HK1176009A - Liquid formulations for long-acting erythropoietin conjugate - Google Patents

Description

Liquid formulations of long acting erythropoietin conjugates

Technical Field

The present invention relates to a liquid formulation for ensuring long-term storage stability of long-acting erythropoietin conjugates, in which erythropoietin, a non-peptide polymer and an immunoglobulin Fc fragment are covalently linked and which exhibits an extended duration of action compared with the wild type.

Background

Erythropoietin (EPO) is a glycoprotein consisting of 165 amino acid residues which acts as a cytokine for erythrocyte precursors in the bone marrow and is thus responsible for controlling erythropoiesis (erythrocyte production). EPO is synthesized primarily by kidney cells and produced in small amounts by the liver. In chronic renal failure, it can be seen that loss of kidney function is often accompanied by a decrease in, for example, EPO levels, and concomitantly a decrease in the production of erythrocytes. EPO is now used for the treatment of anemia arising from chronic kidney disease and other serious conditions and is administered to patients who are prepared for surgery (Mijake et al, J.biol.chem.25: 5558-5564, 1977; Eschbach et al, New Engl.J.Med.316: 73-78, 1987; Sanford B.K, Blood, 177: 419-434, 1991; PCT WO 85-02610).

Human urinary EPO was first purified by Miyake et al from patients with aplastic anemia (Miyake et al, j.biol.chem., 252: 5558, 1977), but the amount of EPO from this source was insufficient for the treatment of anemia. Since the identification and cloning of the human EPO gene and the expression of recombinant EPO protein have been disclosed in the publication of U.S. Pat. No.4,703,008, the mass production of EPO has been accomplished by a variety of different genetic manipulations.

Since polypeptides are easily denatured due to their low stability, degraded by proteolytic enzymes in blood, and easily passed through the kidney or liver, it is necessary to frequently administer protein drugs (including polypeptides as pharmaceutically effective ingredients) to patients in order to maintain desired blood level concentrations and titers. However, frequent administration of such protein drugs causes pain to the patient, particularly in the case of administration by injection.

To solve these problems, many efforts have been made to improve the serum stability of protein drugs and to maintain the drugs in the blood at high levels for a longer period of time, thereby maximizing the drug efficacy of the drugs. For use in long acting formulations, protein drugs must be formulated to be highly stable and their potency maintained at a sufficiently high level without eliciting an immune response in the patient.

To stabilize proteins and prevent enzymatic degradation and clearance by the kidneys, polymers with high solubility, such as polyethylene glycol (PEG), are commonly used to chemically modify the protein drug surface. PEG stabilizes the protein and prevents hydrolysis by binding to a specific region or regions of the target protein without causing serious side effects (Sada et al, J.fermentation Bioengineering 71: 137-139, 1991). However, although pegylation can enhance protein stability, it also has problems, such as greatly reducing the potency of a physiologically active protein. In addition, the yield decreases with increasing PEG molecular weight due to decreased protein reactivity.

An alternative method for improving the in vivo stability of a physiologically active protein is to link a gene of a physiologically active protein to a gene encoding a protein having high serum stability by a gene recombination technique, and culture cells transfected with the recombinant gene to produce a fusion protein. For example, fusion proteins can be prepared by: albumin, a protein known to be most effective in enhancing protein stability, or a fragment thereof is conjugated to a physiologically active protein of interest by gene recombination (PCT publication nos. WO 93/15199 and WO 93/15200, european patent publication No.413,622).

Another approach is the use of immunoglobulins. Human growth hormone was conjugated to bovine serum albumin or mouse immunoglobulin by using a cross-linking agent as described in U.S. patent No.5,045,312. The conjugates have enhanced activity compared to unmodified growth hormone. Carbodiimide or glutaraldehyde is used as a cross-linking agent. However, such low molecular weight cross-linking agents that bind non-specifically to peptides do not allow the formation of homogeneous conjugates and are even toxic in vivo. Furthermore, the patent indicates that the increase in activity is due solely to chemical coupling with growth hormone. The method of this patent does not guarantee enhancement of the activity of various kinds of polypeptide drugs, and thus the patent does not even recognize factors related to the stability of proteins, such as duration, half-life in blood, and the like.

One recently proposed pharmaceutical formulation is a long-acting protein pharmaceutical formulation with improved in vivo duration and stability. For use in long-acting pharmaceutical formulations, protein conjugates were prepared by covalently linking physiologically active polypeptides, non-polypeptide polymers and immunoglobulin Fc fragments (korean patent nos. 10-0567902 and 10-0725315).

In this method, EPO can be used as a physiologically active polypeptide to provide long-acting EPO conjugates. In order to apply long-acting EPO conjugates to pharmaceutical products, it is necessary to maintain their efficacy in vivo while inhibiting physicochemical changes such as denaturation, aggregation, adsorption or hydrolysis caused by light, heat or additives during storage and transportation. Long-acting EPO conjugates are more difficult to stabilize than the EPO polypeptide itself due to their increased volume and molecular weight.

Generally, proteins have a very short half-life and when exposed to inappropriate temperatures, water-air interfaces, high pressures, physical/mechanical stresses, organic solvents, microbial contamination, etc., denaturation occurs, such as monomer aggregation, aggregate precipitation, and adsorption onto container surfaces. After denaturation, proteins lose their physicochemical properties and physiological activity. Once denatured, the protein can hardly recover its original properties, because denaturation is irreversible. Particularly in the case where the proteins (such as EPO) to be administered are in trace amounts of several hundred micrograms per injection, the losses occurring when they lose stability and are therefore adsorbed onto the container surface are relatively large. In addition, the adsorbed protein is easily aggregated during denaturation, and the aggregate of denatured protein acts as an antigen when administered into the body (unlike the in vivo synthetic protein). Therefore, the protein must be administered in a stable form. Many studies have been performed to prevent denaturation of proteins in solution (John Geigert, J.Parenteral Sci. Tech., 43 (5): 220- & 1989; David Wong, pharm. Tech., 34-48, 1997; Wei Wang., int.J.Pharm., 185: 129- & 188, 1999; Willem Norde, adv.Colloid Interface Sci., 25: 267- & 340, 1986; Michelle et al, int.J.Pharm.120: 179- & 188, 1995).

Lyophilization was applied to some protein drugs to achieve stability goals. However, lyophilized products have the inconvenience that they must be re-dissolved in water for injection for use. In addition, since the production process thereof includes a lyophilization process, a large investment is required on a large-capacity lyophilizer. It has been proposed to break down proteins by using a spray dryer. However, this method is uneconomical due to low yield. In addition, the spray drying process exposes the protein to high temperatures, thus negatively affecting protein stability.

