the advantage of obtaining a high purity product without the use of immunoaffinity chromatography. As the present invention, the purified starting material raw FSH consists in the harvest of cell cultures containing recombinant FSH.

In a preferred embodiment, antioxidants or free amino acids or dipeptides with antioxidant and scavenging action are included in some or all steps of the purification process according to the invention. More precisely, the antioxidant is present in any buffer used for purification and/or concentration and/or filtration of rhFSH. Antioxidants prevent oxidation of FSH during processing. A preferred antioxidant is L-methionine. The L-methionine concentration used is preferably at or about 10-100 mM. Other examples of antioxidants include t-butyl-4-methoxy-phenol, 2, 6-bis (1, 1-dimethylethyl) -4-methylphenol; potassium or sodium metabisulfite and sodium bisulfite. Examples of free amino acids and dipeptides with antioxidant and scavenging action are histidine, taurine, glycine, alanine, carnosine, anserine, 1-methylhistidine or combinations thereof.

The starting material is typically first clarified, and then optionally concentrated (e.g., by ultrafiltration) and/or buffer exchanged (e.g., by a diafiltration step) prior to collection in the first chromatography step.

In the chromatography step, polymer-based and agarose-based resins may be used. Membrane chromatography may also be used, in which the resin is replaced by a functionalized membrane.

The 3 purification steps of the invention (i.e., dye affinity chromatography, hydrophobic interaction chromatography, and reverse phase chromatography) are described in detail below.

Dye affinity chromatography step (1)

The method of the invention comprises a dye affinity chromatography step (1). In a preferred embodiment, dye affinity chromatography, i.e., Ciba blue F3G-A, is performed using a resin having dye compounds known to those skilled in the art as immobilized ligands. The term "immobilized" is understood by those skilled in the art to mean derivatizing the ligand in the sense of chemical attachment to the resin. A particularly preferred resin is blue Sepharose FF (Amersham Biosciences Inc.). The technical characteristics of blue sepharose FF are as follows:

it will be appreciated that other resins having similar characteristics may be used in the process. Other resins include: toyopearl AF-Blue-hc-650m (Tosoh bioscience), Toyopearl SuperButyl550, Toyopearl Phenyl 650, Blue Cellthru BigBead (Sterogene), Swell Gel Blue (Pierce), Ciba Crom Blue 3 GA-Sepharose 100(Sigma), Affi-Gel Blue (BioRad), Econo-Pac Blue cards (Bio-Rad), Blue Sepharose HP (Amersham), Ciba Blue 3GA (Sigma).

The elution step of dye affinity chromatography is preferably performed with a phosphate buffer, particularly preferably sodium phosphate. The pH of the eluent is preferably about 6.0 to 11.5, more preferably about 6.5 to 8, and particularly preferably about 7.0. Additional buffers suitable for maintaining a pH of 7.0 include the following: MES, Bis-Tris, ADA, PIPES, ACES, BES, MOPS, TES, HEPES. The elution buffer for dye affinity chromatography preferably contains a salt to increase conductivity, preferably NaCl.

In a particularly preferred embodiment, the buffer contacted with the product in the dye affinity chromatography steps (equilibration, wash and elution) contains an antioxidant, such as L-methionine. Other examples of antioxidants include t-butyl-methoxyphenol, 2, 6-bis (1, 1-dimethylethyl) -4-methylphenol; potassium or sodium metabisulfite and sodium bisulfite.

Hydrophobic interaction chromatography step (2)

The method also includes hydrophobic interaction chromatography (2). In a preferred embodiment, hydrophobic interaction chromatography is performed using a resin such as Toyopearl butyl 650M (from Tosoh Biosep).

It will be appreciated that step (2) may also be carried out with other resins having similar characteristics. Alternative resins are: fast phenyl sepharose 6(low sub); fast phenyl sepharose 6(high sub); quick butyl sepharose 4; quick octyl sepharose 4; high performance phenyl sepharose; SOURCE 15 ETH; SOURCE 15 ISO; SOURCE 15PHE, these resins were from Amersham Biosciences (800) 526-3593; (seewww.amershambiosciences.com). Other resins are: hydrocell C3 or C4; hydrocell Phenyl from BioChrom Labs (812) 234-; (seewww.biochrom.com)。

The binding of HIC resin is achieved in a buffer with high conductivity obtained by the addition of salt (e.g., NaCl, (NH)₄)₂SO₄Or Na₂SO₄). Elution during the hydrophobic interaction chromatography step preferably reduces the conductivity of the mobile phase (reduces the salt concentration) and the buffer pH is about 6 to 8, preferably about 6.5 to 7.5, and most preferably about 7. A particularly preferred system comprises a sodium phosphate buffer at a pH of about 7, and ammonium sulfate. Other buffers are mentioned above.

