Protein liquid preparation and method for producing same

Abstract

According to one embodiment of the present invention, a liquid preparation containing high concentrations of efrapegrastim and a method for producing the same can provide a liquid preparation that contains high concentrations of protein, but has excellent solubility and stability, reduces irritation / pain at the administration site or discomfort to the patient, and allows for patient-friendly injection.

Description

[Technical field]

[0001] The present invention relates to a liquid protein formulation and a method for producing the same.

[0002] This application claims priority to Korean Patent Application No. 10-2021-0011802, filed with the Korean Intellectual Property Office on January 27, 2021, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0003] Granulocyte-colony stimulating factor (G-CSF) is a cytokine that directs the division and differentiation of bone marrow stem cells and white blood cells, and plays a role in promoting cell division and differentiation outside the bone marrow. It is a glycoprotein with a molecular weight of 18,000 to 19,000 daltons and an isoelectric point (pI) of 6.1 (pI value is 5.5 to 6.1 depending on the degree of glycosylation).

[0004] Recombinant DNA technology has elucidated the molecular and genetic properties of G-CSF, and after the human G-CSF gene was cloned from a cDNA library prepared by isolating mRNA from CHU-2 cells and human bladder cancer cell line 5637, it became possible to produce G-CSF from mammalian and prokaryotic cells.

[0005] It should be noted that, in the commercial viability and efficiency of pharmaceutical protein formulations containing proteins such as G-CSF as described above, formulation stability can also be overcome by including additional molecules in the formulation. Protein stability can be improved by including excipients that interact with the protein and maintain it in a stable, soluble, and non-aggregated state in solution. For example, salt compounds and other inonic species are additives to protein formulations. They help prevent protein denaturation by binding to proteins in a non-specific manner and increasing thermal stability. Salt compounds (e.g., NaCl, KCl) have been successfully used in commercial insulin formulations to prevent aggregation and precipitation. Amino acids (e.g., histidine, arginine) have been shown to reduce alterations in the secondary structure of proteins when used as formulation additives. Other examples of commonly used additives include polyalcoholic substances such as glycerol and sugars, and nonionic (e.g., Tween, Pluronic) surfactants.

[0006] Pharmaceutical additives must be soluble, non-toxic, and used at specific concentrations to provide a stabilizing effect for a particular therapeutic protein. Because the stabilizing effect of an additive is protein- and concentration-dependent, each additive used in a pharmaceutical dosage form must be carefully tested to ensure that it does not induce instability or other adverse effects on the chemical or physical makeup of the dosage form. Components used to stabilize proteins may induce problems related to protein stability over time or due to environmental changes during storage.

[0007] In addition, pharmaceutical preparations of proteins must be formulated to a high concentration in order to improve therapeutic effects. High-concentration protein preparations are advantageous for therapeutic use because they allow smaller volumetric capacity and are more economical to package and store. However, the development of high-concentration protein preparations has many challenges, such as production, stability, and patient pain. For example, protein aggregation or insolubility generally increases with increasing protein concentration in the formulation (Shire, SJ et al., J. Pharm. Sci., 93, 1390 (2004)). Therefore, in high-concentration protein preparations, side effects that do not appear in low-concentration formulations, such as non-native protein aggregation and microparticle formation, may appear even when additives that provide advantageous effects in low-concentration protein preparations are used. In addition, the high viscosity of high-concentration proteins may interfere with the manufacturing process of filtration methods, but may cause pain and further side effects to patients during injection, and may reduce patient compatibility. Therefore, pharmaceutical protein formulations usually require a careful balance of components and concentrations to enhance protein stability, patient affinity and therapeutic requirements while limiting any side effects.

[0008] Therefore, there is a need to develop protein formulations containing high concentrations of non-naturally occurring proteins with a high aggregation potential that are not only useful for therapeutic use, but also have advantages in terms of solubility and stability, and are patient-friendly. Summary of the Invention [Problem to be solved by the invention]

[0009] One embodiment provides a liquid formulation containing a high concentration of eflapegrastim and a buffering agent.

[0010] Another aspect provides a method for producing the liquid formulation.

[0011] Yet another aspect provides an article of manufacture comprising the liquid formulation. [Means for solving the problem]

[0012] One embodiment is a liquid formulation comprising efrapegrastim and a buffering agent, Contains efrapegrastim in concentrations between 6 mg / mL and 150 mg / mL; The patient friendly index (PF) defined by the following formula 1 is 10 or less, Formula 1 PF(patient friendly) index=Osm(mOsm / kg) / 100+MGF(N) In formula 1, Osm is the osmolarity of the liquid formulation, and MGF is the maximum gliding force when the liquid formulation is administered at a speed of 2.835 mm / s using a 29-cage syringe; Osmolality is between 100 mOsm / kg and 1,000 mOsm / kg; When the liquid formulation is administered with a 29-cage syringe at a speed of 2.835 mm / s, the maximum gliding force is 7 N or less, or when the liquid formulation is administered with a 29-cage syringe at a speed of 4.725 mm / s, the maximum gliding force is 10 N or less; Provided is a liquid formulation of efrapegrastim, which has a residual rate of efrapegrastim of 95% or more as measured by reverse phase high performance liquid chromatography (RP-HPLC) or size exclusion chromatography (SE-HPLC) after storage for 4 weeks at 23 to 27°C and 55 to 65% relative humidity.

[0013] In some embodiments, the liquid formulation has a conductivity of 15 mS / cm or less, hi some embodiments, the retention of efrapegrastim is 98% or more.

[0014] In some embodiments, the liquid formulation has a viscosity of 4 cP or less at room temperature between 20°C and 25°C.

[0015] In some embodiments, the buffer has a concentration of about 5 mM to about 100 mM, hi some embodiments, the buffer is citric acid and / or a citrate salt.

[0016] In some embodiments, the liquid efrapegrastim formulation further comprises a stabilizer. In some embodiments, the stabilizer comprises mannitol. In some embodiments, the concentration of mannitol is about 1% to about 20% (w / v) of the liquid formulation.

[0017] In some embodiments, the liquid efrapegrastim formulation further comprises a surfactant. In some embodiments, the surfactant is a polysorbate-based non-ionic surfactant. In some embodiments, the polysorbate-based non-ionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80. In some embodiments, the final concentration of the polysorbate-based non-ionic surfactant after concentrating the liquid formulation is about 0.0001% to about 0.5% (w / v) of the total liquid formulation.

[0018] In some embodiments, the liquid formulation has a pH of about 4 to about 8.

[0019] In some embodiments, the liquid efrapegrastim formulation further comprises a tonicity modifier. In some embodiments, the tonicity modifier is sodium chloride. In some embodiments, the concentration of the tonicity modifier is about 5 mM to about 200 mM.

[0020] In some embodiments, the liquid formulation is pretreated using a purification column, hi some embodiments, the pretreated liquid formulation is concentrated after buffer exchange with a buffer that does not contain a polysorbate-based non-ionic surfactant.

[0021] In another aspect, the disclosure provides a liquid efrapegrastim formulation comprising efrapegrastim, a buffer and a surfactant, wherein: The concentration of efrapegrastim is about 11 mg / mL to about 66 mg / mL, and the concentration of the buffer is about 5 mM to about 100 mM. The concentration of the surfactant after the liquid formulation is concentrated is about 0.001% to about 5% (w / v) of the total liquid formulation, and the concentration of the surfactant after the liquid formulation is concentrated is about 0.001% to about 5% (w / v) of the total liquid formulation.

[0022] In some embodiments, the surfactant is a polysorbate-based non-ionic surfactant.

[0023] In some embodiments, the liquid formulation comprises: About 11 mg / mL to about 66 mg / mL efrapegrastim, about 5 mM to about 100 mM citric acid and / or citrate salt, and about 0.001% to about 5% (w / v) polysorbate-based nonionic surfactant.

[0024] In some embodiments, the polysorbate-based non-ionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0025] In some embodiments, the liquid formulation comprises: About 11 mg / mL to about 66 mg / mL efrapegrastim, about 5 mM to about 100 mM sodium citrate, about 0.001% to about 0.5% (w / v) polysorbate 80, about 1% to about 20% (w / v) mannitol, and about 5 mM to about 200 mM sodium chloride.

[0026] In some embodiments, the osmolality of the liquid formulation is from about 100 mOsm / kg to about 800 mOsm / kg, In some embodiments, the liquid formulation has a conductivity of 15 mS / cm or less.

[0027] Another aspect provides a method for producing the liquid formulation.

[0028] Yet another aspect provides an article of manufacture comprising the liquid formulation.

[0029] In yet another aspect, the disclosure provides a method for preventing, alleviating, or treating neutropenia in a patient with reduced white blood cell production, comprising administering to the patient a therapeutically effective amount of a liquid efrapegrastim formulation described herein.

[0030] In some embodiments, the neutropenia is severe chronic neutropenia or febrile neutropenia.

