HK1175721A - Liquid formulations for long-acting g-csf conjugate - Google Patents

Description

Liquid formulations of long-acting G-CSF conjugates

Technical Field

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

Background

Granulocyte colony stimulating factor (G-CSF) is a cytokine that stimulates bone marrow stem cells and leukocytes to differentiate and proliferate. It is a glycoprotein with a molecular weight in the range of 18,000 to 19,000 Da and a pI of 6.1(5.5-6.1, depending on the degree of glycosylation) (Nomura et al, EMBO J.5 (5): 871) 876, 1986).

The molecular and genetic properties of G-CSF have been discovered by recombinant DNA techniques (Clark and Kaman, Science, 236: 1229-. Since the cloning of the human G-CSF gene from cDNA libraries constructed from mRNA isolated from CHU-2 and human bladder cancer 5637 cell lines (Nagata et al, Nature, 319: 415-418, 1986; Nagata et al, EMBO J., 5 (3): 575-581, 1986; Souza et al, Science, 232: 61-65, 1986), recombinant DNA technology has allowed the production of G-CSF from mammalian cells and prokaryotes. Furthermore, the present inventors found that a modified hG-CSF, in which at least one amino acid residue, particularly the cysteine residue at position 17, is replaced with a different amino acid residue, can be secreted into the periplasm in a large amount without containing a methionine residue at its N-terminus (Korean patent No. 10-356140).

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 kidney, 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 crosslinking 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, G-CSF can be used as a physiologically active polypeptide to provide long-acting G-CSF conjugates. In order to apply the long-acting G-CSF conjugate to a pharmaceutical product, it is necessary to maintain its 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 G-CSF conjugates are more difficult to stabilize than G-CSF polypeptides themselves 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 protein (such as G-CSF) to be administered is in a trace amount of several hundred micrograms per injection, the losses that occur are relatively large when they lose stability and are therefore adsorbed onto the surface of the container. 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 publication No.10-2004-0111351) and albumin-free G-CSF (Korean patent 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 proportions depending on 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 G-CSF conjugates with improved in vivo duration and stability, their molecular weight and volume are considerably different from those of the ordinary G-CSF complexes because they are composed of the physiologically active peptide G-CSF and the immunoglobulin fragment Fc. In addition, the stability of immunoglobulin Fc fragments varies greatly depending on pH. Therefore, the conventional stabilizer for G-CSF cannot be used as it is. Thus, specific stabilizers of different composition than G-CSF stabilizers are needed for long-acting G-CSF conjugates.

The present invention has been made in view of the extensive and intensive studies for developing a stable liquid formulation of a long-acting G-CSF 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 G-CSF conjugate and allows the formation of an economical and stable long-acting G-CSF 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 G-CSF conjugate in which G-CSF, a non-peptidic polymer and an immunoglobulin Fc fragment are covalently linked and a stabilizer free of albumin, wherein the stabilizer comprises a buffer and mannitol.

Solution to the problem

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

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

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 G-CSF, a non-peptidic polymer and an immunoglobulin Fc fragment are covalently linked.

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

Human G-CSF or an analog thereof useful in the present invention may be isolated from a vertebrate or may be chemically synthesized. Alternatively, G-CSF or an analog thereof may be obtained from a prokaryote or eukaryote transformed with a gene encoding G-CSF 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 G-CSF or analog thereof may be glycosylated with mammalian or eukaryotic sugars or may be non-glycosylated. Upon expression, the recombinant G-CSF or analog thereof may comprise an initial methionine residue (position 1). Preferably, recombinant human G-CSF (HuG-CSF) is prepared using CHO cells as a host. Recombinant human G-CSF (HuG-CSF) prepared using E.coli as a host cell is suitable for use in the present invention. In a preferred embodiment of the invention, the recombinant human G-CSF is a mutant: (¹⁷Ser-G-CSF), in which position 17 is a serine residue instead of wild-type cysteine, and the recombinant human G-CSF can be expressed as disclosed in korean patent No. 10-356140.