In a particularly preferred embodiment, the buffer in which the product is contacted in step (2) (equilibration, wash, elution) of the HIC comprises an antioxidant, such as L-methionine, other antioxidants being mentioned above. Reversed phase chromatography step (3)

The method of the invention also comprises a reverse phase chromatography step (RPC) (3). RPC is preferably performed with a resin such as SOURCE30RPC (from Amersham Biosciences). It will be appreciated that step (3) may also be carried out with other resins known to those skilled in the art to have similar characteristics.

Chromatography is preferably carried out in a weakly basic pH-buffered mobile phase, e.g., at about pH7-8.5, preferably at about pH 7.5 or 7.6. In a preferred embodiment, the buffer is ammonium acetate. Other suitable buffers having a pH of about 7.6 include: BES, MOPS, phosphate, TES, HEPES. The buffer solution used in this step also comprises an organic modifier, the concentration of which is adjusted at different phases of the chromatography step (column loading, washing, elution and regeneration). In a preferred embodiment, the organic modifier is an organic solvent that is readily soluble in water, preferably an alcohol (e.g., methanol, ethanol, etc.). 2-propanol (isopropanol) is particularly preferred.

In a particularly preferred embodiment, the buffer contacted with the product in step (3) (equilibration, washing, elution) of the RPC comprises an antioxidant, such as L-methionine, other antioxidants being mentioned above. Optional additional purification step 0-ion exchange chromatography

In addition to the three main purification steps-outlined above-the present invention may also comprise further purification steps.

In one embodiment, the purification process of the invention comprises a preliminary step (0) of ion exchange chromatography, preferably with a strong anion exchange resin, particularly preferably a quaternary ammonium resin, such as Q sepharose FF (from Amersham Biosciences), having the following characteristics:

the ion exchange type: strong anions

Total capacity (mmol/ml): 0.18-0.25

Exclusion limit (globin): 4X 10⁶

Particle form: spherical, diameter 45-165 μm

The particle structure is as follows: crosslinked agarose, 6%

pH operation stability: 2-12

pH cleaning stability: 1-14

At 25 ℃ 1 bar 15cm bed height,

linear flow rate of XK50/30 column: 400-700cm/h

Alternatively, the ion exchange resin step (0) may be performed using a resin such as Fractogel EMD TMAEHICAP (from Merck KGaA, Darmstadt Germany), or a resin having the following properties:

the ion exchange chromatography step is preferably performed in a weakly basic buffer (e.g., about 7.2 to 9.0, or about 8.0 to 9.0, and most preferably about 8.5). Suitable buffers include, for example, boric acid buffer, triethanolamine/iminodiacetic acid Tris, ammonium acetate, Tris (hydroxymethyl) methylglycine, bicine, TES, HEPES, TAPS. Sodium borate having a pH of about 8.5 is preferred. Elution from the ion exchange resin is carried out by adding a salt, preferably NaCl, to increase the conductivity of the mobile phase. In a particularly preferred embodiment, the buffer in which the product is contacted in ion exchange chromatography (equilibration, wash, elution) contains an antioxidant, preferably L-methionine. Other antioxidants are mentioned above.

Thus, in a preferred embodiment, the method of the present invention comprises the steps of:

(0) anion exchange chromatography, preferably on a strong anion exchange resin, [ preferably a quaternary ammonium resin, e.g., Q sepharose FF or Fractogel EMDTMAE ];

(1) dye affinity chromatography [ preferably on blue sepharose FF ];

(2) hydrophobic interaction chromatography [ preferably on Toyopearl butyl 650M ];

(3) reverse phase chromatography [ preferably on Source30RPC ].

Optional additional purification step (-1) -Ultrafiltration/diafiltration

An ultrafiltration step may be performed to concentrate the crude FSH before the ion exchange chromatography (0) is performed. Ultrafiltration (or diafiltration) preferably uses a cut-off membrane of about 3-10kD, preferably 8 kD. Optional additional purification step (4) -anion exchange chromatography

In another preferred embodiment, the method of the invention may further comprise a second anion exchange chromatography (4). The resin is preferably Fractogel EMD TMAE HICAP (from Merck KGaA, Darmstadt Germany), or a resin with similar properties as mentioned above. Alternatively, the second anion exchange chromatography can be performed on Q sepharose FF or a resin of similar properties as mentioned above.