[0031] In some embodiments, the liquid efrapegrastim formulation is administered after the patient has been treated with adjuvant or neoadjuvant chemotherapy. In some embodiments, the liquid efrapegrastim formulation is administered 1-5 days after the patient has been treated with adjuvant or neoadjuvant chemotherapy. In some embodiments, the adjuvant or neoadjuvant chemotherapy is a combination of docetaxel and cyclophosphamide.

[0032] In some embodiments, the second dose of the liquid efrapegrastim formulation is administered between 15 and 25 days after the first dose of the liquid efrapegrastim formulation is administered to the patient.

[0033] In some embodiments, the therapeutically effective amount is a unit dosage form selected from 25 μg / kg, 50 μg / kg, 100 μg / kg, and 200 μg / kg.

[0034] In some embodiments, the therapeutically effective amount is 13.2 mg of the liquid efrapegrastim formulation in a 0.6 mL dosage volume.

[0035] In some embodiments, the method further comprises administering to the patient a therapeutically effective amount of a second agent, hi some embodiments, the second agent is an anti-cancer agent.

[0036] In some embodiments, the liquid efrapegrastim formulation is administered to the patient within about 6 hours, about 5 hours, about 2 hours, or about 1 hour of the end of chemotherapy. Effect of the Invention

[0037] According to one embodiment of a liquid formulation containing a high concentration of efrapegrastim and a method for producing the same, although it contains a high concentration of protein, it has the effect of providing a liquid formulation that has excellent solubility and stability, reduces irritation / pain at the administration site or discomfort to the patient, and allows for patient-friendly injection. [Brief description of the drawings]

[0038] [Figure 1] 1 shows the results of a change in the residual rate of efrapegrastim depending on the concentration of a polysorbate-based nonionic surfactant in a liquid preparation according to one embodiment, as confirmed by reverse phase high performance liquid chromatography (RP-HPLC). [Diagram 2] 1 shows the results of size exclusion chromatography (SE-HPLC) confirming the change in the residual rate of efrapegrastim depending on the concentration of a polysorbate-based nonionic surfactant in a liquid preparation according to one example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Reference will now be made in detail to the embodiments illustrated in the accompanying drawings, in which like reference numerals refer to like elements throughout. In this regard, the embodiments may have different forms and are not to be construed as limited to the description set forth herein. Accordingly, the embodiments are described below with reference to the drawings only to explain aspects of the present specification. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. When preceding a list of elements, a phrase such as "at least one" modifies the entire list of elements and not each individual element of the list.

[0040] One embodiment provides a liquid formulation containing a high concentration of eflapegrastim and a buffering agent.

[0041] Efrapegrastim As used herein, the term "efrapegrastim" refers to the International Nonproprietary Name (INN) of a long-acting human granulocyte-colony stimulating factor (hG-CSF) conjugate that contains a variant of hG-CSF (WHO Drug Information Volume 29, 2015). The efrapegrastim is also a conjugate in which a physiologically active peptide, a granulocyte-colony stimulating factor, a biodegradable polymer, and an immunoglobulin Fc region are linked together.

[0042] Also, the immunoglobulin Fc useful in the present specification has the sequence of human immunoglobulin Fc or a closely related analog thereof, and may be of animal origin such as cow, goat, pig, mouse, rabbit, hamster, rat or guinea pig. Also, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE or IgM, or a combination thereof, or a hybrid thereof. Specifically, the immunoglobulin Fc region is derived from IgG or IgM, which are the most abundant in human blood, and more specifically, is derived from IgG, which is known to improve the half-life of ligand-binding proteins. The immunoglobulin Fc may be produced by treating natural IgG with a specific protease, or may be produced from a cell transformed using recombinant technology. Specifically, the immunoglobulin Fc is a recombinant human immunoglobulin Fc produced from an E. coli transformant.

[0043] In addition, IgG can also be divided into subclasses IgG1, IgG2, IgG3, and IgG4, and in the present invention, combinations or hybrids thereof are also possible. Specifically, the subclasses are IgG2 and IgG4, and more specifically, the Fc region of IgG4 that has almost no effector function such as complement dependent cytotoxicity (CDC). That is, the immunoglobulin Fc region for drug carriers in the present specification is a non-glycosylated Fc region derived from human IgG4. A human-derived Fc region is more desirable than a non-human-derived Fc region that may cause an undesirable immune reaction, such as acting on an antigen in the human body and generating a new antibody against it.

[0044] Efrapegrastim as used herein is prepared by binding the hG-CSF variant to the immunoglobulin Fc region. The binding method used here may be to crosslink the hG-CSF variant to the immunoglobulin Fc region using a non-peptide polymer, or to prepare a fusion protein in which the hG-CSF variant is linked to the immunoglobulin Fc region using recombinant technology. The non-peptide polymer used in crosslinking may be selected from the group consisting of biodegradable polymers such as polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, polylactic acid (PLA) and polylactic-co-glycolic acid (PLGA), lipid polymers, chitins, hyaluronic acid, and combinations thereof. Derivatives of those already known in the art and derivatives that can be easily prepared at the technical level of the art are also included within the scope of the present specification.

[0045] The hG-CSF variants herein may be extracted from mammals or chemically synthesized. They may also be obtained from prokaryotes or eukaryotes transformed with DNA encoding the hG-CSF variants using recombinant gene techniques, but may also use Escherichia coli (e.g., E. coli) or yeast (e.g., S. cerevisiae) as hosts, as well as mammalian cells (e.g., Chinese hamster ovary cells, monkey cells). Depending on the host used, the hG-CSF variant expression product may or may not be glycosylated with mammalian or other eukaryotic carbohydrates. When expressed in prokaryotes, the hG-CSF variant expression product may also contain an initial methionine residue (position 1). The hG-CSF variants suitable for the present invention are also those produced using Escherichia coli as a host cell.

[0046] In one embodiment, the efrapegrastim comprises a recombinant human granulocyte colony-stimulating factor derivative 17,65 Ser-G-CSF, in which the 17th cysteine ​​and 65th proline residues of native G-CSF are replaced with serine, and the 1st threonine is deleted. As mentioned above, the non-native protein of the efrapegrastim may provide additional protein aggregation potential and any side effects compared to the native protein or 17Ser-G-CSF. The protein aggregation is a common problem in protein solutions, causing an increase in protein concentration or viscosity. The present disclosure provides a means to achieve a high concentration, low aggregation protein formulation. The formulations of the present disclosure can achieve a stable high concentration of protein in solution, which is also advantageous for therapeutic purposes.

[0047] In addition, the function and physiological activity of a protein such as a polypeptide are determined by the three-dimensional structure of the protein, and if the part of the three-dimensional structure of the protein that is related to the function is different, the protein will not be able to perform its original specific function. For example, it is a known fact that even if there is a difference in the amino acid sequence, if that amino acid corresponds to a functional site in the three-dimensional structure of the protein and the three-dimensional structure of that site is changed, the function of the protein will be affected. In addition, in the pharmaceutical formulation field, the most common discussion of protein formulations and peptide dosage forms is related to the physicochemical stability of the drug, and in fact, the properties of the drug are important in determining the appropriate formulation for successful delivery and stability. The first step in the development of a protein drug formulation includes a thorough characterization of the drug properties and stability in different formulations, and a person of ordinary skill in the art would begin by considering the physicochemical properties of the protein, such as the isoelectric point, molecular weight, and overall amino acid composition (Jeffrery L. et al., 1994). In other words, a unique solution to the stability approach is required not only for different protein drugs (e.g., IL-1β), but also for protein-stabilized formulations that exhibit different physicochemical properties from the native protein, even if only one amino acid sequence differs in the native protein.

[0048] The liquid preparation of the present invention has a technical feature in that it can exhibit high stability even under high concentration conditions for efrapegrastim containing 17,65 Ser-G-CSF having additional amino acid modifications compared to native human G-CSF or 17Ser-G-CSF.

[0049] As used herein, the term "high concentration" refers to a dose that can increase the beneficial effects of therapeutic use. For example, high concentration efrapegrastim may be contained in the formulation at a concentration of 6mg / mL, 7mg / mL, 8mg / mL, 9mg / mL, 10mg / mL, 11mg / mL, 12mg / mL, 13mg / mL, 14mg / mL, 15mg / mL, 16mg / mL, 17mg / mL, 18mg / mL, 19mg / mL or 20mg / mL or more. Specifically, high-concentration efrapegrastim is 6 mg / mL to 150 mg / mL, 10 mg / mL to 150 mg / mL, 11 mg / mL to 150 mg / mL, 10 mg / mL to 100 mg / mL, 11 mg / mL to 100 mg / mL, 10 mg / mL to 80 mg / mL, 11 mg / mL to 70 mg / mL, 12 mg / mL to 70 mg / mL, 14 mg / mL to 70 mg / mL, 11 mg / mL or 66 mg / mL, 12 mg / mL to 66 mg / mL, 13 mg / mL to 66 mg / mL, 14 mg / mL to 66 mg / mL, 15 mg / mL to 66 mg / mL, 16 mg / mL to 66 mg / mL, 17 mg / mL to 66 mg / mL, 18 mg / mL to 66 mg / mL, 19 mg / mL to 66 mg / mL, or 20 mg / mL to 66 mg / mL.

