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WO2007010552A2 - N- terminal peg conjugate of erythropoietin - Google Patents

N- terminal peg conjugate of erythropoietin Download PDF

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Publication number
WO2007010552A2
WO2007010552A2 PCT/IN2006/000103 IN2006000103W WO2007010552A2 WO 2007010552 A2 WO2007010552 A2 WO 2007010552A2 IN 2006000103 W IN2006000103 W IN 2006000103W WO 2007010552 A2 WO2007010552 A2 WO 2007010552A2
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WO
WIPO (PCT)
Prior art keywords
epo
conjugate
erythropoietin
peg
mpeg
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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PCT/IN2006/000103
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French (fr)
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WO2007010552A3 (en
WO2007010552B1 (en
Inventor
Subhash V. Kapre
Umesh S. Shaligram
Kwang Nho
Gunwon Bae
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Serum Institute of India Pvt Ltd
Sunbio Inc
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Serum Institute of India Pvt Ltd
Sunbio Inc
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Publication of WO2007010552A3 publication Critical patent/WO2007010552A3/en
Publication of WO2007010552B1 publication Critical patent/WO2007010552B1/en
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a conjugated Erythropoietin and a method for producing thereof.
  • Erythropoiesis is the production of red blood cells, which occurs to offset cell destruction. Erythropoiesis is a controlled physiological mechanism that enables sufficient red blood cells to be available for proper tissue oxygenation.
  • Naturally occurring human erythropoietin (hEPO) is produced in kidney and is the humoral plasma factor which stimulates red blood cell production (Carnot, P and Deflandre, C (1906) CR. Acad. Sci. 143: 432; Erslev, A J (1953 Blood 8: 349; Reissmann, K R (1950) Blood 5: 372; Jacobson, L O, Goldwasser, E, Freid, W and Plzak, L F (1957) Nature 179: 6331-4).
  • Naturally occurring EPO stimulates the division and differentiation of committed erythroid progenitors in the bone marrow and exerts its biological activity by binding to receptors on erythroid precursors (Krantz, B S (1991) Blood 77: 419).
  • Erythropoietin has been manufactured biosynthetically using recombinant DNA technology (Egrie, J C, Strickland, T W, Lane, J et al. (1986) Immunobiol. 72: 213-224) and is the product of a cloned human EPO gene inserted into and expressed in the ovarian tissue cells of the Chinese hamster (CHO cells).
  • the carbohydrate groups account for approximately 40% of the molecular weight that glycosylate the protein at glycosylation sites on the protein (Sasaki, H, Bothner, B, Dell, A and Fukuda, M (1987) J. Biol. Chem. 262: 12059).
  • EPO is used in the treatment of anemia in chronic renal failure patients (CRF) (Eschbach, J W, Egri, J C, Downing, M R et al. (1987) NEJM
  • PEG polyethylene glycol compound
  • European Patent Application EP 651,761 discloses the selective modification of recombinantly produced polypeptides at terminal alpha- carbon reactive groups.
  • the first step in the method is to form a recombinantly produced polypeptide so that it is protected at the terminal alpha-carbon reactive group with a biologically added protecting group.
  • the biologically added protecting group is preferably an amino acid, peptide and/or polypeptide that contains at least one site that is cleavable enzymatically or chemically, and preferably has a sequence that is not present in the sequence of the desired polypeptide.
  • the biologically protected polypeptide is reacted with chemical protecting agents to protect the side chain groups and then is cleaved with a cleavage reagent specific for the biologically added protecting group.
  • chemical protecting agents to protect the side chain groups and then is cleaved with a cleavage reagent specific for the biologically added protecting group.
  • a polypeptide is produced having an unprotected N-terminal amino group and protected side chain reactive groups.
  • the unprotected N-terminal amino group is modified with a modifying agent to form an N-terminally modified and side-chain- protected polypeptide. It is then deprotected to form an N-terminally modified polypeptide.
  • EP 651 ,761 suggests that any sequence of amino acids may be attached as biologically added protecting groups.
  • EPO is expressed with a leader signal sequence, which is cleaved off by a signal peptidase in order to yield the processed, mature EPO.
  • signal peptidases recognize only restricted amino acid residues at the P1' and P3' cleavage site (R. E. Dalbey et al. Protein Sci. 6, 1129 (1997).
  • a biologically added protecting peptide has to be built up from an N- terminal amino acid sequence of at least three amino acids for cleavage of the signal sequence, followed by an amino acid sequence for enzymatic or chemical removal of the protecting group. If the recognition sequences of both the signal peptidase and the cleavage protease are identical or closely related, then the sequence of the biologically added protecting group can be reduced to a few amino acids.
  • N-terminal selective modification is obtained by chemoselective ligation to an aldehyde (or ketone)-functionalized target macromolecule (European Patent Application EP 788,375; Gaertner, H F, Offord, R E, Bioconjugate Chem., 7 (1), 38-44 (1996)).
  • this method only works for N-terminal serines or threonines.
  • the Roche's US patent application US 2002/0115833 although discloses the N-terminally pegylated EPO, it specifically defines their formulation of PEG-EPO being pegylated specifically by PEG-NHS (PEG-N-hydroxy succinimide).
  • PEG-NHS PEG-N-hydroxy succinimide
  • the PEG-NHS reacts non-specifically to all lysines, so all lysines needs to be protected by a process called citraconylation.
