HK1033328B - Erythropoietin derivatives - Google Patents

Description

Erythropoietin conjugates

Technical Field

The present invention relates to erythropoietin conjugates.

Background

Erythropoiesis (Erythropoiesis) refers to the production of red blood cells to compensate for the loss of cells. Erythropoiesis is a controlled physiological mechanism that provides sufficient red blood cells for tissue oxygenation. Naturally occurring human erythropoietin (hEPO) is produced by the kidney and is a humoral factor that stimulates red Blood cell production (Carnot, P and Deflandre, C (1906) C.R.Acad.Sci.143: 432; Erslev, AJ (1953 Blood 8: 349; Reissmann, KR (1950) Blood 5: 372; Jacobson, LO, Goldwasser, E, Freid, Wand Plzak, LF (1957) Nature (Nature) 179: 6331-4). naturally occurring EPO stimulates division and differentiation of targeted erythroid progenitors in the bone marrow and exerts its biological activity by binding to receptors on erythroid precursors (Krantz, BS (1991) Blood 77: 419).

Erythropoietin (Egrie, JC, Strickland, TW, Lane, J et al (1986) immunobiol. 72: 213-224), which is the product of a cloned human EPO gene inserted and expressed in Chinese hamster ovary cells (CHO cells), has been produced by biosynthesis using recombinant DNA techniques. The primary structure of the major, fully processed form of hEPO is shown in SEQ ID NO: 1. in Cys⁷-Cys¹⁶¹And Cys²⁹-Cys³³There are two disulfide bonds between them. The molecular weight of the polypeptide chain of EPO without a glycosyl group is 18,236da. Of the intact EPO molecule, about 40% of the molecular weight is due to the carbohydrate groups on the protein which glycosylate the protein at the glycosylation sites (Sasaki, H)Bothner, B, Dell, a and Fukuda, M (1987) j.biol.chem.262: 12059).

Because human erythropoietin is essential in red blood cell production, this hormone can be used to treat blood disorders characterized by low or defective hematopoiesis. Clinically, EPO is used to treat anemia In patients with Chronic Renal Failure (CRF) (Eschbach, JW, Egri, JC, Downning, MR et al (1987) NEJM 316: 73-78; Eschbach, JW, Abdulhadi, MH, Brown, JK et al (1989) Ann. Intern. Med.111: 992; Egrie, JC, Eschbach, JW, McGuire, T, Adamson, JW (1988) Kidney Intl.33: 262; Lim, VS, Degowin, RL, Zavala, D et al (1989) Ann. Intern. Med.110: 108. 114), and to treat anemia In patients with AIDS and cancer In chemotherapy (Danna, RP, Rudnick, SA, Abels, RI: MB, Garnik, Garnihrin. interferon, Eriger.324; Australin. Yincher. 324: 16. Yinge. Yingqing, Mar, Yingying, III, Marx, et al, 1990). However, the bioavailability of commercially available protein therapeutics, such as EPO, is limited due to its short plasma half-life and susceptibility to protease degradation. These disadvantages limit their potential for clinical use.

Disclosure of Invention

The present invention provides an erythropoietin conjugate, said conjugate comprising an erythropoietin glycoprotein having at least one free amino group and having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells, the erythropoietin glycoprotein being selected from the group consisting of human erythropoietin and analogs thereof, the human erythropoietin analog having the sequence of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites or a rearrangement of at least one glycosylation site; said glycoprotein is conjugated with "n" units of formula-CO- (CH)₂)_x-(OCH₂CH₂)_mThe poly (ethylene glycol) groups of-OR are covalently linked, wherein the-CO (i.e., carbonyl) of each poly (ethylene glycol) group is linked to the amino groupOne formation of an amide bond; wherein R is lower alkyl; x is 2 or 3; m is from about 450 to about 900; n is 1 to 3; and n and m are selected such that the molecular weight of the conjugate minus the erythropoietin glycoprotein is from 20 kilodaltons to 100 kilodaltons. The invention also provides compositions comprising the conjugates described herein, wherein the percentage of conjugates in the composition where n is 1 is at least ninety percent.

The conjugates of the invention have an increased half-life in circulation and an increased residence time in plasma and an increased clinical activity in vivo compared to unmodified EPO (i.e., EPO without PEG attachment) and conventional PEG-EPO conjugates. The conjugates of the invention have the same utility as EPO. In particular, the conjugates of the invention are useful in treating patients by stimulating the division and differentiation of committed erythroid progenitors in the bone marrow in the same manner as EPO treats patients.

The present invention provides a conjugate comprising an erythropoietin glycoprotein having at least one free amino group and having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells, the erythropoietin glycoprotein selected from the group consisting of human erythropoietin and analogs thereof having the sequence of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites or a rearrangement of at least one glycosylation site; said glycoprotein is conjugated with "n" units of formula-CO- (CH)₂)_x-(OCH₂CH₂)_m-the poly (ethylene glycol) groups of OR are covalently linked, wherein the-CO (i.e. carbonyl group) of each poly (ethylene glycol) group forms an amide bond with one of said amino groups; wherein R is lower alkyl; x is 2 or 3; m is from about 450 to about 900; n is 1 to 3; and n and m are selected such that the molecular weight of the conjugate minus the erythropoietin glycoprotein is from 20 kilodaltons to 100 kilodaltons.