[0050] As used herein, the term "stable" may mean that a protein substantially retains its physical and / or chemical and / or biological stability during storage. Typically, such a formulation is considered stable if the loss of active ingredient is less than a certain amount, for example, less than 10%, less than 7%, less than 5%, less than 4% or less than 3%, under a certain storage condition for a certain period of time.

[0051] Concentration and residual rate of efrapegrastim As mentioned above, an increase in the concentration of efrapegrastim may have a negative effect on the survival rate. In addition, efrapegrastim may have an unpredictable effect on the survival rate in terms of being a further mutant compared to natural hG-CSF. Therefore, the stability of the liquid formulation is also evaluated with aggregate formation of the protein drug as a major factor. Protein drugs will form aggregates when subjected to shear stress or other physical or chemical environments, and such aggregate formation is a factor that affects the decrease in bioavailability, such as reduced efficacy, and is therefore a major component to be considered in the development of liquid formulations.

[0052] According to one embodiment, the liquid formulation has a residual rate of a protein (e.g., efrapegrastim) of 95% or more, 96% or more, 97% or more, or 98% or more after a 4-week storage test, as measured by reversed-phase high-performance liquid chromatography (RP-HPLC) or size exclusion-high performance liquid chromatography (SE-HPLC) at 23 to 27° C. and 55 to 65% relative humidity (RH). Here, the residual rate refers to the relative ratio of the purity at a specific time point to the initial protein purity, and the residual rate of a protein (e.g., efrapegrastim) in the liquid formulation at week n is also defined by Equation 2. Equation 2 Residual rate at nth week (%) = purity at nth week / initial purity x 100

[0053] In one embodiment, the RP-HPLC measurement is performed on a liquid formulation sample using a suitable column (e.g., a C4 column (particle size 5 μm, interior diameter×length: 4.6 mm×250 mm)) at 40 to 80° C. In summary, the HPLC conditions are as follows: an eluent linear gradient system with a flow rate of 0.5 to 2.0 mL / min (preferably 1.0 mL / min) can be used; mobile phase A contains 0.05 to 1.0% trifluoroacetosan (preferably 0.1%) and 10 to 40% acetonitrile (preferably 20%); and mobile phase B contains 0.05 to 1.0% trifluoroacetosan (preferably 0.1%) and 60 to 95% acetonitrile (preferably 80%). The detector is set at 214 nm.

[0054] In one embodiment, the SE-HPLC measurement is performed on a liquid formulation sample using an appropriate column (e.g., Protein LW-803 column (particle size 5 μm, interior diameter×length: 8.0 mm×300 mm)). In summary, the HPLC conditions are an isocratic gradient system with a flow rate of 0.3 to 1.2 ml / min (preferably 0.6 ml / min), and the mobile phase contains 10 to 10 mM sodium phosphate, 50 to 300 mM sodium chloride, or 1 to 15% isopropyl alcohol. The detector is set at 214 nm.

[0055] For example, in the liquid formulation of the present specification, under the above-mentioned conditions, 95% or more, 96% or more, 97% or more, or 98% or more of the initial purity of the protein drug is maintained in monomeric form without forming aggregates or degradants. In other words, in the liquid formulation of the present specification, under the above-mentioned conditions, 5% or less, for example 4% or less, or 3% or less of the initial content of the protein drug is converted into aggregate or degradant form.

[0056] In general, if the residual rate of the protein (e.g., efrapegrastim) is maintained at about 95% under accelerated conditions (25±2°C / 60±5% RH) after a 4-week storage test, the formulation is deemed to be stable. If the residual rate of the protein is maintained at 97% or more under accelerated conditions (25±2°C / 60±5% RH) after a 4-week storage test, the formulation is deemed to have excellent stability. If the residual rate of the protein is maintained at 98% or more under accelerated conditions (25±2°C / 60±5% RH) after a 4-week storage test, the formulation is deemed to have very excellent stability.

[0057] Without being bound by any particular theory, it is believed that the liquid formulation of the present invention contains a high concentration of efrapegrastim, which may increase the residual rate of efrapegrastim after long-term storage due to protein-protein interactions. The specific amino acid sequence of the hG-CSF variant may also affect the increased residual rate, which may be due to electrostatic bonds between charged amino acids or interactions between amino acid residues due to chemical structures.

[0058] In order to ensure the stability of the formulation, in addition to the efrapegrastim and buffer substance, other ingredients or substances known in the art may be selectively contained in the liquid formulation within a range that does not impair the efficacy of the liquid formulation of the present invention.

[0059] Stabilizers In one embodiment, the liquid formulation also includes a stabilizer.

[0060] The term "stabilizer" may refer to an excipient that improves or enhances stability. Examples of the stabilizer include mannitol, sorbitol, dextrose, trehalose, sucrose, raffinose, maltose, benzyl alcohol, biotin, bisulfite compounds, boron compounds, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ascorbic acid and its esters, carotenoids, calcium citrate, acetyl-L-camitin, chelating agents, chondroitin, chromium, citric acid, coenzyme Q-10, cysteine, cysteine ​​hydrochloride, 3-dehydroshikimic acid (DHS), EDTA (ethylenediaminetetraacetic acid, disodium edetate), vitamin A and its esters, vitamin B and its esters, vitamin C and its esters, vitamin D and its esters, vitamin E and its esters, such as vitamin E acetate, zinc, and any combination thereof. The stabilizer may be present in an amount of 0.2 to 30% (w / v), 0.5 to 30% (w / v), 0.5 to 20% (w / v), 0.5 to 10% (w / v), 1 to 30% (w / v), 1 to 25% (w / v), 1 to 20% (w / v), 1 to 15% (w / v), 2 to 20% (w / v), 2 to 15% (w / v), or 2 to 10% (w / v) of the liquid formulation.

[0061] In one embodiment, the stabilizer is substantially free of albumin. Human serum albumin, which can be used as a protein stabilizer, is produced from human blood, so there is a possibility of contamination with human-derived pathogenic viruses, and gelatin and bovine serum albumin may cause diseases or induce allergic reactions in some patients. The albumin-free stabilizer of the present specification does not contain heterologous proteins such as human-derived or animal-derived serum albumin or purified gelatin, and is therefore free from the worry of viral infection.

[0062] As used herein, "substantially does not comprise" means that the referenced material is included to such an extent that it does not contribute to the formulation or activity of the composition, or to the properties or activity of the formulation, or is not included at all.

[0063] Without being bound by any particular theory, it is possible that the above-mentioned substances play a role in improving the stability of the formulation. Therefore, the residual rate of efrapegrastim changes due to the change in the physical or chemical environment caused by the use of specific amounts of these substances.

[0064] Surfactants In one embodiment, the liquid formulation also includes a surfactant.

[0065] In this specification, the term "surfactant" generally includes preparations that protect proteins from air / solution interface-induced deformation forces and solution / surface-induced deformation forces. For example, the surfactant can protect proteins from aggregation. Suitable surfactants include polysorbate-based nonionic surfactants, such as polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. Examples of the surfactants also include poloxamers, such as poloxamer 188, twins, such as twins 20 and 80, polyoxyethylene alkyl ethers, Triton X-100, Brij 30, or Brij 35.

[0066] When the polysorbate-based nonionic surfactant has a final concentration of 5% (w / v) or more in the total solution, it significantly affects the stability of the liquid formulation containing efrapegrastim, and the residual rate can be used as the stability evaluation measure. For example, if the residual rate of the protein is maintained at 97% or more under accelerated conditions (25±2°C / 60±5% RH) after a 4-week storage test, such a formulation is considered to have excellent stability.

[0067] Since first being approved in Europe, polysorbate-based nonionic surfactants have been widely used as additives in the pharmaceutical and cosmetic fields, but recently, some reports have shown that they have a negative effect on the human body. For example, it has been reported that polysorbate 80 can induce anaphylaxis when injected into the human body (Palacios Castano MI et al., Anaphylaxis Due to the Excipient Polysorbate 80, 2016). Therefore, it is necessary to adjust the concentration of polysorbate-based nonionic surfactants within a range that does not affect the stability of protein drugs and does not cause discomfort to patients when injected, and it is necessary to technically prevent the concentration of polysorbate-based nonionic surfactants from inevitably becoming high during the liquid formulation manufacturing process.