As an alternative to overcoming this limitation, stabilizers have emerged, which, when added to solutions of proteins, can inhibit physicochemical changes of protein drugs and maintain in vivo efficacy even after long-term storage. The stabilizer includes saccharide, amino acid, protein, surfactant, polymer and salt. In particular, human serum albumin has been widely used as a stabilizer for a variety of protein drugs and its performance has been demonstrated (Edward Tarelli et al, Biologicals, 26: 331-346, 1998).

Typical purification processes for human serum albumin include inactivation of biological contaminants, such as mycoplasmas, prions, bacteria and viruses, or screening or examining one or more biological contaminants or pathogens. However, there is always a risk that the patient is in contact with biological contaminants because they are not completely removed or inactivated. For example, human blood from donors is screened to see if it contains certain viruses. However, this process is not always reliable. In particular, certain viruses present in very small numbers cannot be detected.

Recently, alternatives to human serum albumin have been proposed, including recombinant albumin (Korean patent laid-open No.10-2004-0111351) and erythropoietin without albumin (Korean patent laid-open Nos. 10-0560697 and 10-0596610).

Even with albumin-free stabilizers, different proteins can be gradually inactivated due to their chemical differences, as they have different ratios and conditions during storage. The effect of stabilizers on protein shelf life varies from protein to protein. That is, different stabilizers may be used in different ratios according to the physicochemical properties of the protein of interest.

In addition, when different stabilizers are used simultaneously, adverse effects may be caused due to their competition and mishandling. Combinations of different stabilizers also cause different effects, as they cause the properties or concentration of the protein to change during storage. Since each stabilizer suitably exerts its stabilizing effect in a specific concentration range, many efforts have to be made to carefully combine the kinds and concentrations of the different stabilizers.

In particular, for long-acting EPO conjugates with improved in vivo duration and stability, their molecular weight and volume are considerably different from those of the ordinary erythropoietin complex because they are composed of physiologically active peptides EPO, non-peptide polymers and immunoglobulin fragments Fc. Thus, specific stabilizers different from the EPO stabilizer composition are needed for long-acting EPO conjugates.

The present invention has been made through extensive and intensive studies to develop a stable liquid formulation of a long-acting EPO conjugate, which can maintain drug efficacy for a long period of time without viral infection, resulting in the finding that a stabilizer comprising a buffer of a specific pH range and mannitol at a high concentration imparts enhanced stability to the long-acting EPO conjugate and allows the formation of an economical and stable long-acting EPO conjugate liquid formulation.

Disclosure of Invention

Technical problem

It is therefore an object of the present invention to provide a liquid formulation comprising a long-acting erythropoietin conjugate in which EPO, a non-peptide polymer and an immunoglobulin Fc fragment are covalently linked, and a stabilizer free of albumin, wherein the stabilizer consists of a buffer and mannitol.

Solution to the problem

According to one embodiment thereof, the present invention provides a liquid formulation comprising a long-acting erythropoietin conjugate in which EPO, a non-peptide polymer and an immunoglobulin Fc fragment are covalently linked, and a stabilizer that is free of albumin, wherein the stabilizer comprises a buffer and mannitol.

The term "long-acting erythropoietin conjugate" or "long-acting EPO conjugate" as used herein means a protein construct in which physiologically active EPO, one or more non-peptide polymers and one or more immunoglobulin Fc fragments are covalently linked and which has an extended duration of action compared to its native form of EPO.

The term "long acting" as used herein means an extended duration of action compared to the native form. The term "conjugate" means a construct in which EPO, a non-peptidic polymer and an immunoglobulin Fc fragment are covalently linked.

For use in the present invention, EPO has the amino acid sequence of human erythropoietin or a closely related analog. The EPO that can be used in the present invention can be a natural protein or a recombinant protein. Furthermore, EPO can be a mutant with inserted, deleted or inserted amino acids, provided that the mutation has no significant effect on its original biological activity.

Human EPO or analogs thereof useful in the present invention can be isolated from vertebrates or can be chemically synthesized. Alternatively, EPO or an analog thereof can be obtained from a prokaryote or eukaryote transformed with a gene encoding EPO or an analog thereof using genetic recombination techniques. For this, enterobacteria such as e.coli, yeast cells such as brewers yeast (s.cerevisiae), or mammalian cells such as chinese hamster ovary cells, monkey cells may be used as host cells. Depending on the host cell, the recombinant EPO or analog thereof may be glycosylated with mammalian or eukaryotic sugars or may be non-glycosylated. Upon expression, the recombinant EPO or analog thereof may contain an initial methionine residue (position 1). Preferably, recombinant human epo (huepo) is produced using CHO cells as a host.

For use in the present invention, an immunoglobulin Fc fragment has the amino acid sequence of a human immunoglobulin Fc fragment or a closely related analog thereof. Fc fragments can be obtained from native forms isolated from animals including cows, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs. Furthermore, the immunoglobulin Fc fragment may be an Fc fragment from IgG, IgA, IgD, IgE and IgM, or made from combinations or hybrids thereof. Preferably, it is derived from the protein IgG or IgM that is most abundant in human blood, and most preferably from IgG that is known to enhance the half-life of ligand binding proteins. Herein, the immunoglobulin Fc may be obtained from a natural immunoglobulin by isolating intact immunoglobulins of human or animal organisms and treating them with proteolytic enzymes, or it may be a recombinant or derivative thereof obtained from transformed animal cells or microorganisms. Preferred is recombinant human immunoglobulin Fc produced by e.

On the other hand, IgG is classified into IgG1, IgG2, IgG3 and IgG4 subtypes, and the present invention includes combinations and hybrids thereof. Preferred are the IgG2 and IgG4 subtypes, most preferred is the Fc fragment of IgG4 with little effector function, such as CDC (complement dependent cytotoxicity). That is, the most preferred immunoglobulin Fc fragment as a drug carrier of the present invention is the non-glycosylated Fc fragment of human IgG 4. Human-derived Fc fragments are preferred over non-human-derived Fc fragments, which can act as antigens and elicit undesirable immune responses in humans, such as the production of new antibodies against the antigens.

Long-acting EPO conjugates useful in the invention are prepared by linking EPO to an immunoglobulin Fc fragment. In this regard, EPO and the immunoglobulin Fc fragment may be crosslinked by a non-peptide polymer, or a fusion protein may be formed using recombinant techniques.