Anion exchange chromatography, dye affinity chromatography, Hydrophobic Interaction Chromatography (HIC), reverse phase chromatography and second anion exchange chromatography may be performed in any order, although it is preferred to perform the anion exchange chromatography first. The steps of remaining dye affinity chromatography, Hydrophobic Interaction Chromatography (HIC), RPC and optionally second anion exchange chromatography may be performed in any order, although preferably in the following order:

(0) anion exchange chromatography, (1) dye affinity chromatography, (2) Hydrophobic Interaction Chromatography (HIC), (3) reverse phase chromatography RPC, and (4) a second anion exchange chromatography.

Optional additional purification step (5) -Ultrafiltration/diafiltration

In another preferred embodiment, after the above chromatography step (in particular after the reverse phase chromatography step), the FSH sample is subjected to a concentration step. Said steps preferably combine ultrafiltration with diafiltration to obtain a bulk product of the desired composition. Ultrafiltration (or diafiltration) preferably uses a cut-off membrane of about 3-10kD, preferably 8 kD.

In a particularly preferred embodiment, the following steps are carried out in the following order:

(-1) Ultrafiltration (preferably with a cut-off membrane of about 8 kD)

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

(1) dye affinity chromatography (preferably with a blue sepharose FF column);

(2) hydrophobic Interaction Chromatography (HIC) (preferably using a butyl 650M column);

(3) reverse Phase Chromatography (RPC) (preferably with Source30RPC column)

(4) Anion exchange chromatography, strongly basic anion exchange resin (preferably using TMAEhicap column)

(5) Ultrafiltration (preferably with a 5kD cut-off membrane).

It may be desirable to subject the FSH sample to nanofiltration, particularly as a viral clearance step; that is, the risk of contamination of the FSH preparation with virus or viroid particles from the cell culture is reduced. Nanofiltration may be performed at any step in the purification process, however, it is particularly preferred to perform nanofiltration after the second ion exchange chromatography, or after reverse phase chromatography or after hydrophobic interaction chromatography. More than one nanofiltration may be performed, such as two nanofiltration passes.

In a particularly preferred embodiment, the method of the invention comprises the steps of:

(-1) Ultrafiltration (preferably with a cut-off membrane of about 8 kD)

(0) Anion exchange chromatography (preferably with Q sepharose FF);

(1) dye affinity chromatography (preferably with blue sepharose FF);

(2) hydrophobic Interaction Chromatography (HIC) (preferably with butyl 650M);

(3) reverse Phase Chromatography (RPC) (preferably with Source30 RPC);

(4) anion exchange chromatography, strongly basic anion exchange resin (preferably with TMAEhicap);

(4') nanofiltration;

(5) ultrafiltration (preferably with a 5kD cut-off membrane).

The present invention has the advantage that the purification method does not have a high-price immunoaffinity chromatography step, and provides FSH with high purity and high specific bioactivity. Also, the purified FSH of the present invention is free of unwanted impurities (e.g., immunoglobulins filtered from the resin) added as a result of immunoaffinity chromatography.

Storage/lyophilization process

The liquid composition containing purified FSH obtained from the purification process as described above may be stored frozen as is or after purification, the eluate may be lyophilized ("freeze-dried") to remove the solvent. The resulting liquid or lyophilized product is referred to as "FSH stock".

FSH formulations

The FSH or FSH variant of the invention or the FSH or FSH variant purified according to the method of the invention may be formulated for injection, either intramuscularly or subcutaneously, preferably subcutaneously. The FSH formulation may be lyophilized, in which case it is dissolved in water for injection prior to injection. The FSH formulation may also be a liquid formulation, in which case it may be injected directly without any dissolution.

FSH formulations may be single or multiple dose. If multiple doses are used, it is preferred to include a bacteriostatic agent such as benzyl alcohol, m-cresol, thymol or phenol, preferably benzyl alcohol or m-cresol. The single dose formulation may also contain a bacteriostatic agent.

The FSH of the present invention may be formulated with known excipients and stabilizers, such as sucrose and mannitol. Antioxidants such as methionine may also be included. Further, a surfactant such as TWEEN (preferably TWEEN 20) or polyether (preferably polyether F68) may be contained.