For use in the present invention, the 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 G-CSF conjugates useful in the invention are prepared by linking G-CSF and an immunoglobulin Fc fragment together. In this regard, G-CSF and an immunoglobulin Fc fragment may be cross-linked by a non-peptidic 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, 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 G-CSF conjugate used in the present invention may be prepared using genetic engineering techniques, as disclosed in korean patent No. 10-0725315.

The liquid formulation of the invention comprises a therapeutically effective amount of a long-acting G-CSF conjugate. Typically, a therapeutically effective amount of G-CSF is about 300mcg per disposable bottle. The concentration of the long-acting G-CSF conjugate for use in the invention is about 7mg/ml to 22mg/ml, preferably about 11mg/ml to 22 mg/ml.

The term "stabilizer" as used herein means a substance that allows for the safe storage of long-acting G-CSF 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%). G-CSF is understood to be stable when it retains 90% or more of its original activity (preferably 95% or more of its original activity) after storage at 10 ℃ for 2 years, at 25 ℃ for 6 months or at 40 ℃ for one to two weeks. For proteins such as G-CSF, its storage stability is important to inhibit the possible production of G-CSF-like antigenic material and to ensure accurate administration. During storage, unless the G-CSF in the preparation aggregates or breaks to form antigenic material, a loss of about 10% of the G-CSF activity is understood to be permitted by administration.

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

In addition, the stabilizer of the present invention is preferably free of 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.

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 G-CSF conjugate. 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 which the concentration of phosphate is preferably in the range of 5 to 100mM, more preferably 10 to 50 mM. buffer preferably the pH is 4.0 to 8.0, more preferably the pH is 5.0 to 7.0, most preferably the pH is 5.0 to 6.0.

In one embodiment, the stability of the long-acting G-CSF conjugate is evaluated based on the pH of the buffer used. At pH 5.5, it was found that citrate buffer at pH 5.5 ensured a higher stability of long-acting G-CSF than pH6.0 (see tables 2 and 4). From these results, it can be understood that the long-acting G-CSF conjugate of the present invention is stabilized to various degrees depending on the pH value of the buffer, and exhibits peak stability at a certain pH. In particular, it was found that long-acting G-CSF conjugates in which Fc (stable at neutral pH) is linked to G-CSF have reduced storage stability when the liquid formulation comprises a buffer at low pH. Neulasta, a commercially available G-CSF drug fused to PEG, employs an acetate buffer at pH 4 as a stabilizer. However, the use of conventional stabilizer compositions and pH in long-acting G-CSF conjugates is not recommended not only because the long-acting G-CSF conjugates of the invention are both larger in molecular weight and volume than wild-type G-CSF, but also because the immunoglobulin Fc is stable in the neutral pH range.

Mannitol, a sugar alcohol, is used in the stabilizer of the present invention because it acts to enhance the stability of the long-acting G-CSF conjugate. Mannitol is preferably used in a concentration of 1 to 20% (w/v), more preferably 3 to 10% (w/v), most preferably 5 to 7% (w/v), based on the total volume of the liquid formulation.

According to one embodiment of the invention, the storage stability of the long-acting G-CSF conjugate shows a large increase when mannitol is used as a stabilizer in the presence of citrate buffer compared to when sorbitol is used (see table 6). These data show the specificity of mannitol as a long-acting G-CSF conjugate stabilizer compared to other stabilizers, indicating that different stabilizers are required depending on the target to be stabilized.

The stabilizers also comprise other sugar alcohols, provided that they do not reduce the stabilizing effect of the combination of mannitol and buffer on long-acting G-CSF conjugates.

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 functions not only to maintain a proper osmotic pressure when the long-acting G-CSF conjugate in the liquid formulation enters the body, but also to further stabilize the long-acting G-CSF conjugate in the liquid formulation. Examples of isotonic agents include water-soluble inorganic salts. Among these are 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.

Preferred examples of sugars that may also be included to increase the storage stability of the long-acting G-CSF 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 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 0.001 to 0.05% (w/v).