The non-peptidic polymer used for cross-linking may be selected from the group consisting of biodegradable polymers, lipid polymers, chitin, hyaluronic acid, and combinations thereof. The biodegradable polymer may be selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran (dextran), polyvinyl ethyl ether (polyvinyl ethyl ether), PLA (polylactic acid), PLGA (polylactic-glycolic acid), and combinations thereof. Most preferred is poly (ethylene glycol) (PEG), preferably polyethylene glycol. Derivatives thereof well known in the art and readily preparable by those skilled in the art are also included within the scope of the present invention.

The long-acting EPO conjugate used in the present invention can be prepared using genetic engineering techniques, as disclosed in Korean patent No. 10-0725315.

The liquid formulation according to the invention comprises a therapeutically effective amount of a long-acting EPO conjugate. Typically, a therapeutically effective amount of EPO is 2,000 to 10,000 International Units (IU) per disposable bottle. The concentration of the long-acting EPO conjugate used in the present invention is about 1 to 5000. mu.g/ml, preferably about 50 to 3000. mu.g/ml.

The term "stabilizer" as used herein means a substance that allows for the safe storage of long-acting EPO conjugates. The term "stable" means that the loss of active ingredient over a certain period of time under storage conditions is not more than a predetermined proportion (usually up to 10%). EPO retains 90% or more of its original activity (preferably 95% or more of its original activity) when stored at 10 ℃ for 2 years, at 25 ℃ for 6 months, or after one to two weeks at 40 ℃, is understood to be stable. For proteins such as EPO, the storage stability is important to inhibit the possible production of EPO-like antigenic substance and to ensure accurate administration. During storage, unless the EPO in the formulation aggregates or breaks to form antigenic material, a loss of about 10% of EPO activity is understood to be tolerated by administration.

The stabilizers used in the present invention comprise a buffer solution formulated to impart stability to the long-acting EPO conjugate and mannitol.

Furthermore, the stabilizer of the present invention preferably does not contain albumin. Human serum albumin, which is prepared from human blood and is used as a protein stabilizer, has a possibility of being contaminated with pathogenic viruses of human origin. Gelatin or bovine serum albumin can cause disease or cause allergic reactions in some patients. The stabilizer of the present invention has no problem of virus infection because it does not contain serum albumin or heterologous protein (such as purified gelatin) derived from human or animal.

Mannitol, a sugar alcohol, is used in the stabilizers of the present invention because it acts to enhance the stability of long-acting EPO conjugates. Mannitol is used at a concentration of preferably 1 to 20% (w/v), more preferably 3 to 12% (w/v) and most preferably 5 to 10% (w/v) based on the total volume of the liquid formulation.

According to one embodiment of the present invention, when mannitol is used as a stabilizer in the presence of a phosphate buffer solution, the storage stability of the long-acting EPO conjugate shows a great increase compared to when a conventional stabilizer comprising sorbitol, maltose, PEG400 and amino acids is used (see table 1). When applied to the present invention, it was found that maltose used as a stabilizer in Japanese patent laid-open publication No.2009-249292 decreased the stability of long-acting EPO conjugates over an extended storage period (see Table 8).

These data reveal the specificity of mannitol as a stabilizer for long-acting EPO conjugates compared to other stabilizers, indicating that different stabilizers are required depending on the target for which stabilization is desired.

The buffer solution in the stabilizer plays a role in maintaining the pH of the liquid formulation constant to prevent pH fluctuation, thereby stabilizing the long-acting EPO conjugate. The buffer solutions useful in the present invention may comprise pharmaceutically acceptable pH buffering agents including basic salts (sodium or potassium phosphates, hydrogen or dihydrogen salts thereof), sodium citrate/citric acid, sodium acetate/acetic acid, and combinations thereof. Suitable for use herein are citrate buffers, phosphate buffers, tartrate buffers, carbonate buffers, succinate buffers and acetate buffers, preferably phosphate buffers and citrate buffers, more preferably phosphate buffers. In the phosphate buffer, the concentration of phosphate is preferably in the range of 5 to 100mM, more preferably 10 to 50 mM. The pH of the buffer is preferably 4.0 to 8.0, more preferably 5.0 to 7.0.

In another embodiment of the present invention, the stabilizer useful in the present invention may further comprise at least one ingredient selected from the group consisting of isotonic agents (isotonics agents), polyols, sugars, nonionic surfactants and neutral amino acids, in addition to the buffer solution and mannitol.

The isotonic agent not only plays a role in maintaining proper osmotic pressure when the long-acting EPO conjugate in the liquid preparation enters into the body, but also plays a role in further stabilizing the long-acting EPO conjugate in the liquid preparation. Examples of isotonic agents include water-soluble inorganic salts. Which include sodium chloride, sodium sulfate, sodium citrate, calcium chloride, and combinations thereof. Most preferred is sodium chloride.

Preferably, the concentration of the isotonic agent is about 5 to 200 mM. Within this range, the concentration of the isotonic agent may be adjusted according to the kind and amount of the contained component so that the liquid preparation is isotonic.

According to one embodiment of the invention, the effect of different kinds of salts on the stability of long-acting EPO conjugates was evaluated in the presence of buffer solutions. As a result, liquid formulations comprising sodium sulfate, sodium chloride, sodium citrate, or a combination of sodium sulfate and sodium chloride along with phosphate buffer solutions were found to increase the stability of long-acting EPO conjugates compared to liquid formulations without salt (see table 2). As can be understood from these data, the long-acting EPO conjugates of the present invention are stabilized to varying degrees depending on the kind of salt used, and some salts exhibit peak stability.

Preferred examples of sugars that may also be included to increase the storage stability of the long-acting EPO conjugate include monosaccharides (such as mannose, glucose, fructose, and xylose) and polysaccharides (such as lactose, maltose, sucrose, raffinose, and dextran). In the liquid formulation, the sugar is preferably used in an amount of 1 to 20% (w/v) and more preferably in an amount of 5 to 20% (w/v). Examples of polyols useful in the present invention include propylene glycol, low molecular weight polyethylene glycols, glycerin, and low molecular weight polypropylene glycols. They may be used alone or in combination. And their concentration in the liquid formulation is preferably about 1 to 15% (w/v), more preferably about 5 to 15% (w/v).

For nonionic surfactants, they reduce the surface tension of the protein solution to prevent the protein from being adsorbed or aggregated on hydrophobic surfaces. Polysorbate (polysorbate) based nonionic surfactants and poloxamer (poloxamer) based nonionic surfactants are suitable for use in the present invention. They may be used alone or in combination. Polysorbate-based nonionic surfactants are preferred. Among these are polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80, with polysorbate 80 being more preferred.

