MXPA00001208A - Chemical modification of proteins to improve biocompatibility and bioactivity - Google Patents

Abstract

The present invention broadly relates to chemical modification of biologically active proteins or analogs thereof. More spcecifically, the present invention describes novel methods for site-specific chemical modification of various proteins, and resultant compositions having improved biocompatibility and bioactivity.

Description

CHEMICAL MODIFICATION OF PROTEINS TO IMPROVE THEIR BIOCOMPATTBILITY AND BIOACTIVITY FIELD OF THE INVENTION The present invention relates broadly to the chemical modification of biologically active proteins or analogs thereof (the term "protein" as used herein is synonymous with "polypeptide" or "peptide" unless otherwise indicated). More specifically, the present invention describes new methods for site-specific chemical modifications of various proteins, and resulting compositions.

BACKGROUND OF THE INVENTION Due to recent advances in genetics and cellular engineering technologies, proteins known to exhibit various pharmacological actions in vivo are capable of production in large quantities for pharmaceutical applications. Such proteins include erythropoietin (EPO), granulocyte colony stimulation factor (G-CSF), interferons (alpha, beta, gamma, consensus), REF .: 32364 tumor necrosis factor binding protein (TNFbp), 1-interleukin receptor antagonist (IL-lra), brain-derived neurotrophic factor (BDNF), keratinocyte growth factor (KGF), cell factor of stem or strain (SCF), megakaryocyte growth differentiating factor (MGDF), osteoprotegerin (OPG), neurotrophic factor derived from cell lines (GDNF) and obesity protein (OB protein). The OB protein can also be referred to herein as leptin. The stimulation factor of the granulocyte colonies (G-CSF) is a glycoprotein which induces the differentiation of hemopoietic precursor cells to neutrophils, and stimulates the activity of mature neutrophils. The recombinant human G-CSF (rhG-CSF), expressed in E. coli, contains 175 amino acids, has a molecular weight of 18,798 Da, and is biologically active. Commonly, Filgrastim, a recombinant G-CSF, is available for therapeutic use. The structure of G-CSF under various conditions has been studied extensively; Lu et al., J. Biol. Chem. Vol. 267, 8770-8777 (1992), and the three-dimensional structure of rhG-CSF has been recently determined by x-ray crystallography. G-CSF is an element of a class of growth factors that divide a common structure motif from a beam of four a-helices with two common transverse connections; Hill et al., P.N.A.S. USA, Vol. 90, 5167-5171 (1993). This family includes GM-CSF, growth hormone, 2-interleukin, 4-interleukin, and interferon-β. The extension of the secondary structure is sensitive to the pH of the solvent, where the protein acquires an even higher degree of alpha helix content of acidic pH; Lu et al., Arch. Biochem. Biophys., 286, 81-92 (1989). Leptin is active in vivo in both ob / ob mutant mice (obese mice due to a defect in the production of the OB gene product) as well as in normal, wild-type mice. The biological activity manifests itself in, among other things, weight loss. See generally, Barinaga, "Obese" Protein Sli s Mice, Science 2j59: 475-476 (1995) and Friedman, "The Alphabet of Weight Control," Nature 385 ': 119-120 (1997). It is known, for example, that in ob / ob mutant mice, the administration of leptin results in a decrease in the levels of insulin in the blood and levels of glucose in the blood. It is known that the administration of leptin results in a decrease in body fat. This is observed in both ob / ob mutant mice, as well as in normal non-obese mice. Pelleymounter et al., Science 269: 540-543 (1995); Halaas et al., Science 269: 543-546 (1995). See also, Campfield et al., Science 269: 546-549 (1995) (peripheral and central dose administration in microgram of reduced feed supply with leptin and body weight of ob / ob mice and obese mice induced by diet but not in obese db / db mice.) In none of these reports have toxicities been observed, even at higher doses. Weight loss experiments induced by preliminary leptin in animal models predicts the need for a high concentration leptin formulation with chronic administration for the effective treatment of obesity in humans. Dosages in the milligram range of protein per kilogram of body weight, such as .5 or 1.0 mg / kg / day or lower, are desirable for the injection of therapeutically effective amounts in larger mammals, such as humans. An increase in protein concentration is therefore necessary to avoid the injection of large volumes, which can be uncomfortable or possible damage to the patient. Unfortunately, for the preparation of a pharmaceutical composition for injection in humans, it has been observed that the amino acid sequence of leptin is insoluble at physiological pH at relatively high concentrations, such as above about 2 mg of active protein / milliliter of liquid. The poor solubility of leptin under physiological conditions appears to contribute to the formation of leptin precipitates at the injection site in a concentration-dependent manner when high dosages are administered in a formulation at low pH. An inflammatory response is associated with the leptin precipitates observed at the injection site when an infiltrate of mixed cells is included characterized by the presence of eosinophilic cells, macrophages and giant cells. To date, there are no reports of stable preparations of human OB protein at concentrations of at least about 2 mg / ml at physiological pH, and in addition, there are no reports of stable concentrations of active human OB protein at least about 50. mg / ml or higher. The development of leptin forms which could be allowed for high dosages without the aforementioned problems, could be of great benefit. Therefore, it is an object of the present invention to provide improved forms of leptin by means of chemical modifications of specific site of the protein. There are several methods of chemical modification of useful therapeutic proteins which have been reported. One such method, succinylation, involves the conjugation of one or more portions of succinyl to a biologically active protein. Classic approaches to succinylation traditionally employ alkaline reaction conditions with very large excesses of succinic anhydride. The resulting succinyl protein conjugates are typically modified at multiple sites, frequently show altered tertiary and quaternary structures, and are occasionally inactivated. The properties of several succinylated proteins are described in Holcenberg et al., J. Biol. Chem, 250: 4165-4170 (1975), and WO 88/01511 (and references cited here), published on March 10, 1988. Importantly, none of the references cited describe methods wherein the biologically active protein is exclusively monosuccinylated at the N-terminus of the protein, and wherein the resulting composition exhibits improved solubility and improved injection site toxicities. Diethylenetriaminepentaacetic acid anhydride (DTPA = and ethylene diaminetetraacetic acid dianhydride (later referred to as EDTA) has been used classically to introduce metal chelation sites into proteins for the purpose of radiolabelling.Similar to succinylation, modification with DTPA and / or EDTA typically occurs at multiple sites throughout the molecule and changes the charge and isoelectric point of the modified protein.To date, there are no reports of monomers of the DTPA and / or EDTA protein and dimers that exhibit improved solubility and site toxicities. improved injection.

