JP4933439B2 - Purification method of FSH - Google Patents

Description

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

  Follicle stimulating hormone (FSH) is an injectable protein that falls into the gonadotrophin class. FSH is used in the treatment of infertility and reproductive disorders in both female and male patients.

  Originally, FSH is produced by the pituitary gland. For pharmaceutical use, FSH can be produced recombinantly (rFSH) or isolated from postmenopausal female urine (uFSH).

  FSH is used in female patients in ovulation induction (OI) and superovulation induction (COH) for assisted reproductive medicine (ART). In a typical therapy for ovulation induction, patients are administered daily injections of FSH or variants (about 75-300 IU FSH / day) over a period of about 6-12 days. In a typical treatment for superovulation induction, the patient is FSH or a variant (approximately 150-600 IU FSH / day injection is administered daily) for a period of about 6-12 days.

  FSH is also used to induce spermatogenesis in men suffering from sperm reduction. Treatment with 150 IU FSH 3 times a week in combination with 2500 IU hCG twice a week successfully achieves improved sperm count in men with hypogonadotropic hypogonadism [Burgues et al .; Subcutaneous self- administration of highly purified follicle stimulating hormone and human chorionic gonadotrophin for the treatment of male hypogonadotrophic hypogonadism.Spanish Collaborative Group on Male Hypogonadotrophic Hypogonadism; Hum. Reprod .; 1997, 12, 980-6 ].

  Due to the importance of FSH in the treatment of pregnancy disorders, the provision of high purity and high specific activity FSH is desirable. FSH treatment requires repeated injections. Highly purified FSH preparations can be administered subcutaneously, allowing self-administration by the patient, thus improving patient convenience and compliance.

Lynch et al. [The extraction and purification of human pituitary follicle-stimulating hormone and luteinising hormone; Acta Endocrinologica, 1988, 288, 12-19] describe a method for purifying human pituitary FSH. The methods include anion and cation exchange chromatography, immunoaffinity extraction, and size exclusion chromatography. The method results in pituitary FSH with a specific activity of 4990 IU (immunoassay) / mg with 16 IU / mg LH. The protein content was measured either by dry weight or in solution at absorption at 280 nm (assuming 1 g / l A ²⁸⁰ _{1 cm} is 1).

  WO 98/20039 (IBSA lnstitut Biochimique SA) describes a method for the purification of human urine FSH starting with a urine extract called human menopausal gonadotropin (hMG). The method uses ion exchange chromatography on a weakly basic anion exchange resin of the DEAE type, followed by affinity chromatography on a resin having an anthraquinone derivative as a ligand. The method is that LA is not present and produces urinary FSH with a specific activity of 6,870 IU (immunoassay) / mg. The protein content was measured by assuming that an aqueous solution of 1 mg / ml protein had an absorbance of 0.62 at 277 nm in a quartz cuvette with a length of 1 cm.

  WO 00/63248 (Instituto Massone SA) describes a method for purifying gonadotropins containing FSH from human urine. The method comprises the following steps: ion exchange chromatography on a strong cation resin of the sulfopropyl type, and hydrophobic interaction chromatography (HIC). According to reports, a FSH preparation with a specific activity of 8,400 IU / mg (Steel man-Pohley method: Assay of the follicle stimulating hormone based on the augmentation with human chorionic gonadotrophin; Endocrinology; 1953, 53, 604-616) And LH (rat seminal vesicle weight gain method: Van Hell H, Matthijsen R & GA Overbeek; Acta Endocrinol, 1964, 47, 409) per 75 IU FSH. Protein content was determined by the Lowry method [O. H. Lowry et al., J. Biol. Chem., 1951, 193, 265].

US Pat. No. 5,990,288 (Musick et al.) Describes a method for purifying FSH from biological samples such as human pituitary or human postmenopausal urine. The method uses the steps of cation exchange chromatography in Fractogel EMD SO ₃ -650M, followed by dye affinity chromatography on Mimic Orange 1 resin, followed by hydrophobic interaction chromatography on Bakerbond Wide Por HI-propyl resin. . The method comprises human pituitary FSH with a specific activity of 7,066 IU (immunoassay) / mg, and LH of 1 IU (immunoassay) / mg or less, and human urine FSH with a specific activity of 6,298 IU (immunoassay) / mg. And LH of 3 IU (immunoassay) / mg or less. The protein content was measured by absorption at 280 nm (assuming 1 g / l A ²⁸⁰ _{1 cm} is 1).