  • An object of the present invention is to provide a conjugate of Erythropoietin glycoprotein having higher bioactivity.
  • a conjugate comprises an Erythropoietin glycoprotein having N-terminal ⁇ - amino group and aldehyde derivatives of polyethylene glycol, said erythropoietin glycoprotein being selected from the group consisting of human erythropoietin and analogs thereof, said aldehyde derivative of methoxy polyethylene glycol (mPEG) of the formula:
  • R alkyl group of formula -(CH 2 ) n -
  • n 2 to 4.
  • the polyethylene glycol according to the present invention is a mono or branched polyethylene glycol preferably monoPEG and molecular weight of the PEG is about 20 to 60 kda.
  • the R is preferably ethyl, propyl or butyl.
  • Said erythropoietin according to the present invention is a recombinant erythropoietin (rhEPO) produced in animal cells.
  • a process for making the conjugate of the present invention comprises steps of: expressing, fermenting, and purifying rhEPO protein in recombinant animal cells and reacting EPO with PEG-Aldehyde to obtain N- terminally pegylated EPO by reductive amination reaction and then further purified to obtain pure N-terminally pegylated EPO.
  • PEG - Aldehyde added at an about molar ratio of ⁇ 10:2 preferably at 7.5:1 molar ratio of PEG: EPO.
  • the fermentation step according to the present invention is serum free.
  • a pharmaceutical composition comprises the N-pegylated Erythropoietin conjugate of the present invention and a pharmaceutically acceptable excipient.
  • FIG 1 Shows the SDS-PAGE Analysis of native EPO (left lane), PEG- lys-EPO (center lane), and PEG-N-EPO (right lane).
  • FlG 2 Shows the Rat Reticulocyte Count Bioassay with Native EPO, PEG-N-
  • FIG 3 Shows the Quantitation of Pegylated-N-EPO by LC-MS
  • FIG 4 shows the tabular representation of peptides generated by digestion of EPO and PEG-N-EPO with endoproteinase Lys-C.
  • FIG 5 shows the peptide mapping chromatogram of EPO and PEG-N-
  • 0 C refers to degrees Celsius
  • mmol refers to mill mole or mill moles
  • mg refers to milligrams
  • ⁇ g refers to micrograms
  • ml or mL refers to milliliters
  • ⁇ l or ⁇ L refers to micro liters.
  • EPO erythropoietin
  • huEPO erythropoietin
  • EPO is a glycoprotein hormone that is secreted by the human kidney, which stimulates formation of erythrocytes (erythropoiesis) in human bone marrow.
  • the amino acid sequence of the predominant allelic variant of the protein portion of erythropoietin is known.
  • EPO consists of 165 amino acids, is comprised of about 40% carbohydrate, by mass, and has a total molecular weight of approximately 30.6 kDa.
  • the carbohydrate structure of EPO is heterogeneous, whereas the amino acid sequence of the predominant human allelic variant is not. Therefore, these terms refer to a heterogeneous group of EPO or huEPO molecules.
  • PEG-N-EPO means an N-terminally pegylated EPO
  • PEG-Lys-EPO means Lysine pegylated EPO
  • CHO cell means Chinese Hamster Ovary cell.
  • rhEPO means Recombinant Erythropoietin.
  • Erythropoietin compound means a glycosylated protein having nearly the same amino acid sequence as EPO, and having the ability to increase hematocrit when properly administered to a mammal, but differing from EPO in having one or more amino acid modifications.
  • An amino acid modification may be an insertion, a deletion, a replacement, or an inversion of one or more amino acids.
  • Erythropoietic activity refers to the ability of a compound to stimulate erthyropoiesis. Erythropoietic activity can be assessed in vitro, as well as in vivo.
  • Erythropoietic activity generally refers to the ability of a compound to cause an increase in hematocrit levels from an established baseline when administered by an acceptable route of administration at effective doses. In vitro activity can be determined
  • the invention provides a conjugated erythropoietin compound comprising an aldehyde group of PEG molecule primarily attached to N- terminal amino acid and having formula 1 :
  • the conjugate of the present invention can be represented by the formula 2:
  • R alkyl group of formula -(CH 2 )n.
  • the erythropoietin glycoprotein is recombinant EPO being selected from the group consisting of human erythropoietin glycoprotein and analog thereof.
  • the recombinant EPO (rhEPO) produced by growing recombinant human EPO synthesized by recombinant CHO cells is secreted to the culture medium which is then purified by different high performance chromatography steps.
  • the purified EPO thus obtained has similar biological and chemical characteristics as compared to International Standards.
  • Recombinant EPO may be prepared via expression in
  • Expression of proteins, including EPO, by endogenous gene activation is well known in the art and is disclosed, for example in U.S. Pat. Nos. 5,733,761 , 5,641 ,670, 5,733,746, 5,994,122,
  • the preferred EPO species for the preparation of erythropoietin glycoprotein products are human EPO species.
  • the aldehyde derivative of PEG has following formula:
  • R alkyl group of formula -(CHa) n -
  • the polyethylene glycol according to the present invention is a mono or branched polyethylene glycol preferably monoPEG and molecular weight of the PEG is about 20 to 60 kda.