In a particularly preferred multi-dose formulation, the FSH produced by the method of the present invention is dissolved in water and formulated into an injection with sucrose, phosphate buffer (pH7), polyether F68, methionine and m-cresol or benzyl alcohol.

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

indications of

The FSH of the present invention is suitable for use in a variety of therapies requiring FSH. Is particularly suitable for subcutaneous administration in ovulation induction, controlled ovarian hyperstimulation and oligospermia treatment in the control assisted reproductive technology. It can be used in combination with other gonadotropins such as LH and hCG, or with compounds that increase the response to FSH, such as clomiphene, and aromatase inhibitors such as anastrozole, letrozole, fadrozole and YM-511.

The sequence is as follows:

SEQ ID NO. 1: a human glycoprotein alpha subunit;

SEQ ID NO. 2: hFSH beta subunit

SEQ ID NO. 3: hFSH beta subunit variant 1

SEQ ID NO. 4: hFSH beta subunit variant 2

SEQ ID No. 5: hFSH beta subunit variant 3

Follicle stimulating hormone, or FSH, as used herein refers to human FSH (hfsh) proposed as the full-length mature protein. FSH is a dimer containing both the human glycoprotein alpha subunit and the human FSH beta subunit. The protein sequence of the alpha subunit of human glycoprotein is SEQ ID NO.1, and the protein sequence of the beta subunit of human FSH is SEQ ID NO. 2.

The term "recombinant" as used herein refers to a preparation of FSH produced by recombinant DNA techniques (see, e.g., WO 85/01958). An example of a method for expressing FSH using recombinant technology is the transfection of eukaryotic cells with DNA sequences encoding the α and β subunits of FSH, as described in EP 0211894 and EP 0487512, each subunit having a single promoter, whether on one or both vectors. The DNA encoding FSH may be a cDNA or it may contain introns. Another example of a method for generating FSH by recombinant technology is the insertion of a heterologous regulatory fragment by homologous recombination technology into the endogenous sequence encoding one or both of the subunits of FSH in an operably linked manner, as described in European patent No. EP 0505500 (Applied research systems ARS Holding NV). Some of the methods disclosed in WO 99/57263(Transkaryotic therapeutics) are also contemplated in which one subunit is inserted into the cell heterologously and the other subunit is inserted into a heterologous regulatory fragment via homologous recombination to activate the genomic sequence for expression. The method of the invention may be used to purify FSH expressed by any of the above methods.

Expression of "recombinant cell" refers to a cell produced by insertion of a heterologous DNA, including any of the genetic manipulation methods mentioned above.

FSH is preferably recombinantly produced in Chinese Hamster Ovary (CHO) cells transfected with DNA containing one or more vectors encoding the human glycoprotein alpha subunit and the FSH beta subunit. The DNA encoding the alpha and beta subunits may be present in the same or different vectors.

The expression "FSH variant" is intended to include molecules which differ from human FSH in their amino acid sequence, glycosylation pattern or linkage between subunits, but which exhibit FSH activity. Examples include CTP-FSH, a long-acting modified recombinant FSH consisting of a wild-type α subunit and a hybrid β subunit, wherein the carboxy-terminal peptide of hCG is fused at the C-terminus of the FSH β subunit, e.g., lapart et al; endocrinology; 1992, 131, 2514 and 2520; or Klein et al; development and characterization of a long-acting recombinanthFSH agonist; human reprod.2003, 18, 50-56 ]. Also included is single chain CTP-FSH, a single chain molecule consisting of the following sequences (from N-terminus to C-terminus):

βFSH

βhCG-CTP(113-145)

αFSH

wherein β FSH represents the FSH β subunit, β hCG CTP (113-145) represents the hCG carboxy-terminal peptide, and α FSH represents the FSH α subunit, as described in Klein et al [ pharmaceuticals and pharmacodynics of single-chain-interacting hormone binding the human chloronic gonadotrophin carboxypeptide in thermal peptides monkey; fetility & sterilite; 2002, 77, 1248-1255]. Other examples of FSH variants include FSH molecules with introduced glycosylation sites in the alpha and/or beta subunits, as shown in WO 01/58493(Maxygen), and FSH molecules with S-S bonds between subunits, as shown in WO 98/58957. Other examples of FSH variants include chimeric molecules comprising a FSH sequence and a hCG or LH sequence, such as those described in WO 91/169922 and WO 92/22568.