The use of high concentrations of nonionic surfactants is not recommended because if the nonionic surfactants are present at high concentrations, UV spectroscopy or isoelectric focusing methods are disturbed, making it difficult to accurately assess the concentration or stability of the protein. Accordingly, the liquid formulation of the present invention may comprise a nonionic surfactant at a preferred concentration of 0.1% (w/v) or less, and more preferably 0.01 to 0.05% (w/v).

In one embodiment of the invention, the effect of nonionic surfactants on protein stability in the presence of phosphate buffers was analyzed. In storage periods as short as one week, the non-ionic surfactant was found to have little effect on the stability of the long-acting EPO conjugates (see table 3). Furthermore, when polysorbate 80, a non-ionic surfactant, was used, it was found that a stabilizer comprising 0.01% (w/v) polysorbate 80 more stabilized the long-acting EPO conjugate during storage than a stabilizer comprising 0.1% polysorbate 80 (see table 4).

Amino acids also act as stabilizers for liquid formulations, which in solution act to attract more water molecules to the EPO environment, such that the EPO outermost hydrophilic amino acid molecule is further stabilized (Wang, Iht. J. Pharm. 185: 129-188, 1999). In this regard, the charged amino acids may cause electrostatic attraction with EPO to promote EPO aggregation. Therefore, neutral amino acids (e.g., glycine, alanine, leucine, and isoleucine) are added as stabilizing components. In liquid formulations, the neutral amino acid is preferably used at a concentration of 0.1 to 10% (w/v).

In one embodiment of the invention, maltose ensures a higher storage stability of the long-acting EPO conjugate when used in combination with glycine than when used alone. However, it was observed that treatment with mannitol at concentrations as high as 3 to 12% (w/v) provided higher stability even in the absence of neutral amino acids than treatment with maltose in combination with glycine (see table 10).

Thus, a liquid formulation for providing a long-acting EPO conjugate of high stability can be prepared using mannitol at a high concentration even when a neutral amino acid is not added. However, mannitol concentrations in excess of 20% (w/v) exceed the upper isotonic limit. Thus, mannitol is used in a liquid formulation at a concentration of 1 to 20%, preferably at a concentration of 3 to 12% (w/v), more preferably at a concentration of 5 to 10% (w/v).

In addition to the above-mentioned components including a buffer, an isotonic agent, a sugar alcohol, a neutral amino acid and a non-ionic surfactant, the liquid formulation of the present invention may optionally contain other components known in the art as long as they do not reduce the effect of the present invention.

According to a preferred embodiment of the present invention, the liquid formulation does not comprise albumin and may comprise a buffer solution, mannitol, an isotonic agent and a non-ionic surfactant.

More specifically, the present invention provides a liquid formulation comprising a long-acting EPO conjugate and a stabilizer comprising a phosphate or citrate buffer, mannitol, an isotonic agent selected from the group consisting of sodium chloride, sodium sulfate, sodium citrate, and combinations thereof, and polysorbate 80. Preferably, the liquid formulation comprises phosphate or citrate buffer solution at a concentration of 5 to 100mM, mannitol at a concentration of 1 to 20% (w/v), an isotonic agent at a concentration of 5 to 200mM, and polysorbate 80 at a concentration of 0.001 to 0.05% (w/v), wherein the isotonic agent is selected from sodium chloride, sodium sulfate and sodium citrate. More preferably, the liquid formulation comprises a phosphate buffer solution at a concentration of 5 to 100mM, mannitol at a concentration of 3 to 12% (w/v), sodium chloride at a concentration of 100 to 200mM, and polysorbate 80 at a concentration of 0.001 to 0.05% (w/v). Most preferably, the liquid formulation comprises sodium citrate buffer at a concentration of 10mM (pH6.5), mannitol at a concentration of 5 to 10% (w/v), sodium at a concentration of 100 to 200mM, and polysorbate 80 at a concentration of 0.001 to 0.05% (w/v), and does not comprise neutral amino acids.

In one embodiment of the invention, the EPO storage stability of a long-acting EPO conjugate liquid formulation comprising sodium phosphate buffer solution (pH6.5) at a concentration of 10mM, mannitol at a concentration of 5 to 10% (w/v), sodium chloride at a concentration of 100 to 200mM, and polysorbate 80 at a concentration of 0.001% to 0.05% (w/v) is compared to the known EPO formulation recormon (roche). EPO was found to be more stable in the liquid formulations of the invention than in the commercial formulations (see tables 6 and 14).

The EPO liquid formulations of the present invention were also compared with the EPO storage stability of other formulations such as Ananesp (an anemia treating agent produced by Amgen), Enbrel (TNFR-Fc) (a rheumatoid arthritis treating agent produced by Amgen), and PBS alone. As a result, the long-acting EPO conjugate liquid formulation of the present invention showed higher stability than any other liquid formulation (see table 17).

In another embodiment, the long-term stability of the long-acting EPO conjugate liquid formulations of the present invention was determined and found to stabilize the long-acting EPO conjugate for 12 months and ensure at least 92.5% activity even after 6 months of storage under accelerated conditions (see tables 19 to 21).

It will be appreciated from this data that liquid formulations comprising a buffer and mannitol at a concentration of 1 to 20% (w/v) can stably store long-acting EPO conjugates therein for 12 months or more even in the absence of neutral amino acids.

Advantageous effects of the invention

Since it does not contain human serum albumin and other potential factors harmful to the body, the long-acting EPO conjugate liquid formulation in which EPO is linked to an immunoglobulin Fc fragment and has a larger molecular weight and a longer duration of action than EPO in its native form does not have the problem of viral infection and ensures excellent storage stability of the long-acting EPO conjugate. The liquid formulation of the present invention can provide excellent storage stability of EPO even when no neutral amino acid is contained, thereby being economically more advantageous than other stabilizers and lyophilizates.

Drawings

FIG. 1 is a graph showing the stability of EPO in different long-acting EPO conjugate liquid formulations and in a commercially available EPO formulation, Recormon, when analyzed by reverse phase chromatography weekly over a period of four weeks of storage at 40 ℃.

Figure 2 is a graph showing the stability of long-acting EPO conjugates in a liquid formulation comprising phosphate buffer at pH6.5, sodium chloride, mannitol, and polysorbate 80 when analyzed by reverse phase chromatography and size exclusion chromatography every two months for a storage period of 12 months at 4 ℃.