Polysorbate 20 and polysorbate 80 were compared for storage stability of the long-acting G-CSF conjugate. The long-acting G-CSF conjugate was found to have increased stability in the presence of polysorbate 80 compared to polysorbate 20 (see table 8). The G-CSF medicinal preparation Neulasta fused with PEG adopts polysorbate 20. However, the liquid formulation of the present invention ensures higher storage stability for long-acting G-CSF conjugates when containing polysorbate 80 than when containing polysorbate 20. From these data it will be appreciated that different surfactants are required depending on the target to be stabilized.

In one embodiment of the invention, the long-acting G-CSF conjugate remains more stable than 0.01% (w/v) polysorbate 80 in a liquid formulation containing 0.005% (w/v) polysorbate 80 as the non-ionic surfactant during storage at 40 ℃ for a period of 4 weeks (see table 10).

Amino acids can also act as stabilizers for liquid formulations, which in solution act to attract more water molecules to the periphery of the G-CSF, such that the outermost hydrophilic amino acid molecules of the G-CSF are further stabilized (Wang, int.J.Pharm.185: 129-188, 1999). In this regard, the charged amino acids promote aggregation of G-CSF by causing electrostatic attraction with G-CSF. Therefore, neutral amino acids (such as 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 present invention, the stabilizer comprising mannitol in a concentration of 3 to 12% (w/v) based on the total volume of the liquid formulation enables the long-acting G-CSF conjugate having a large molecular weight Fc to be stably stored for 4 weeks even without addition of neutral amino acids (see table 12). Thus, a liquid formulation for providing high stability to a long-acting G-CSF conjugate 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. Therefore, mannitol is used at a concentration of 1 to 20% (w/v), preferably at a concentration of 3 to 10% (w/v), more preferably at a concentration of 5 to 7% (w/v) of the liquid formulation.

In addition to the above 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.

In more detail, the present invention provides a liquid formulation comprising a long-acting G-CSF 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 a phosphate or citrate buffer solution at a concentration ranging from 5 to 100mM and a pH ranging from 5 to 7, mannitol at a concentration ranging from 1 to 20% (w/v), an isotonic agent at a concentration ranging from 5 to 200mM, and polysorbate 80 at a concentration ranging from 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 citrate buffer solution at a concentration ranging from 5 to 100mM and pH from 5 to 6, mannitol at a concentration ranging from 1 to 10% (w/v), sodium chloride at a concentration ranging from 100 to 200mM, and polysorbate 80 at a concentration ranging from 0.001 to 0.05% (w/v). Most preferably, the liquid formulation comprises citrate buffer at a concentration of 20mM (pH 5.2-5.8), mannitol at a concentration of 3 to 7% (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), and wherein no neutral amino acid is used.

In one embodiment of the invention, the G-CSF storage stability of a long-acting G-CSF conjugate liquid formulation comprising sodium citrate buffer solution (pH 5.5), 5% (w/v) mannitol, 150mM sodium chloride and 0.005% (w/v) polysorbate 80 was compared to the known G-CSF formulation neuasta (amgen). G-CSF was found to be more stable in the liquid formulations of the invention than in the commercial formulations comprising sodium citrate at pH 4 (see table 14).

In another embodiment, the long-term storage stability of the long-acting G-CSF conjugate liquid formulation of the present invention, which comprises citric acid buffer at pH 5.5, mannitol, sodium chloride and polysorbate 80, was determined and found to be stable for 6 months for long-acting G-CSF conjugates and to ensure at least 96.6% activity even after 6 months of storage under accelerated conditions (see table 16).

It will be appreciated from the data that liquid formulations comprising a buffer at a pH range of 5 to 6 and mannitol at a concentration of 1 to 20% (w/v) can stably store long-acting G-CSF conjugates therein for 12 months or more.

Advantageous effects of the invention

As previously mentioned, the stabilizers of the invention comprising a buffer and mannitol are specific for long-acting G-CSF conjugates. The long-acting G-CSF conjugate liquid formulation of the present invention does not have the problem of viral infection and ensures excellent storage stability of the long-acting G-CSF conjugate, which is linked to an immunoglobulin Fc fragment and has a larger molecular weight and longer duration of action than the native form of G-CSF, since it does not contain human serum albumin and other potential factors harmful to the body.