DESCRIPTION OF THE INVENTION The present invention relates to substantially homogeneous preparations of chemically modified proteins, for example, leptin and G-CSF, and methods for this. Unexpectedly, chemical modification of the specific leptin site demonstrates advantages in bioavailability and biocompatibility that are not seen in other leptin species. Importantly, the methods described herein are broadly applicable to other proteins (or analogs thereof), as well as leptin. Accordingly, as described below in more detail, the present invention has a number of aspects that relate to chemically modifying proteins (or analogs thereof) as well as specific modifications of specific proteins. In one aspect, the present invention relates to a substantially homogeneous preparation of mono-succinylated leptin (or analogs thereof) and relates to methods. Importantly, the method described results in a high yield of monosuccinylated protein which is modified exclusively at the N-terminus, thereby providing processing advantages when compared to other species. And, in spite of the modest N-terminal modification, the monosubstituted succinyl leptin unexpectedly demonstrates: 1) a substantial improvement in solubility; 2) preservation of secondary structure, in vitro receptor binding activity and bioefficacy in vivo; and 3) improvement of the various reactions at the injection site observed with administration of high concentrations of unmodified leptin. In another aspect, the present invention relates to a substantially homogeneous preparation of mono-succinylated G-CSF, and an analog thereof, and related methods. Importantly, monosubstituted succinyl-G-CSF and monosubstituted succinyl-G-CSF analogue unexpectedly demonstrate a substantial improvement in solubility, physical stability at 4 ° C and 37 ° C, and preservation of bioactivity in vitro. In another aspect, the present invention relates to substantially homogeneous preparations of DTPA-leptin monomers and dimers and related methods. When reacted with leptin at neutral pH and a low stoichiometric excess of DTPArprotein, this reagent unexpectedly forms a unique bond between the N-terminals of two leptin molecules in high yield.

When the monosubstituted DTPA-leptin monomer and dimer are isolated, both show substantially increased solubilities relative to the unmodified protein. Both forms also demonstrate preservation of in vitro receptor binding activity and bioefficacy in vivo. Significantly, the dimeric form of monosubstituted DTPA leptin does not precipitate when injected at a high concentration in PBS and shows strong improvement in injection site reactions over those observed with unmodified leptin. In still another aspect, the present invention relates to substantially homogeneous preparations of monomers and dimers of EDTA (EDTA2) -leptin dianhydride and related methods. Similar to DTPA in the structure, EDTA reticles leptin efficiently through N-terminal when it allows to react at neutral pH in a substoichiometric excess. The isolated EDTA2-leptin dimers demonstrate dramatically improved solubility relative to unmodified leptin and maintains full in vitro receptor binding activity and bioactivity in vivo. In addition, the EDTA-leptin conjugate is not precipitated at the injection site when dosed at high concentration in PBS and demonstrated substantial improvement in the adverse injection site reactions observed with unmodified leptin.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a chromatogram of a separation separation chromatography of succinylated leptin anions. The absorbance at 280 nm is plotted against the elution volume in mL. The monosuccinylated leptin peak is marked by (*).

Figure 2 is an IEF-PAGE gel at pH 3-7 demonstrating unmodified leptin (line 2), succinylated leptin (line 3), DTPA-modified leptin dimer (line 4) and EDTA2-modified leptin dimer (line 5) The lines or routes 1 and 6 are isoelectric point markers.

Figure 3 is a chromatogram of separation by size exclusion chromatography of leptin dimer and monomer cross-linked by DTPA. The absorbance at 280 nm is plotted against the volume of elution in mL. The dimeric form of monosubstituted DTPA-leptin is marked by (*).

Figure 4 is a 4-20% SDS-PAGE gel showing the unmodified leptin (lane 2), succinylated leptin (lane 3), DTPA modified leptin dimer (lane 4) and EDTA-modified leptin dimer (route 5) Routes 1 and 6 are molecular weight markers.

Figure 5 is a chromatogram of a separation by size exclusion chromatography of the monomer and leptin dimer crosslinked by EDTA2. The absorbance at 280 nm is plotted against the volume of elution in mL. The dimeric form of monosubstituted EDTA2-leptin is marked by (*).

Figure 6 is a reversed-phase HPLC chromatogram of retention time shifts showing the digests of Lys-C resulting in chemical modifications of the N-terminal peptide (M1-K6) by succinic anhydride.

Figure 7 is a reverse phase HPLC chromatogram of retention time shifts showing the digests of Lys-C resulting from chemical modifications of the N-terminal peptide (M1-K6) by DTPA.

Figure 8 is a reversed-phase HPLC chromatogram of retention time shifts showing the digests of Lys-C resulting from chemical modifications of the N-terminal peptide (M1-K6) by EDTA2.

Figure 9 shows the Far-UV spectra of unmodified native leptin and monosuccinylated leptin. Both samples are at 0.25 mg / mL in saline buffered with phosphate at room temperature.

Figure 10 is a graph showing the in vitro receptor binding of unmodified leptin (- * -), succinylated leptin (-B-), DTPA-leptin dimer (-A-) or EDTA2-leptin dimer (- • -) by displacement of radiolabeled human leptin from the immobilized human leptin receptor. The ligand concentration (ng / mL) is broken against the% binding or binding of the ligand.

Figure 11 is a graph showing weight loss in mice that have been treated with unmodified leptin (- * -), succinylated leptin (-U-), DTPA-leptin (-A-) dimer or DTPA monomer -leptin (-x-). Mice were dosed daily at 10 mg / kg delivered at 2 mg / mL in PBS. The time (days) is plotted against% of weight loss.

Figure 12 is a graph showing weight loss in mice that have been treated with either 20 mg / mL of unmodified leptin (-A-), 2 mg / mL of unmodified leptin (- * -), 20 mg / mL of EDTA2-leptin dimer (-H-) or 2 mg / mL of EDTA-leptin dimer (-x-). Mice were dosed daily at 100 mg / kg delivered at 20 mg / mE or 10 mg / kg delivered at 2 mg / mL in PBS (unmodified leptin dosed at 100 mg / kg and 20 mg / mL was formulated at pH 4.0, acetate buffer due to its poor solubility in PBS). Time (days) is plotted against% loss in weight.

Figure 13 is a graph showing in vitro bioactivity of unmodified G-CSF (- * -) and succinylated G-CSF (-B-). CPM-BGND is plotted against performance (ng / cavity).