The FSH variants referred to herein also include a carboxy-terminal deletion of the beta subunit of the full-length mature protein shorter than SEQ ID No. 2. The carboxy-terminal deletion of the human FSH β subunit in SEQ IDS No.3, 4 and 5. It is understood that the carboxy-terminal variant of the beta chain forms a dimer with a known alpha subunit, thereby forming an FSH variant heterodimer.

In a preferred embodiment, FSH is recombinantly produced in CHO cells in serum-containing or serum-free medium.

In a preferred embodiment, the purified FSH produced according to the method of the present invention is suitable for subcutaneous administration, and the patient may be self-administered.

Expression of "unprocessed recombinant FSH" refers to the cell culture supernatant in recombinant cells expressing FSH prior to any chromatography steps being taken. The expression includes the crude form of the supernatant (isolated from the cells) as well as the concentrated and/or filtered and/or ultrafiltered supernatant.

The term "biological activity" in relation to FSH activity refers to the ability of an FSH preparation to elicit a biological response associated with FSH, such as ovarian weight as obtained in the Steelman-Pohley Assay [ Assay of the nesting hormone base on the evaluation with human chorionic gonadotropin; endocrinology; 1953, 53, 604, 616], or follicular growth in female patients. Follicle growth in female patients can be estimated by ultrasound, e.g., based on the number of follicles after stimulation at 8 th scale with a mean diameter of approximately 16 mm. Biological activity may be estimated by a standard acceptable for FSH.

The amount of LH in the FSH preparation may be determined, for example, by LH specific immunoassays, such as the Delfia hLH Spec (Wallac Oy, Turku, Finland).

The term "specific activity" with respect to FSH refers to the biological activity of the preparation expressed as IU, as measured according to accepted FSH bioassays such as the Steeelman Pohley bioassay, divided by the weight of protein, as determined according to one of the assays for total protein content, such as the Lowry assay [ o.h.lowry, n.j.rosebrough, a.l.farr and r.j.randall (1951) j.biol.chem.193: 265 of a nitrogen-containing gas; hartree E.E, (1972), anal. biochem.48: 422; j.r.dulley and p.a.grieve (1975) anal.biochem.64: 136], Bradford assay [ Bradford, M.M, (1976) anal. biochem.72, 248], or absorbance at 280 nm.

Preferably, the specific activity of the FSH of the invention is greater than or about 8000IU/mg, more preferably greater than or about 9000IU/mg, still more preferably greater than or about 10000IU/mg, most preferably greater than or about 14000IU/mg, wherein the biological activity is determined by Steelman-Pohley assay and the protein content is determined by SE-HPLC.

The purity of the FSH sample may be analyzed at various stages of the purification process, for example, using the techniques listed below:

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

As mentioned above, FSH is a heterodimeric glycoprotein and contains an α subunit and a β subunit. These subunits can be dissociated, which can be monitored by the amount of free alpha subunit in the sample. Alternatively, the FSH subunits could be oxidized, oxidized contaminants quantified by RP-HPLC, and free subunits estimated by SDS-PAGE.

r-hFSH quantification: immunoassay method

The amount of FSH in the sample may be measured using an immunoassay specific for FSH, e.g., DELFIA FSH immunoassay.

Total protein: bradford assay, Lowry assay, absorbance at 280nm

The total protein content of any protein preparation can be determined using techniques such as the Bradford assay, Lowry assay, absorbance at 280nm, and the like.

Isomeric reforming form: IEF

As mentioned above, FSH is a glycoprotein with multiple oligosaccharide residues bound at different positions in two subunits. The oligosaccharide residues may be branched to varying degrees or capped with sialic acid residues. Sialic acid residues are negatively charged (at neutral pH). Different capping and mixtures of species with different isoelectric Points (PI) together lead to heterogeneity. This can be assessed using charge-based separation techniques and the like, such as electro-focusing (IEF).

Host Cell Protein (HCP)

Host cell proteins can be analyzed using ELISA assays. For example, the antibody may be "mock cultured" with a host cell cultured without the FSH gene.

Examples

The invention will be illustrated by two embodiments.

Fig. 1 and 2 illustrate the respective flows of the two embodiments described. The resulting purified r-hFSH is referred to as "r-hFSH total".