  • the R is preferably ethyl, propyl or butyl or n is 2 to 4.
  • the invention is also directed to a process of preparing a PEG-N- EPO conjugate by expressing, fermenting, and purifying rhEPO protein in recombinant animal cells preferably fermentation is serum free, the reaction of EPO with mPEG-Aldehyde preferably with Propionaldehyde to obtain N-terminally pegylated EPO.
  • EPO reacts with mPEG- propionaldehyde by the reductive amination reaction.
  • the addition of mPEG-propionaldehyde is at about molar ratio of 10:2, preferably at 7.5:1 molar ratio of PEG: EPO.
  • the PEG-N-EPO is further subjected to purification.
  • erythropoietic compound having the PEG - N- Erythropoietin which is an N-terminally pegylated EPO, or erythropoietin analog along with the pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carriers include adjuvants, excipients, surfactants, and the like that are commonly known and used by the person skilled in the art for the preparation of pharmaceutical compositions.
  • compositions may include various dosage forms including topical, injectable, and the like.
  • a method of treating anemia using erythropoietic compound or a pharmaceutical composition containing the erythropoietic compound of the present invention is provided.
  • the present PEG-N-EPO conjugate Compared to unmodified EPO (i.e., EPO without a PEG attached) and conventional PEG-EPO conjugates, the present PEG-N-EPO conjugate have an increased circulating half-life and plasma residence time, decreased clearance, and increased clinical activity in vivo.
  • the conjugate of this invention have the same uses as EPO.
  • the conjugate of this invention are useful to treat patients by stimulating the division and differentiation of committed erythroid progenitors in the bone marrow in the same way EPO is used to treat patients.
  • conjugate of this invention can be used in the same manner as unmodified EPO.
  • conjugate of this invention have an increased circulating half-life and plasma residence time, decreased clearance, and increased clinical activity in vivo.
  • N-terminal .alpha. -amino group refers to the N-terminal amino residue of a peptide, i.e. that end of a peptide or protein chain having an amino acid with a free alpha-amino (NH. sub.2--) group.
  • the glycoprotein of the conjugate as defined above is a human erythropoietin.
  • Human erythropoietin and analogous proteins as defined above can be expressed by endogenous gene activation.
  • EP-A 0 267 678 discloses an ion exchange chromatography on S-Sepharose, a preparative reverse phase HPLC on a C. sub.8 column and a gel filtration chromatography for the purification of EPO produced in serum-free culture after dialysis.
  • the gel filtration chromatography step can be replaced by ion exchange chromatography on S-Sepharose fast flow. It is also proposed that a dye chromatography on a Blue Trisacryl column be carried out before the ion exchange chromatography.
  • a process for the purification of recombinant EPO is also described by Nobuo, I. et al., J. Biochem. 107 (1990) 352-359.
  • EPO is treated with a solution of Tween.RTM. 20, phenylmethylsulfonyl fluoride, ethylmaleimide, pepstatin A, copper sulfate and oxamic acid prior to the purification steps.
  • EPOsf erythropoietin in a serum free fermentation process
  • PEG-N-EPO is an N-terminally pegylated EPO produced by the following procedure. A 6 mg of EPO was dissolved in 20 mM phosphate buffer containing 150 mM NaCI with pH adjusted at 5.0. The EPO solution was then mixed with mPEG-propionaldehyde 2OK (M.W.: 20,000, manufactured by SunBio, Inc.). The amount of mPEG- propionaldehyde added was 7.5: 1 molar ratio of PEG: EPO. After addition of the PEG, the reaction mixture was stirred well to facilitate the complete dissolution of PEG powder into the EPO solution. After the PEG addition, 100 mM of sodium cyanoborohydride was added and mixed well. The reaction was allowed to continue for 18 hours. In the meanwhile the preparation of the chromatography process was done.
  • chromatography purification is to obtain pure mono- PEG-EPO.
  • HiTrap Q-sepharose chromatography column (Amersham Pharmacia) was packed and equilibrated with 10 mM Tris buffer of pH 8.0. Then the reaction mixture was loaded onto the column, which was then washed with 30 CV (column volume) of 10 mM Tris buffer of pH 8.0 to wash off all remaining free PEG that had not reacted with EPO.
  • the column was eluted with 40 CV of 25 mM NaCI in 10 mM Tris buffer of pH 8.0 to elute di-PEG-EPO (EPO with two PEGs attached to it).
  • the next step was to elute with 40 CV of 65 mM NaCI in 10 mM Tris buffer of pH 8.0 to elute mono-PEG-EPO (EPO with one PEG attached to it).
  • the next step was to elute with 12 CV of 200 mM NaCI in 10 mM
  • Tris buffer of pH 8.0 to elute native EPO which was followed by 8 CV of 1 M NaCI in 10 mM Tris buffer of pH 8.0 to elute any remaining bound material.
  • the collected mono-PEG-EPO fractions were then loaded onto Superdex 200 chromatography (Amersham Pharmacia) to further refine the purity of mono-PEG-EPO.
  • the column was eluted with 20 mM phosphate buffer containing 150 mM NaCI of pH 7.0 to elute the pure mono-PEG-EPO.
  • the collected fractions of mono-PEG-EPO was then concentrated to the final concentration of 65 ⁇ g/ml in buffer of 20 mM phosphate, 150 mM NaCI and pH 7.0.