Figure 14 is a graph showing the results of the physical stability test of unmodified G-CSF at 4 ° C (- * -), succinylated G-CSF at 4 ° C (-1-), G-CSF not modified at 37 ° C (-A-), and succinylated G-CSF at 37 ° C (- • -). The protein concentration (mg / mL) is plotted against time (hours).

Figure 15 is a graph showing in vitro bioactivity of unmodified G-CSF (- * -), unmodified G-CSF (C17A) (- «-) and G-CSF-succinylated (C17A) (-A-) . CPM-BGND is plotted against performance (ng / cavity).

DETAILED DESCRIPTION The present invention relates to substantially homogeneous preparations of chemically modified proteins, and methods thereof. "Substantially homogeneous" as used herein means that the only chemically modified proteins observed are those that have a "modifying" portion (eg, DTPA, EDTA, succinyl). The preparation may contain unreacted protein (i.e., missing or deficient modifier portion). When determined by peptide planimetry and N-terminal sequencing, a subsequent example provided for a preparation which is at least 90% modified protein, and at most 10% unmodified protein. Preferably, the chemically modified material is at least 95% of the preparation (as in the working example below) and more preferably, the chemically modified material is 99% of the preparation or more. The chemically modified material has biological activity. The preparations of monosuccinylated leptin, leptin DTPA, and leptin EDTA2"substantially homogeneous", present, provided herein are those which are sufficiently homogeneous to exhibit the advantages of a homogeneous preparation, for example, ease in clinical application in the quality to predict of pharmacokinetics from batch to batch. As used herein, biologically active agents refer to recombinants or proteins that are naturally present, whether human or animal, useful for prophylaxis, therapeutic application or diagnosis. The biologically active agent can be natural, synthetic, semi-synthetic or derivatives thereof. In addition, the biologically active agents of the present invention may be perceptible. A wide range of biologically active agents are contemplated. This includes but is not limited to hormones, cytokines, hematopoietic factors, growth factors, anti-obesity factors, trophic factors, anti-inflammatory factors, and enzymes (see also U.S. Patent No. 4,695,463 for additional examples of useful biologically active agents). One skilled in the art will be able to easily adapt a desired biologically active agent to the compositions of the present invention. Such proteins may include but are not limited to interferons (see, U.S. Patent Nos. 5,372,808, 5,541,293 4,897,471, and 4,695,623 for this are incorporated by reference including drawings), interleukins (see, U.S. Patent No. 5,075,222, whereby they are incorporated as reference including drawings), erythropoietins (see, U.S. Patent Nos. 4,703,008, 5,441,868, 5,618,698, 5,547,933, and 5,621,080, this is incorporated by reference including the drawings), stimulation factors of the granulocyte colony (see, US Patent Nos. 4,810,643 , 4,999,291, 5,581,476, 5,582,823, and PCT Publication No. 94/17185, whereby it is incorporated by reference including drawings), Stem Cell or Stem Cell Factor (PCT Publication Nos. 91/05795, 92/17505 and 95 / 17206, incorporated by reference including drawings), and leptin (OB protein) (see PCT Publications Nos. 96/40912, 96/05309, 97/00128, 97/01010 and 97/06816 incorpo prayed here for reference including figures). PCT Publication No. WO 96/05309, published February 22, 1996, entitled, "Modulators of Body Weight, Corresponding Nucleic Acids and Proteins, and Diagnostic and Therapeutic Uses Thereof" fully describe the OB protein and related methods and compositions, and Incorporates here as a reference. An amino acid sequence for human OB protein is described in WO 96/05309 Seq. ID Nos. 4 and 6 (on pages 172 and 174 of this publication), and the first amino acid residue of the mature protein is in position 22 and it is a residue of valine. The mature protein is 146 residues (or 145 if the glutamine at position 49 is absent, Sec. ID No. 4). In general, the G-CSF useful in the practice of this invention may be an isolated form of mammalian organisms or, alternatively, a product of synthetic chemical procedures or expression of the prokaryotic or eukaryotic host of exogenous DNA sequences obtained by genomic or cloning of cDNA or by DNA synthesis. Suitable prokaryotic hosts include several bacteria (e.g., E. coli); Suitable eukaryotic hosts include yeast (e.g., S. cerevisiae) and mammalian cells (e.g., Chinese hamster ovary cells, monkey cells). Depending on the host employed, the expression product of G-CSF can be glycosylated with mammals or other eukaryotic carbohydrates, or it can be non-glycosylated. The product of the G-CSF expression may also include an initial methionine amino acid residue (in the -1 position). the present invention contemplates the use of any and all forms of G-CSF, although recombinant G-CSF, especially derived E. coli, is preferred, for, among other things, greater commercial viability. Certain analogs of G-CSF have been reported to be biologically functional, and these can also be chemically modified. G-CSF analogs are reported in U.S. Patent No. 4,810,643. Examples of other G-CSF analogues that have been reported to have biological activity are those described in AU-76380/91, EP 0 459 630, EP 0 272 703, EP 0 473 268 and EP 0 335 423, although the representation is made with respect to the activity of each analog described as reported. See also AU-A-10948/92, PCT US94 / 00913 and EP 0 243 153. Generally, the G-CSFs and analogs thereof useful in the present invention can be ascertained by practicing chemical modification procedures as described herein and testing the product results in the desired biological characteristic, such as the biological activity assays provided herein. Of course, if one wishes when treating non-human mammals, one can use recombinant non-human G-CSFs, such as murine, bovine, canine, etc., recombinants. See PCT WO 9105798 and PCT WO 8910932, for example. In addition, biologically active agents may also be included but not limited to insulin, gastrin, prolactin, adrenocorticotropic hormone (ACTH), hormone for thyroid stimulation (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), gonadotropin human chorionic (HCG), motilin, interferons (alpha, beta, gamma), interleukins (IL-1 to IL-12), tumor necrosis factor (TNF), tumor necrosis factor binding protein (TNF-bp) ), brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), fibroblast growth factors (FGF), neurotrophic growth factor (NGF), bone growth factors such as osteoprotegerin (OPG), insulin-like growth factors (IGFs), macrophage colony stimulation factor (M-CSF), granulocyte macrophage colony stimulation factor (GM-CSF), growth factor megakaryocyte derivative (MGDF), keratinocyte growth factor (KGF), thrombopoietin, platelet-derived growth factor (PGDF), colony simulation growth factors (CSFs), bone morphogenetic protein (BMP), dismutase of superoxide (SOD), plasminogen activator. of tissue (TPA), urokinase, streptokinase and kallikrein. The term "proteins", as used herein, includes peptides, polypeptides, consensus molecules, analogs, derivatives or combinations thereof. In general, pharmaceutical compositions comprising effective amounts of chemically modified proteins, or derivative products, together with diluent, preservatives, solubilizers, emulsifiers, adjuvants and / or pharmaceutically acceptable carriers necessary for administration are included by the invention. (See PCT 97/01331 incorporated by reference.) The optimal pharmaceutical formulation for a desired biologically active agent will be determined by one skilled in the art depending on the route or route of administration and dosage desired. Exemplary pharmaceutical compositions are described in Remington's Pharmaceutical Sciences (Marck Publishing Co., 18th Ed., Easton, PA, pp. 1435-1712 (1990)). The pharmaceutical compositions of the present invention can be administered by oral and non-oral preparations (e.g., intramuscular, subcutaneous, transdermal, visceral, IV (intravenous), IP (intraperitoneal), intraarticular, placement in the ear or ear, ICV (intracerebralventricular), IP (intraperitoneal), intraarterial, intrathecal, intracapsular, intraorbital, injectable, pulmonary, nasal, rectal, and uterine-transmucosal preparations).