Chiba et al. [Isolation and partial characterization of LH, FSH and TSH from canine pituitary gland; Endocrinol. J., 1997, 44, 205-218] are published in Concanavalin (Con) A affinity chromatography, hydrophobic interaction chromatography (HIC). ), And techniques for purifying canine pituitary gonadotropins, eg, FSH, using immobilized metal ion chromatography on Cu ⁺⁺ . The resulting FSH is a specific activity of 2.17 IU / g protein using a radioreceptor assay for FSH to measure biological activity and a BioRad protein assay kit (BioRad Laboratories CA USA) to measure protein content. It has been reported to have

  WO 88/10270 (Instituto di Ricerca Cesare Serono SPA) describes a method for purifying human FSH from urine. The method involves immunochromatography with an FSH-specific immobilized antibody that binds to Sepharose 4B by divinylsulfone followed by reverse phase HPLC. The resulting FSH is free of LH and other urine proteins and has a lyophilized powder with a specific activity of 6,200 IU / mg (Steelman-Pohley method). Because of its purity, the preparation is the first FSH preparation suitable for subcutaneous administration.

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

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

In a first aspect, the present invention is a method for purifying recombinant human FSH or FSH variants starting from a liquid containing crude FSH comprising the following steps (which may be performed in any order): :
(1) dye affinity chromatography;
(2) hydrophobic interaction chromatography; and (3) reverse phase chromatography;
A method comprising the steps of:

Abbreviations The following abbreviations are used in the description of the invention.
DF: Diafiltration;
FSH: follicle stimulating hormone;
r-FSH: recombinant FSH;
hFSH: human FSH;
r-hFSH: Recombinant human FSH
BV: Total volume DEAE: Diethylaminoethyl ELISA: Enzyme-linked immunoassay DAC: Dye affinity chromatography IMAC: Immobilized metal ion affinity OD: Absorbance HIC: Hydrophobic interaction chromatography HPLC: High performance liquid chromatography IRMA: Immunoradiation measurement Method KD or kD: kilodalton HCP: host cell protein, host cell derived protein used for expression of FSH IPC: process control IEF: isoelectric focusing PES: polyethersulfone RP-HPLC: reverse phase high speed Liquid Chromatography Q FF: Anion exchange in Q Sepharose FF RT: Room temperature UF: Ultrafiltration WFI: Water for injection

Detailed Description of the Invention The present invention is a method for purifying recombinant human FSH or recombinant FSH variant starting from a liquid containing crude FSH, which may be carried out in any of the following steps:
(1) dye affinity chromatography;
(2) hydrophobic interaction chromatography; and (3) reverse phase chromatography;
A method comprising the steps of:

  The purification method of the present invention yields a high purity recombinant FSH bulk that can be formulated into a final pharmaceutical, eg, gonal F (Serono). This has the advantage of producing high purity without the use of immunoaffinity chromatography. The crude FSH that forms the starting material for purification according to the present invention is present in cell culture harvests containing recombinant FSH.

  In a preferred embodiment, an antioxidant or a free amino acid or dipeptide having an antioxidant effect and a scavenger effect is included in certain or all steps of the purification method according to the invention. More precisely, the antioxidant is present in any buffer used to purify and / or concentrate and / or filter r-hFSH. The oxidizing agent prevents oxidation of FSH during processing. A preferred antioxidant is L-methionine. Preferably L-methionine is used at a concentration of about 10-100 mM. Further examples of antioxidants include t-butyl-4-methoxy-phenol, 2,6-bis (1,1-dimethylethyl) -4-methylphenol; bimetallic potassium or sodium bisulfite, sodium bisulfite . Examples of advantageous amino acids and dipeptides with antioxidant and scavenging effects are histidine, taurine, glycine, alanine, carnosine, anserine, 1-methylhistidine, or combinations thereof.

  Typically, the starting material is first identified and then optionally concentrated (eg, by using ultrafiltration) and / or buffered before being captured in the first chromatographic step. Are exchanged (for example, a diafiltration step).

  In the chromatography step, polymer type and agarose type resins can be used. Also, membrane chromatography in which the resin is replaced with a functional membrane can be used.

  The three purification steps of the present invention (ie, dye affinity chromatography, hydrophobic interaction chromatography, reverse phase chromatography) are outlined in more detail below.

Dye affinity chromatography step (1)
The method of the present invention includes the step of dye affinity chromatography (1). In a preferred embodiment, the dye affinity chromatography step can be performed using a resin having a staining compound well known to those skilled in the art, ie Cibacron Blue F3G-A, as the immobilized ligand. The term “immobilization” is well understood by those skilled in the art and means that the ligand is derivatized in the sense of chemically binding to the resin. A particularly preferred resin is Blue Sepharose FF (obtained from Amersham Biosciences Inc.). The technical features of Blue Sepharose FF are as follows:

  It is understood that the method can be performed with other resins having similar properties. Examples of other resins are: Toyopearl AF-blue-HC-650M (Tosoh Bioscience), Toyopearl SuperButyl 550, Toyopearl Phenyl 650, Blue Cellthru BigBead (Sterogene), SluGel , Affi-Gel Blue (BioRad), Econo-Pac blue cartridge (Bio-Rad), Blue sepharose HP (Amersham), Cibacron Blue 3GA (Sigma).

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

  In a particularly preferred embodiment, the product-containing buffer (equilibration, washing and elution) for the dye affinity chromatography step comprises an antioxidant, for example L-methionine. Examples of further antioxidants include t-butyl-4-methoxyphenol, 2,6-bis (1,1-dimethylethyl) -4-methylphenol; potassium or sodium bimetabisulfite, sodium bisulfite.