Example 1 (see FIG. 1)

Step (1): dye affinity chromatography on blue agarose gel

FSH purification starting material was prepared from cell culture collections containing recombinant FSH, that is, FSH produced recombinantly in CHO cells in serum-containing or serum-free culture media. The dye affinity column (blue Sepharose FF resin) was first equilibrated with a low conductivity buffer containing L-methionine at pH 8.5. The FSH-containing liquid is then added directly to the resin. After loading, the resin-unbound starting material is washed out with equilibration buffer. Finally, FSH was eluted by washing with pH 7.0 sodium phosphate buffer containing NaCl and L-methionine. The total eluate was directly taken to the next step. This step is carried out at 2-8 ℃.

Step (2): hydrophobic Interaction Chromatography (HIC) on Toyopearl butyl 650M

The eluate from blue Sepharose FF from step (1) was loaded onto a Toyopearl butyl 650M column, equilibrated with a pH 7.0 sodium phosphate buffer containing ammonium sulfate and L-methionine. The resin unbound material was washed out with equilibration buffer. FSH was eluted with the same buffer (but containing a lower concentration of ammonium sulfate). The eluate was directly taken to the next step. This step is carried out at room temperature.

And (3): reverse phase chromatography on Source30RPC

The HIC eluate (from step (2)) is first conditioned by the addition of IPA (isopropanol). A Source30RPC column was equilibrated with a pH 7.6 ammonium acetate buffer containing L-methionine and 2-propanol at a concentration equivalent to the adjusted charge. After washing the unbound charge out with equilibration buffer, the resin was washed with ammonium acetate buffer at pH 7.6 containing L-methionine and increasing concentrations of 2-propanol. The FSH is finally eluted with further increasing concentrations of 2-propanol. The total eluate is finally diluted with an aqueous solution of L-methionine with stirring. The total dilution goes to the next step. This step is carried out at room temperature.

Through the above steps, the purification factor, that is, the ratio of the purity of FSH in the purified sample to the purity of FSH in the starting material (raw FSH), is about 40.000. Example 2 (see FIG. 2)

Step (-1): concentrated r-hFSH ultrafiltration/diafiltration

All operations were carried out under refrigerated conditions (2-8 ℃). The raw FSH forming the starting material to be purified is derived from a cell culture harvest containing recombinant FSH.

Purification

The raw r-hFSH was filtered through a 0.5 μm wide filter (e.g. Pall Profile II filter or equivalent).

Ultrafiltration

The purified crude liquid was first concentrated by ultrafiltration through a 10kD polyethersulfone membrane. The concentrated retentate is then diafiltered in at least 5 diafiltration volumes of borate buffer containing L-methionine as antioxidant at pH 8.6. The conductivity and pH of the retentate were measured to monitor the diafiltration process. The retentate is then further concentrated before the system is drained. Finally the ultrafiltration unit is rinsed with diafiltration buffer and the rinse is mixed with the reconstituted retentate. The total solution goes to the next step.

Filtration

The concentrated product was filtered through a 0.2 μm polyethersulfone filter membrane (or equivalent).

Step (0): anion exchange chromatography on Q Sepharose FF

The filtered liquid was applied to a strong anion exchange (Q Sepharose FF) resin and equilibrated with sodium borate buffer containing L-methionine at pH 8.5. After packing, the column was rinsed with equilibration buffer and all unbound charge was washed out. The column was then eluted with sodium borate buffer at pH 8.5 containing NaCl (to increase conductivity) and L-methionine (as an antioxidant). The total eluate collected was subjected to dye affinity chromatography.

Step (1) dye affinity chromatography on blue Sepharose

Dye affinity chromatography column (blue sepharose FF resin) was first equilibrated with elution buffer from Q sepharose FF resin. The eluate obtained is added directly to the resin. After loading, unbound charge is washed out with equilibration buffer. FSH is finally eluted by washing the column with sodium borate at pH 7.0 containing NaCl and L-methionine. The total eluate was directly taken to the next step. This step is carried out at 2-8 ℃.

Step (2): hydrophobic interaction chromatography on Toyopearl butyl 650M

The eluate from blue Sepharose FF was loaded onto a Toyopearl butyl 650M column and equilibrated with a pH 7.0 sodium phosphate buffer containing ammonium sulfate and L-methionine. The material not bound to the resin was washed out with equilibration buffer. FSH was eluted with the same buffer (but at a lower ammonium sulfate concentration). The eluate was directly taken to the next step. This step is carried out at room temperature.