Therapeutic uses of the compositions of the present invention depend on the biologically active agent used. One skilled in the art will be able to easily adapt a desired biologically active agent to the present invention for its intended therapeutic uses. Therapeutic uses for agents are described in greater detail in the following publications incorporated herein by reference including drawings. Therapeutic uses include but are not limited to uses for interferon-like proteins (see, U.S. Patent Nos. 5,372,808, 5,541,293, incorporated herein by reference including drawings), interleukins (see, U.S. Patent No. 5,075,222 incorporated by reference including drawings), erythropoietins (see, U.S. Patent Nos. 4,703,008, 5,441,868, 5,618,698, 5,547,933, and 5,621,080 incorporated herein by reference including drawings), granulocyte colony stimulation factors (see, U.S. Patent Nos. 4,999,291, 5,581,476, 5,582,823, 4,810,643, and Publication of PCT No. 94/17185, incorporated by reference including drawings), stem or stem cell factor (PCT Publication Nos. 91/05795, 92/17505 and 95/17206, incorporated by reference including drawings), and the OB protein ( see PCT publication nos. 96/40912, 96/05309, 97/00128, 97/01010 and 97/06816 incorporated by reference including figures). In addition, the present compositions can also be used to produce one or more medicaments for the treatment or improvement of the conditions it is intended to treat the biologically active agent. The main modality of the method for treating the substantially homogenous preparation of monoinylated protein comprises: (a) reacting a protein with 3-7 times the molar excess of inic anhydride; (b) stirring the reaction mixture 2-16 hours at 4 ° C; (c) dialyzing the mixture against 20mM Tris-HCl, pH 7.2; Y (d) Isolate the monoinylated protein. Optionally, the method may comprise, until after step (b), the steps of: adding solid hydroxylamine to the mixture while maintaining a pH above 6.5 until the hydroxylamine is completely dissolved, followed by raising the pH to 8.5 , using 5N NaOH, followed by stirring the mixture for another 1-2 hours at 4 ° C. The general process is shown schematically in Example 1. The main modality of the method for producing the substantially homogeneous preparation of DTPA protein comprises: (a) reacting a protein with 1-5 times the molar excess of DTPA; (b) stirring the reaction mixture 2-16 hours at 4 ° C; (c) dialyzing the mixture against 20mM Tris-HCl, pH 7.2; and (d) isolate the DTPA protein. The general process is shown schematically in Example 1. The main modality of the method for producing the substantially homogeneous preparation of EDTA protein comprises: (a) reacting a protein with 0.5-5 times the molar excess of EDTA2; (b) stirring the reaction mixture 2-16 hours at 4 ° C; (c) filtering the reaction mixture; and (d) concentrating the reaction mixture; and (e) isolating the EDTA protein. The general process is shown schematically in Example 1. The following examples are offered to fully illustrate the invention, but without being constructed as limiting the scope thereof. Example 1 describes the preparation monomers and dimers of monoinylated leptin, monosubstituted DTPA leptin, and EDTA2 leptin monomers and dimers. Example 2 describes the physiochemical characterization of the modified leptin species prepared in Example 1. Example 3 describes the receptor binding studies performed on the modified leptin species prepared in Example 1. Example 4 describes the solubility test performed in the modified leptin species prepared in Example 1. Example 5 describes the in vivo bioactivity studies performed on the modified leptin species prepared in Example 1. Example 6 describes the evaluation of the injection site performed on the species of modified leptin prepared in Example 1. Example 7 describes the preparation of monosuccinylated G-CSF and monosuccinylated G-CSF analog (C17A) and then describes the results of the in vitro bioactivity test, solubility test test, and physical stability test for the preparations.