Hydrophobic interaction chromatography step (2)
The method also includes the step of hydrophobic interaction chromatography (2). In a preferred embodiment, hydrophobic interaction chromatography is performed on a resin such as, for example, Toyopearl Butyl 650M (obtainable from Tosoh Biosep Inc.).

  It is understood that step (2) can be performed using other resins having similar properties. Other resins that can be used are: Phenyl Sepharose 6 Fast Flow (high sub); Butyl Sepharose 4 Fast Flow; Octyl Sepharose 4 Fast Flow; Phenyl Sepharose 4 Fast Flow; Phenyl Sepharose 4 Fast Flow; Phenyl Sepharose 4 Fast Flow; . These are all derived from Amersham Biosciences (800) 526-3593; see www.amershambiosciences.com. Further resins are from BioChrom Labs Inc. (812) 234-2558: Hydrocell C3 or C4; Hydrocell Phenyl; (see www.biochrom.com).

Binding in the HIC resin is achieved in a buffer with high conductivity obtained through the addition of a salt (eg, NaCl, (NH ₄ ) ₂ SO ₄ or Na ₂ SO ₄ ). The elution in the hydrophobic interaction chromatography step is preferably performed using a buffer having a pH of about 6 to about 8, more preferably about 6.5 to about 7.5, and most preferably about 7. This is done by reducing the conductivity of the mobile phase. A particularly preferred system contains sodium phosphate buffering at a pH of about 7 and ammonium sulfate. Other buffers are described above.

  In a particularly preferred embodiment, the product-containing buffer for HIC step (2) (equilibration, washing, elution) contains an antioxidant, for example L-methionine. Other antioxidants are described above.

Reversed phase chromatography step (3)
The method of the present invention also comprises the step of reverse phase chromatography (RPC) (3). The RPC is preferably performed using a resin such as, for example, SOURCE 30 RPC (which can be obtained from Amersham Biosciences). It is understood that step (3) can be performed using other resins well known to those skilled in the art having similar properties.

  The chromatography is preferably carried out using a mobile phase that buffers at weakly alkaline pH, for example at about 7-8.5, more preferably about 7.5 or 7.6.

  In a preferred embodiment, the buffering species is ammonium acetate. Other buffers suitable for a pH of about 7.6 include: BES, MOPS, phosphate, TES, HEPES. The buffer solution used for the process may also contain organic modifiers and concentrations adjusted for different stages of the chromatography process (addition, washing, elution and regeneration). In a preferred embodiment, the organic modifier is a water miscible organic solvent, preferably an alcohol (eg, methanol, ethanol, etc.), most preferably 2-propanol (isopropanol).

  In a particularly preferred embodiment, the product-containing buffer for the RPC process (equilibration, washing, elution) contains an antioxidant, for example L-methionine. Other antioxidants are described above.

Optional Additional Purification Steps 0-Ion Exchange Chromatography In addition to the three main purification steps outlined above, the present invention may include additional purification steps.

In one embodiment, the purification method of the invention comprises an ion with a strong anion exchange resin, particularly preferably a quaternary ammonium resin, such as Q Sepharose FF (obtainable from Amersham Biosciences), having the following properties: Including preparatory steps for exchange chromatography (0):
Ion exchanger type: Strong anion Total volume (mmol / ml): 0.18-0.25
Exclusion limit (globular protein): 4 × 10 ⁶
Bead form: spherical, diameter 45-165 μm
Bead structure: Cross-linked agarose, 6%
Operational pH stability: 2-12
Washing pH stability: 1-14
Linear flow rate at 25 ° C., 1 bar, 15 cm bed height, XK 50/30 column:
400-700cm / h

Alternatively, the ion exchange chromatography step (0) can be performed using, for example, a resin such as Fractogel EMD TMAE HICAP (obtainable from Merck KGaA, Darmstadt Germany) or a resin having similar properties. . See below:

  The ion exchange chromatography step preferably comprises a buffer having a weak alkaline pH (eg, about 7.2 to about 9.0, or about 8.0 to about 9.0, most preferably about 8.5). Is done using. Suitable buffers include, for example, borate buffer, triethanolamine / iminodiacetic acid tris, ammonium acetate, tricine, bicine, TES, HEPES, TAPS. 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 through the addition of a salt, preferably NaCl. In a particularly preferred embodiment, the product-containing buffer for ion exchange chromatography (equilibration, washing, elution) contains an antioxidant, preferably L-methionine. Other antioxidants are described above.

Thus, in a preferred embodiment, the method of the invention comprises the following steps:
(0) preferably an anion exchange chromatography on a strong anion exchange resin [preferably a quaternary ammonium resin such as Q Sepharose FF or Fractogel EMD TMAE];
(1) Dye affinity chromatography [preferably in Blue Sepharose FF];
(2) hydrophobic interaction chromatography [preferably in Toyopearl Butyl 650M]; and (3) reverse phase chromatography [preferably in Source 30 RPC],
Comprising.