  • EPO conjugates can be prepared using mPEG-butyraldehyde and mPEG-pentaldehyde.
  • the lysine-52 Pegylated EPO was produced and purified as described in the US patent US 2002/0115833 A1.
  • the result for the peptide mapping Analysis of PEG-N-EPO shows the digestion of native EPO and PEG-N-EPO by lys-C enzyme (Fig. 4) yielded 9 peptide fragments. Of these, 5 peptide fragments, L1 (AA 1- 20), L3 (AA 46-52), L6 (AA 117-140), L7 (AA 141-152), and L9 (156- 166) were quantitatively obtained and analyzed (Fig 3).
  • the peptide mapping analysis of PEG-N-EPO shows that the peptide fragment (L1) containing the N-terminal has been Pegylated.

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Abstract

The present invention refers to conjugate of erythropoietin having higher bioactivity comprises an Erythropoietin glycoprotein having N-terminal α - amino group and aldehyde derivatives of polyethylene glycol, said erythropoietin glycoprotein being selected from the group consisting of human erythropoietin and analogs thereof, said aldehyde derivative of methoxy polyethylene glycol of the formula: PEG-R-CHO wherein R= alkyl group of formula -(CH2)n wherein n=2 to 4. The formula of the conjugate of the present invention is: PEG-R-CONH-EPO wherein R=alkyl group of formula -(CH2)n wherein n=2 to 4. Said erythropoietin according to the present invention is a recombinant erythropoietin (rhEPO) produced in animal cells; said erythropoietin conjugate is prepared by expressing, fermenting, and purifying rhEPO protein in recombinant animal cells and reacting EPO with PEG- Aldehyde to obtain N-terminally pegylated EPO by reductive amination reaction and then further purified to obtain pure N-terminally pegylated EPO.

Description

TITLE
N- TERMINAL PEG CONJUGATE OF ERYTHROPOIETIN
FILED OF INVENTION
The present invention relates to a conjugated Erythropoietin and a method for producing thereof.
BACKGROUND OF THE INVENTION
Erythropoiesis is the production of red blood cells, which occurs to offset cell destruction. Erythropoiesis is a controlled physiological mechanism that enables sufficient red blood cells to be available for proper tissue oxygenation. Naturally occurring human erythropoietin (hEPO) is produced in kidney and is the humoral plasma factor which stimulates red blood cell production (Carnot, P and Deflandre, C (1906) CR. Acad. Sci. 143: 432; Erslev, A J (1953 Blood 8: 349; Reissmann, K R (1950) Blood 5: 372; Jacobson, L O, Goldwasser, E, Freid, W and Plzak, L F (1957) Nature 179: 6331-4). Naturally occurring EPO stimulates the division and differentiation of committed erythroid progenitors in the bone marrow and exerts its biological activity by binding to receptors on erythroid precursors (Krantz, B S (1991) Blood 77: 419).
Erythropoietin has been manufactured biosynthetically using recombinant DNA technology (Egrie, J C, Strickland, T W, Lane, J et al. (1986) Immunobiol. 72: 213-224) and is the product of a cloned human EPO gene inserted into and expressed in the ovarian tissue cells of the Chinese hamster (CHO cells). In the intact EPO molecule, the carbohydrate groups account for approximately 40% of the molecular weight that glycosylate the protein at glycosylation sites on the protein (Sasaki, H, Bothner, B, Dell, A and Fukuda, M (1987) J. Biol. Chem. 262: 12059). Because human erythropoietin is essential in red blood cell formation, the hormone is useful in the treatment of blood disorders characterized by low or defective red blood cell production. Clinically, EPO is used in the treatment of anemia in chronic renal failure patients (CRF) (Eschbach, J W, Egri, J C, Downing, M R et al. (1987) NEJM
316: 73-78; Eschbach, J W, Abdulhadi, M H, Browne, J K et al. (1989) Ann. Intern. Med. 111: 992; Egrie, J C, Eschbach, J W, McGuire, T, Adamson, J W (1988) Kidney Intl. 33: 262; Lim, V S, Degowin, R L, Zavala, D et al. (1989) Ann. Intern. Med. 110: 108-114) and in AIDS and cancer patients undergoing chemotherapy (Danna, R P, Rudnick, S A,
Abels, R I In: M B, Gamick, ed. Erythropoietin in Clinical Applications. An International Perspective. New York, N.Y.: Marcel Dekker; 1990: p. 301-324). However, the bioavailability of commercially available protein therapeutics such as EPO is limited by their short plasma half-life and susceptibility to protease degradation. These shortcomings prevent them from attaining, maximum clinical potency.
Common approaches for preparing pegylated proteins yield mixtures of mono- and oligo-pegylated proteins. Moreover, the polyethylene glycol compound (PEG) is bound at several positions of the proteins depending on the amount and reactivities of the available reactive groups on the protein surface. Such a mixture may have severe shortcomings: PEG may be bound at positions which interact with the protein specific receptor and conclusively reduce or even prohibit therapeutic efficacy. To solve this drawback either separation or purification of active ingredients of such a mixture or a selective synthetic route to avoid formation is required. Avoiding formation of any mixtures would greatly simplify the synthesis of a pure active pharmaceutical ingredient in terms of a single positional isomer in essentially higher yields. This is particularly true because separation of positional isomers of PEG-protein mixtures may not be possible using common production scale-synthesis.