EXAMPLE 1 This example describes the preparation of monosuccinylated leptin, monomers and monomers of monosubstituted DTPA leptin monomers and dimers, and leptin EDTA2 monomers and dimers. 1. Monosuccinilated leptin The succinylation method of the protein of the present invention can generally be shown as follows: Protein - (SjHj + - Recombinant human methionyl leptin protein (rhu-met-leptin) prepared as described in Materials and Methods, infra) at 2-3 mg / mL in 20 mM NaHP0, pH 7.0, are reacted with 3-7 times of molar excess of solid succinic anhydride (Sigma Chemical, St. Louis, MO), with a preferred 5-fold molar excess, and the reaction is stirred 2-16 hours at 4 ° C. The solid hydroxylamine (Sigma Chemical, St. Louis, MO) is then added to the reaction while maintaining the pH above 6.5. After the hydroxylamine has completely dissolved the pH rises to 8.5 using NaOH N and the reaction is allowed to stir another 1-2 hours at 4 ° C (The hydroxylamine step can be omitted with a small decrease in performance). Finally, the reaction is dialyzed against 20mM Tris-HCl, pH 7.2. Monosuccinylated rhu-met-leptin is isolated by anion exchange chromatography with a High Performance Sepharose Q column (Pharmacia, Piscataway, NJ) in 20 mM Tris, pH 7.2, with a gradient of 0-0.5M NaCl (see Figure 1) . The product is recognized in the eluent by an isoelectric shift of -0.7 Pl units observed with isoelectric focusing (IEF) PAGE using 5% polyacrylamide, gel at pH 3-7 (Novex, Inc., San Diego CA) (Figure 2 ). The final recovery of monosuccinylated rhu-met-leptin is typically 45-47%. 2. Monomers and dimers of monosubstituted DTPA leptin The method of modifying DTPA of the present invention can generally be shown as follows: Recombinant human methionyl leptin protein (rhu-met-leptin) (prepared as described in Materials and Methods, infra) at 2-3 mg / mL in 20mM NaHP04, pH 7.0, is reacted with a molar excess of 1- 5 times of solid DTPA (Sigma Chemical, St. Louis, MO), with 2-3 times the preferred molar excess, and the reaction is stirred 2-16 hours at 4 ° C. Finally, the reaction is dialyzed against 20mM Tris-HCl, pH 7.2. DTPA modified rhu-met-leptin is isolated by anion exchange chromatography with a High Performance Sepharose Q column (Pharmacia, Piscataway, NJ) in 20mM Tris, pH 7.2, with a NaCl gradient of 0-0.5M. Alternatively, the monomeric and dimeric forms of rhu-metleptin monosubstituted DTPA or rhu-met-leptin are separated by size exclusion chromatography on a Sephacryl 100 column (Pharmacia, Piscataway, NJ) in PBS (Life Technologies, Grand Island , NY) (see Figure 3). The products are recognized in the eluent by an isoelectric shift observed with monomeric DTPA leptin by isoelectric focusing (IEF) PAGE using 5% polyacrylamide, gel at pH 3-7 (Novex, Inc., San Diego CA) (Figure 2 ) or the increased mass of a crosslinked dimer observed with SDS-PAGE using a 4-20% polyacrylamide gel (Novex, Inc., San Diego CA) (see Figure 4). The final recovery of the DTPA-rhu-met-leptin dimer is approximately 30%. 3. Monomers and dimers of monosubstituted EDTA leptin The EDTA modification method of the present invention can generally be shown as follows: Dimero Recombinant human methionyl leptin protein (rhu-met-leptin) (prepared as described in Materials and Methods, infra) at 2-3 mg / mL in 20mM NaHP04, pH 7.0, is reacted with a molar excess of 0.5- 5 times of EDTA2 (Aldrich Chemical Co., Milwaukee, Wl) either as a solid or dissolved in DMSO, with 0.75 times the molar excess of EDTA in preferred DMSO, and the reaction is stirred 2-16 hours at 4 ° C . The reaction is then filtered through a 0.45 micron filter (Nalgene), concentrated by agitated cells on lOkDa molecular weight cut-off membrane at ~ 20 mg / mL and the monomeric and dimeric forms of rhu-met-leptin of EDTA2. monosubstituted then separated by size exclusion chromatography on a Sephacryl 100 column (Pharmacia, Piscataway, NJ) balanced in PBS (see Figure 5). Alternatively, the reaction can be purified by hydrophobic interaction chromatography using a High Performance Phenyl Sepharose column (Pharmacia, Piscataway, NJ) eluted with a gradient of 0.8-0M ammonium sulfate in 20 mM NaHP04, pH 7.0. The products are recognized in the eluent by an isoelectric shift observed with the rhu-met-leptin of monomeric EDTA2 by isoelectric focusing (IEF) PAGE using a 5% polyacrylamide, gel at pH 3-7 (Novex, Inc., San Diego CA) (Figure 2) or the mass increase of a crosslinked dimer observed with SAD-PAGE using a 4-20% polyacrylamide gel (Novex, Inc., San Diego CA) (Figure 4). The final recovery of the rhu-met-leptin dimer of EDTA2 exceeds 50%.

EXAMPLE 2 This example describes the physiochemical characterization of the leptin conjugates prepared in Example 1. The modification of succinyl-leptin, monomers and dimers of DTPA-leptin, and EDTA2-leptin monomers and dimers were evaluated by a combination of peptide planimetry of digesta of Lys-C in the reverse phase HPLC, mass spectrography of MALDI-TOF and peptide sequencing. The digests of Lys-C of unmodified leptin and several modified leptins were made by the reaction of 100 μg of protein with 4 μg of endoproteins Lys-C (Boehringer Mannheim) in 50 mM Tris-HCl, pH 8.5 (200 μl) for four hours at room temperature. Peptide maps of several samples were generated by reverse phase HPLC on a 5 μC4 column of 4.6 x 250 mm (Vydak, Hesperia, CA) balanced in 0.1% trifluoroacetic acid (TFA) with elution on a gradient of acetonitrile from 0. 90% (see Figures 6-8). As evidenced by the traces shown in Figures 6-8, only the N-terminal peptide (M1-K6) shows any change in retention time as a result of chemical modification. These results indicate that the lysine at position 6 is not modified and accessible to the digestion of Lys-C and suggests that chemical modification occurs at the N-terminal α-amine. The N-terminal modification is further supported by N-terminal sequencing efforts which indicate that the N-terminus is blocked (data not shown). Mass determinations for succinyl leptin and dimers of DTPA and EDTA2 are made on a Kompact Maldi IV (Kratos, Ramsey, NJ) using a 12 pmol sample in a sinapinic acid matrix. Each conjugate indicates a single chemical modification per molecule.

Table 1 Expected Mass More Linker Mass Measure Conjugate (Da) (Da) (Da) Leptin not modified 16, 157 0 16,156 Succinyl-leptin 16, 258 101 16,254 Dimer of DTPA-leptin 32, 671 357 32,705 Dimer of EDTA2-leptin 32, 570 256 32,509 In addition to the previous analysis, the effects on the secondary structure of succinyl-leptin were evaluated using circular dichroism spectroscopy. The Far-UV circular dichroism spectrum of unmodified and succinylated leptin in phosphate buffered saline was collected using a 0. 05 cm cell in a Jasco J-170 circular dichroism spectrophotometer (Jasco, Tokyo, Japan). The spectrum is shown in Figure '9 and demonstrates that the secondary structure of succinylated leptin is preserved.

In sum, the data of Example 2 confirm the modification of succinyl-leptin, monomers and dimers of DTPA-leptin, and monomers and dimers of EDTA-leptin in the N-terminus, as well as the preservation of secondary structure with succinyl-leptin .