Optional Further Purification Step (-1)-Ultrafiltration / Diafiltration Prior to the ion exchange chromatography (0) step, it is desirable to perform an ultrafiltration step to concentrate the crude FSH. . Ultrafiltration (or diafiltration) is preferably performed using a membrane having a cutoff of about 3-10 kD, most preferably about 8 kD.

Optional further purification step (4)-anion exchange chromatography In a further preferred embodiment, the process of the invention also comprises a second step of anion exchange chromatography (4). Preferred resins are Fractogel EMD TMAE HICAP (obtainable from Merck KGaA, Darmstadt Germany), or resins with similar properties, as described above. Alternatively, the second step of anion exchange chromatography can be performed on Q Sepharose FF, or other resins with similar properties, as described above.

The steps of anion exchange chromatography, dye affinity chromatography, hydrophobic interaction chromatography (HIC), reverse phase chromatography, and the second step of anion exchange chromatography may be performed in any order, It is preferred to perform the anion exchange chromatography step first. The remaining steps of the dye affinity chromatography, hydrophobic interaction chromatography (HIC), RPC step, and optional second step of anion exchange chromatography can be performed in any order, but It is preferable to follow the order shown in:
(0) anion exchange chromatography, (1) dye affinity chromatography, (2) interaction chromatography (HIC), (3) reverse phase chromatography RPC; and (4) second anion exchange chromatography.

Optional further purification step (5)-Ultrafiltration / diafiltration In a further preferred embodiment, after any chromatography step (especially after the reverse phase chromatography step), the FSH sample is subjected to a concentration step. Call. Preferably, the process is performed using ultrafiltration in combination with diafiltration to obtain a bulk having the desired composition. The ultrafiltration (diafiltration) is preferably performed using a membrane having a cutoff of about 3-10 kD, most preferably about 5 kD.

In a particularly preferred embodiment, the following steps are performed in the order shown below;
(-1) ultrafiltration (preferably with a membrane having a cut-off of about 8 kD),
(0) anion exchange chromatography (preferably using a Q Sepharose FF column);
(1) Dye affinity chromatography (preferably using a Blue Sepharose FF column);
(2) Interaction chromatography (HIC) (preferably using a Butyl 650M column);
(3) Reversed phase chromatography (preferably using a Source 30 RPC column);
(4) Anion exchange chromatography on a strongly basic anion exchange resin (preferably using a TMAE hicup resin); and (5) Ultrafiltration (preferably with a membrane having a 5 kD cut-off).

  It is desirable to subject the FSH sample to a nanofiltration step, particularly as a virus clearance step, i.e. to reduce the risk of FSH contamination by viruses or virus-like particles from cell culture. Nanofiltration can be carried out at any stage of the purification process, however, it is particularly preferred to carry out after the second step of ion exchange chromatography, after reverse phase chromatography or after interaction chromatography. Nanofiltration can be performed one or more times, for example, twice.

In a particularly preferred embodiment, the method of the invention comprises the following steps:
(-1) ultrafiltration (preferably with a membrane having a cut-off of about 8 kD),
(0) anion exchange chromatography (preferably with a Q Sepharose FF column);
(1) Dye affinity chromatography (preferably, using a Blue Sepharose FF column);
(2) Interaction chromatography (HIC) (preferably with a Butyl 650M column);
(3) Reversed-phase chromatography (RPC) (preferably with a Source 30 RPC column);
(4) Anion exchange chromatography on a strongly basic anion exchange resin (preferably with TMAE high cup resin);
(4 ′) Nanofiltration (5) Ultrafiltration (preferably with a membrane having a cut-off of 5 kD).

  An advantage of the present invention is that the purification method avoids costly immunoaffinity chromatography steps and provides a high degree of FSH purity and specific biological activity. Also, the purified FSH of the present invention does not contain undesired impurities (eg, immunoglobulins leached from the resin) that are added by immunoaffinity chromatography.

Storage / Freeze Drying The liquid composition resulting from the purification process as described above and containing the purified FSH can be frozen as is or after purification and the eluate is lyophilized to remove the solvent ( Freeze drying). The resulting liquid or lyophilized product is termed “FSH bulk”.

FSH formulation The FSH or FSH variant of the present invention, or the purified product according to the method of the present invention, can be formulated for injection intramuscularly or subcutaneously, preferably subcutaneously. The FSH formulation can be freeze-dried, in which case it is dissolved in water for injection just prior to injection. The FSH formulation may also be a liquid formulation, in which case it can be directly injected without prior dissolution.

  The FSH formulation may be a single dose or multiple doses. In the case of multiple administration, it should preferably contain a bacteriostatic agent such as benzyl alcohol, meta-cresol, thymol, or phenol, preferably benzyl alcohol or meta-cresol. Single dose formulations may also contain a bacteriostatic agent.