Several methods have been proposed for the selective modification of recombinantly produced polypeptides.
European Patent Application EP 651,761 discloses the selective modification of recombinantly produced polypeptides at terminal alpha- carbon reactive groups. The first step in the method is to form a recombinantly produced polypeptide so that it is protected at the terminal alpha-carbon reactive group with a biologically added protecting group. The biologically added protecting group is preferably an amino acid, peptide and/or polypeptide that contains at least one site that is cleavable enzymatically or chemically, and preferably has a sequence that is not present in the sequence of the desired polypeptide.
Once formed, the biologically protected polypeptide is reacted with chemical protecting agents to protect the side chain groups and then is cleaved with a cleavage reagent specific for the biologically added protecting group. By these means, a polypeptide is produced having an unprotected N-terminal amino group and protected side chain reactive groups. The unprotected N-terminal amino group is modified with a modifying agent to form an N-terminally modified and side-chain- protected polypeptide. It is then deprotected to form an N-terminally modified polypeptide. EP 651 ,761 suggests that any sequence of amino acids may be attached as biologically added protecting groups.
However, in mammalian expression systems, EPO is expressed with a leader signal sequence, which is cleaved off by a signal peptidase in order to yield the processed, mature EPO. Such signal peptidases recognize only restricted amino acid residues at the P1' and P3' cleavage site (R. E. Dalbey et al. Protein Sci. 6, 1129 (1997). Thus, a biologically added protecting peptide has to be built up from an N- terminal amino acid sequence of at least three amino acids for cleavage of the signal sequence, followed by an amino acid sequence for enzymatic or chemical removal of the protecting group. If the recognition sequences of both the signal peptidase and the cleavage protease are identical or closely related, then the sequence of the biologically added protecting group can be reduced to a few amino acids.
In another method, N-terminal selective modification is obtained by chemoselective ligation to an aldehyde (or ketone)-functionalized target macromolecule (European Patent Application EP 788,375; Gaertner, H F, Offord, R E, Bioconjugate Chem., 7 (1), 38-44 (1996)). However, this method only works for N-terminal serines or threonines.
In yet another method, selective modification at N-terminal alanine is accomplished by transamination of alanine to pyruvate (European Patent Applications EP 964,702 and EP 605,963). The disadvantage of this method is that the EPO derivative obtained possessed reduced in vitro activity. Also the transformation agents Cu.sup.2+/glyoxylic acid/NaOAc are likely to produce side reactions within the EPO molecule.
Site-specific N-terminal modification was also shown by transglutaminase-mediated incorporation of poly (ethylene glycol) derivatives (Sato, H., Yamamoto, K, Hayashi, E, Takahara, Y,
Bioconjugate Chem. 11 (4), 502-509 (2000)). But this method showed only low yields and needs incorporation of a peptide tag at the N- terminus and hence modifies the polypeptide structure.
Modification with glyoxylyl-based labelling reagents also enables selective N-terminal modification (Zhao, Z G, Im, J S, Clarke, D F, Bioconjugate Chem. 10, 424-430 (1999) but is restricted to cysteine.
The Roche's US patent application US 2002/0115833 although discloses the N-terminally pegylated EPO, it specifically defines their formulation of PEG-EPO being pegylated specifically by PEG-NHS (PEG-N-hydroxy succinimide). The PEG-NHS reacts non-specifically to all lysines, so all lysines needs to be protected by a process called citraconylation.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a conjugate of Erythropoietin glycoprotein having higher bioactivity.
According to one of the embodiment of the present invention, a conjugate comprises an Erythropoietin glycoprotein having N-terminal α - amino group and aldehyde derivatives of polyethylene glycol, said erythropoietin glycoprotein being selected from the group consisting of human erythropoietin and analogs thereof, said aldehyde derivative of methoxy polyethylene glycol (mPEG) of the formula:
mPEG-R-CHO
wherein R= alkyl group of formula -(CH2)n-
wherein n=2 to 4
The formula of the conjugate of the present invention is:
mPEG-R-CONH-EPO wherein R=alkyl group of formula -(CH2V
wherein n= 2 to 4.
The polyethylene glycol according to the present invention is a mono or branched polyethylene glycol preferably monoPEG and molecular weight of the PEG is about 20 to 60 kda. The R is preferably ethyl, propyl or butyl.
Said erythropoietin according to the present invention is a recombinant erythropoietin (rhEPO) produced in animal cells.
According to another embodiment of the present invention, a process for making the conjugate of the present invention comprises steps of: expressing, fermenting, and purifying rhEPO protein in recombinant animal cells and reacting EPO with PEG-Aldehyde to obtain N- terminally pegylated EPO by reductive amination reaction and then further purified to obtain pure N-terminally pegylated EPO. PEG - Aldehyde added at an about molar ratio of ~ 10:2 preferably at 7.5:1 molar ratio of PEG: EPO.
The fermentation step according to the present invention is serum free.
In yet another embodiment of the present invention, a pharmaceutical composition comprises the N-pegylated Erythropoietin conjugate of the present invention and a pharmaceutically acceptable excipient.