EXAMPLE 3 This example describes the receptor binding studies in each of the leptin conjugates prepared in Example 1. Each of the leptin conjugates prepared in Example 1 is evaluated using an in vitro receptor binding assay which measures the relative affinity of leptin conjugates based on their ability to displace the radiolabeled human leptin from a human leptin receptor expressed in the immobilized cell membranes. As evidenced by the data in Figure 10, the chemically modified isoforms, succinyl-, DTPA-, and EDTA2-leptin each show relative affinities for human leptin receptors equal to unmodified leptin over the full range of ligand binding ( -1-100 ng / mL), with ED50 's of approximately 10 ng / mL.

The data of Example 3 thus show that monosubstituted succinyl leptin, monosubstituted DTPA-leptin dimer, and EDTA2-leptin dimer demonstrate preservation of receptor binding activity in vitro when compared to unmodified leptin.

EXAMPLE 4 This example describes the solubility test performed on each of the leptin conjugates prepared in Example 1. The leptin conjugates are dialysed in PBS and then concentrated with CentriPrep concentrators., cutting the molecular weight of lOkDa (A ico) to the point that the precipitate is observed. The sample was clarified by centrifugation and the protein concentration of the conjugate in the determined supernatant. The samples were then kept at room temperature ("22 ° C) for 48 hours and at regular centrifuged time points and the protein concentration of the conjugate in the supernatant • that is determined again. The solubility of the conjugate protein in PBS is defined as the protein concentration of the stable state at room temperature observed in the supernatant after centrifugation (see Table 2).

Table 2 Sample Maximum Solubility in PBS (mg / ml) Unmodified leptin 3.2 Succinyl leptin 8.4 DTPA-leptin 31.6 EDTA2-leptin 59.9 The data in Table 2 show that monosubstituted succinyl leptin, monosubstituted DTPA-leptin, and monosubstituted EDTA2-leptin have substantially improved solubility when compared to unmodified leptin, with monosubstituted EDTA2-leptin showing dramatically improved solubility.

EXAMPLE 5 This example describes the in vivo bioactivity studies performed on the leptin conjugates prepared in Example 1. The described leptin conjugates were tested in animal models both mice and dogs to determine the bioefficacy relative to unmodified leptin. The mice were injected daily for 5-7 days with monosubstituted succinyl leptin, DTPA-leptin dimer, DTPA-leptin monomer and EDTA-leptin dimers in dosages of 1, 10 and 50 mg / kg body weight. The bioefficacy was measured as a percentage of weight loss from day 0, normalized for vehicle control alone and compared to the weight loss observed with the unmodified protein. All samples for dosages of 1 and 10 mg / kg were formulated in PBS at 0.2 mg / ml respectively. Higher dosages were formulated in PBS at 20-50 mg / ml for chemically modified forms, however, the solubility limits of unmodified leptin need their formulation at high concentrations in an acetate buffer at pH 4. In addition, dogs they were injected with 0.05, 0.15 and 0.5 mg / kg daily dosages of succinyl-leptin at 5 mg / ml for 28 days while weight loss was observed followed by a recovery period. Bioactivity, when judged by drug-induced weight loss in animal models, for succinyl leptin was equivalent to unmodified leptin in both dogs and mice (Figure 11). Similarly, both the DTPA-leptin monomers and dimers and EDTA2-leptin dimers cause equivalent weight losses in mice when compared to unmodified leptin (Figures 11 and 12). The data of Figures 11 and 12 show that monosubstituted succinyl leptin, monosubstituted DTPA-leptin monomers and dimers, and EDTA2-leptin dimer demonstrate bioefficacy preservation in vivo when compared to unmodified leptin.

EXAMPLE 6 This example describes the evaluation of the injection site performed on the leptin conjugates prepared in Example 1. The tissue sections of the injection sites of three mice of each dosage group were examined histochemically. The pathologies of the injection site which were identified and recorded were necrosis, suppurative (infiltrate of mixed cells composed of sphinophils and neutrophils), mononuclear cells (macrophages), leptin precipitates (characterized as fine ppt or deposits / large groupings) and giant cells. Each reaction was recorded using the following gradient system: 0. 5 Minimum change 1.5 2 light change 2.5 3 Moderate change 3.5 4 Change marked 4.5 5 Massive change The averaged sum of the records for each animal was used to define a total biocompatibility record using the following registry key: 0 -. 0 - 2 Normal 3 - 5 Minimum 6 - 10 Light 11 - 20 Moderate 21 - 30 Marking > 30 Severe Although high concentrations of succinyl-leptin were marginally soluble in PBS at pH 7.0, for the purpose of injection site testing, samples of succinyl-leptin at 20 mg / ml of soluble remaining in PBS at pH 7.2 and at 50 mg / ml in PBS at pH 7.5. Table 3 shows the evaluation of the injection site compared to unmodified leptin at 50 mg / mL delivered in acetate buffer at pH 4.0 against monosubstituted succinyl leptin at 50 mg / mL in pH 7.5, PBS, after 7 days.

Table 3 Mono Volume Dose. Prcip. Deposit Treatment mg / Jcg piL Nßcr. Fine sup. Grand * G ga te Cells Amount absorber 0 20 0 0.5 1 0 0 1 of acetate 0 20 0 0 0.5 0 0 0 0 20 0 0.5 1 0 0 0 Leptin no 50 20 0 3 2 1 4 1 modified 50 20 0 2.5 2 1 4 2.5 50 20 0 1.5 2 0 1.5 1 Shock absorber 0 20 0 0.5 0.5 0 0 0 of PBS 0 20 0 0.5 0.5 0 0 0 0 20 0 0 0 0 0 0 Succ-Leptin 50 20 0 1 1.5 0 0 0 50 20 0 2 1 0 0.5 0.5 50 20 0 1.5 0.5 0 0 0 As shown in Table 3, monosubstituted succinyl leptin, at high concentration dosages, shows improvement in each category of injection site pathology in relation to unmodified leptin, with the most dramatic improvement observed with almost elimination. Complete of leptin precipitates and giant cells at the injection sites. Table 4 shows the evaluation of the injection site comparing unmodified leptin with 43 mg / mL delivered in acetate buffer at pH 4.0 against monosubstituted succinyl leptin at 43 mg / mL in pH 4.0, acetate buffer, after 7 days .