  The FSH of the present invention can be formulated with known excipients and stabilizers such as sucrose and mannitol. It may also comprise an oxidant such as methionine. This may further comprise a surfactant, for example 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 dissolved in water for injection along with sucrose, phosphate buffer (pH 7), pluronic F68, methionine, and meta-cresol or benzyl alcohol. By doing so, it can be prescribed.

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

The FSH of the present invention is suitable for use in all processes where FSH is indicated. This is particularly suitable for subcutaneous administration in the treatment of ovulation induction, superovulation induction, and sperm reduction for assisted reproductive medicine. This can be used in combination with other gonadotropins such as LH and hCG. It can also be used with additional compounds that increase the response to FSH, such as clomiphene citrate, aromatase inhibitors such as anastrozole, letrozole, fadrozole, and YM-511.

  As used herein, follicle stimulating hormone, or FSH, refers to human FSH (hFSH) produced as a full-length mature protein. FSH is a dimer composed of human glycoprotein α-subunit and human FSHβ-subunit. The protein sequence of the human glycoprotein α subunit is provided by SEQ ID NO: 1 and the protein sequence of the human FSHβ subunit is given in SEQ ID NO: 2.

  The use of the term “recombinant” refers to a preparation of FSH produced through the use of recombinant DNA technology (see, eg, WO85 / 01958).

  One example of a method for expression of FSH using recombinant technology is the FSH alpha provided in one or two vectors with each subunit having separate promoters, as described in EP0211894 and EP0487512. By transfection of eukaryotic cells with DNA sequences encoding-and β-subunits. The DNA encoding FSH may be cDNA or it may contain introns.

  Other examples of the use of recombinant technology to produce FSH are described in EP 0505500 (Applied Research Systems ARS Holding NV), wherein the heterologous regulatory segment encodes an endogenous component encoding one or both subunits of FSH. By the use of homologous recombination to introduce in operative linkage to the sex sequence. Also, for example, the method described in WO99 / 57263 (Transkaryotic Therapies), wherein one subunit is inserted heterologously into the cell, and the other subunit is a heterologous regulatory segment by homologous recombination. Also contemplated are methods expressed by activation of genomic sequences by insertion of. The methods of the invention can be used to purify FSH expressed using any of these and other methods.

  The expression “recombinant cell” means a cell produced by insertion of heterologous DNA, including any of the methods of genetic manipulation described above.

  Preferably, the FSH comprises DNA, a Chinese hamster ovary (CHO) transfected with one or more vectors comprising DNA encoding the human glycoprotein α-subunit and the β-subunit of FSH. Produced recombinantly in cells. The DNA encoding the α and β subunits may be present in the same or different vectors.

ここで、βＦＳＨは、Klein ら[Pharmacokinetics and pharmacodynamics of single-chain recombinant human follicle-stimulating hormone containing the human chorionic gonadotrophin carboxyterminal peptide in the rhesus monkey, Fertility & Sterility; 2002, 77, 1248-1255]により記載されるとおり、ＦＳＨのβ−サブユニットを示し、βｈＣＧ ＣＴＰ（１１３−１４５）はｈＣＧのカルボニル末端ペプチドを示し、そしてαＦＳＨはＦＳＨのα−サブユニットを示す。ＦＳＨ変異体のほかの例は、ＷＯ０１／５８４９３(Maxygen)において開示される、α−及び／又はβ−サブユニット中に組み込まれる更なる糖鎖付加部位を有するＦＳＨ分子、及びＷＯ９８／５８９５７に開示される、サブユニット間Ｓ−Ｓ結合を伴うＦＳＨ分子を含む。ＦＳＨ変異体の更なる例は、例えば、ＷＯ９１／１６９２２及びＷＯ９２／２２５６８に開示される、ＦＳＨ由来の配列及びｈＣＧ又はＬＨ由来の配列を含んで成るキメラ分子を含む。 The expression “FSH variant” is meant to encompass molecules that differ in amino acid sequence, glycosylation pattern, or binding between subunits derived from human FSH and exhibit FSH activity. Examples are described in LaPoIt et al .; Endocrinology; 1992, 131, 2514-2520; or Klein et al .; Development and characterization of a long-acting recombinant hFSH agonist; Human Reprod. 2003, 18, 50-56 Thus, CTP-FSH, including a long-acting modified recombinant FSH consisting of a hybrid β subunit in which the wild-type α subunit and the carboxy terminal peptide of hCG are fused to the C-terminus of the β subunit of FSH. Also includes single chain CTP-FSH, single chain molecules consisting of the following sequences (from N-terminal to C-terminal):

Here, βFSH is described by Klein et al. [Pharmacokinetics and pharmacodynamics of single-chain recombinant human follicle-stimulating hormone containing the human chorionic gonadotrophin carboxyterminal peptide in the rhesus monkey, Fertility &Sterility; 2002, 77, 1248-1255] As shown, the β-subunit of FSH, βhCG CTP (113-145) indicates the carbonyl terminal peptide of hCG, and αFSH indicates the α-subunit of FSH. Other examples of FSH variants are disclosed in WO 01/58493 (Maxygen), FSH molecules having additional glycosylation sites incorporated into α- and / or β-subunits, and disclosed in WO 98/58957. FSH molecules with intersubunit S—S bonds are included. Further examples of FSH variants include chimeric molecules comprising FSH-derived sequences and hCG or LH-derived sequences, as disclosed, for example, in WO 91/16922 and WO 92/22568.