Figure 3 is a graph showing the stability of long-acting EPO conjugates in a liquid formulation comprising phosphate buffer at pH6.5, sodium chloride, mannitol, and polysorbate 80 when analyzed in an in vitro assay every two months for a period of 12 months of storage at 4 ℃.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

The invention will be better understood by reference to the following examples which are set forth to illustrate, but are not to be construed as limiting the invention.

Example 1: construction of Long-acting EPO conjugates

<1-1> preparation of immunoglobulin Fc fragment Using immunoglobulin

The immunoglobulin Fc fragment that can be used in the present invention is a human non-glycosylated IgG4Fc fragment, which can be expressed from an e.coli transformant as described in korean patent No. 725314.

<1-2> preparation of recombinant human erythropoietin

Human EPO used in this example was obtained as disclosed in Korean patent No. 880509. For this, an animal cell line transfected with a vector capable of greatly enhancing gene amplification efficiency by artificially weakening the dihydrofolate reductase gene promoter, which is a transcription control sequence of the gene, is cultured to express the human EPO protein. Only highly glycosylated proteins are purified for use.

<1-3> preparation of long-acting EPO conjugates Using immunoglobulin Fc fragments

The long-acting EPO conjugates in this example are constructs of human erythropoietin covalently linked to an immunoglobulin Fc fragment by a non-peptide polymer. Which is obtained as described in korean patent nos. 725315 and 775343.

Example 2: determination of Long-acting EPO conjugate stability dependent on different stabilizers

The ability of different stabilizers including sugars, sugar alcohols, polyols and amino acids to stabilize long-acting EPO conjugates was determined in the presence of phosphate buffer.

For this assay, sodium phosphate buffer was used as phosphate buffer, mannitol or sorbitol as sugar alcohol, histidine or methionine as sugar alcohol, maltose as sugar, and PEG400 as polyol.

The compositions listed in Table 1 were analyzed by reverse phase chromatography after storage at 40 ℃ for one week. The results are summarized in Table 1 below. The retention of the long acting EPO conjugate compared to its initial value is expressed as RPC (%) (area%/initial area%).

TABLE 1

[ Table 1]

[ Table ]

Numbering	EPO	Buffering agent	Stabilizer	RPC(％)
					1	200μg/ml	10mM sodium phosphate, pH6.5	1% mannitol	95.2
2	200μg/ml	10mM sodium phosphate, pH6.5	1mM histidine	86.2
					3	200μg/ml	10mM sodium phosphate, pH6.5	1mM methionine	76.1
4	200μg/ml	10mM sodium phosphate, pH6.5	10% maltose	93.5
					5	200μg/ml	10mM sodium phosphate, pH6.5	1％PEG 400	92.8
6	200μg/ml	10mM sodium phosphate, pH6.5	1% sorbitol	92.7

As is evident from the data in table 1, the use of mannitol as a stabilizer keeps the long-acting EPO conjugate most stable.

Example 3: determination of salt-dependent Long-acting EPO conjugate stability

The ability of different salts to stabilize long-acting EPO conjugates in the presence of phosphate buffer was determined as follows. Salts such as basic and inorganic salts not only act as pH buffers to impart additional pH stability to the long-acting conjugate, but also act as isotonic agents to maintain a suitable osmotic pressure.

The compositions listed in Table 2 were analyzed by reverse phase chromatography after storage at 40 ℃ for one week. For this assay, sodium phosphate buffer (pH6.5) was used as the buffer and copper (II) chloride, sodium sulfate, sodium citrate and sodium carbonate were used as the salts. The results are summarized in Table 2 below. The retention of long-acting EPO conjugate compared to its initial value is expressed as RP-HPLC (%).

TABLE 2

[ Table 2]

[ Table ]

As is evident from the data in table 2, the stability of the long acting EPO conjugates in the presence of phosphate buffer is increased when sodium sulfate, sodium chloride, sodium citrate, or a combination of sodium sulfate and sodium chloride is used compared to these controls, all without addition. In contrast, the stability of long-acting EPO conjugates is reduced in the presence of copper (II) chloride compared to controls.

It will be appreciated from the data that the long-acting EPO conjugates of the invention are stabilized to varying degrees depending on the type of salt used and that certain salts exhibit greater stability.

Example 4: determination of Long-acting EPO conjugate stability dependent on non-ionic surfactants

The ability of different non-ionic surfactants to stabilize long-acting EPO conjugates in the presence of phosphate buffer was determined as follows.

For this assay, polysorbate 80 was used as a non-ionic phosphate buffer, and other reagents, including salts, sugar alcohols, and sugars, were used in appropriate combinations that were shown to provide long-acting EPO conjugate stability.

For simplicity, sodium chloride was selected from the validated salts including: sodium chloride, sodium sulfate and sodium citrate. Mannitol, which ensures the maximum stability as demonstrated in example 1, was also used. The long-acting EPO conjugates of the present invention were stored in the compositions listed in Table 3 at 40 ℃ for one week and then analyzed by reverse phase chromatography under the same conditions that EPO was set to 200. mu.g/ml in 10mM sodium phosphate buffer (pH 6.5). The results are summarized in Table 3 below. The retention of long-acting EPO conjugate compared to its initial value is expressed as RP-HPLC (%).

TABLE 3

[ Table 3]

[ Table ]

In the presence of phosphate buffer, as shown in table 3, EPO does fluctuate only slightly, regardless of the presence of the non-ionic surfactant, indicating that the non-ionic surfactant does have a significant effect on EPO stability over a period of as little as one week.

Experiments were also conducted to determine the effect of nonionic surfactant concentration on the stability of long-acting EPO conjugates. The long-acting EPO conjugates of the present invention were stored in the compositions listed in Table 4 at 40 ℃ for two weeks and then analyzed by reverse phase chromatography under the same conditions that EPO was set to 200. mu.g/ml in 10mM sodium phosphate buffer (pH 6.5). The results are summarized in Table 4 below. The retention of long-acting EPO conjugate compared to its initial value is expressed as RP-HPLC (%).

TABLE 4

[ Table 4]

[ Table ]

Numbering	Surface active agent	Salt (salt)	Stabilizer	1W(％)	2W(％)
						1	0.1% polysorbate 80	150mM NaCl	5% mannitol	95.9	91.8
2	0.01% polysorbate 80	150mM NaCl	5% mannitol	98.4	93.7

For a storage time of 2 weeks, as shown in table 4, the long acting EPO conjugate was found to be more stable when supplemented with 0.01% polysorbate 80 than when supplemented with 0.1% polysorbate 80 in a liquid formulation comprising 5% mannitol and 150mM sodium chloride in 10mM sodium phosphate buffer (ph 6.5).