It has been found that the conjugates of the invention can be used in the same way as unmodified EPO. However, the conjugates of the invention have an extended half-life in the circulation and residence time in plasma, decreased clearance, and increased clinical activity in vivo. Because of these improved properties, the conjugates of the invention may be administered once a week, rather than three times a week as with unmodified EPO. The reduction in frequency of administration makes it easier for the patient to coordinate, improves the therapeutic effect, and improves the quality of life of the patient. Conjugates having the molecular weight and linkage structure of the conjugates of the invention have been found to have improved potency, stability, AUC, circulating half-life, and reduced cost compared to conventional conjugates of EPO linked to poly (ethylene glycol).

The conjugates of the invention can be administered to a patient in the same manner as EPO in a therapeutically effective amount. A therapeutically effective amount is the amount of conjugate required to cause the bone marrow to increase production of reticulocytes and red blood cells in vivo. The exact amount of conjugate will depend on the exact type of disease to be treated, the condition of the patient to be treated, and the preferred amounts of other ingredients in the composition required for the factor. For example, it may be administered, for example, once a week at 0.01 to 10 micrograms, preferably 0.1 to 1 micrograms, per kilogram of body weight.

The pharmaceutical composition containing the conjugate can be formulated into an effective dosage form to be used in various ways, thereby being administered to a patient suffering from a blood disease in which the production of red blood cells is too low or deficient. The average therapeutically effective amount of the conjugate may vary, and in particular should be based on the recommendations of a physician.

Erythropoietin glycoprotein produced according to the present invention may be prepared as a pharmaceutical composition suitable for injection using pharmaceutically acceptable carriers according to methods known in the art. Suitable compositions are described, for example, in WO97/09996, WO97/40850, WO98/58660, and WO 99/07401. Pharmaceutically acceptable carriers for formulating the products of the invention include human serum albumin, human plasma protein and the like. The compounds of the invention may be formulated in 10mM sodium/potassium phosphate buffer, pH7, containing an osmotic agent, such as 132mM sodium chloride. The pharmaceutical composition may optionally contain a preservative. The pharmaceutical composition may contain varying amounts of erythropoietin, for example 10-1000 micrograms/ml, for example 50 micrograms or 400 micrograms.

The term "erythropoietin" or "EPO" refers to a glycoprotein having the amino acid sequence shown in (SEQ ID NO: 1) or (SEQ ID NO: 2) or an amino acid sequence substantially homologous thereto, the biological properties of which are associated with the stimulation of red blood cell production and the stimulation of division and differentiation of committed erythroid progenitors in the bone marrow. This term as applied herein includes such an artificially modified protein, for example by site-directed mutagenesis or by accidental mutation. These terms also include analogs having from 1 to 6 additional glycosylation sites, analogs having at least one additional amino acid at the carboxy-terminus of the glycoprotein, wherein the additional amino acid comprises at least one glycosylation site, and analogs comprising an amino acid sequence in which at least one glycosylation site is rearranged. These terms include naturally and recombinantly produced human erythropoietin.

Erythropoietin conjugates of the invention can be represented by formula I:

P-[NHCO-(CH₂)_x-(OCH₂CH₂)_m-OR]_n (I)

wherein x, m, n and R are as defined above. In formula I, P is the residue of the erythropoietin glycoprotein described herein, (i.e., without the amino group forming an amide bond with the carbonyl group shown in formula I), which has in vivo biological activity causing bone marrow cells to increase production of reticulocytes and red blood cells.

In a preferred embodiment of the invention, R is methyl. Preferably m is from about 650 to about 750, and n is preferably 1.

In the most preferred embodiment of the invention, R is methyl, m is from about 650 to about 750, and n is 1, i.e., the conjugate described above has the formula:

[CH₃O(CH₂CH₂O)_mCH₂CH₂CH₂CO-NH]_n-P

wherein m is 650 to 750, n is 1 and P is as defined above. Preferably, m has an average value of about 680.

Preferably the glycoprotein of the conjugate as defined above is human erythropoietin. The human erythropoietin and analogs thereof can be expressed by activation of endogenous genes. Preferably the human erythropoietin glycoprotein SEQ ID NO: 1 and SEQ ID NO: 2, most preferably those of SEQ ID NO: 1.

Furthermore, P may be selected from the group consisting of residues of erythropoietin and its analogs with 1 to 6 additional glycosylation sites. The preparation and purification of EPO is known in the prior art as described below. EPO refers to a natural or recombinant protein, preferably a human protein, which can be obtained from any conventional source, e.g. tissue, protein synthesis, cell culture with natural or recombinant cells. Any protein having EPO activity, such as muteins or otherwise modified proteins, are included within the scope of the present invention. Recombinant EPO can be prepared by expression in CHO-, BHK-or HeLa cell lines, by recombinant DNA techniques or by activation of endogenous genes. Expression of proteins, including EPO, by activation of endogenous genes is a technique known in the art and is disclosed, for example, in U.S. patent nos.5,733,761, 5,641,670, and 5,733,746, and in international patent application nos. WO 93/09222, WO 94/12650, WO 95/31560, WO 90/11354, WO 91/06667, and WO 91/09955, the contents of which are incorporated herein by reference. The preferred EPO for use in the preparation of erythropoietin glycoprotein products is human EPO. Preferably the EPO is a peptide having SEQ ID NO: 1 or SEQ ID NO: 2, more preferably having the sequence of SEQ ID NO: 1, human EPO having the amino acid sequence shown in fig. 1.