  The FSH variants referred to herein also include a carboxy-terminal deletion of the β subunit that is shorter than the full-length mature protein of SEQ ID NO: 2. Carboxy terminal deletions of the human β subunit are provided in SEQ ID NOs: 3, 4, and 5. The carboxy-terminal variant of the β chain is understood to form a complex with a known α subunit to form an FSH variant heterodimer.

  In a preferred embodiment, FSH is produced recombinantly in CHO cells in serum 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, allowing self-administration by the patient.

  The expression “crude recombinant FSH” means a cell culture supernatant derived from a recombinant cell that expresses FSH before going through any chromatography step. The expression encompasses the bulk of the supernatant (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 biological responses related to FSH, such as the increase in ovarian weight in the Steelman-Pohley assay [Assay of the follicle stimulating hormone based on the augmentation with human chorionic gonadotrophin; Endocrinology; 1953, 53, 604-616], or the ability of an FSH formulation to induce follicular growth in a supernatant patient. Follicular growth in female patients can be assessed by ultrasound, for example, for the number of follicles having an average diameter of about 16 mm on the 8th day of stimulation. Biological activity is assessed with respect to accepted standards for FSH.

  The LH content in the 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” for FSH refers to biological assays recognized for FSH, such as the Steelman Pohley bioassay [divided by the amount of protein measured by the assay for total protein content], such as the Lowry assay [OH Lowry , NJ Rosebrough, AL Farr and RJ Randall (1951) J. Biol. Chem. 193: 265; Hartree EE (1972). Anal. Biochem. 48: 422; JR Dulley and PA Grieve (1975) Anal. Biochem. 64: 136], meaning the biological activity in the IU of the preparation in the Bradford assay [Bradford, MM (1976) Anal. Biochem. 72, 248] or by absorbance at 280 nm.

  Preferably, the FSH obtained according to the present invention has a specific activity of about 8000 IU / mg or more, more preferably about 9000 IU / mg or more, even more preferably about 10,000 IU / mg or more, even more preferably about 14000 IU / mg or more. Have. Here the biological activity is measured by the Steelman-Pohley bioassay and the protein content is measured by SE-HPLC.

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

r-hFSH quantification / free α subunit / purity / oxidized form: RP-HPLC
As described above, FSH is a heterodimeric glycoprotein composed of α- and β-subunits. Some subunit dissociation occurs and can be monitored by noting the amount of free α subunit present in the sample. Furthermore, the FSH subunit can be oxidized. Oxidative contaminants can be quantified using RP-HPLC, while free subunits can be assessed using SDS-PAGE.

r-hFSH quantification / immunoassay The FSH content in a sample can be measured using an FSH-specific immunoassay, for example, the DELFIA FSH immunoassay.

Total protein: Bradford assay, Lowry assay, absorbance at 280 nm As with any protein preparation, the total protein content may be measured using techniques such as Bradford assay, Lowry assay or by absorbance at 280 nm. it can.

Isoform pattern: IEF
As mentioned above, FSH is a glycoprotein having multiple oligosaccharide residues attached at various locations in both subunits. The oligosaccharide residues have different degrees of branching and may be capped with sialic acid residues. Sialic acid is negatively charged (at neutral pH). Differences in capping lead to inhomogeneities with types of mixtures having different isoelectric points (pI). This can be assessed, for example, using techniques that separate based on charge, such as isoelectric focusing (IEF).

Host cell protein (HCP)
Host cell proteins can be analyzed using an ELISA assay. For example, the antibody can be raised to a “pseudo-culture medium” that is a culture medium for host cells that do not have the FSH gene.

  The invention will now be described by way of two examples.

  Corresponding flowcharts illustrating the above two examples are shown in FIGS. The resulting purified r-hFSH is referred to as “r-hFSH bulk”.

Example 1 (see Figure 1)
Step (1): Dye affinity chromatography on Blue Sepharose The FSH starting material for purification is recombinant FSH, ie FSH recombinantly produced in CHO cells either in serum medium or in serum-free medium. From a cell culture broth containing. A dye affinity chromatography column (Blue Sepharose FF resin) is equilibrated with a low conductivity buffer at pH 8.5 containing L-methionine. After addition, unbound material is washed away using equilibration buffer. Finally, FSH is eluted by flushing the column with sodium phosphate at pH 7.0 containing NaCl and L-methionine. Treat the elution pool directly to the next step. This process is performed at 2-8 degreeC.

Step (2): Hydrophobic Interaction Chromatography (HIC) on Toyopearl Butyl 650M
The Blue Sepharose FF eluate from step (1) is added to a Toyopearl Butyl 650M column equilibrated against pH 7.0 sodium phosphate buffer containing ammonium sulfate and L-methionine. Wash away unbound material with equilibration buffer. FSH is eluted with the same buffer, but at a lower concentration of ammonium sulfate. The eluate is processed in the next step. The process is performed at room temperature.