Example 5: comparative long-acting EPO conjugate liquid formulations (I)

In terms of storage stability, commercial liquid formulations of EPO, recormon (roche), were compared with the long-acting EPO conjugate liquid formulations of the present invention. The composition of Recormon (although still to be demonstrated) includes sodium phosphate as a buffer, polysorbate 20 as a surfactant, sodium chloride as a salt, and urea, CaCl, glycine, leucine, isoleucine, threonine, glutamic acid, and phenylalanine as stabilizers.

For the long-acting EPO conjugate liquid formulation of the present invention, polysorbate 80 at varying concentrations ranging from 0.1 to 0.005% is used as a surfactant, sodium chloride at a concentration ranging from 150 to 200mM is used as a salt, and mannitol or urea at varying concentrations ranging from 1 to 10% (w/v) is used as a stabilizer. The long-acting EPO conjugates of the invention were stored in the compositions listed in table 5 at 40 ℃ for 2 weeks and subsequently analyzed by reverse phase chromatography and size exclusion chromatography (SE-HPLC). The results are summarized in Table 6 below. The retention of the long-acting EPO conjugate compared to its initial value is expressed as RP-HPLC (%) and SE-HPLC (%).

TABLE 5

[ Table 5]

[ Table ]

TABLE 6

[ Table 6]

[ Table ]

For a two week storage time, all liquid formulations of the present invention ensured higher stability than Recormon, except formulation No.5 containing 0.1% polysorbate 80 as a surfactant and formulation No.7 containing urea as a stabilizer, as shown in table 6.

Example 6: studies of stabilizers capable of imparting storage stability to long-acting EPO conjugates

To investigate stabilizers capable of stabilizing long-acting EPO conjugates during long-term storage, long-acting EPO conjugate liquid formulations were prepared using the components of table 7 below. In this regard, glycine and methionine were added separately to the surfactant-sodium chloride-maltose composition to examine the effect of amino acids on long-term storage stability. In the liquid formulation, the EPO concentration was set to 200. mu.g/ml in 10mM sodium phosphate buffer (pH 6.5).

The long-acting EPO conjugate liquid formulations were analyzed weekly by reverse phase chromatography after 4 weeks of storage at 40 ℃. The results are summarized in Table 8 below. The retention of long-acting EPO conjugate compared to its initial value is expressed as RP-HPLC (%).

TABLE 7

[ Table 7]

[ Table ]

Numbering	Surface active agent	Salt (salt)	Stabilizer
				1	0.005% polysorbate 80	200mM NaCl	10% maltose

2	0.005% polysorbate 80	200mM NaCl	10% maltose + 1% glycine
				3	0.005% polysorbate 80	200mM NaCl	10% maltose +1mM methionine

TABLE 8

[ Table 8]

[ Table ]

For a storage time of 4 weeks, the long acting EPO conjugate was found to be more stable in a liquid formulation comprising sodium chloride in combination with maltose and glycine as stabilizers, as shown in table 8. After 2 weeks of storage, similar storage stability was detected between liquid formulations in which only 10% maltose was used and in combination with 1% glycine, respectively. However, the stability after 4 weeks of storage was significantly reduced using only 10% maltose. In contrast, 10% maltose in combination with 1% glycine was found to maintain high stability after 4 weeks of storage.

Referring to the comparison between liquid formulations No.2 and No. 3, methionine significantly reduced the stability of the long-acting EPO conjugate, indicating that neutral amino acids (especially glycine) contributed greatly to the long-term storage stability of the long-acting EPO conjugate.

Example 7: determination of mannitol and maltose for Long-term storage stability of Long-acting EPO conjugates

Three liquid formulations were prepared as given in table 9 below: a liquid formulation comprising maltose-glycine in example 6 which shows the highest stability of long-acting EPO conjugates; the same liquid formulation except that mannitol was used instead of maltose; and liquid formulations comprising mannitol only. Their long-term storage stability was determined. In the liquid formulation, the concentration of EPO was set to 200. mu.g/ml in 10mM sodium phosphate buffer (pH 6.5).

The long-acting EPO conjugate liquid formulations were analyzed weekly by reverse phase chromatography after 4 weeks of storage at 40 ℃. The results are summarized in Table 10 below. The retention of long-acting EPO conjugate compared to its initial value is expressed as RP-HPLC (%). The data in Table 10 are also plotted in FIG. 1, where Rocormon values for a commercial EPO preparation are extrapolated.

TABLE 9

[ Table 9]

[ Table ]

Numbering	Surface active agent	Salt (salt)	Stabilizer
				1	0.005% polysorbate 80	200mM NaCl	10% maltose, 1% glycine
2	0.005% polysorbate 80	200mM NaCl	10% mannitol, 1% glycine
				3	0.005% polysorbate 80	200mM NaCl	10% mannitol

Watch 10

[ Table 10]

[ Table ]

For a storage time of 5 weeks, the long-acting EPO conjugate was found to be more stable in a liquid formulation comprising mannitol-glycine as a stabilizer than in a liquid formulation comprising maltose-glycine as a stabilizer, as shown in table 10. Comparable storage stability was detected in liquid formulations containing only mannitol.

These results are quite contrary to the following facts: the liquid formulation comprising maltose alone is significantly reduced in storage stability compared to the liquid formulation comprising maltose and mannitol, indicating that mannitol can ensure long-term storage stability of long-acting EPO conjugates even without the aid of neutral amino acids. It was also found that the use of mannitol in concentrations as high as 5 to 15% (w/v) allows the EPO conjugate to be stored and maintain high stability even in the absence of neutral amino acids.

Example 8: long-term storage stability assay buffers for long-acting EPO conjugates

The ability of the buffer to stabilize long-acting EPO conjugates was determined. The relationship between the stability of long-acting EPO conjugates and the salt and sugar alcohol dosages was also studied as follows.

First, the effect of buffering agents on the stability of long-acting EPO conjugates was investigated using liquid formulations (nos. 1 and 2) formulated with 0.01% polysorbate, 150mM sodium chloride and phosphate or citrate buffers as shown in table 11. The liquid formulation contained a high concentration (5%) of mannitol as a stabilizer, but did not contain any amino acids, and the long-acting EPO conjugate was added at a concentration of 200 μ g/ml. The formulations were stored at 40 ℃ for 4 weeks, during which time the long-acting EPO conjugate liquid formulations were analyzed by reverse phase chromatography at weeks 1 and 4. The results are summarized in Table 12 below (Nos. 1 and 2). The retention of long-acting EPO conjugate compared to its initial value is expressed as RP-HPLC (%).