In yet another embodiment of the present invention is to provided a method of treating anemia in a patient afflicted with chronic renal failure (CRF) resulting from chemotherapy, comprising administering to said patient an effective amount of conjugate of the present invention. DESCRIPTION OF DRAWINGS
FIG 1: Shows the SDS-PAGE Analysis of native EPO (left lane), PEG- lys-EPO (center lane), and PEG-N-EPO (right lane).
FlG 2 Shows the Rat Reticulocyte Count Bioassay with Native EPO, PEG-N-
EPO and PEG-lys-EPO.
FIG 3 Shows the Quantitation of Pegylated-N-EPO by LC-MS
FIG 4 shows the tabular representation of peptides generated by digestion of EPO and PEG-N-EPO with endoproteinase Lys-C.
FIG 5 shows the peptide mapping chromatogram of EPO and PEG-N-
EPO digested with endoproteinase Lys-C.
DEFINITION
For purposes of the present invention, as disclosed and claimed herein, the
' following terms and abbreviations are defined below. The terms and abbreviations used in this document have their normal meanings unless otherwise designated.
For example, "0C" refers to degrees Celsius; "mmol" refers to mill mole or mill moles; "mg" refers to milligrams;"μg" refers to micrograms; "ml or mL" refers to milliliters; and "μl or μL" refers to micro liters.
"Erythropoietin" means human erythropoietin, and is abbreviated herein as "EPO" or "huEPO." EPO is a glycoprotein hormone that is secreted by the human kidney, which stimulates formation of erythrocytes (erythropoiesis) in human bone marrow. The amino acid sequence of the predominant allelic variant of the protein portion of erythropoietin is known. EPO consists of 165 amino acids, is comprised of about 40% carbohydrate, by mass, and has a total molecular weight of approximately 30.6 kDa. The carbohydrate structure of EPO is heterogeneous, whereas the amino acid sequence of the predominant human allelic variant is not. Therefore, these terms refer to a heterogeneous group of EPO or huEPO molecules.
"PEG-N-EPO" means an N-terminally pegylated EPO; "PEG-Lys-EPO" means Lysine pegylated EPO.
"CHO cell" means Chinese Hamster Ovary cell.
"rhEPO" means Recombinant Erythropoietin.
"Erythropoietin compound" means a glycosylated protein having nearly the same amino acid sequence as EPO, and having the ability to increase hematocrit when properly administered to a mammal, but differing from EPO in having one or more amino acid modifications. An amino acid modification may be an insertion, a deletion, a replacement, or an inversion of one or more amino acids.
"Erythropoietic activity" refers to the ability of a compound to stimulate erthyropoiesis. Erythropoietic activity can be assessed in vitro, as well as in vivo.
Erythropoietic activity generally refers to the ability of a compound to cause an increase in hematocrit levels from an established baseline when administered by an acceptable route of administration at effective doses. In vitro activity can be determined
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a conjugated erythropoietin compound comprising an aldehyde group of PEG molecule primarily attached to N- terminal amino acid and having formula 1 :
mPEG-R-CHO
wherein R=alkyl group of formula -(CH2)n- wherein n= 2 to 4
In the most preferred embodiment of the present invention the conjugate of the present invention can be represented by the formula 2:
mPEG-R-CONH-EPO
wherein R=alkyl group of formula -(CH2)n.
wherein n=2 to 4
The erythropoietin glycoprotein is recombinant EPO being selected from the group consisting of human erythropoietin glycoprotein and analog thereof. The recombinant EPO (rhEPO) produced by growing recombinant human EPO synthesized by recombinant CHO cells is secreted to the culture medium which is then purified by different high performance chromatography steps. The purified EPO thus obtained has similar biological and chemical characteristics as compared to International Standards.
Alternatively the Recombinant EPO may be prepared via expression in
CHO, BHK, COS, HeLa or PER.C6 cell lines or other appropriate cell lines of animal or human origin, by recombinant DNA technology or by endogenous gene activation. Expression of proteins, including EPO, by endogenous gene activation is well known in the art and is disclosed, for example in U.S. Pat. Nos. 5,733,761 , 5,641 ,670, 5,733,746, 5,994,122,
5,733,761 , 5,641 ,670, 5,981 ,214 and 5,272,071 , and international patent publication WO 90/11354, the contents of each of which are incorporated herein by reference. The preferred EPO species for the preparation of erythropoietin glycoprotein products are human EPO species. According to the present invention the aldehyde derivative of PEG has following formula:
mPEG-R-CHO
wherein R=alkyl group of formula -(CHa)n-
wherein n=2 to 4
The polyethylene glycol according to the present invention is a mono or branched polyethylene glycol preferably monoPEG and molecular weight of the PEG is about 20 to 60 kda. The R is preferably ethyl, propyl or butyl or n is 2 to 4.
The invention is also directed to a process of preparing a PEG-N- EPO conjugate by expressing, fermenting, and purifying rhEPO protein in recombinant animal cells preferably fermentation is serum free, the reaction of EPO with mPEG-Aldehyde preferably with Propionaldehyde to obtain N-terminally pegylated EPO. EPO reacts with mPEG- propionaldehyde by the reductive amination reaction. The addition of mPEG-propionaldehyde is at about molar ratio of 10:2, preferably at 7.5:1 molar ratio of PEG: EPO. The PEG-N-EPO is further subjected to purification.