In one embodiment, P may be a residue of a glycoprotein analog having 1 to 6 additional glycosylation sites. Glycosylation of proteins with one or more oligosaccharide groups occurs at specific sites on the polypeptide backbone and greatly affects the physical properties of the protein, such as protein stability, secretion, subcellular localization and biological activity. Glycosylation is generally of two types. O-linked oligosaccharides are attached to serine or threonine residues and N-linked oligosaccharides are attached to asparagine residues. One type of oligosaccharide present in both N-linked and O-linked oligosaccharides is N-acetylneuraminic acid (sialic acid), a family of amino sugars containing 9 or more carbon atoms. Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides, and, since it carries a negative charge, it confers acidity to the glycoprotein. Human erythropoietin has 165 amino acids and contains three N-linked and one O-linked oligosaccharide chains that account for about 40% of the total molecular weight of the glycoprotein. N-linked glycosylation occurs at asparagine residues at positions 24, 38, and 83, and O-linked glycosylation occurs at serine residue at position 126. The oligosaccharide chains are modified with terminal sialic acid residues. Cleavage of all sialic acid residues from glycosylated erythropoietin enzyme results in loss of activity in vivo, but not in vitro, because sialylation of erythropoietin prevents its binding by liver binding proteins and subsequent clearance.

The glycoproteins of the present invention include analogs of human erythropoietin which have one or more changes in the amino acid sequence of human erythropoietin, resulting in an increased number of sialic acid attachment sites. These glycoprotein analogs can be generated by site-directed mutagenesis, e.g., by addition, deletion, or substitution of amino acid residues to increase or alter the sites available for glycosylation. Glycoprotein analogs having sialic acid levels above that of human erythropoietin are produced by increasing glycosylation sites without affecting the secondary or tertiary structure required for the biological activity of the protein. The glycoproteins of the present invention also include analogs with increased levels of carbohydrate linkages at glycosylation sites, which typically involve the substitution of one or more amino acids adjacent to an N-linked or O-linked site. The glycoproteins of the present invention also include analogs having one or more amino acids extending from the termini of erythropoietin and providing at least one additional carbohydrate site. The glycoproteins of the present invention also include analogs having an amino acid sequence that includes a rearrangement of at least one glycosylation site. This rearrangement of glycosylation sites involves the deletion of one or more glycosylation sites in human erythropoietin and the addition of one or more non-naturally occurring glycosylation sites. Increasing the number of carbohydrate chains on erythropoietin, and thus increasing the number of sialic acids per erythropoietin molecule, can bring several advantages, such as increased stability, increased resistance to protease cleavage, reduced immunogenicity, increased serum half-life, and increased biological activity. Erythropoietin analogs with additional glycosylation sites are disclosed in Elliot, European patent application 640619, published 3/1/1995.

In a preferred embodiment, the glycoprotein of the present invention comprises an amino acid sequence comprising at least one additional glycosylation site, such as, but not limited to, erythropoietin comprising the sequence of human erythropoietin modified by a modification selected from the group consisting of:

Asn³⁰Thr³²；

Asn⁵¹Thr⁵³，

Asn⁵⁷Thr⁵⁹；

Asn⁶⁹；

Asn⁶⁹Thr⁷¹；

Ser⁶⁸Asn⁶⁹Thr⁷¹；

Val⁸⁷Asn⁸⁸Thr⁹⁰；

Ser⁸⁷Asn⁸⁸Thr⁹⁰；

Ser⁸⁷Asn⁸⁸Gly⁸⁹Thr⁹⁰；

Ser⁸⁷Asn⁸⁸Thr⁹⁰Thr⁹²；

Ser⁸⁷Asn⁸⁸Thr⁹⁰Ala¹⁶²；

Asn⁶⁹Thr⁷¹Ser⁸⁷Asn⁸⁸Thr⁹⁰；

Asn³⁰Thr³²Val⁸⁷Asn⁸⁸Thr⁹⁰；

Asn⁸⁹Ile⁹⁰Thr⁹¹；

Ser⁸⁷Asn⁸⁹Ile⁹⁰Thr⁹¹；

Asn¹³⁶Thr¹³⁸；

Asn¹³⁸Thr¹⁴⁰；

Thr¹²⁵(ii) a And

Pro¹²⁴Thr¹²⁵。

as used herein, a symbol used to indicate a modification of an amino acid sequence means that the position of the corresponding unmodified protein (e.g., hEPO of SEQ ID NO: 1 or SEQ ID NO: 2) indicated by the superscript number is changed to the amino acid immediately preceding the respective superscript number.

The glycoprotein may also be an analog having at least one additional amino acid at the carboxy terminus of the glycoprotein, wherein the additional amino acid includes at least one glycosylation site, i.e., the conjugate described above also refers to a compound wherein the glycoprotein has a sequence comprising the sequence of human erythropoietin and a second sequence at the carboxy terminus of the sequence of human erythropoietin, wherein the second sequence comprises at least one glycosylation site. The additional amino acid may comprise a peptide fragment derived from the carboxy terminus of human chorionic gonadotropin. Preferably the glycoprotein is an analogue selected from the group consisting of: (a) having the amino acid sequence Ser Ser Ser Ser SerLys Ala Pro Pro Pro Ser Leu ProSer Pro Ser Arg Leu Pro Gly Pro Ser AspTrh Pro Ile Leu Pro Gln (SEQ ID NO: 3) human erythropoietin, extending from the carboxy terminus; (b) also contains Ser⁸⁷Asn⁸⁸Thr⁹⁰The analog of (a) of EPO; and (c) further contains Asn³⁰Thr³²Val⁸⁷Asn⁸⁸Thr⁹⁰The analog of (a) of EPO.