The following liquid formulations were tested for their effect on the stability of long-acting EPO conjugates: the same liquid formulation containing mannitol, the same liquid formulation containing citrate buffer instead of phosphate buffer, and the same liquid formulation containing only half the amount of salt and sugar alcohol as in example 7.

In the liquid formulation, the concentration of the long-acting EPO conjugate was set to 200 μ g/ml and the stabilizers as shown in table 11 below were used. The long-acting EPO conjugate liquid formulations were stored at 40 ℃ and analyzed by reverse phase chromatography at weeks 1 and 4. The results are summarized in Table 12 below. The retention of long-acting EPO conjugate compared to its initial value is expressed as RP-HPLC (%).

TABLE 11

[ Table 11]

[ Table ]

Watch 10

[ Table 10]

[ Table ]

As is apparent from the data in table 12, if the liquid formulation contains mannitol therein, the storage stability of the long-acting conjugate is ensured at a desired level regardless of the buffer used therein. These results indicate that typical buffers other than phosphate buffers can be used to prepare liquid formulations in which long-acting EPO conjugates can be stably stored for long periods of time.

Example 9: comparison of the storage stability of Long-acting EPO conjugates between liquid formulations (II)

In terms of storage stability, liquid formulations prepared with phosphate buffer (pH6.5), sodium chloride, mannitol and polysorbate 80, all of which have demonstrated stabilizing ability in examples 2 to 8, were compared with the commercially available EPO liquid formulation recormon (roche). The compositions of the liquid formulations of the present invention and Recormon are shown in table 13 below. The long-acting EPO conjugate liquid formulations were analyzed by reverse phase chromatography and size exclusion chromatography at week 2 and 4 after 4 weeks storage at 40℃ for 4 weeks. The results are summarized in Table 14 below. The retention of the long-acting EPO conjugate compared to its initial value is expressed as RP-HPLC (%) and SE-HPLC (%).

Watch 13

[ Table 13]

[ Table ]

TABLE 14

[ Table 14]

[ Table ]

As is apparent from the data in table 14, the liquid formulation containing mannitol as a stabilizer at a high concentration ensures higher storage stability of EPO than Recormon containing various neutral amino acids. From these results, it is understood that the liquid formulation of the present invention can specifically ensure excellent storage stability of the long-acting EPO conjugate.

Example 10: comparing storage stability between multiple liquid formulations

With respect to storage stability, liquid formulations prepared with phosphate buffer (pH6.5), sodium chloride, mannitol and polysorbate 80, all of which have demonstrated stabilizing ability in examples 2 to 8, were compared with liquid formulations prepared by a combination of different commercially available formulations employing long-acting EPO conjugates.

The liquid formulations used in this example are summarized in table 15 below. In Table 15, No.1 is Aranesp (manufactured by Amgen), which is currently used as a therapeutic agent for anemia; no.2 is a liquid formulation prepared with a stable composition comprising phosphate buffer (pH6.5), sodium chloride, mannitol, and polysorbate 80; no. 3 is the same liquid formulation as Aranesp except that the drug is replaced with a long-acting EPO conjugate; no.4 is the same liquid formulation as etanercept (TNFR-Fc) (rheumatoid arthritis therapeutic agent produced by Amgen) except that the drug is replaced with a long-acting EPO conjugate; no.5 is a liquid formulation containing only PBS.

The long-acting EPO conjugate liquid formulations were analyzed weekly by reverse phase chromatography and size exclusion chromatography, stored at 40 ℃ for three weeks. The results are summarized in Table 17 below. The retention of the long-acting EPO conjugate compared to its initial value is expressed as RP-HPLC (%) and SE-HPLC (%).

Watch 15

[ Table 15]

[ Table ]

TABLE 16

[ Table 16]

[ Table ]

Batch number	HM10760A B10098 LGL211 (batch of research centre)
		Temperature of	40℃
Storage conditions	Glass syringe, 500. mu.l
		Analytical method	SEC、RPC
Sampling frequency	Initial, week 1, week 2, all 3 weeks

TABLE 17

[ Table 17]

[ Table ]

As is apparent from the data in table 17, aggregation was observed during 3 weeks of storage for all liquid formulations except for the liquid formulation of the present invention comprising 10mM sodium phosphate buffer (pH6.5), 0.005% polysorbate 80, 10% mannitol, and 200nM sodium chloride. Thus, the liquid formulation of the present invention comprising 10mM sodium phosphate buffer (pH6.5), 0.005% polysorbate 80, 10% mannitol and 200nM sodium chloride is the most promising agent for stable storage of long-acting EPO conjugates for long periods of time.

Example 11: determination of Long-term storage stability and accelerated stability of Long-acting EPO conjugate liquid formulations

To examine its long-term storage stability and accelerated stability, it was demonstrated that a long-acting EPO conjugate liquid formulation prepared from a stabilizer comprising phosphate buffer (pH6.5), sodium chloride, mannitol and polysorbate 80, which ensures maximum storage stability, was stored at 4 ℃ for 12 months, during which the stability of the long-acting EPO conjugate was analyzed. The detailed storage conditions are summarized in Table 18 below. The results of the analysis are given in table 19 and fig. 2. In Table 19, the retention of the long-acting EPO conjugates compared to their initial values is expressed as RP-HPLC (%) and SE-HPLC (%).

In addition, the storage stability of the long-acting EPO conjugate liquid formulations was determined in vitro during storage at 4 ℃ for a period of 12 months (fig. 3).

The potency of the long-acting EPO conjugates used in this example was measured in vitro in a TF-1 cell line (erythroleukemia cells, ATCC CRL 2003). After thawing from the nitrogen storage tank, the TF-1 cells were cultured to a predetermined extent and counted. A predetermined ratio of a mixture of BRP-EPO and long-acting EPO conjugate was placed in a 96-well plate at 50 μ L/well. Cells were diluted to a concentration of 40000 cells/mL in assay medium and then plated onto 96-well plates at 50. mu.l/well. CO at 37 deg.C₂After 72 hours incubation in the incubator, 15 μ LCellTiter 96 Aqueous One Solution Reagent (PROME GA, G358B) was added to each well of the 96-well plate. Subjecting it to CO at 37 ℃₂Incubate again in the incubator for 4 hours. The stain was gently removed with a pipette and then absorbance at 490nm was measured to calculate EC 50. Specific activity was obtained from the calculated EC50 values.