Table 4 Dosage Volume Pracip. Mark- Treatment mg / kg s_I Necr. Delete Mono. Biocomp. Reaction Shock absorber 0 20 0 1 1 0 2 Normal acetate 0 20 0 0 0 0 2 Normal 0 20 0 0 0.5 0 2 Normal Leptin no 43 20 2 4 3 3 27 Marking ULOQlIlCaQa 43 20 1 3 3 2 27 Marking 43 20 1. 5 3. 5 2.5 3 27 Marking Succ-leptin 43 20 0. 5 2 1.5 0 10 Light 43 20 0. 5 1. 5 1.5 0 10 Light 43 20 0 1 1 0 10 Light The data in Table 4 show that, surprisingly, it was also observed that high concentrations of monosubstituted succinyl leptin could be supplied at pH 4, acetate buffer and still demonstrates the dramatic improvements in injection site reactions observed when succinyl- monosubstituted leptin in PBS. Table 5 shows the evaluation of the injection site compared to unmodified leptin at 20 mg / mL delivered in acetate buffer at pH 4.0, against monosubstituted DTPA-leptin dimer at 20 mg / mL in PBS, after 7 days.

Table 5 Dosage Volume Prcip. Brand - Treatment mg / kg mi. Naox. Delete Mono. Biocomp. Reaction Shock absorber 0 80 0 0.25 1 0 3 Minimum of acetate 0 80 0 0 0.5 0 1 Normal 0 80 0.25 0.25 1 0 4 Minimal Leptin no 20 80 0 2.5 2.5 2 16 Moderate modified 20 80 0.25 3.5 3 2 22 Marked 20 80 0.25 3 3 2.5 21 Marked Shock absorber 0 80 0 0 0 0 0 Normal of PBS 0 80 0 0 0.5 0 1 Normal 0 80 0 0 0 0 0 Normal Dimero of 20 80 0.25 1.5 2 0 11 Moderate DTPA-leptin 20 80 0 1 1.5 0 7 Light 20 80 0 1.5 2 - 0 10 Light Table 6 shows the evaluation of the injection site comparing unmodified leptin at 30 mg / mL delivered in acetate buffer at pH 4.0 against EDTA2-leptin monosubstituted dimer at 20 mg / mL in PBS, after 7 days.

Table 6 Dose Volume Precip. Marca¬ Treatment mg / kst Nßcr. Delete Mono. Bioconp. Reaction Shock absorber 0 100 0 0.25 1 0 3 Minimum of acetate 0 100 0 0. 5 1 0 4 Minimum 0 100 0 0.5 1 0 4 Minimum Leptin not 100 100 0. 5 2 3 3 18 Moderate modified 100 100 0 2 3 3 18 Moderate 100 100 0 2 2. 5 2 14 Moderate Shock absorber 0 100 0 0 0. 5 0 1 Normal of PBS 0 100 0 0.25 0.25 0 1 Normal 0 100 0 0.25 0.25 0 1 Normal Dimer of 100 100 0 1. 5 2 0 9 Light EDTA-leptin 100 100 0 1. 5 1. 5 0 8 Light 100 100 0. 5 2. 5 3 0 16 Moderate As shown in Tables 5 and 6, the DTPA-leptin dimers (Table 5) or EDTA2-leptin dimers (Table 6) can be administered to mice at high concentrations in PBS demonstrating the same improvement in site pathology. injection as observed with succinyl-leptin. However, these conjugates are substantially more soluble in pH 7, PBS and therefore provide a coarser or resistant formulation in this buffer. In sum, the data of Example 6 show that monosubstituted succinyl leptin, monosubstituted DTPA-leptin monomers and dimers, and EDTA2-leptin monomers and dimers are not precipitated at the injection site when dosed at high concentrations, and Importantly, they demonstrate substantial improvement in adverse injection site reactions observed with unmodified leptin.

Example 7 This example describes the preparation of monosuccinylated G-CSF and monosuccinylated G-CSF analog (C17A) and then describes the results of the in vitro bioactivity test, solubility test test and physical stability test for G-CSF preparations. . The recombinant human methionyl-G-CSF protein (rhu-met-G-CSF) and G-CSF analogue (C17A) (prepared as described in Materials and Methods, infra) at 2-3 mg / L in 20mM NaHP04 , pH 7.0, is reacted with 3-7 times the molar excess of succinic anhydride (Sigma Chemical, St.

Louis, MO), with a preferred molar excess of 5 times, and the reaction was stirred 2-16 hours at 4 ° C. Solid hydroxylamine . { Sigma Chemical, St. Louis, MO) was then added to the reaction while maintaining the pH above 6.5. After the hydroxylamine was completely dissolved the pH was raised to 8.5 using 5N NaOH and the reaction was allowed to stir another 1-2 hours at 4 ° C (the hydroxylamine step can be omitted with a small decrease in yield). Finally, the reaction was dialyzed against 20 mM Tris-HCl, pH 7.2.

Monosuccinylated rhu-met-G-CSF (and the analog) is isolated by anion exchange chromatography with a High Performance Sepharose Q column (Pharmacia, Piscataway, NJ) in 20 mM Tris, pH 7.2, with a NaCl gradient of 0 -0.5M. The product is recognized in the eluent by an isoelectric shift of -0.7 pl units observed with isoelectric focusing PAGE (IEF) using a 5% polyacrylamide gel, pH 3-7 (Novex, Inc., San Diego CA). Finally, the recovery of monosuccinylated rhu-met-G-CSF (and the analogue) is typically 45-47%. Samples of unmodified G-CSF and succinylated rhu-met-G-CSF were tested in an in vitro bioassay in which the proliferation of G-CSF-dependent murine hematopoietic progenitor cells is measured as a function of recovery of radiolabeled thymidine and concentration of G-CSF. The total preservation of bioactivity is demonstrated (see Figures 13 and 15). The solubility of succinylated isoforms was compared to unmodified rhu-met-G-CSF and rhu-met-G-CSF analogue. The G-CSF samples were dialyzed in PBS and then concentrated with CentriPrep concentrators, lOkDa molecular weight cutoff (Amicon) to the point where precipitates are observed. The sample was clarified by centrifugation and the concentration of conjugated protein in the determined supernatant. The samples were then kept at 37 ° C for 22 hours and at points of regular centrifuged periods and the protein concentration of the conjugate in the supernatant was determined again. The solubility of the protein in PBS is thus defined as the concentration of stable state protein at room temperature observed in the supernatant after centrifugation (see Table 7).