The glycoprotein may also be an analog having an amino acid sequence that includes a rearrangement of at least one glycosylation site. This rearrangement may involve deletion of any of the N-linked carbohydrate sites in human erythropoietin and addition of an N-linked carbohydrate site at position 88 of the amino acid sequence of human erythropoietin. Preferably, the erythropoietin is an analog selected from the group consisting of: gln²⁴Ser⁸⁷Asn⁸⁸Thr⁹⁰EPO；Gln³⁸Ser⁸⁷Asn⁸⁸Thr⁹⁰EPO; and Gln⁸³Ser⁸⁷Asn⁸⁸Thr⁹⁰EPO。

As used herein, "lower alkyl" refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms. Examples of lower alkyl groups include methyl, ethyl and isopropyl. According to the invention, R is any lower alkyl. Conjugates wherein R is methyl are preferred.

The symbol "m" denotes the presence of a poly (oxyethylene) group (OCH)₂CH₂) The number of oxyethylene residues in the resin. One single PEG subunit of ethylene oxide has a molecular weight of about 44 daltons. Thus, the molecular weight of the conjugate (excluding the molecular weight of EPO) is dependent on the number of "m". In the conjugates of the invention, "m" is from about 450 to about 900 (corresponding to a molecular weight of about 20kDa to about 40 kDa), preferably from about 650 to about 750 (corresponding to a molecular weight of about 30 kDa). The number m is chosen such that the resulting conjugate of the invention has a physiological activity comparable to that of unmodified EPO, which activity may represent the same, higher activity as that of the corresponding unmodified EPO, or a part of this activityAnd (4) dividing. Molecular weight is "about" a value that means the molecular weight is within a reasonable range of this amount as determined by conventional analytical techniques. The "m" is selected so that each poly (ethylene glycol) group covalently linked to the erythropoietin glycoprotein has a molecular weight of from about 20kDa to about 40kDa, preferably about 30 kDa.

In the conjugates of the invention, the number "n" is the number of polyethylene glycol groups covalently attached to the free amino groups of erythropoietin (including the epsilon amino group and/or the amino-terminal amino group of lysine) via an amide linkage. The conjugates of the invention have one, two, or three PEG groups per molecule of EPO. "n" is an integer from 1 to 3, preferably "n" is 1 or 2, most preferably "n" is 1.

The compounds of formula I can be prepared from known polymeric materials by condensation of a compound of formula II with erythropoietin glycoprotein:

wherein R and m are as described above. Compounds of formula II wherein x is 3 are α -lower alkoxy of poly (ethylene glycol), butyric acid succinimidyl ester (lower alkoxy-PEG-SBA). Compounds of formula II wherein x is 2 are α -lower alkoxy of poly (ethylene glycol), succinimidyl propionate (lower alkoxy-PEG-SPA). Any conventional method of reacting an activated ester with an amine to form an amide may be used. In the above reaction, the exemplified succinimidyl ester is a leaving group which causes amide formation. Methods for producing conjugates with proteins using succinimidyl esters of compounds of formula II are disclosed in U.S. patent No.5,672,662, granted 9/30 1997 (Harris, et al).

Human EPO contains 9 free amino groups, the amino-terminal amino group plus the epsilon-amino group of 8 lysine residues. When the PEGylation reagent (pegylation reagent) was bound to the SBA compound of formula II, it was found that at pH7.5, a protein to PEG ratio of 1: 3, and a reaction temperature of 20-25 deg.C, a mixture of mono-, di-, and traces of tri-PEGylated products was produced. When the PEGylation reagent is a SPA compound of formula II, under similar conditions except that the protein to PEG ratio is 1: 2, a predominantly mono-PEGylated product is produced. PEGylated EPO can be administered as a mixture or as different PEGylated products separated by cation exchange chromatography. By manipulating the reaction conditions (i.e., ratio of reactants, pH, temperature, protein concentration, reaction time, etc.), the different pegylated products can be varied.

Human Erythropoietin (EPO) is a human glycoprotein that stimulates erythropoiesis. Their preparation and therapeutic use are described, for example, in U.S. patent nos.5,547,933 and 5,621,080, EP-B0148605, Huang, s.l., proc.natl.acad.sci.usa (1984) 2708-. Erythropoietin for therapeutic use can be produced recombinantly (EP-B0148605, EP-B0209539 and Egrie, J.C., Strickland, T.W., Lane, J.et al (1986) immunobiol. 72: 213-224).

Methods for expressing and preparing erythropoietin in serum-free medium are described, for example, in WO 96/35718, Burg, published at 11/14/1996. And European patent application No.513738, Koch, published as 6/12/1992. In addition to the above-mentioned documents, it is known that serum-free fermentation of recombinant CHO cells containing EPO gene can be carried out. The process is described, for example, in EP-A0513738, EP-A0267678 and Kawamoto, T.et al, Analytical biochem 130(1983)445-453, EP-A0248656, Kowar, J. and Franek, F., Methods in enzymology 421(1986)277-292, Bavister, B., Expcology 271(1981)45-51, EP-A0481791, EP-A0307247, EP-A0343635, WO 88/00967.

In EP-A0267678 an ion exchange chromatography on S-Sepharose, a chromatography on C is described₈Preparative reverse phase HPLC on a column and aGel filtration chromatography EPO prepared in serum-free culture was purified after dialysis. In this respect, the gel filtration chromatography step can be replaced by ion exchange chromatography on fast flow S-Sepharose fast flow. It has also been proposed to perform dye chromatography on a blue Trisacryl column prior to ion exchange chromatography.