Watch 18

[ Table 18]

[ Table ]

Batch number	HM10760A B10098 LGL071
		Concentration of	0.352mg/ml protein
Temperature of	4℃
		Storage conditions	Glass syringe 500ml
Analytical method	SEC、RPC
		Formulation of	10mM sodium phosphate (pH 6.5)/0.005% polysorbate 80/10% mannitol/200 mM NaCl
Sampling frequency	Initial, 1, 3,6, 9, 12 months

Watch 19

[ Table 19]

[ Table ]

As shown in table 19, long-acting EPO conjugates were found to be very stable in liquid formulations of the invention comprising stabilizer compositions over 12 months.

Furthermore, as described above, long-acting EPO conjugate liquid formulations containing the same stabilizer composition were stored at 4 ℃ for 12 months and then at 25 ℃ for 6 months, during which the storage stability of the samples was analyzed. The results are summarized in tables 20 and 21 below. In tables 20 and 21, the retention of the long-acting EPO conjugate compared to its initial value is expressed as RP-HPLC (%), SE-HPLC (%), protein content (%), and biological inert activity (%).

Watch 20

[ Table 20]

[ Table ]

TABLE 21

[ Table 21]

[ Table ]

As is apparent from the data in tables 20 and 21, long-acting EPO conjugates remained highly stable in the liquid formulations of the present invention comprising the stabilizer composition for 12 months and were found to have 92.5% of the initial activity in the liquid formulations even after 6 months of storage under accelerated conditions. Thus, the long-acting EPO conjugate liquid formulation of the present invention exhibits effective storage stability.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Industrial applicability

The liquid formulation of the present invention for specifically ensuring the storage stability of long-acting EPO conjugates is free from the problem of viral infection because it does not contain human serum albumin. It is simple in composition and therefore has advantages over other stabilizers or lyophilized formulations. Furthermore, the liquid formulation can be used as an effective pharmaceutical system because it contains a long-acting EPO conjugate having a longer duration of action than other native forms and maintaining high protein activity for a long time.

Claims (28)

1. A liquid formulation of a long-acting Erythropoietin (EPO) conjugate comprising a therapeutically effective amount of a long-acting erythropoietin conjugate and an albumin-free stabilizer, wherein EPO, a non-peptide polymer and an immunoglobulin Fc fragment are covalently linked in the conjugate and the stabilizer comprises a buffer and mannitol.

2. The liquid formulation according to claim 1, wherein the concentration of mannitol ranges from 1 to 20% (w/v) based on the total volume of the liquid formulation.

3. The liquid formulation according to claim 1, wherein the buffer is selected from the group consisting of citrate, phosphate, tartrate, carbonate, succinate, lactate and acetate buffers.

4. The liquid formulation according to claim 1, wherein the concentration of the buffer ranges from 5 to 100 mM.

5. The liquid formulation according to claim 1, wherein the buffer has a pH ranging from 4 to 8.

6. The liquid formulation according to claim 1, wherein the albumin-free stabilizer further comprises an ingredient selected from the group consisting of isotonic agents, polyols, sugars, non-ionic surfactants, neutral amino acids, and combinations thereof.

7. The liquid formulation according to claim 6, wherein the isotonic agent is a salt selected from the group consisting of sodium chloride, sodium sulfate, sodium citrate, and combinations thereof.

8. The liquid formulation according to claim 6, wherein the concentration of the isotonic agent ranges from 5 to 200 mM.

9. The liquid formulation according to claim 6, wherein the non-ionic surfactant is a polysorbate-based or poloxamer-based non-ionic surfactant.

10. The liquid formulation according to claim 9, wherein the polysorbate-based nonionic surfactant is selected from polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80.

11. The liquid formulation according to claim 6, wherein the concentration of the nonionic surfactant ranges from 0.001 to 0.05% (w/v) based on the total volume of the liquid formulation.

12. The liquid formulation according to claim 6, wherein the sugar is selected from the group consisting of mannose, glucose, fucose, xylose, lactose, maltose, sucrose, raffinose, dextran, and combinations thereof.

13. The liquid formulation of claim 6, wherein the concentration of the sugar ranges from 1 to 20% (w/v) based on the total volume of the liquid formulation.

14. The liquid formulation according to claim 6, wherein the polyol is selected from the group consisting of propylene glycol, low molecular weight polyethylene glycol, glycerin, low molecular weight polypropylene, and combinations thereof.

15. The liquid formulation according to claim 6, wherein the concentration of the polyhydric alcohol in the liquid formulation ranges from 1 to 15% (w/v).

16. The liquid formulation according to claim 6, wherein the neutral amino acid is selected from the group consisting of glycine, alanine, leucine, isoleucine, and combinations thereof.

17. The liquid formulation according to claim 6, wherein the concentration of the neutral amino acid in the liquid formulation ranges from 0.1 to 10% (w/v).

18. The liquid formulation according to claim 6, wherein the albumin-free stabilizer comprises mannitol in an amount of 3 to 12% (w/v) and does not comprise a neutral amino acid.

19. The liquid formulation according to claim 1, wherein the albumin-free stabilizer comprises phosphate buffer at a concentration of 5 to 100mM, mannitol at a concentration of 1 to 20% (w/v), sodium chloride at a concentration of 5 to 200mM, and polysorbate 80 at a concentration of 0.001 to 0.05% (w/v).

20. The liquid formulation according to claim 1, wherein the EPO is a mutant EPO protein modified by a wild-type EPO by substitution, deletion or insertion of one or more amino acids, or a peptide analog having similar activity to the wild-type EPO.

21. The liquid formulation according to claim 1, wherein the concentration of the long-acting EPO conjugate is between 1 and 500 μ g/ml.

22. The liquid formulation according to claim 1, wherein the immunoglobulin Fc fragment is selected from the group consisting of IgG, IgA, IgD, IgE, IgM and combinations thereof.

23. The liquid formulation according to claim 22, wherein the immunoglobulin Fc fragment is a hybrid fragment consisting of domains from different sources of IgG, IgA, IgD, IgE and IgM.

24. The liquid formulation according to claim 22, said immunoglobulin Fc fragment being a dimeric or multimeric form of a single chain immunoglobulin consisting of domains of the same origin.

25. The liquid formulation according to claim 22, wherein the immunoglobulin Fc fragment is an IgG4Fc fragment.

26. The liquid formulation according to claim 25, wherein the immunoglobulin Fc fragment is a human aglycosylated IgG4Fc fragment.

27. The liquid formulation according to claim 1, wherein the non-peptidic polymer is selected from the group consisting of biodegradable polymers, lipid polymers, chitin, hyaluronic acid, and combinations thereof.

28. The liquid formulation according to claim 27, wherein the biodegradable polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, PLA (polylactic acid) and PLGA (polylactic-glycolic acid).