Step (3): Reversed Phase Chromatography in Source 30 RPC First, the HIC eluate (derived from step (2)) is prepared by adding IPA (isopropanol). A Source 30 RPC column is equilibrated against ammonium acetate buffer at pH 7.6 containing L-methionine and 2-propanol at the same concentration as the conditioned additive material. After flushing away unbound material with equilibration buffer, the resin is washed with pH 7.6 ammonium acetate buffer containing L-methionine and an elevated concentration of 2-propanol. Further, the FSH is finally eluted by increasing the concentration of 2 propanol. Finally, the elution pool is diluted with water containing L-methionine while stirring. The diluted pool is processed in the next step. The process is performed at room temperature.

  In accordance with the procedure described above, the ratio of purification factors—ie, FSH purity in the purified sample and FSH purity in the starting material (crude FSH) is about 40.000.

Example 2 (see Figure 2)
Step (-1): Ultrafiltration / diafiltration of concentrated r-hFSH All operations were performed under refrigerated centrifugation conditions (2-8 ° C). The crude FSH that forms the starting material for purification is derived from a cell culture harvest containing recombinant FSH.

Clarification Crude r-hFSH was filtered through a 0.5 μm depth filter (eg, a Pall Profile II filter or equivalent).

Ultrafiltration First, an unpurified product clarified by ultrafiltration was concentrated using a 10KD polyethersulfone membrane. The concentrated retentate was then diafiltered with at least 5 diavolume borate buffer at pH 8.5 containing L-methionine as an antioxidant. To monitor the progress of diafiltration, the retentate conductivity and pH were measured. The retentate was then concentrated prior to discharging the system. The ultrafiltration unit was finally flushed with diafiltration buffer and the rinse was mixed with the recovered retentate. The pool was used for the next step.

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

Step (0): Anion Exchange in Q Sepharose FF A strong anion exchange (Q Sepharose FF) resin in which the filtered material was then equilibrated against a sodium borate buffer at pH 8.5 containing L-methionine. Applied to. After the addition, the column was rinsed with equilibration buffer to allow all unbound material to flow. The column was then eluted with a sodium borate buffer at pH 8.5 containing NaCl (to increase conductivity) and L-methionine (as an antioxidant). The recovered elution pool was processed in dye affinity chromatography.

Step (1): Dye affinity chromatography in Blue Sepharose First, a dye affinity chromatography column (Blue Sepharose FF resin) was equilibrated with an elution buffer derived from the Q-Sepharose FF step. The capture eluate was then applied directly to the resin. After addition, unbound material was washed away using equilibration buffer. Finally, FSH was eluted by flushing the column with sodium phosphate at pH 7.0 containing NaCl and L-methionine. The elution pool was processed directly in the next step. The process was performed at 2-8 ° C.

Step (2): Hydrophobic Interaction Chromatography on Toyopearl Butyl 650M The Blue Sepharose FF eluate was applied to a Toyopearl Butyl 650M column equilibrated against sodium phosphate buffer pH 7.0 containing ammonium sulfate and L-methionine. Added. Unbound material was washed away with equilibration buffer. FSH was eluted with the same buffer, but with a low concentration of ammonium sulfate. The eluate was processed in the next step. The process was performed at room temperature.

Step (3): Reverse Phase in Source 30 RPC First, an HIC eluate (derived from Step (2)) was prepared by adding IPA (isopropanol). The Source 30 RPC column was equilibrated against pH 7.6 ammonium acetate buffer containing L-methionine and 2-propanol at the same concentration as the adjusted additive. After running off unbound material with equilibration buffer, the resin is washed with pH 7.6 ammonium acetate buffer containing L-methionine and increasing concentrations of 2-propanol. Further, the FSH is finally eluted by increasing the concentration of 2 propanol. Finally, the elution pool is diluted with water containing L-methionine while stirring. The diluted pool is processed in the next step. The process was performed at room temperature.

Step (4): Anion exchange chromatography on Fractogel EMD TMAE hicap resin First, a Fractogel EMD TMAE hicap column was equilibrated with sodium borate buffer at pH 8.5 containing L-methionine. Diluted post-RPC material (from step (3)) was added to the column. Unbound material was run using equilibration buffer. FSH is eluted from the column with increasing salt concentration linearly. The process was performed at 2-8 ° C.

Step (4 ′): Nanofiltration The eluate from the Fractogel EMD-TMAE step (4) was applied directly to a 20 nm nanofiltration apparatus at 3 bar pressure under a nitrogen atmosphere. The filtrate is processed in the next step. This operation was performed in 2-8.

Step (5): Bulk Ultrafiltration The nanofiltered FSH material was concentrated by tangential flow filtration on a 5KD polyethersulfone membrane. After the retentate reached approximately half of the initial volume, the material was buffer exchanged by diafiltration against WFI.