One method of purifying recombinant EPO is described in Nobuo, I.et al, J.biochem.107(1990) 352-359. In this method, EPO is treated with Tween * 20, phenylmethylsulfonyl chloride, ethylmaleimide, pepstatin A, copper sulfate and oxamic acid prior to the purification step. Publications including WO 96/35718, Burg, published at 11/14/1996, disclose a method for the preparation of erythropoietin in a serum-free fermentation process (EPOsf).

The specific activities of EPO and the EPO conjugates of the invention can be determined by a variety of known methods. The biological activity of the purified EPO protein of the present invention, administration of the EPO protein to a patient by injection results in bone marrow cells increasing production of reticulocytes and red blood cells as compared to a control group without injection. The biological activity of the EPO protein, or a fragment thereof, obtained or purified in accordance with the present invention may be determined in accordance with Annable, et al, Bull.Wld.Hlth.org. (1972) 47: 99-112 and Pharm, Europa Spec, Issue Erythropoietin BRP Bio 1997 (2). Another method for determining the activity of EPO protein, the normocythaemic mouse assay, is described in example 4.

The invention provides a composition consisting of the conjugate. A conjugate comprising at least 90% mono-PEG, i.e. wherein n is 1, was prepared by the method shown in example 5. In general, mono-PEG conjugates of erythropoietin glycoproteins are desirable because of their higher biological activity compared to di-PEG conjugates. The percentage of mono-PEG conjugate and the ratio of mono-and di-PEG products can be controlled by either reducing the percentage of mono-PEG in the composition by collecting wider fractions around the elution peak or increasing the percentage of mono-PEG in the composition by collecting narrower fractions. About 90% of the mono-PEG conjugates is a better balance between yield and activity. Compositions in which, for example, at least 92% or at least 96% of the conjugates are mono-PEG products (n equals 1) may sometimes be desired. In one embodiment of the invention, the percentage of conjugates wherein n is 1 is between 90% and 96%.

The invention also provides a pharmaceutical composition comprising a conjugate or composition as described above and a pharmaceutically acceptable carrier.

The conjugates and compositions of the invention are particularly useful in the preparation of medicaments for the treatment or prophylaxis of diseases associated with anemia in chronic renal failure patients (CRF), AIDS, and in the treatment of cancer patients undergoing chemotherapy.

Another embodiment of the present invention is a method for preventing and/or treating anemia in patients with Chronic Renal Failure (CRF), AIDS and cancer undergoing chemotherapy comprising the step of administering to the patient a composition as described above.

The present invention also relates to a process for the preparation of the above compound comprising reacting a compound of formula II:

condensation with an erythropoietin, wherein R, m and x are as defined above.

The invention also relates to the above-mentioned compounds for use in the treatment of anemia associated with chronic renal failure patients (CRF), AIDS and cancer patients undergoing chemotherapy.

The invention is further described with reference to the following examples, which do not limit the scope of the invention.

Detailed Description

Examples

Example 1: fermentation and purification of human EPO

a) Preparation and fermentation of inoculum

A vial of Working Cell Bank from an EPO-producing CHO Cell line (available as ATCC CRL8695, published in EP 411678(Genetics Institute)) was removed from the gas phase in a liquid nitrogen tank. Cells were transferred to glass roller bottles and cultured in bicarbonate buffered medium in a humidified carbon dioxide incubator. Typical serum-free media for inoculum preparation and fermentation are disclosed in European patent application 513738, Koch, published as 6.12.1992 or WO 96/35718, Burg, published as 11.14.1996, e.g.DMEM/F12 (e.g.JRH Biosciences/Hazleton Biologics, Denver, US, order No.57-736), also containing sodium bicarbonate, L + glutamine, D + glucose, recombinant insulin, sodium selenite, diaminobutane, hydrocortisone, ferrous sulfate (II), asparagine, aspartic acid, serine and a stabilizer for mammalian cells, e.g.polyvinyl alcohol, methylcellulose, dextran, polyethylene glycol, Pluronic F68, plasma expanders Polyminnesin peptide (HEMACCEL *) or polyvinylpyrrolidone (WO 96/35718).

The presence of contaminating microorganisms was examined microscopically and the cell density was examined. These tests are performed in each classification step.

After the initial growth period, the cell culture is diluted with fresh medium to the initial cell density and another growth cycle is performed. This procedure was repeated until a culture volume of about 2 liters per glass spinner flask was reached. After approximately 12 doublings, 1 to 5 liters of culture were obtained, which were then used as inoculum for a 10 liter seed fermentor.

After 3-5 days, a 10 liter culture of the fermentor may be used as inoculum for a 100 liter seed fermentor.

After a further 3-5 days, a 100 liter culture of the fermenter can be used as inoculum for the 1000 liter production fermenter.

b) Harvesting and cell separation

A batch refeed process is used, i.e. when the desired cell concentration is reached, about 80% of the culture is harvested. The remaining culture was refilled with fresh medium and cultured until the next harvest. One production cycle is composed of up to 10 subsequent harvests: 9 partial harvests and 1 complete harvest at the end of the fermentation. Harvesting is carried out every 3-4 days.

The harvest after the assay was transferred to a cooled vessel. The cells are removed by centrifugation or filtration and discarded. The supernatant containing EPO from the centrifugation step was filtered in-line and collected into a second cooled vessel. The harvest was processed separately during the purification process.