Drawings

Figure 1 is a graph showing the stability of G-CSF in long-acting G-CSF conjugate liquid formulations and in PEG fusion G-CSF formulations neuasta using SE-HPLC weekly when stored at 40 ℃ for a period of two weeks, wherein buffers of different pH values were used in the long-acting G-CSF conjugate liquid formulations.

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 G-CSF 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 granulocyte colony stimulating factor

The recombinant human G-CSF used in this example is the mutant strain: (¹⁷Ser-G-CSF), in which position 17 is serine, not wild-type cysteine, and can be expressed from an e.coli transformant as described in korean patent No. 356140.

<1-3> preparation of Long-acting G-CSF conjugate Using immunoglobulin Fc fragment

The long-acting G-CSF conjugate in this example is a construct of human granulocyte colony-stimulating factor covalently linked to an immunoglobulin Fc fragment by a non-peptidic polymer. And it is obtained as described in korean patent nos. 725315 and 775343.

Example 2: determination of Long-acting G-CSF conjugate stability depending on buffer pH

To formulate a liquid formulation for stabilizing the G-CSF conjugate prepared in example 1, experiments were conducted to change the effect of buffer pH on the stability of the long-acting G-CSF conjugate.

For this assay, the long-acting G-CSF conjugate liquid formulation of table 1 was prepared with a stabilizer composition comprising mannitol as a stabilizer, polysorbate 80 as a surfactant, and a sodium citrate solution at pH 5.0, 5.5, or 6.0 as a buffer. After storage at 40 ℃ for two weeks, it was analyzed by size exclusion chromatography. The results are summarized in Table 2 below. The retention of the long-acting G-CSF conjugate compared to its initial value is expressed as SE-HPLC (%).

TABLE 1

[ Table 1]

[ Table ]

TABLE 2

[ Table 2]

[ Table ]

As is evident from the data in table 2, the sodium citrate buffer at pH 5.0 caused protein precipitation in the first week and no precipitation was detected when using the sodium citrate buffer at pH 5.5 or 6.0. It is also understood that the stability of long-acting G-CSF is further increased at pH 5.5 than in citrate buffer at pH 6.0.

The following experiments were performed with buffers having finely divided pH values. Long-acting G-CSF conjugate liquid formulations were prepared with the stabilizer compositions listed in Table 3 below and stored at 40 ℃ for two weeks, followed by analysis by reverse phase chromatography and size exclusion chromatography (SE-HPLC). The results are summarized in Table 4 below. The retention of the long-acting G-CSF conjugate compared to its initial value is expressed as SE-HPLC (%).

TABLE 3

[ Table 3]

[ Table ]

TABLE 4

[ Table 4]

[ Table ]

After two weeks of storage in sodium citrate buffer at pH 5.2 to 5.5, long acting G-CSF conjugates were found to retain about 90% or more of the initial activity as shown in table 4.

As can be understood from the results, the long-acting G-CSF conjugate of the present invention is stabilized to various degrees depending on the pH of the buffer used, with peak stability over a range of pH. In particular, the storage stability of long-acting G-CSF conjugates in which Fc (stable at neutral pH) is linked to G-CSF was found to be reduced in liquid formulations containing low pH buffers.

Example 3: determination of Long-acting G-CSF conjugate stability depending on sugar alcohol

The ability of sugar alcohols (e.g., sorbitol and mannitol) to stabilize long-acting G-CSF conjugates was determined as follows:

long-acting G-CSF conjugate liquid formulations were prepared with a stabilizer composition comprising mannitol or sorbitol as a sugar alcohol, sodium chloride as an isotonic agent and polysorbate as a surfactant as listed in table 5 below, and analyzed by size exclusion chromatography (SE-HPLC) after storage for 4 weeks at 40 ℃. The results are summarized in Table 6 below. The retention of the long-acting G-CSF conjugate compared to its initial value (area%/initial area%) was expressed as SE-HPLC (%).