Sample purity The purity of the FSH sample after the purification step was measured.

Sample Bioactivity The biological activity of purified r-hFSH was measured using the Steelman-Pohley ovarian weight gain method. Specific activity was calculated using the biological activity divided by the FSH content measured by the SE-HPLC method as follows.

The specific activity of the final bulk obtained was typically 10000-17000 IU / mg. Representative values for two samples of final bulk FSH obtained according to the method of Example 2 are shown in Table 1.

FIG. 1 illustrates a purification method comprising the present invention, ie the following steps: • Dye affinity chromatography, • Hydrophobic interaction chromatography, • Reversed phase chromatography. FIG. 1 shows a specific embodiment of the invention, namely the following steps: • Ultrafiltration / diafiltration • Anion exchange chromatography • Dye affinity chromatography • Hydrophobic interaction chromatography • Reversed phase 2 shows a flow chart of a purification method comprising: chromatography, • anion exchange chromatography, • nanofiltration • ultrafiltration / diafiltration.

Sequence SEQ ID NO: 1: Human glycoprotein α-subunit;
SEQ ID NO: 2: hFSHβ-subunit SEQ ID NO: 3: hFSHβ-subunit variant 1
SEQ ID NO: 4: hFSHβ-subunit variant 2
SEQ ID NO: 5: hFSHβ-subunit mutant 3

Claims (17)

A method for purifying a recombinant FSH or FSH variant, wherein the FSH-containing liquid is performed in the following order :
(0) anion exchange chromatography;
(1) dye affinity chromatography;
(2) hydrophobic interaction chromatography;
(3) reverse phase chromatography; and
(4) anion exchange chromatography;
A method comprising subjecting to .   The method according to claim 1, wherein the dye affinity chromatography in step (1) is performed with a resin having immobilized Cibacron Blue F3G-A.   The method according to claim 1 or 2, wherein the resin used in the dye affinity chromatography in step (1) is Blue Sepharose FF.   4. The dye affinity chromatography of step (1) is performed using sodium phosphate buffer at a pH of about 6.5 to 11.5 as an eluent. the method of.   The method according to any one of claims 1 to 4, wherein the hydrophobic interaction chromatography (HIC) of step (2) is performed using Toyopearl Butyl 650M or a resin having similar properties. .   The method according to any one of claims 1 to 5, wherein the hydrophobic interaction chromatography in step (2) is performed using sodium phosphate / ammonium sulfate as an eluent.   The method according to any one of claims 1 to 6, wherein the reverse phase chromatography step of step (3) is performed using SOURCE 30 RPC as a resin.   The method according to any one of claims 1 to 7, wherein the reverse phase chromatography step of step (3) is performed using ammonium acetate with 2-propanol as an eluent. The method as described in any one of Claims 1-8 by which the anion exchange chromatography of a process (0) is performed by Q Sepharose FF resin or Fractogel EMD TMAE HiCap resin. The method according to any one of claims 1 to 9 , wherein the ion exchange chromatography in the step (0) is performed using a borate buffer as an eluent. The method of claim 10 , wherein the borate buffer has a pH of about 8.5. The method according to any one of claims 1 to 11 , wherein the second step of the anion exchange chromatography in the step (4) is performed using Fractogel EMD TMAE HiCap resin or Q Sepharose FF resin. The method according to any one of claims 1 to 12 , wherein the second step of the anion exchange chromatography of step (4) is performed using a borate buffer and a gradient of increasing NaCl concentration. Comprises the step of nanofiltration step (4 '), said step is carried out after the second step of anion-exchange chromatography step (4) The method according to any one of claims 1 to 13. 15. The method according to claim 14 , comprising a step of ultrafiltration (5) after the step of nanofiltration (4 '). 16. A method according to any one of claims 1 to 15 , wherein any eluent and / or buffer may comprise an antioxidant, in particular L-methionine. A method for purifying human recombinant FSH comprising the steps of:
(-1) ultrafiltration;
(0) Anion exchange chromatography on Q Sepharose FF with borate / NaCl, L-methionine at a pH of about 8.5 as eluent;
(1) subjecting the eluate of step (0) to dye affinity chromatography in Blue Sepharose FF and phosphate, NaCl, L-methionine at a pH of about 7 as eluent;
(2) subjecting the eluate of step (1) to interaction chromatography on Toyopearl Butyl 650M with phosphate, ammonium sulfate, L-methionine at a pH of about 7 as eluent;
(3) subjecting the eluate of step (2) to a reverse phase chromatography step in Source 30 RPC with ammonium acetate, L-methionine, with 2-propanol at a pH of about 7.6 as eluent;
(4) subjecting the eluate of step (3) to an anion exchange chromatography step on Fractogel EMD TMAE hicap resin with borate, L-methionine and NaCl at a pH of about 8.5;
(4 ′) a step of subjecting the eluate of step (4) to a nanofiltration step; and (5) a step of subjecting the permeate of step (4 ′) to an ultrafiltration step;
Comprising a method.

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