A typical procedure for the purification of EPO-proteins is described in WO 96/35718, Burg, published 1996, 11/14. The purification process is described below.

a) Blue Sepharose Chromatography (Blue Sepharose Chromatography)

Blue Sepharose (Pharmacia) is composed of agarose beads to the surface of which a Cibacron Blue dye is covalently attached. EPO can be enriched in this step due to its stronger binding to blue agarose than most non-protein contaminants, some proteinaceous impurities and PVA. Elution of the blue agarose beads was performed by increasing the salt concentration as well as the pH.

The column was loaded with 80-100 liters of blue agarose, regenerated with NaOH, and equilibrated with equilibration buffer (sodium chloride/calcium and sodium acetate). The acidified and filtered fermenter supernatant was charged. After loading was complete, the column was washed first with a similar buffer containing a higher sodium chloride concentration and then with a Tris-base buffer. The product was eluted with Tris-base buffer and collected as a single fraction according to the total elution profile.

b) Butyl Toyopearl chromatography

Butyl Toyopearl 650C (Toso Haas) is a polystyrene-based matrix to which fatty Butyl-residues are covalently coupled. Since EPO binds more strongly to this gel than most impurities and PVA, elution was performed with a buffer containing isopropanol.

The column was loaded with 30-40 liters of Butyl Toyopearl 650C, regenerated with NaOH, washed with Tris-base buffer and equilibrated with Tris-base buffer containing isopropanol.

The blue agarose eluate was adjusted to the concentration of isopropanol in the column equilibration buffer and loaded onto the column. The column was then washed with equilibration buffer containing an increased isopropanol concentration. The product was eluted with elution buffer (Tris-base buffer with high isopropanol content) and collected as a single fraction according to the total elution profile.

c) Hydroxypatite Ultrogel chromatography

Hydroxyaptite Ultrogel (Biosepra) is composed of Hydroxyapatite, which is incorporated in agarose to improve mechanical properties. EPO has a low affinity for hydroxyapatite and can therefore elute at low phosphate concentrations compared to protein impurities.

The column was loaded with 30-40 liters of Hydroxypatite Ultrogel (Biosepra), regenerated with potassium phosphate/calcium chloride buffer and NaOH, and then with Tris-base. Equilibration was then performed with a Tris-base buffer containing low concentrations of isopropanol and sodium chloride.

The eluate of Brtyl Toyopearl chromatography containing EPO was loaded onto the column. The column was then washed with equilibration buffer and Tris-base buffer without isopropanol and sodium chloride. The product was washed with Tris-base buffer containing low concentrations of potassium phosphate and collected as a single fraction according to the total elution profile.

d) Reverse phase HPLC on Vydac C4

RP-HPLC material Vydac C4(Vydac) is composed of silica gel particles carrying a C4-alkane chain on their surface. The separation of EPO from protein impurities is based on the difference in the strength of hydrophobic interactions. Elution was performed in a gradient of acetonitrile in diluted trifluoroacetic acid.

Preparative HPLC was performed using a stainless steel column (loaded with 2.8 to 3.2 liters of Vydac C4 silica gel). The Hydroxyaptite Ultrogel eluate was acidified by addition of trifluoroacetic acid and loaded onto a Vydac C4 column. For washing and elution, a gradient of acetonitrile in diluted trifluoroacetic acid was used. Fractions were collected and immediately neutralized with phosphate buffer. EPO fractions within the IPC limits were pooled.

e) DEAE Sepharose chromatography

DEAE Sepharose (Pharmacia) material consists of diethylaminoethyl groups covalently attached to the surface of agarose beads. The binding of EPO to DEAE groups is mediated by ionic interactions. Acetonitrile and trifluoroacetic acid are not retained as they flow through the column. After these materials are washed, trace impurities are removed by washing the column with low pH acetate buffer. The column is then washed with neutral phosphate buffer and EPO is eluted with gradually increasing ionic strength buffer.

The column was packed with DEAE Sepharose fast flow. The column volume was adjusted to ensure that the EPO loading was in the range of 3-10mg EPO/ml gel. The column was washed with water and equilibration buffer (sodium/potassium phosphate). The combined HPLC eluent fractions were loaded and the column was washed with equilibration buffer. The column was then washed with wash buffer (sodium acetate buffer) and then with equilibration buffer. EPO was then eluted with elution buffer (sodium chloride, sodium/potassium phosphate) and collected as a single fraction according to the total elution profile.

The eluent from the DEAE sepharose column is adjusted to a specific conductivity. The resulting drug was sterile filtered, filled into teflon bottles, and stored at-70 ℃.

Example 2: PEGylation of EPO with mPEG-SBA

EPO purified according to the serum-free method of example 1 (EPOsf) was determined to be homogeneous by analytical methods and showed a typical isoform consisting of 8 isoforms. Has a specific activity of 190,000IU/mg as determined by normocythaemic mice. The PEGylation reagent used is methoxy-PEG-SBA, which is one in which R is methyl; x is 3; and m is from 650 to 750 (average about 680, corresponding to an average molecular weight of about 30 kDa).