[0068] In one embodiment, the polysorbate-based nonionic surfactant is present in a final concentration of 0.0001 to 5% (w / v), 0.0001 to 0.5% (w / v), 0.0001 to 0.05% (w / v), 0.0001 to 0.005% (w / v), 0.0001 to 0.0005% (w / v), 0.00 1 to 5% (w / v), 0.001 to 0.5% (w / v), 0.001 to 0.05% (w / v), 0.001 to 0.005% (w / v), 0.01 to 5% (w / v), 0.01 to 0.5% (w / v), 0.01 to 0.05% (w / v), 0.1 to 5% (w / v), or 0.1 to 0.5% (w / v) ), for example, 0.0001 to 4.5% (w / v), 0.0001 to 0.45% (w / v), 0.0001 to 0.045% (w / v), 0.0001 to 0.0045% (w / v), 0.0001 to 0.00045% (w / v), 0.001 to 4.5% (w / v), 0.001 to 0.4 It may also be present at 5% (w / v), 0.001 to 0.045% (w / v), 0.001 to 0.0045% (w / v), 0.01 to 4.5% (w / v), 0.01 to 0.45% (w / v), 0.01 to 0.045% (w / v), 0.1 to 4.5% (w / v), or 0.1 to 0.45% (w / v).

[0069] In the present specification, the term "final concentration" is a concept distinct from the stated concentration, and means the actual concentration substantially contained in the liquid formulation. For example, in the process of concentrating efrapegrastim to a target concentration after buffer replacement in the preparation of a liquid formulation, the final concentration of the surfactant in the solution may be higher than the stated concentration. For example, in an embodiment in which a polysorbate-based nonionic surfactant is used as the surfactant, if the stated concentration is 0.005% (w / v), the polysorbate-based surfactant is concentrated together with efrapegrastim, and its actual or final concentration in the formulation may exceed at least 1,000 times 0.005% (w / v). For example, the final concentration (i.e., actual concentration) of polysorbate 80 of 0.005% (w / v) or 0.01% (w / v) described in the examples of Korean Patent Publication No. 10-1340710 (published on December 12, 2013), which describes a liquid formulation containing a granulocyte-colony stimulating factor conjugate other than efrapegrastim, may exceed a minimum of 5% (w / v) or 10% (w / v). In contrast, when the final concentration of a polysorbate-based nonionic surfactant is expressed as 0.005% (w / v) in the present specification, it means that the formulation is obtained by exchanging the buffer with a buffer material that is substantially free of the polysorbate-based nonionic surfactant, for example, by diafiltration, concentrating the buffer material to the target efrapegrastim concentration, and then spiking the polysorbate-based nonionic surfactant to 0.005% (w / v) until the concentration reaches 0.005% (w / v). Thus, the liquid formulation according to one embodiment is a liquid formulation that is pretreated using a purification column, or a liquid formulation that is pretreated and then concentrated after exchanging the buffer with a buffer material that is substantially free of the polysorbate-based nonionic surfactant. Thus, without being limited by a particular theory, the liquid formulation of the present specification may provide remarkable formulation stability and optional improved properties of a liquid formulation containing a non-native protein that has a high aggregation potential, even at a high concentration, compared to conventional liquid formulations.

[0070] Tonicity Modifier In one embodiment, the liquid formulation also includes a tonicity modifying agent.

[0071] As used herein, the term "tonicity modifier" may refer to a compound or compounds that may be used to adjust the tonicity of a liquid formulation.

[0072] The tonicity modifier may be one or more selected from the group consisting of pharma- ceutically acceptable salts, sugars, and amino acids. Specifically, the tonicity modifier may be one or more selected from the group consisting of sodium chloride, sodium phosphate, sodium succinate, sodium sulfate, potassium chloride, magnesium chloride, magnesium sulfate, magnesium chloride, etc., and more specifically, sodium chloride, etc. The tonicity modifier may be one or more selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, and polysaccharides, for example, one or more selected from the group consisting of trehalose, sucrose, mannitol, sorbitol, fructose, maltose, lactose, dextran, etc. The tonicity modifier may be one or more selected from the group consisting of proline, alanine, arginine (e.g., L-arginine), asparagine, aspartic acid (e.g., L-aspartic acid), glycine, serine, lysine, histidine, etc.

[0073] The concentration of the tonicity modifier for stabilization of the formulations herein is also an amount capable of maintaining or adjusting the osmolarity range, e.g., 1 to 600 mM, 5 to 600 mM, 5 to 400 mM, 5 to 300 mM, 10 to 400 mM, 10 to 300 mM, 10 to 200 mM, 20 to 400 mM, 20 to 200 mM, 30 to 400 mM, 30 to 200 mM, 50 to 600 mM, 50 to 400 mM, 80 to 400 mM, 80 to 200 mM, 100 to 400 mM, 100 to 300 mM, or 100 to 200 mM.

[0074] A tonicity modifier may be added in an amount sufficient to provide an appropriate osmotic pressure, as described below.

[0075] Buffer substances and pH In the present specification, the term "buffer substance" may refer to one or more components that can protect the solution against changes in pH when added to an aqueous solution, when an acid or alkali is added, or when diluted with a solvent. The buffer substance may be any substance that can bring the pH of the composition to a specific range for stabilization of the formulation, such as an organic acid buffer or an inorganic acid buffer, such as an organic acid, an inorganic acid, or a salt of an organic acid or an inorganic acid. More specifically, the buffer may be one or more organic or inorganic acids selected from the group consisting of succinic acid, acetic acid, citric acid, histidine, phosphoric acid, glycine, lactic acid, tris, bistris, etc., or one or more selected from the group consisting of sodium salts of the organic or inorganic acids, succinate, acetate, citrate, phosphate, lactate, etc. More specifically, examples of said buffer substances include alkali salts (sodium acetate or potassium acetate, or their hydrogen or dihydrogen salts), sodium citrate / citric acid, sodium acetate / acetic acid, and any other pharma- ceutically acceptable pH buffer substances known in the art, and mixtures thereof may also be used.

[0076] In one embodiment, the concentration of the buffering agent is 5 to 100 mM, 5 to 80 mM, 10 to 80 mM, 10 to 60 mM, 10 to 50 mM, or even 15 to 25 mM.

[0077] In one embodiment, the specified pH range of the liquid formulation is 4 to 8, 5 to 8, 5 to 7, or 5 to 6, and in a specific embodiment, even 5.5.

[0078] The buffer substance may be dissolved in a liquid medium such as water and used in a solution having a pH of 4-8, 5-8, 5-7, or 5-6.

[0079] Osmolarity Based on various literature, the osmolality of the drug is adjusted to 300±30 mOsm / kg, but due to the need to use various excipients, it is also manufactured into a hypertonic solution in the technical industry. In intravenous or intravascular administration, the upper limit of osmolality is generally proposed to not exceed 1,000 mOsm / kg, and since the osmolality of serum is about 285 mOsm / L, the lower limit of osmolality is generally proposed to exceed 100 mOsm / L or 200 mOsm / L. Therefore, it is generally expected that patients can tolerate 100 to 1,000 mOsm / kg, 200 to 1,000 mOsm / kg, 100 to 800 mOsm / kg, or 200 to 800 mOsm / kg. In one embodiment, the osmolality of the liquid formulation to show excellent stabilizing effect is also 400 to 800 mOsm / kg.

[0080] The osmotic pressure can be measured using a measurement method and a measurement device known in the art.

[0081] Without being bound by any particular theory, it is speculated that the osmotic pressure of the liquid formulation of the present invention is affected by the concentration of efrapegrastim or by the concentration of the stabilizer or tonicity modifier that is also included in addition. The stability of the liquid formulation of the present invention is increased by a certain concentration range of efrapegrastim and / or a hG-CSF variant having a specific amino acid sequence, so that the composition of the liquid formulation that may affect stability can be adjusted more flexibly compared to the composition of a conventional liquid formulation. Thus, the composition for the liquid formulation to have the osmotic pressure to achieve the effect of the present invention can be adjusted more flexibly. For example, the concentration of the stabilizer or tonicity modifier that is also included in addition to the stability of the liquid formulation can be relatively low to adjust the osmotic pressure.

[0082] Conductivity In other embodiments, the formulations herein have a conductivity of 20 mS / cm, 19 mS / cm, 18 mS / cm, 17 mS / cm, 16 mS / cm, or 15 mS / cm or less. Intermediate ranges of the above recited numerical values, e.g., 1 to 20 mS / cm, are intended to be included in the present invention. Numerical ranges using combinations of the above recited numerical values, e.g., as upper and / or lower values, are intended to be included. Also included herein are numerical values ​​within the recited numerical values, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mS / cm, etc.

[0083] The term "conductivity" as used herein refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In general, electrical conductivity and specific conductivity are measures of a material's ability to conduct an electric current. In a solution, electric current flows by ion transport. Thus, by increasing the amount of ions present in an aqueous solution, the solution has a higher conductivity. The unit of measurement for conductivity is mmhos (mS / cm) and can be measured using commercially available conductivity meters.