TABLE 5

[ Table 5]

[ Table ]

TABLE 6

[ Table 6]

[ Table ]

As is evident from the data in table 6, the replacement of sorbitol with mannitol as a stabilizer keeps the long-acting G-CSF conjugate more stable.

Example 4: determination of Long-acting G-CSF conjugate stability depending on nonionic surfactant species

The ability of a number of different nonionic surfactants to stabilize long-acting G-CSF conjugates in the presence of sodium citrate buffer was determined as follows:

for this assay, two nonionic surfactants were compared, polysorbate 80 and polysorbate 20, the latter contained in Neulasta (a commercially available liquid formulation of PEG fused G-CSF). Other agents that were shown to provide stability to long acting G-CSF conjugates in examples 2 and 3 were used in appropriate combinations, including sodium citrate buffer at pH 5.5 and mannitol. Long-acting G-CSF conjugate liquid formulations were prepared with stabilizer compositions comprising different types of polysorbates (as listed in table 7 below) and analyzed by size exclusion chromatography (SE-HPLC) after 4 weeks of storage at 40 ℃. The results are summarized in Table 8 below. The retention of the long-acting G-CSF conjugate compared to its initial value is expressed as SE-HPLC (%).

TABLE 7

[ Table 7]

[ Table ]

TABLE 8

[ Table 8]

[ Table ]

Under the same conditions, polysorbate 80 ensures higher storage stability of long-acting G-CSF conjugates compared to polysorbate 20, as shown in table 8. Until the end of two weeks of storage at 40 ℃, no significant difference in long-acting G-CSF storage stability between the two polysorbates could be detected. However, at the fourth week, the nonionic surfactants showed a significant difference in storage stability, although they were very similar in structure.

Example 5: determination of Long-acting G-CSF conjugate stability in relation to non-Ionic surfactant concentration

In example 4, polysorbate 80 was evaluated as more effective than polysorbate 20 in stabilizing long-acting G-CSF. In this example, the effect of polysorbate 80 concentration on the stability of long-acting G-CSF conjugates was tested. For this purpose, long-acting G-CSF conjugate liquid formulations were prepared with the stabilizer compositions listed in table 9 below and analyzed by size exclusion chromatography after 4 weeks of storage at 40 ℃. The results are summarized in Table 10 below. The retention of the long-acting G-CSF conjugate compared to its initial value is expressed as SE-HPLC (%).

TABLE 9

[ Table 9]

[ Table ]

Watch 10

[ Table 10]

[ Table ]

During storage at 40 ℃ for a period of 4 weeks, long-acting G-CSF conjugates were found to be more stable in the liquid formulation containing 0.005% polysorbate 80 than in the liquid formulation containing 0.01% polysorbate 80, as shown in table 10.

Example 6: determining long-acting G-CSF conjugate stability in relation to amino acids

The ability to stabilize long-acting G-CSF conjugates using amino acids as stabilizers was determined. Experiments to evaluate the storage stability of long-acting G-CSF were performed with stabilizers comprising sodium citrate buffer (pH 6.0), mannitol and the neutral amino acid glycine.

Long-acting G-CSF conjugate liquid formulations were prepared with the stabilizer compositions listed in table 11 below and analyzed by size exclusion chromatography after 4 weeks of storage at 40 ℃. The results are summarized in Table 12 below. The retention of the long-acting G-CSF conjugate compared to its initial value (area%/initial area%) was expressed as SE-HPLC (%).

TABLE 11

[ Table 11]

[ Table ]

TABLE 12

[ Table 12]

[ Table ]

Even in the absence of the neutral amino acid glycine, as shown in table 12, the liquid formulation comprising mannitol at a high concentration (5% (w/v)) ensured storage stability of the long-acting G-CSF conjugate at a level similar to that of the liquid formulation obtained when the neutral amino acid was used.