PEGylation reaction

To 100mg of EPOSf (9.71ml of 10.3mg/ml EPOSf stock solution, 5.48 micromoles) was added 10ml of 0.1M potassium phosphate buffer containing 506mg of 30kDa methoxy-PEG-SBA (16.5 micromoles) (obtained from Shearwater Polymers, Inc., Huntsville, Alabama), pH7.5 and mixed at room temperature (20-23 ℃) for 2 hours. The final protein concentration was 5mg/ml and the ratio of protein to PEG reagent was 1: 3. After 2 hours, the reaction was stopped by adjusting the pH to 4.5 with glacial acetic acid and stored at-20 ℃ until purification.

Purification of

1. Conjugate mixture: about 28ml of SP-SEPHAROSE FF (sulfo-propyl cation exchange resin) was loaded onto an AMICON glass column (2.2X 7.5cm) and equilibrated with 20mM acetate buffer pH4.5 at a flow rate of 150 ml/hour. 6ml of the reaction mixture containing 30mg of protein was diluted 5-fold with equilibration buffer and applied to the column. The non-adsorbed material was washed with buffer and the adsorbed PEG conjugate mixture was eluted from the column with 0.175m nacl in equilibration buffer. The unmodified EPOsf remained on the column and was eluted with 750mM NaCl. The column was re-equilibrated with the initial buffer. Samples were analyzed by SDS-PAGE and their extent of PEGylation determined. The 0.175M NaCl eluate was found to contain mono-, as well as di-and traces of tri-PEGylated (pegylated) product, whereas the 750mM NaCl eluate contained unmodified EPOSf

2. di-PEG and mono-PEG-EPOsf: the purified conjugate mixture eluted from the column in the previous step was diluted 4-fold with buffer and reloaded onto the column and washed as described above. The di-PEG-EPOSf and mono-PEG-EPOSf were eluted from the column with 0.1M NaCl and 0.175M NaCl, respectively. Elution was also performed with 750mM NaCl, eluting any unmodified EPOsf.

Alternatively, the reaction mixture was diluted 5-fold with acetate buffer and loaded onto an SP-Sepharose column (approximately 0.5mg protein/ml gel). The column was washed and mono-PEG-EPOsf, di-PEG-EPOsf and unmodified EPOsf were eluted as described previously.

Results

PEG-EPOSf was synthesized by chemical coupling of a linear PEG molecule, and had a number average molecular weight of 30 kDa. PEG-EPOSf is derived from the reaction between the primary amino group of EPOSf and a succinimidyl ester derivative of 30kDa PEG-butanoic acid to give an amide bond.

The results are summarized in table 1. The purified conjugate mixture consisting of mono-and-PEG-EPOsf was analyzed by SDS-PAGE to be free of unmodified EPOsf. The conjugate mixture accounted for 23.4mg or 78% of the starting material. Cation exchange chromatography to separate mono-and di-PEG-EPOSf showed that the ratio of mono-to di-PEG in the conjugate mixture was almost 1: 1. After the reaction was complete, the ratio of the various components was 40: 38: 20 (%) mono: di: unmodified. The overall yield is almost quantitative.

TABLE 1 results of EPOSfPEG

Sample protein (mg) yield (%)

Rxn.Mix. 30 100

Mono-12.040

Two-11.438

Non-modified 6.020

Conjugate mixture 23.478

Example 3: PEGylation of EPO with mPEG-SPA

Another aliquot of EPOsf used in example 2 was reacted with 30kDa methoxy-PEG-SPA (Shearwater Polymers, inc., Huntsville, Alabama). The reaction was carried out at a protein: reagent ratio of 1: 2, and purification was carried out according to the method of example 2. The product was prepared mainly except for mono-pegylation.

Example 4: determination by Normocythaemic mouse assay

In vivo Activity of PEGylated EPO

The normocythamic mouse bioassay is one of the methods known in the prior art (pharm. europaspec. issue erythropoetin BRP Bio 1997(2)) and the monopoietins of ph. eur. BRP. Samples were diluted with BSA-PBS. Normal healthy mice, 7-15 weeks old, were administered 0.2ml of an EPO-fraction containing non-PEGylated EPO or tri-, di-or mono-PEGylated (pegylated) EPO of example 2 or 3, intraperitoneally. Blood was drawn through the tail vein puncture and diluted to make 1 microliter of blood present in 1ml of 0.15 micromolar acridine orange staining solution over a period of 6 days. The dyeing time is 3 to 10 minutes. Reticulocyte counts were performed by analyzing red fluorescence histograms under a fluorescence microscope in a flow cytometer. Reticulocyte counts are expressed in absolute values (every 30,000 blood cells analyzed). For the values given, each group consisted of 5 mice per day, and the mice were bled only once.

In another experiment, mice were administered a single dose of unmodified EPO (25ng EPO), the PEG (sba) -EPO mixture of example 2 (10ng conjugate), mono-and di-pegylated EPO of example 2 (10ng conjugate), PEG (spa) -EPO of example 3 (10ng conjugate), and buffer. The results are shown in Table 2. The results show that the pegylated EPO product has superior activity and long half-life with the same dose (10ng) per mouse compared to unmodified EPO at a dose of 25ng, as indicated by a significant increase in reticulocyte count and a change in the maximum value of reticulocyte count.