And (3): reverse phase chromatography on Source30RPC

The HIC eluate (from step (2)) is first conditioned by the addition of IPA (isopropanol). A Source30RPC column was equilibrated with a pH 7.6 ammonium acetate buffer containing L-methionine and 2-propanol at a concentration equivalent to the adjusted charge. After washing the unbound charge out with equilibration buffer, the resin was washed with ammonium acetate buffer at pH 7.6 containing L-methionine and increasing concentrations of 2-propanol. The FSH is finally eluted with a further increase in the concentration of 2-propanol. The total eluate is finally diluted with an aqueous solution of L-methionine with stirring. The total dilution goes to the next step. This step is carried out at room temperature.

And (4): anion exchange chromatography on Fractogel EMD TMAE hicap resin

The Fractogel EMD TMAE hicap column was first equilibrated with sodium borate buffer containing L-methionine at pH 8.5. The RPC-treated material (from step (3)) was diluted and packed onto the column. Unbound charge was washed out with equilibration buffer. FSH eluted from the column with a linear increase in salt concentration. This step is carried out at 2-8 ℃.

Step (4'): nanofiltration

The eluate from the Fractogel EMD TMAE hicap step (4) was directly fed to a 20nm nanofiltration unit and subjected to nanofiltration with a nitrogen pressure of 3 bar. The filtrate was taken to the next step. This step is carried out at 2-8 ℃.

And (5): total material ultrafiltration

The nanofiltered FSH material was concentrated by tangential flow through a 5KD polyethersulfone membrane. When the retentate reached half the initial volume, the material was buffer exchanged by WFI diafiltration.

Purity of the sample

The purity of the FSH samples after these purification steps was determined as follows:

purification step	Purity of
		Step (-1): ultrafiltration/diafiltration	Determination of 19% FSHFSH Total protein content by RP-HPLC determined by Bradford Assay
Step (0): anion exchange chromatography on Q Sepharose FF	Determination of 44% FSHFSH by RP-HPLC Total protein content by Bradford assay
		Step (1): dye affinity chromatography on blue agarose gel	68% FSHFSH by RP-HPLC; total protein content was determined by absorption at 280nm or approximately 420' 000ppm HCP; FSH content was determined by RP-HPLC; host cell content was determined by ELISA
Step (2): hydrophobic interaction chromatography on Toyopearl butyl 650M	Impurity amount: 3400ppm FSH content determined by RP-HPLC; host cell content was determined by ELISA
		And (3): reverse phase chromatography on Source30RPC	Impurity amount: 170ppm FSH content by RP-HPLC; host cell content was determined by ELISA
Step (4) anion exchange chromatography on Fractogel EMD TMAE hicap resin	Impurity amount: the content of < 80ppm FSH is determined by RP-HPLC; host cell content was determined by ELISA

Biological Activity of the sample

Purified r-hFSH bioactivities were measured by Steelman-Pohley ovarian weight gain method. The specific activity can be calculated by dividing the biological activity by the FSH content determined by the SE-HPLC method. As follows.

The final specific activity of the total feed is generally between 10000 and 17000 IU/mg. The final total FSH values for the two examples obtained according to the method of example 2 are shown in table 1.

TABLE 2 Total specific Activity of purified r-hFSH of the present invention

Analysis of	Sample 1	Sample 2
			Protein concentration (mg/ml) determined by SE-HPLC	0.61	0.54
Specific Activity (bioactivity/SE-HPLC)	12’600IU/mg	14’600IU/mg

Sequence listing

<110> Alies trade company, Inc. (ARES TRADING S.A.)

<120> method for purifying FSH

<130>WO 1001

<160>5

<170> PatentIn version 3.1

<210>1

<211>91

<212>PRT

<213> Intelligent people

<400>1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Tyr Asn Arg Val Thr Val Met Gly Gly Phe Val Glu Ash His Thr Ala

65 70 75 80

Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser

85 90

<210>2

<211>129

<212>PRT

<213> Intelligent people

<400>2

Met Lys Thr Leu Gln Phe Phe Phe Leu Phe Cys Cys Trp Lys Ala Ile

1 5 10 15

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

20 25 30

Glu Glu Cys Arg Phe Cys Ile Ser Ile Ash Thr Thr Trp Cys Ala Gly

35 40 45

Tyr Cys Tyr Thr Arg Asp Leu Val Tyr Lys Asp Pro Ala Arg Pro Lys

50 55 60

Ile Gln Lys Thr Cys Thr Phe Lys Glu Leu Val Tyr Glu Thr Val Arg

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Glu

<210>3

<211>108

<212>PRT

<213> Intelligent people

<400>3

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105

<210>4

<211>106

<212>PRT

<213> Intelligent people

<400>4

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Cys Gly Lys Cys Asp Ser Asp Ser Thr Asp Cys Thr Val Arg Gly Leu