According to another embodiment of the present invention provides a pharmaceutical composition having the erythropoietic compound having the PEG - N- Erythropoietin which is an N-terminally pegylated EPO, or erythropoietin analog along with the pharmaceutically acceptable carrier.
The pharmaceutically acceptable carriers include adjuvants, excipients, surfactants, and the like that are commonly known and used by the person skilled in the art for the preparation of pharmaceutical compositions.
The pharmaceutical compositions may include various dosage forms including topical, injectable, and the like.
According to another embodiment of the present invention, a method of treating anemia using erythropoietic compound or a pharmaceutical composition containing the erythropoietic compound of the present invention is provided.
Compared to unmodified EPO (i.e., EPO without a PEG attached) and conventional PEG-EPO conjugates, the present PEG-N-EPO conjugate have an increased circulating half-life and plasma residence time, decreased clearance, and increased clinical activity in vivo. The conjugate of this invention have the same uses as EPO. In particular, the conjugate of this invention are useful to treat patients by stimulating the division and differentiation of committed erythroid progenitors in the bone marrow in the same way EPO is used to treat patients.
It has been found that the conjugate of this invention can be used in the same manner as unmodified EPO. However, the conjugate of this invention have an increased circulating half-life and plasma residence time, decreased clearance, and increased clinical activity in vivo.
Because of these improved properties, the conjugate of this invention can be administered once weekly instead of the three times weekly for unmodified EPO. Decreased frequency of administration is expected to result in improved patient compliance leading to improved treatment outcomes, as well as improved patient quality of life. Compared to conventional conjugates of EPO linked to poly(ethylene glycol). The term "N-terminal .alpha. -amino group" refers to the N-terminal amino residue of a peptide, i.e. that end of a peptide or protein chain having an amino acid with a free alpha-amino (NH. sub.2--) group.
Preferably, the glycoprotein of the conjugate as defined above is a human erythropoietin. Human erythropoietin and analogous proteins as defined above can be expressed by endogenous gene activation. Preferred human erythropoietin glycoproteins.
Methods for the expression and preparation of erythropoietin in serum free medium are described for example in WO 96/35718, to Burg published Nov. 14 1996, and in European Pat. Publication No. 513 738, to Koch published Jun. 12 1992. In addition to the publications mentioned above, it is known that a serum-free fermentation of recombinant CHO cells which contain an EPO gene can be carried out. Such methods are described for example in EP-A 0 513 738, EP-A 0 267 678 and in a general form by Kawamoto, T. et al., Analytical
Biochem. 130 (1983) 445-453, EP-A 0 248 656, Kowar, J. and Franek, F., Methods in Enzymology 421 (1986) 277-292, Bavister, B., Exp. Zoology 271 (1981) 45-51 , EP-A 0 481 791 , EP-A 0 307 247, EP-A 0 343 635, WO 88/00967.
Purification methods for erythropoietin and derivatives of EPO are also known in the art EP-A 0 267 678 discloses an ion exchange chromatography on S-Sepharose, a preparative reverse phase HPLC on a C. sub.8 column and a gel filtration chromatography for the purification of EPO produced in serum-free culture after dialysis. The gel filtration chromatography step can be replaced by ion exchange chromatography on S-Sepharose fast flow. It is also proposed that a dye chromatography on a Blue Trisacryl column be carried out before the ion exchange chromatography.
A process for the purification of recombinant EPO is also described by Nobuo, I. et al., J. Biochem. 107 (1990) 352-359. In the Nobuo process EPO is treated with a solution of Tween.RTM. 20, phenylmethylsulfonyl fluoride, ethylmaleimide, pepstatin A, copper sulfate and oxamic acid prior to the purification steps.
It is also known to prepare erythropoietin in a serum free fermentation process (EPOsf). See, e.g. WO 96/35718 (Burg), published November 1996.
The invention will be better understood by the following examples which illustrate but do not limit the invention described herein.
EXAMPLE 1
Preparation and Purification of N - Terminally Pegylated EPO
PEG-N-EPO is an N-terminally pegylated EPO produced by the following procedure. A 6 mg of EPO was dissolved in 20 mM phosphate buffer containing 150 mM NaCI with pH adjusted at 5.0. The EPO solution was then mixed with mPEG-propionaldehyde 2OK (M.W.: 20,000, manufactured by SunBio, Inc.). The amount of mPEG- propionaldehyde added was 7.5: 1 molar ratio of PEG: EPO. After addition of the PEG, the reaction mixture was stirred well to facilitate the complete dissolution of PEG powder into the EPO solution. After the PEG addition, 100 mM of sodium cyanoborohydride was added and mixed well. The reaction was allowed to continue for 18 hours. In the meanwhile the preparation of the chromatography process was done.
The purpose of chromatography purification is to obtain pure mono- PEG-EPO. HiTrap Q-sepharose chromatography column (Amersham Pharmacia) was packed and equilibrated with 10 mM Tris buffer of pH 8.0. Then the reaction mixture was loaded onto the column, which was then washed with 30 CV (column volume) of 10 mM Tris buffer of pH 8.0 to wash off all remaining free PEG that had not reacted with EPO.