[0084] Maximum sliding force and viscosity As used herein, the term "maximum gliding force (MGF)" refers to the maximum force applied when administering a drug using a syringe, and is affected by the viscosity of the formulation, the injection speed, and the syringe characteristics. The rheological properties of protein formulations are also affected by the concentration of the protein. A fine needle of 29G or more substantially increases the maximum gliding force. As a result, the aggregation phenomenon caused by high concentration of protein and the resulting increase in viscosity cause an increase in the maximum gliding force, and in some cases, the drug must be administered using a needle of less than 29G to reduce the maximum gliding force, which causes discomfort to the patient. It has been reported that patients can generally tolerate a maximum gliding force of about 15N to 20N (Development of Syringeability Guide for Subcutaneous Protein Formulations, L. Joseph et al., Pfizer Global Research & Development, 2010).

[0085] In one embodiment, the liquid formulation contains a sufficiently high concentration of epilurapegrastim for therapeutic effect, yet has a maximum gliding force of 7N, 6N, 5N, 4N, or 3N or less when administered with a 29-cage syringe at a speed of 2.835 mm / s, or 10N, 9N, 8N, 7N, 6N, 5N, 4N, 3.5N, or 3N or less when administered with a 29-cage syringe at a speed of 4.725 mm / s. The maximum gliding force can be measured using a gliding force measuring device known in the art, for example, a rheometer available from DAEGO TRADING CO. (Seoul, Korea). In one embodiment, the liquid formulations herein have a viscosity of less than 10 cP, 9 cP, 8 cP, 7 cP, 6 cP, 5 cP, 4 cP, 3 cP, 3.5 cP, 3 cP, 2.5 cP, or 2 cP at room temperature between 20° C. and 25° C. Viscosity can be measured using methods and devices known in the art.

[0086] As mentioned above, the maximum sliding force is affected by the viscosity of the formulation, and the viscosity, i.e., the rheological properties of the protein formulation, are affected by the concentration. Therefore, the maximum sliding force is affected by the residual ratio, concentration, or buffer substance of the protein drug (efrapegrastim), and may also be affected by the surfactant, stabilizer, or tonicity modifier that is additionally included.

[0087] Patient-friendly formulation In the case of protein drugs, they are required to be formulated at high concentrations for therapeutic effects, but generally, problems such as aggregation, insolubility, and degradation are increased as the protein concentration increases. Therefore, in pharmaceutical protein formulations, one of the technical problems that must be solved in the art is to balance the components and concentrations to improve stability and therapeutic requirements while limiting any side effects. To this end, the present invention has been able to produce a formulation that contains a high concentration of active ingredient while having high formulation stability and solubility. In addition to the surprising advances of the present invention in protein formulations for therapeutic use, irritation / pain and discomfort at the administration site when such liquid formulations of protein drugs are administered to individuals remain a problem that must be solved.

[0088] Osmolality, and administration site irritation / pain Osmotic changes in tissues and cells are perceived as danger signals in the human body, activating dendritic cells and stimulating immune and inflammatory responses (Gallo and Gallucci, 2013). It has been reported that hypertonicity in the gastrointestinal tract of human infants can cause necrotizing colitis (Atakent et al., 1984). It is already known that the presence of pain receptors is responsible for the sensation of pain induced by a variety of events, including injections. The sensation of peripheral pain is mediated through afferent fibers (sensory nerve fibers) called nociceptors (Brazeau at al., 1998). Functionally, the pain receptors are classified into two main types: polymodal nociceptors, which respond to chemical stimuli, and mechanothermal nociceptors, which respond to mechanical and thermal stimuli. Therefore, the sensitivity of pain receptors to pain depends not only on the type of chemical, but also on the injection site, injection speed, and injection volume. Hypertonic solutions (or preservative solutions) can draw water out of cells (or cause water absorption into cells), activating compression (or stretch)-sensitive channels and causing pain.

[0089] Maximum gliding force and viscosity, and patient discomfort There is interest in higher concentration protein formulations to reduce injection volume (drug injection volume at injection) and to secure storage space. The development of high concentration protein formulations has several practical problems in terms of stability, production, and supply, due to the tendency of proteins to aggregate at high concentrations. The physical properties of high concentration protein formulations affect their ability to be easily transferred, and such high concentration solutions sometimes exhibit high viscosity, preventing the solution from passing through a syringe needle. Therefore, there is a correlation between material viscosity and protein concentration, and the higher the concentration of the protein formulation, the higher the viscosity, causing discomfort to the patient. Therefore, the development of formulations that can be more acceptable to patients while increasing the therapeutic effect through increasing the concentration and decreasing the viscosity of the protein formulation represents an advanced development in the design of protein formulations.

[0090] In order to administer a protein formulation with a thinner needle in order to reduce the maximum gliding force and enhance the therapeutic effect, the formulation must contain a high concentration of protein while having little aggregation and low viscosity, but these conflicting values ​​(high concentration of protein and low viscosity) must be resolved.

[0091] Patient friendly index (PF) As mentioned above, the production of high-concentration protein formulations induces considerable problems related to opalescence, aggregation, and precipitation. In addition to the possibility of non-native protein aggregation and particulate formation, reversible self-binding can occur, which can lead to increased viscosity and other properties that complicate delivery by injection. High viscosity can also complicate the production of high-concentration proteins by filtration. Therefore, the development of stable and patient-friendly formulations requires careful consideration of various factors in combination. That is, the residual rate, which is a factor related to the stability of the formulation, is affected by stabilizers and surfactants, which in turn affect the viscosity of the material and affect the maximum sliding force. In addition, the osmotic pressure is affected by tonicity modifiers and buffer substances, which can affect the conductivity.

[0092] In one embodiment, the present disclosure provides a liquid formulation that satisfies a patient affinity index of the following formula 1 within a specific range: Formula 1 PF(patient friendly) index=Osm(mOsm / kg) / 100+MGF(N) In formula 1, Osm is the osmolarity value of the liquid formulation, and MGF is the maximum gliding force value when the liquid formulation is administered at a speed of 2.835 mm / s using a 29-gauge syringe.

[0093] The patient affinity index is also 10 or less when considering the values ​​of the appropriate osmotic pressure and the appropriate maximum gliding force. If the patient affinity index is more than 10, the patient's discomfort will increase rapidly due to the osmotic pressure difference with body fluids and / or the high maximum gliding force. If the patient affinity index is less than 3, the patient's discomfort will increase rapidly due to the osmotic pressure difference with body fluids and / or the low maximum gliding force.

[0094] Specifically, the patient affinity index of a liquid preparation is 3 to 10, 5 to 10, 6 to 10, or 6 to 9. Considering that the maximum gliding force, which is affected by viscosity, is at least 1N in most cases, when the osmotic pressure is 1,000 mOsm / kg, the patient affinity index exceeds 10, making patient-friendly injection difficult. Also, considering that the maximum gliding force is almost always 1N or more, when the osmotic pressure is less than 200 mOsm / kg, the patient affinity index does not exceed 3, making patient-friendly injection difficult.

[0095] Therefore, liquid formulations falling within the range of the patient affinity index have low viscosity, low gliding force, and an appropriate osmotic pressure range that take into account the high concentration protein formulations, and by solving the technical problems mentioned above, it is possible to perform patient-friendly injection while maintaining high dosage form stability.

[0096] The liquid formulation of the present invention may contain efrapegrastim at a concentration of 11 to 66 mg / mL and a buffer substance at a concentration of 5 to 100 mM, or may contain efrapegrastim at a concentration of 11 to 66 mg / mL, a buffer substance at a concentration of 5 to 100 mM, and a polysorbate-based nonionic surfactant at a concentration of 0.001 to 5% (w / v).The liquid formulation of the present invention may also contain efrapegrastim at a concentration of 11 to 66 mg / mL, a buffer substance at a concentration of 5 to 100 mM, a stabilizer at a concentration of 1 to 20% (w / v), a surfactant at a concentration of 0.001 to 5% (w / v), and a tonicity modifier at a concentration of 5 to 200 mM. For example, a liquid formulation of the invention may contain efrapegrastim in a concentration of 11 to 66 mg / mL and sodium citrate in a concentration of 5 to 100 mM, or may contain efrapegrastim in a concentration of 11 to 66 mg / mL, sodium citrate in a concentration of 5 to 100 mM, and polysorbate 80 in a concentration of 0.001 to 5% (w / v). The liquid formulation of the present invention also contains efrapegrastim at a concentration of 11 to 66 mg / mL, sodium citrate at a concentration of 5 to 100 mM, mannitol at a concentration of 1 to 20% (w / v), polysorbate 80 at a concentration of 0.001 to 5% (w / v), and sodium chloride at a concentration of 5 to 200 mM, or contains efrapegrastim at a concentration of 11 to 66 mg / mL, sodium citrate at a concentration of 5 to 100 mM, mannitol at a concentration of 1 to 20% (w / v), polysorbate 80 at a concentration of 0.001 to 0.5% (w / v), and sodium chloride at a concentration of 5 to 200 mM.

[0097] The injection volume of the liquid formulation of the present invention may be adjusted appropriately to minimize irritation / pain at the administration site or patient discomfort. For example, the liquid formulation may have an injection volume of 0.2 to 1.2 mL.