85 90 95

Gly Pro Ser Tyr Cys Ser Phe Gly Glu Met

100 105

<210>5

<211>110

<212>PRT

<213> Intelligent people

<400>5

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Claims (22)

1. A method of purifying recombinant human FSH or a FSH variant, said method comprising subjecting a FSH-containing liquid to the following steps, optionally in the order listed:

(1) dye affinity chromatography;

(2) hydrophobic interaction chromatography; and

(3) reversed phase chromatography.

2. The method of claim 1, wherein the dye affinity chromatography of step (1) uses a resin with immobilized cibacron blue F3G-a.

3. The method according to claim 1 or 2, wherein the resin used for dye affinity chromatography in step (1) is blue sepharose FF.

4. The method of claim 1, wherein the dye affinity chromatography of step (1) uses a sodium phosphate buffer at or about pH 6.5 to 11.5 as the eluent.

5. The method of claim 1, wherein the hydrophobic interaction chromatography of step (2) is performed using Toyopearl butyl 650M.

6. The method of claim 1, wherein the hydrophobic interaction chromatography of step (2) uses sodium/ammonium phosphate as eluent.

7. The method of claim 1, wherein the reverse phase chromatography of step (3) uses Source30RPC as a resin.

8. The method of claim 1, wherein the reverse phase chromatography of step (3) uses ammonium acetate containing 2-propanol as an eluent.

9. The method of claim 1, wherein the steps are performed in the following order:

(1) dye affinity chromatography;

(2) hydrophobic interaction chromatography; and

(3) reversed phase chromatography.

10. The method of claim 1, further comprising an anion exchange chromatography step (0).

11. The method of claim 10, wherein the anion exchange chromatography step is performed prior to steps (1), (2), and (3).

12. The method of claim 10, wherein the anion exchange chromatography of step (0) is performed using Q sepharose FF resin or Fractogel EMD TMAE HiCap resin.

13. The method of claim 10, wherein the anion exchange chromatography of step (0) uses borate buffer as eluent.

14. The method of claim 13, wherein the borate buffer has a pH of 8.5 or about 8.5.

15. The method of claim 10, further comprising a second step of the anion exchange chromatography step (4).

16. The method of claim 15, wherein the second step of anion exchange chromatography step (4) is performed after steps (1), (2), and (3).

17. The method of claim 15, wherein the second anion exchange chromatography step (4) is performed using Fractogel EMD TMAE HiCap resin or Q sepharose FF resin.

18. The method of claim 15, wherein the second anion exchange chromatography step (4) uses borate buffer and increases the NaCl concentration gradient.

19. The method according to claim 15, characterized in that it comprises a nanofiltration step (4') after the second anion exchange chromatography step (4).

20. The method according to claim 19, characterized in that it comprises an ultrafiltration step (5) after the nanofiltration step (4').

21. The method of claim 1, wherein any eluent and/or buffer contains L-methionine as an antioxidant.

22. A method for purifying human recombinant FSH, said method comprising subjecting FSH to the steps of:

-ultrafiltration;

(0) anion exchange chromatography on Q Sepharose FF with borate/NaCl, L-methionine, pH 8.5 or approximately 8.5 as eluent;

(1) after the elution step (0), performing a dye affinity chromatography step on blue sepharose FF using phosphate, NaCl, L-methionine as eluent at a pH of 7 or about 7;

(2) after the elution step (1), a hydrophobic interaction chromatography step is carried out on Toyopearl butyl 650M using phosphate, ammonium sulfate, L-methionine as eluent at pH7 or about 7;

(3) after the elution step (2), a reverse phase chromatography step is carried out on Source30RPC using ammonium acetate, L-methionine, 2-propanol, pH 7.6 or about 7.6 as eluent;

(4) after elution step (3), an anion exchange chromatography step on Fractogel EMD TMAE hicap resin with borate, L-methionine, pH 8.5 or about 8.5, and NaCl;

(4') performing nanofiltration after the elution step (4); and

(5) after step (4'), ultrafiltration is carried out.