TABLE 2

	EPO (unmodified)	30kDaSPAPEG	Single 30KSBA	Two 30KSBA	PEG-EPOSBA conjugate mixtures	Control buffer
							72 hours	1000	1393	1411	994	1328	857
96 hours	500	1406	1501	926	1338	697
							120 hours	About 200	1100	1182	791	944	701
144 hours	About 0	535	607	665	660	708

Example 5: preparation of predominantly mono-PEG-EPO

PEGylation reaction

To 100mg (5.48. mu. mol) of EPOSf in 100mM potassium phosphate buffer pH7.5 prepared according to the method of example 1 was added 329mg (10.96. mu. mol) of 30kDa PEG-SBA reagent dissolved in 3ml of 1mM HCl. Sufficient 100mM potassium phosphate buffer pH7.5 was added to make the volume of the reaction mixture 20 ml. The final protein concentration was 5mg/ml and the ratio of protein to PEG reagent was 1: 2. The reaction mixture was mixed at ambient temperature (20-22 ℃) for 2 hours. After 2 hours, the reaction was stopped by adjusting the pH to 4.5 with glacial acetic acid and stored at-20 ℃ until purification.

Purification of

The reaction mixture in the above step was diluted 1: 5 with 10mM sodium acetate, pH4.5, and loaded onto 300ml of SP-Sepharose FF (sulfopropyl cation exchange resin) packed in a 4.2X 19cm column. The column was previously equilibrated with the same buffer. The column effluent was monitored at 280nm with a Gilson UV monitor and recorded with a Kipp and Zonen recorder. The column is washed with 300ml or 1 bed volume of equilibration buffer to remove excess reagents, reaction by-products and oligomeric PEG-EPO. The di-PEG-EPO was then removed by washing with 2 bed volumes of 100mM NaCl. The mono-PEG-EPO was then eluted with 200mM NaCl. During elution of the mono-PEG-EPO, the first 50ml protein peak was discarded and the mono-PEG-EPO was collected as a 150ml fraction. The unmodified EPOsf which remained on the column was eluted with 750mM NaCl. All elution buffers were prepared in equilibration buffer. All eluted samples were analyzed by SDS-PAGE and by high performance Size Exclusion Chromatography (SEC). The mono-PEG-EPO pool obtained from the 150ml fraction, which contained no detectable unmodified EPOSf, was then concentrated to about 4.5 to 7.5mg/ml and filtered into storage buffer, 10mM potassium phosphate, 100mM NaCl, pH 7.5. Concentration/filtration was performed using Millipore Labscale^TMTFF systems and Millipore Pellicon XL Biomax 50 membranes trapping 50kDa molecules were performed at ambient temperature. The concentrated mono-PEG-EPO was sterile filtered and stored at-20 ℃.

About 75% of EPOsf was pegylated (pegylated). After purification, the overall yield was about 30% mono-PEG-EPO, no detectable unmodified EPOsf, and about 25% di-PEG-EPO. The remaining proteins were oligomeric and non-pegylated EPOsf. The mono-PEG-EPO pool obtained from the 150ml fraction contained about 90% mono-PEG-EPO and about 10% di-PEG-EPO.

Sequence listing

(1) Total information:

(i) the applicant:

(A) name: hoffman Rackey Ltd

(B) Street: 124 Grenzachlasse

(C) City: basle

(E) The state is as follows: switzerland

(F) And E, postcode: CH-4070

(G) Telephone: (61)6881111

(H) Faxing: (61)6881395

(I) Electric transmission: 962292 hlr ch

(ii) The invention name is as follows: erythropoietin conjugates

(iii) Number of sequences: 3

(V) computer readable form

(A) Type of medium: flexible disk

(B) IBM PC compatibility of computer

(C) Operating system word

(D) Software: patentin Release 2.0

<170>PatentIn ver.2.0

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Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu

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Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His

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Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe

35 40 45

Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp

50 55 60

Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu

65 70 75 80

Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp

85 90 95

Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu

100 105 110

Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala

115 120 125

Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val

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Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala

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Cys Arg Thr Gly Asp

165

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Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu

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Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His

20 25 30

Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe

35 40 45

Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp

50 55 60

Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu

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Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp

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Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu

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Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala

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Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val

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Cys Arg Thr Gly Asp Arg

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Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro Ser Arg

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Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

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Claims (9)

1. A conjugate comprising an erythropoietin glycoprotein having at least one free amino group and having the in vivo biological activity of causing bone marrow cells to increase production of reticulocytes and red blood cells, the erythropoietin glycoprotein selected from the group consisting of human erythropoietin and analogs thereof having the sequence of human erythropoietin modified by the addition of from 1 to 6 glycosylation sites or a rearrangement of at least one glycosylation site; said glycoprotein being covalently linked to a poly (ethylene glycol) group of the formula

-CO-(CH₂)_x-(OCH₂CH₂)_m-OR, wherein-CO of the poly (ethylene glycol) group forms an amide bond with said free amino group; wherein

R is lower alkyl having 1 to 6 carbon atoms;

x is 2 or 3; and is

m is from 450 to 900.

2. The conjugate of claim 1, having the formula:

P-NHCO-(CH₂)_x-(OCH₂CH₂)_m-OR (I)

wherein x, m and R are as defined in claim 1 and P is a glycoprotein residue which does not contain a free amino group forming an amide bond.

3. The conjugate of claim 2, wherein the glycoprotein is human erythropoietin.

4. The conjugate of claim 3, wherein the human erythropoietin glycoprotein is expressed by endogenous gene activation.

5. The conjugate of claim 3, wherein the glycoprotein has the amino acid sequence of SEQ ID NO: 1.

6. The conjugate according to claim 5, wherein R is methyl.

7. The conjugate according to claim 5, wherein x is 3.

8. The conjugate according to claim 7, wherein the molecular weight is from 20 kilodaltons to 40 kilodaltons.

9. The conjugate according to claim 7, wherein the molecular weight is 30 kilodaltons.