[0098] Yet another aspect provides an article of manufacture comprising the liquid formulation.

[0099] In another embodiment herein, an article of manufacture is provided that contains a drug product and provides instructions for its use. The article of manufacture includes a container. Suitable containers can include, for example, bottles, vials, syringes, and test tubes. The container can also be formed from a variety of materials, such as glass, plastic, or metal. EXAMPLES

[0100] The present invention will be described in more detail below through examples. However, these examples are merely for illustrative purposes and the scope of the present invention is not limited to these examples.

[0101] Example 1. Analysis of patient friendly injectable liquid formulations In this embodiment, various variables of the above-mentioned manufactured formulations that affect patients when administered are utilized to derive a patient-friendly injection formulation of the final liquid formulation. For this purpose, as described above, the optimum value of osmotic pressure was determined to be 100 to 1,000 mOsm / kg, or preferably 200 to 1,000 mOsm / kg, based on the description in "Tolerability of hypertonic injectables, Wei Wang, International Journal of Pharmaceutics 490 (2015) 308-315" and "Tonicity Agents Clarity - American Pharmacists Association" and applied to the formulation of the present invention. The optimum value of maximum gliding force was calculated to be 5N or less, based on the description in "Development of Syringeability Guide for Subcutaneous Protein Formulations, L. Joseph et al., Pfizer Global Research & Development, 2010".

[0102] Considering the mutual complementarity between these values, a parameter was introduced and shown in the following Equation 1. When the following Equation 1 is satisfied, it can be known that a liquid formulation with high patient friendliness can be produced, and the result value of Equation 1 was named the patient friendly index.

[0103] Formula 1 Patient friendly index=Osm(mOsm / kg) / 100+MGF(N) Osm: Osmotic pressure of liquid preparations MGF: Maximum gliding force when administering liquid formulations at a velocity of 2.835 mm / s with a 29-cage syringe

[0104] The patient affinity index was determined to be 3 to 10, taking into consideration the values ​​of the appropriate osmotic pressure and the appropriate maximum gliding force. Considering that the maximum gliding force is at least 1N or more in most cases, when the osmotic pressure of the liquid preparation exceeds 1,000mOsm / kg, the patient affinity index exceeds 10, making patient-friendly injection difficult. Also, considering that the maximum gliding force is at least 1N or more in most cases, when the osmotic pressure is less than 200mOsm / kg, the patient affinity index does not exceed 3, making patient-friendly injection difficult. In other words, when the patient affinity index is 3 to 10, it is considered to be an excellent patient-friendly injection preparation.

[0105] Therefore, liquid formulations falling within the range of the patient affinity index have low viscosity, low gliding force, and appropriate osmotic pressure ranges that take into account the high concentration protein formulations, and are therefore expected to be able to be injected with patient affinity while maintaining high formulation stability by solving the technical problems mentioned above.

[0106] Example 2. Stability analysis of liquid formulations The stability of the high-concentration protein formulation was measured based on the residual rate after a 4-week storage test. Specifically, to measure the residual rate of the liquid formulation, the residual rate of efrapegrastim was measured by RP-HPLC and SE-HPLC after a 4-week storage test under accelerated conditions (25±2°C / 60±5% RH). The residual rate of efrapegrastim in the liquid formulation at the nth week was calculated by Equation 2. Equation 2 Residual rate at nth week (%) = purity at nth week / initial purity x 100

[0107] Purity is the relative proportion of the main peak by HPLC.

[0108] HPLC was performed using an Agilent 1200 series, and RP-HPLC was performed using a Phenomenex J upiter C4 column at 60°C. A 2-eluent linear gradient system with a flow rate of 1.0 mL / min was used, with mobile phase A being 20% ​​acetonitrile containing 0.1% trifluoroacetic acid, and mobile phase B being 80% acetonitrile containing 0.1% trifluoroacetic acid. After initial stabilization with 76% mobile phase A and 24% mobile phase B for a minimum of 1 hour, the linear gradient system was used with 24 to 60% mobile phase B for 0 to 15 minutes, 60 to 73% mobile phase B for 15 to 48 minutes, and 73 to 100% mobile phase B for 48 to 75 minutes. Then, re-equilibration was performed with 24% mobile phase B for 75 to 85 minutes. The sample injection amount was set to 20 μg, the detector was set to 214 nm wavelength, and the whole process was controlled by Agilent Chemstation software.

[0109] SE-HPLC was performed at room temperature using a Shodex Protein KW-803 column. An isocratic gradient system with a flow rate of 0.6 mL / min was used, and the mobile phase consisted of 50 mM sodium phosphate, 150 mM sodium chloride, and 5% isopropyl alcohol. After a minimum of 1 hour of stabilization, the measurement was performed for 60 minutes, the sample injection amount was set to 20 μg, the detector was set to 214 nm wavelength, and the entire process was controlled by Agilent Chemstation software.

[0110] Taking into consideration the physicochemical properties of efrapegrastim and common knowledge in the field of protein formulations, it was determined that the drug was stable if the residual rate of efrapegrastim was maintained as follows under accelerated conditions (25±2°C / 60±5% RH) after a 4-week storage test: Maintained at 95% or higher: Stable Maintains 97% or more: Excellent stability Maintains over 98%: Has excellent stability

[0111] Production Example 1-46: Production of a liquid preparation containing efrapegrastim A liquid formulation containing high-concentration efrapegrastim was designed, which is patient-friendly and exhibits dosage form stability as presented in Examples 1 and 2.

[0112] Liquid preparations of Production Examples 1 to 36 were manufactured, which were expected to have dosage form stability and patient-friendly injection based on simulation predictions. In addition, liquid preparations of Production Examples 37 to 39 were manufactured by a conventional manufacturing method in order to know the dosage form stability when a high concentration of a polysorbate-based nonionic surfactant is contained. Furthermore, liquid preparations of Production Examples 40 to 43 were manufactured, which are expected to cause discomfort to patients or have problems in terms of dosage form stability, and liquid preparations of Production Examples 44 to 46 were manufactured in order to confirm the change due to the concentration of the polysorbate-based nonionic surfactant as a factor affecting the residual rate of efrapegrastim, which is the main factor of dosage form stability.

[0113] 1. Preparation Examples 1 to 36 A liquid formulation containing efrapegrastim was prepared as follows.

[0114] First, a liquid formulation was prepared with a composition of 22 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% mannitol, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80. Then, for sample pretreatment, an SQ purification column (Source 15Q, GE Healthcare) was used to remove polysorbate 80 from the liquid formulation prepared above. Then, only the most important fraction was collected from the purification profile. Next, buffer exchange was performed on the pretreated liquid formulation using a filtration method. Specifically, buffer exchange was performed five times in total using a VivaSpin 20 (Sartorius) at 3,700 rpm for 1 hour in a buffer that did not contain polysorbate 80. The buffer-exchanged liquid formulation was then concentrated to about 2 to 3 times the target concentration. Considering the final volume and target concentration, a buffer not containing polysorbate 80 was added to the concentrated liquid formulation. A polysorbate 80 stock concentrated 100 times more than the target concentration was used to spike the liquid formulation so that the final polysorbate concentration (actual concentration) was 0.005% (w / v), thereby producing a liquid formulation containing polysorbate 80 at a final concentration of 0.005% (w / v). In Preparation Examples 2 to 37, liquid formulations with different compositions from the preparation in Preparation Example 1 were produced by the same method as Preparation Example 1.

[0115] That is, the compositions of the liquid preparations of Preparation Examples 1 to 36 are as follows.