Example 7: comparison of storage stability of Long-acting G-CSF conjugates between liquid formulations

In terms of storage stability, a liquid formulation prepared with a stabilizer composition comprising a citric acid buffer (pH 5.5), sodium chloride, mannitol and polysorbate 80 (which have all been demonstrated to have a stabilizing ability in examples 2 to 6) was compared with a commercially available G-CSF liquid formulation neuasta (amgen). The compositions of the liquid formulation of the present invention and Neulasta are shown in Table 13 below. The long-acting G-CSF conjugate liquid formulations were analyzed weekly by reverse phase chromatography and size exclusion chromatography when stored at 40 ℃ for two weeks. The results are summarized in Table 14 below. The retention of the long-acting G-CSF conjugate compared to its initial value was expressed as RP-HPLC (%) and SE-HPLC (%).

Watch 13

[ Table 13]

[ Table ]

TABLE 14

[ Table 14]

[ Table ]

After long-term storage, it is apparent from the data in table 14 that the long-acting G-CSF conjugate liquid formulation of the present invention ensures storage stability comparable to or higher than that provided by neuasta. From these results, it is understood that the liquid formulation of the present invention can specifically ensure excellent storage stability of long-acting G-CSF.

Example 8: determination of Long-term storage stability and accelerated stability of Long-acting G-CSF conjugate liquid formulations

In order to examine its long-term storage stability and accelerated stability, a long-acting G-CSF conjugate liquid formulation prepared from a stabilizer composition comprising a citric acid buffer (pH 5.5), sodium chloride, mannitol and polysorbate 80 was stored at 4 ℃ for 12 months and then at 25 ℃ for 6 months, during which the storage stability of the sample was analyzed, and it was demonstrated in examples 2 to 6 that the maximum storage stability could be ensured. The results are summarized in tables 15 and 16 below. In tables 15 and 16, the retention of the long-acting G-CSF conjugate with respect to its initial value is expressed as RP-HPLC (%), SE-HPLC (%), protein content (%), and biological inert activity (%).

Watch 15

[ Table 15]

[ Table ]

TABLE 16

[ Table 16]

[ Table ]

As is apparent from the data in tables 15 and 16, the long-acting G-CSF conjugate remained highly stable for 6 months in the liquid formulation comprising the stabilizer composition of the present invention and was found to have 92.5% of the initial activity even after 6 months of storage under accelerated conditions in the liquid formulation. Thus, the long-acting G-CSF 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

Since human serum albumin is not contained, the liquid formulation of the present invention for specifically ensuring the storage stability of a long-acting G-CSF conjugate does not have the problem of viral infection. It has simple composition and thus has economic advantages over other stabilizers or lyophilized formulations. In addition, since it contains a long-acting G-CSF conjugate having a longer duration of action than other natural forms and maintaining high protein activity for a long time, the liquid formulation can be used as an effective pharmaceutical system.

Claims (28)

1. A liquid formulation of a long-acting granulocyte colony stimulating factor (G-CSF) conjugate comprising a therapeutically effective amount of a long-acting granulocyte colony stimulating factor conjugate and an albumin-free stabilizer, wherein the conjugate has G-CSF, a non-peptide polymer and an immunoglobulin Fc fragment covalently linked, 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 according to 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 1, wherein the albumin-free stabilizer comprises citrate buffer at a pH ranging from 5 to 8 and at a concentration ranging from 5 to 100, mannitol at a concentration ranging from 1 to 20% (w/v), sodium chloride at a concentration ranging from 5 to 200mM, and polysorbate 80 at a concentration ranging from 0.001 to 0.05% (w/v).

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

20. The liquid formulation of claim 19, wherein the G-CSF derivative is a mutant (G-CSF)¹⁷Ser-G-CSF), wherein position 17 is a serine residue instead of the cysteine residue of wild-type G-CSF.

21. The liquid formulation according to claim 1, wherein the concentration of the long-acting G-CSF conjugate ranges from 7 to 22 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 of different origin selected from the group consisting 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, said single chain immunoglobulin being composed 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 non-glycosylated 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).