Then the column was eluted with 40 CV of 25 mM NaCI in 10 mM Tris buffer of pH 8.0 to elute di-PEG-EPO (EPO with two PEGs attached to it). The next step was to elute with 40 CV of 65 mM NaCI in 10 mM Tris buffer of pH 8.0 to elute mono-PEG-EPO (EPO with one PEG attached to it). The next step was to elute with 12 CV of 200 mM NaCI in 10 mM
Tris buffer of pH 8.0 to elute native EPO, which was followed by 8 CV of 1 M NaCI in 10 mM Tris buffer of pH 8.0 to elute any remaining bound material. The collected mono-PEG-EPO fractions were then loaded onto Superdex 200 chromatography (Amersham Pharmacia) to further refine the purity of mono-PEG-EPO. The column was eluted with 20 mM phosphate buffer containing 150 mM NaCI of pH 7.0 to elute the pure mono-PEG-EPO. The collected fractions of mono-PEG-EPO was then concentrated to the final concentration of 65μg/ml in buffer of 20 mM phosphate, 150 mM NaCI and pH 7.0.
Similarly EPO conjugates can be prepared using mPEG-butyraldehyde and mPEG-pentaldehyde.
The lysine-52 Pegylated EPO was produced and purified as described in the US patent US 2002/0115833 A1.
The in-vivo efficacy and peptide mapping analysis of PEG-N-EPO has been done as shown in Fig. 2, 4 and 5. RESULTS
Results show that the PEG-N-EPO is purified on SDS-PAGE. For the comparison studies with the activities of Native EPO, and PEG-lys-EPO these proteins are also purified and confirmed with the SDS-PAGE as shown in Fig 1.
In vivo Bioassay studies shown that both PEG-N-EPO and PEG-lys - EPO, showed enhanced bioactivity compared to native EPO in terms of reticulocytes percentage in blood sample (Fig 2.). While the reticulocytes percentage of native EPO stayed higher than that of vehicle for 6 days, the reticulocyte percentage of PEG-EPO's stayed higher than that of vehicle for 8-13 days. Regarding the dosage, the 10 μg per rat groups performed better than 5 μg per rat groups. The difference also lies in the performance between the two PEG-EPO groups, in that PEG-N-EPO groups performed better than PEG-lys-EPO groups when compared between the same dosage groups.
The result for the peptide mapping Analysis of PEG-N-EPO shows the digestion of native EPO and PEG-N-EPO by lys-C enzyme (Fig. 4) yielded 9 peptide fragments. Of these, 5 peptide fragments, L1 (AA 1- 20), L3 (AA 46-52), L6 (AA 117-140), L7 (AA 141-152), and L9 (156- 166) were quantitatively obtained and analyzed (Fig 3). The peptide mapping analysis of PEG-N-EPO shows that the peptide fragment (L1) containing the N-terminal has been Pegylated.

Claims

1. A conjugate comprising an Erythropoietin glycoprotein having N- terminal α - amino group and aldehyde derivatives of methoxy polyethylene glycol (mPEG) said erythropoietin glycoprotein being selected from the group consisting of human erythropoietin and analogs thereof, said aldehyde derivative of methoxy polyethylene glycol (mPEG) of the formula
mPEG-R-CHO
wherein R= alkyl group of formula -(CH2)n-
wherein n= 2 to 4.
2. The conjugate as claimed in claim 1 having the formula
mPEG-R-CONH-EPO
wherein R=alkyl group of formula -(CH2Jn-
wherein n=2 to 4.
3. The conjugate as claimed in claim 1 wherein the methoxy polyethylene glycol (mPEG) is a mono or branched polyethylene glycol preferably monoPEG.
4. The conjugate as claimed in claim 1to 3 wherein the molecular weight of mPEG is about 20 to 60 kda.
5. The conjugate as claimed in claim 1 wherein the R is preferably ethyl, propyl or butyl.
6. The conjugate as claimed in claim 1 wherein said erythropoietin is a recombinant erythropoietin (rhEPO) produced in animal cells.
7. A process of making the conjugate of claim 1 comprising : (a) expressing, fermenting, and purifying rhEPO protein in recombinant animal cells.
(b) reacting EPO with mPEG-Aldehyde to obtain N-terminally pegylated EPO.
8. The process of claim 7 further comprises a step of purification of
PEG-N-EPO.
9. The process of claim 7 wherein step b is a reductive amination reaction using mPEG-Aldehyde.
10. The process as claimed in claim 7 wherein the fermentation step is serum free.
11. The process of claim 7 wherein in step b mPEG -Aldehyde added at an about molar ratio of ~ 10:2 preferably at 7.5:1 molar ratio of PEG: EPO.
12. The process of claim 7 wherein the aldehyde is preferably butanaldehyde, propionaldehyde or pentenaldehyde.
13. The process of claim 7 wherein the mPEG is mono or branched PEG.
14. A pharmaceutical composition comprising the conjugate of claim 1 and a pharmaceutically acceptable excipient.
15. A method of treating anemia in a patient afflicted with chronic renal failure (CRF) resulting from chemotherapy, comprising administering to said patient an effective amount of conjugate of claim 1.
PCT/IN2006/000103 2005-03-17 2006-03-16 N- terminal peg conjugate of erythropoietin Ceased WO2007010552A2 (en)

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Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5824784A (en) * 1994-10-12 1998-10-20 Amgen Inc. N-terminally chemically modified protein compositions and methods
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