[0116] [Production Example 1] 22 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) mannitol, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0117] [Production Example 2] 11 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) mannitol, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0118] [Production Example 3] 44 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) mannitol, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0119] [Production Example 4] 66 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) mannitol, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0120] [Production Example 5] 22 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 1% (w / v) mannitol, 10 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0121] [Production Example 6] 22 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 3% (w / v) mannitol, 50 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0122] [Production Example 7] 22 mg / mL efrapegrastim, 20 mM sodium acetate (pH 5.5), 5% (w / v) sorbitol, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0123] [Production Example 8] 44 mg / mL efrapegrastim, 20 mM sodium acetate (pH 5.5), 5% (w / v) sorbitol, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0124] [Production Example 9] The solution contained 66 mg / mL efrapegrastim, 20 mM sodium acetate (pH 5.5), 5 (w / v) sorbitol, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0125] [Production Example 10] 22 mg / mL efrapegrastim, 20 mM sodium acetate (pH 5.5), 5% (w / v) sucrose, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0126] [Production Example 11] 44 mg / mL efrapegrastim, 20 mM sodium acetate (pH 5.5), 5% (w / v) sucrose, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0127] [Production Example 12] 66 mg / mL efrapegrastim, 20 mM sodium acetate (pH 5.5), 5% (w / v) sucrose, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0128] [Production Example 13] 22 mg / mL efrapegrastim, 20 mM sodium acetate (pH 5.5), 3% (w / v) proline, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0129] [Production Example 14] 44 mg / mL efrapegrastim, 20 mM sodium acetate (pH 5.5), 3% (w / v) proline, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0130] [Production Example 15] 66 mg / mL efrapegrastim, 20 mM sodium acetate (pH 5.5), 3% (w / v) proline, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0131] [Production Example 16] 22 mg / mL efrapegrastim, 20 mM histidine (pH 5.5), 5% (w / v) sorbitol, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0132] [Production Example 17] 44 mg / mL efrapegrastim, 20 mM histidine (pH 5.5), 5% (w / v) sorbitol, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0133] [Production Example 18] 66 mg / mL efrapegrastim, 20 mM histidine (pH 5.5), 5% (w / v) sorbitol, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0134] [Production Example 19] 22 mg / mL efrapegrastim, 20 mM histidine (pH 5.5), 5% (w / v) sucrose, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0135] [Manufacturing Example 20] 44 mg / mL efrapegrastim, 20 mM histidine (pH 5.5), 5% (w / v) sucrose, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0136] [Production Example 21] 66 mg / mL efrapegrastim, 20 mM histidine (pH 5.5), 5% (w / v) sucrose, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0137] [Production Example 22] 22 mg / mL efrapegrastim, 20 mM histidine (pH 5.5), 3% (w / v) proline, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0138] [Production Example 23] 44 mg / mL efrapegrastim, 20 mM histidine (pH 5.5), 3% (w / v) proline, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0139] [Production Example 24] 66 mg / mL efrapegrastim, 20 mM histidine (pH 5.5), 3% (w / v) proline, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0140] [Production Example 25] 22 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) mannitol, 20 mM sodium phosphate, and 0.01% (w / v, final concentration) polysorbate 80.

[0141] [Production Example 26] 44 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) mannitol, 20 mM sodium phosphate, and 0.01% (w / v, final concentration) polysorbate 80.

[0142] [Production Example 27] 66 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) mannitol, 20 mM sodium phosphate, and 0.01% (w / v, final concentration) polysorbate 80.

[0143] [Production Example 28] 22 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) mannitol, 25 mM arginine, 20 mM histidine, and 0.2% (w / v, final concentration) polysorbate 80.

[0144] [Production Example 29] 44 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) mannitol, 25 mM arginine, 20 mM histidine, and 0.2% (w / v, final concentration) polysorbate 80.

[0145] [Example 30] 66 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) mannitol, 25 mM arginine, 20 mM histidine, and 0.2% (w / v, final concentration) polysorbate 80.

[0146] [Production Example 31] 22 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 3% (w / v) proline, 150 mM sodium chloride, and 0.01% (w / v, final concentration) polysorbate 20.

[0147] [Manufacturing Example 32] 44 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 3% (w / v) proline, 150 mM sodium chloride, and 0.01% (w / v, final concentration) polysorbate 20.

[0148] [Manufacturing Example 33] 66 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 3% (w / v) proline, 150 mM sodium chloride, and 0.01% (w / v, final concentration) polysorbate 20.

[0149] [Manufacture Example 34] 22 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), and 125 mM sodium chloride

[0150] [Manufacturing Example 35] 44 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), and 125 mM sodium chloride

[0151] [Manufacturing Example 36] 66 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), and 125 mM sodium chloride

[0152] 2. Preparation Examples 37 to 39 The liquid formulation of Preparation Example 37 was prepared with reference to Korean Patent Registration No. 10-1340710, except that efrapegrastim containing an hG-CSF mutant having an amino acid sequence different from the hG-CSF mutant amino acid sequence described in Korean Patent Registration No. 10-1340710 was used as the active ingredient, and was prepared at different concentrations.

[0153] Specifically, a liquid formulation was prepared using 22 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) mannitol, 150 mM sodium chloride, and 0.005% (w / v) (concentration before concentration process) polysorbate 80. Then, buffer exchange was immediately performed using a filtration method without pretreatment of the sample. Specifically, buffer exchange was performed a total of five times for 1 hour at 3,700 rpm using a VivaSpin 20 (Sartorius) in a buffer containing polysorbate 80. The buffer-exchanged liquid formulation was then concentrated to about twice the target concentration. Taking into account the final volume and target concentration, the final liquid formulation was prepared by diluting with a buffer containing all excipients. In the liquid formulation of the above-mentioned Manufacturing Example 37, the above-mentioned concentration of polysorbate 80 is the concentration before the concentration process, and in the following Test Example, polysorbate 80 exceeding a minimum of 5% (w / v (final concentration)) was applied by the concentration process.

[0154] That is, the composition of the liquid preparation of Production Example 37 is as follows.

[0155] [Production Example 37] 22 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) mannitol, 150 mM sodium chloride, and 5% (w / v, final concentration) polysorbate 80.

[0156] In addition, in Preparation Examples 38 and 39, liquid preparations having different compositions from the preparation of Preparation Example 37 were prepared by the same method as Preparation Example 37, as described below.

[0157] [Production Example 38] 31.5 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) mannitol, 150 mM sodium chloride, and 5% (w / v, final concentration) polysorbate 80.

[0158] [Manufacturing Example 39] 40 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) mannitol, 150 mM sodium chloride, and 5% (w / v, final concentration) polysorbate 80.

[0159] 3. Manufacturing Examples 40 to 43 A liquid preparation containing efrapegrastim was produced by the same method as in Preparation Example 1, except that the composition was different from that of the preparation in Preparation Example 1 described above.

[0160] That is, the compositions of the liquid preparations of Preparation Examples 40 to 43 are as follows.

[0161] [Example 40] 22 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), and 0.005% (w / v, final concentration) polysorbate 80

[0162] [Manufacture Example 41] 22 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 10% (w / v) mannitol, 500 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0163] [Example 42] 22 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 20% (w / v) glucose, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0164] [Manufacture Example 43] 22 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) gelatin, 150 mM sodium chloride, and 0.005% (w / v, final concentration) polysorbate 80.

[0165] 4. Preparation Examples 44 to 46 A liquid preparation containing efrapegrastim was produced by the same method as in the above-mentioned Preparation Example 1, but with a different composition from the preparation in the above-mentioned Preparation Example 1.

[0166] That is, the compositions of the liquid preparations of Preparation Examples 44 to 46 are as follows.

[0167] [Manufacture Example 44] 22 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) mannitol, 150 mM sodium chloride, and 0.5% (w / v, final concentration) polysorbate 80.

[0168] [Example 45] 22 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) mannitol, 150 mM sodium chloride, and 2.5% (w / v, final concentration) polysorbate 80.

[0169] [Example 46] 22 mg / mL efrapegrastim, 20 mM sodium citrate (pH 5.5), 5% (w / v) mannitol, 150 mM sodium chloride, and 5% (w / v, final concentration) polysorbate 80.

[0170] Test Example 1. Evaluation of residual rate and patient affinity index of liquid preparations The liquid preparations of Manufacturing Examples 1 to 6 and 37 to 43 were subjected to osmotic pressure measurement, conductivity measurement, viscosity measurement, and maximum gliding force measurement, and then the remaining rate of efrapegrastim after a 4-week storage test under accelerated conditions (25±2°C / 60±5% RH) was measured.

[0171] 1. Liquid formulation test of Production Example 1 The osmotic pressure of the liquid preparation of Production Example 1 was measured using an automatic osmometer (Gonotec, OSMOMAT auto) and was found to be 645 mOsm / kg.

[0172] The conductivity of the liquid preparation of Production Example 1 was measured at room temperature using a conductivity meter (Compact Conductivity Meter EC33, LAQUAtwin, Horiba) according to the manufacturer's instructions, and the conductivity of the liquid preparation of Production Example 1 was 14.37 mS / cm.

[0173] The viscosity of the liquid preparation of Preparation Example 1 was measured at room temperature (20 to 25° C.) using a Vibration viscometer (A&D, SV-1A) and was 1.86 cP.

[0174] In addition, the maximum gliding force of the liquid formulation of Manufacturing Example 1 was measured using a Rheo Meter (Sun scientific, Compac-100) with a 29-cage syringe (length 22.68 mm, based on 400 μL) when the liquid formulation was administered at a speed of 4.725 mm / s (500 μL / 6 sec) and 2.835 mm / s (500 μL / 10 sec). The values ​​were confirmed using Reology Data System Ver 3.0, and the values ​​were 3.099 N and 2.099 N, respectively.

[0175] In addition, in order to measure the residual rate of the liquid preparation of Production Example 1, after a 4-week storage test under accelerated conditions (25±2°C / 60±5% RH), the residual rate of efrapegrastim was measured by RP-HPLC and SE-HPLC.

[0176] The residual rates of efrapegrastim measured by RP-HPLC and SE-HPLC are shown in Table 1 below.

[0177] [Table 1]

[0178] 2. Liquid formulation test of Production Example 2 In the same manner as in Test Example 1 described above, the osmotic pressure, conductivity, viscosity, maximum gliding force and residual rate of the preparation were measured.