Table 7 Sample Maximum Solubility in PBS (mg / ml) Unmodified G-CSF 0.7 Succinyl-G-CSF 8.2 Unmodified G-CSF (C17A) 13.8 Succinyl-G-CSF (C17A) 19.2 The physical stability of unmodified G-CSF and G-CSF (C17A) and succinylates were compared by centrifugation of the samples at 5-6 mg / mL in PBS and the samples were maintained at 4 ° C and 37 ° C. The amount of protein remaining in solution was determined by measuring the concentration spectrophotometrically after centrifugation of the samples. The physical stability (at 4 ° C and 37 ° C) of the succinylated G-CSF is substantially improved relative to unmodified G-CSF (see Figure 14).

Materials and methods 1. Preparation of human recombinant methionyl leptin protein The present recombinant human methionyl leptin (rhu-met-leptin) can be prepared in accordance with the PCT publication incorporated by reference above, WO 96/05309 on pages 151-159. For the present working examples, a rhu-met-leptin was used which was compared to the amino acid sequence on page 158) a lysine at position 35 instead of an arginine, and an isoleucine at position 74 of a isoleucine Other recombinant human leptin proteins can be prepared according to methods generally known in the art of protein expression using recombinant DNA technology Preparation of recombinant human methionyl-G-CSF protein and G-CSF analog (C17A).

The present recombinant human methionyl leptin (rhu-met-G-CSF) can be prepared in accordance with PCT publication WO 94/17185 incorporated by reference above. For the present working examples, an analog of rhu-met-G-CSF, G-CSF (C17A), was also used which has an alanine at position 35 instead of a cysteine. Other recombinant human G-CSF proteins can be prepared according to methods generally known in the art of protein expression using recombinant DNA technology. While the present invention has been described in terms of certain preferred embodiments, it is to be understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all equivalent variations that come within the scope of the invention as claimed.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property

Claims (24)

1. A substantially homogenous preparation of a monosuccinylated protein, characterized in that the monosuccinylated protein is modified exclusively at the N-terminus.

2. A substantially homogeneous preparation, characterized by a protein- (EDTA) dianhydride monomer of ethylenediaminetetraacetic acid.

3. A substantially homogeneous preparation, characterized by a dimer of EDTA protein- (EDTA2) dianhydride.

4. A substantially homogeneous preparation, characterized by a protein-monomer (DTPA) diethylenetriaminepentaacetic acid anhydride

5. A substantially homogeneous preparation, characterized by a protein-DTPA dimer.

6. The substantially homogeneous preparation according to any of claims 1-5, characterized in that the protein is leptin, or an analogue thereof.

7. A monosuccinylated protein produced by a process, characterized in that it comprises the steps of: (a) reacting a protein with 3-7 fold molar excess of succinic anhydride to form a reaction mixture; (b) stirring the reaction mixture 2-16 hours at 4 ° C; (c) dialyzing the reaction mixture against 20mM Tris-HCl, pH 7.2; and (d) isolating the monosuccinylated protein from the reaction mixture, wherein the monosuccinylated protein is modified exclusively at the N-terminus.

8. The monosuccinylated protein according to claim 7, characterized in that the protein is leptin, or an analogue thereof.

9. The monosuccinylated protein according to claim 7, characterized in that the protein is G-CSF 10., or an analogue thereof.

10. A DTPA protein produced by a process characterized in that it comprises the steps of: (a) reacting a protein with 1-5 times molar excess of DTPA to form a reaction mixture; (b) stirring the reaction mixture 2-16 hours at 4 ° C; (c) dialyzing the reaction mixture against 20 mM Tris-HCl, pH 7.2; and (d) isolating the DTPA-protein from the reaction mixture.

11. The DTPA protein according to claim 10, characterized in that the protein is leptin, or an analogue thereof.

12. An EDTA protein produced by a process characterized in that it comprises the steps of: (a) reacting a protein with 0.5-5 times the molar excess of EDTA to form a reaction mixture; (b) stirring the reaction mixture 2-16 hours at 4 ° C; (c) filtering the reaction mixture; (d) concentrating the reaction mixture; and (e) isolating the EDTA protein from the reaction mixture.

13. The EDTA protein according to claim 12, characterized in that the protein is leptin, or an analogue thereof.

14. A method of producing a substantially homogeneous preparation of a monosuccinylated protein characterized in that it comprises the steps of: (a) reacting a protein with 3-7 times the molar excess of succinic anhydride to form a reaction mixture; (b) stirring the reaction mixture 2-16 hours at 4 ° C; (c) raising the pH of the reaction mixture to 8.5 using 5N NaOH; (d) stirring the reaction mixture another 1-2 hours at 4 ° C; (e) dialyzing the reaction mixture against 20mM Tris-HCl, pH 7.2; and (f) isolating the monosuccinylated protein from the reaction mixture.

15. The method according to claim 14, characterized in that it further comprises, until after step (b), the steps of: 1) adding solid hydroxylamine to the mixture while maintaining the pH above 6.5 until the hydroxylamine is completely dissolved; 2) raise the pH to 8.5 using 5N NaOH; and 3) stir the mixture another 1-2 hours at 4 ° C.

16. A method of producing a substantially homogeneous preparation of a DTPA protein characterized in that it comprises the steps of: reacting a protein with 1-5 times the molar excess of DTPA to form a reaction mixture; (b) stirring the reaction mixture 2-16 hours at 4 ° C; (c) dialyzing the reaction mixture against 20mM Tris-HCl, pH 7.2; and (d) isolating the DTPA protein from the reaction mixture.

17. A method for producing a substantially homogeneous preparation of an EDTA2 protein characterized in that it comprises the steps of: (a) reacting a protein with 0.5-5 times the molar excess of 2 EDTA to form a reaction mixture; (b) stirring the reaction mixture 2-16 hours at 4 ° C; (c) filtering the reaction mixture; (d) concentrating the reaction mixture; and (e) isolating the EDTA protein from the reaction mixture.

18. A pharmaceutical composition characterized in that it comprises a monosuccinylated protein.

19. A pharmaceutical composition characterized in that it comprises a monomer of EDTA protein- (EDTA) dianhydride.

20. A pharmaceutical composition characterized in that it comprises an EDTA (EDTA) dianhydride dimer.

21. A pharmaceutical composition characterized in that it comprises a DTPA protein monomer.

22. A pharmaceutical composition characterized in that it comprises a DTPA protein dimer.

23. A pharmaceutical composition according to any of claims 18-22, characterized in that the protein is leptin, or an analogue thereof.

24. A pharmaceutical composition according to any of claims 18-22, characterized in that the protein is G-CSF, or an analogue thereof.