[0179] The formulation of Preparation Example 2 had an osmotic pressure of 643.3 mOsm / kg, a conductivity of 14.80 mS / cm, and a viscosity value of 1.39 cP at room temperature (20 to 25° C.). The maximum gliding force of the formulation of Preparation Example 2 was 2.648 N (4.725 mm / s speed and 29 G syringe) and 2.285 N (2.835 mm / s speed and 29 G syringe).

[0180] Furthermore, the residual rate of the preparation of Production Example 2 under accelerated conditions (25±2° C. / 60±5% RH) is shown in Table 2 below.

[0181] [Table 2]

[0182] 3. Liquid formulation test of Production Example 3 In the same manner as in Test Example 1 described above, the osmotic pressure, conductivity, viscosity, maximum gliding force and residual rate of the preparation were measured.

[0183] The formulation of Preparation Example 3 had an osmotic pressure of 657.7 mOsm / kg, a conductivity of 13.45 mS / cm, and a viscosity value of 2.32 cP at room temperature (20 to 25° C.). The maximum gliding force of the formulation of Preparation Example 3 was 3.177 N (4.725 mm / s speed and 29 G syringe) and 2.775 N (2.835 mm / s speed and 29 G syringe).

[0184] Furthermore, the residual rate of the preparation of Preparation Example 3 under accelerated conditions (25±2° C. / 60±5% RH) is shown in Table 3 below.

[0185] [Table 3]

[0186] 4. Liquid formulation test of Production Example 4 In the same manner as in Test Example 1 described above, the osmotic pressure, conductivity, viscosity, maximum gliding force and residual rate of the preparation were measured.

[0187] The formulation of Preparation Example 4 had an osmotic pressure of 679 mOsm / kg, a conductivity of 12.67 mS / cm, and a viscosity value of 3.54 cP at room temperature (20 to 25° C.). The maximum gliding force of the formulation of Preparation Example 4 was 3.815 N (4.725 mm / s speed and 29 G syringe) and 2.716 N (2.835 mm / s speed and 29 G syringe).

[0188] Furthermore, the residual rate of the preparation of Preparation Example 4 under accelerated conditions (25±2° C. / 60±5% RH) is shown in Table 4 below.

[0189] [Table 4]

[0190] 5. Liquid formulation test of Production Example 5 In the same manner as in Test Example 1 described above, the osmotic pressure, conductivity, viscosity, maximum gliding force and residual rate of the preparation were measured.

[0191] The formulation of Preparation Example 5 had an osmotic pressure of 135.3 mOsm / kg, a conductivity of 4.27 mS / cm, and a viscosity value of 1.40 cP at room temperature (20 to 25° C.). The maximum gliding force of the formulation of Preparation Example 5 was 2.442 N (4.725 mm / s speed and 29 G syringe) and 1.657 N (2.835 mm / s speed and 29 G syringe).

[0192] Furthermore, the residual rate of the preparation of Preparation Example 5 under accelerated conditions (25±2° C. / 60±5% RH) is shown in Table 5 below.

[0193] [Table 5]

[0194] 6. Liquid formulation test of Production Example 6 In the same manner as in Test Example 1 described above, the osmotic pressure, conductivity, viscosity, maximum gliding force and residual rate of the preparation were measured.

[0195] The formulation of Preparation Example 6 had an osmotic pressure of 334.7 mOsm / kg, a conductivity of 7.49 mS / cm, and a viscosity value of 1.50 cP at room temperature (20 to 25° C.). The maximum gliding force of the formulation of Preparation Example 6 was 2.746 N (4.725 mm / s speed and 29 G syringe) and 1.285 N (2.835 mm / s speed and 29 G syringe).

[0196] Furthermore, the residual rate of the formulation of Production Example 6 under accelerated conditions (25±2° C. / 60±5% RH) is shown in Table 6 below.

[0197] [Table 6]

[0198] 7. Liquid formulation test of Manufacturing Example 37 As in the above-mentioned Test Example 1, the residual rate of the formulation of Preparation Example 37 under accelerated conditions (25±2° C. / 60±5% RH) is shown in Table 7 below.

[0199] [Table 7]

[0200] 8. Liquid formulation test of Manufacturing Example 38 As in the above-mentioned Test Example 1, the residual rate of the formulation of Preparation Example 38 under accelerated conditions (25±2° C. / 60±5% RH) is shown in Table 8 below.

[0201] [Table 8]

[0202] 9. Liquid formulation test of Manufacturing Example 39 As in the above-mentioned Test Example 1, the residual rate of the formulation of Preparation Example 39 under accelerated conditions (25±2° C. / 60±5% RH) is shown in Table 9 below.

[0203] [Table 9]

[0204] 10. Liquid formulation test of Manufacturing Example 40 In the same manner as in Test Example 1 described above, the osmotic pressure, conductivity, viscosity, maximum gliding force and residual rate of the preparation were measured.

[0205] The formulation of Preparation Example 40 had an osmotic pressure of 59.3 mOsm / kg, a conductivity of 3.45 mS / cm, and a viscosity value of 1.26 cP at room temperature (20 to 25° C.). The maximum gliding force of the formulation of Preparation Example 40 was 2.471 N (4.725 mm / s speed and 29 G syringe) and 1.834 N (2.835 mm / s speed and 29 G syringe).

[0206] Furthermore, the residual rate of the preparation of Preparation Example 40 under accelerated conditions (25±2° C. / 60±5% RH) is shown in Table 10 below.

[0207] [Table 10]

[0208] 11. Liquid formulation test of Manufacturing Example 41 In the same manner as in Test Example 1 described above, the osmotic pressure, conductivity, viscosity, maximum gliding force and residual rate of the preparation were measured.

[0209] The osmotic pressure of the preparation of Preparation Example 41 was 1721.7 mOsm / kg, the conductivity was 31.20 mS / cm, and the viscosity value at room temperature (20 to 25° C.) was 2.02 cP. The maximum gliding force of the preparation of Preparation Example 41 was 2.952 N (4.725 mm / s speed and 29 G syringe) and 1.922 N (2.835 mm / s speed and 29 G syringe).

[0210] Furthermore, the residual rate of the preparation of Preparation Example 41 under accelerated conditions (25±2° C. / 60±5% RH) is shown in Table 11 below.

[0211] [Table 11]

[0212] 12. Liquid formulation test of Manufacturing Example 42 In the same manner as in Test Example 1 described above, the osmotic pressure, conductivity, viscosity, maximum gliding force and residual rate of the preparation were measured.

[0213] The formulation of Preparation Example 42 had an osmotic pressure of 1964.3 mOsm / kg, a conductivity of 10.56 mS / cm, and a viscosity value of 2.66 cP at room temperature (20 to 25° C.). The maximum gliding force of the formulation of Preparation Example 42 was 7.482 N (4.725 mm / s speed and 29 G syringe) and 5.688 N (2.835 mm / s speed and 29 G syringe).

[0214] Furthermore, the residual rate of the formulation of Preparation Example 42 under accelerated conditions (25±2° C. / 60±5% RH) is shown in Table 12 below.

[0215] [Table 12]

[0216] 13. Liquid formulation test of Manufacturing Example 43 In the same manner as in Test Example 1 described above, the osmotic pressure, conductivity, viscosity, maximum gliding force and residual rate of the preparation were measured.

[0217] The formulation of Preparation Example 43 had an osmotic pressure of 316.3 mOsm / kg, a conductivity of 13.38 mS / cm, and a viscosity value of 64.9 cP at room temperature (20 to 25° C.). The maximum gliding force of the formulation of Preparation Example 43 was 7.482 N (4.725 mm / s speed and 29 G syringe) and 5.688 N (2.835 mm / s speed and 29 G syringe).

[0218] Furthermore, the residual rate of the preparation of Preparation Example 43 under accelerated conditions (25±2° C. / 60±5% RH) is shown in Table 13 below.

[0219] [Table 13]

[0220] 14. Patient Affinity Index Evaluation In order to confirm whether the liquid formulations prepared above conform to Equation 1, the patient affinity index of each formulation was calculated, and the results are shown in Table 14 below.

[0221] [Table 14]

[0222] As shown in Tables 1 to 13 above, in the case of Production Examples 37 to 39, which contain high concentrations of polysorbate-based nonionic surfactants, some data show problems with the residual rate. Also, the liquid preparation of Production Example 43 shows a serious residual rate problem.

[0223] Also, as shown in Table 14, in the case of Preparation Example 43, although it was within the range of the appropriate osmotic pressure, the maximum gliding force was large and still caused pain to the patient, and in the case of Preparation Example 41, although it was within the range of the appropriate maximum gliding force, it was still possible to cause pain to the patient due to the high osmotic pressure. In contrast, it was found that the liquid preparation according to one embodiment was within the range of the appropriate osmotic pressure, had a maximum gliding force of 5N or less, and had a patient affinity index within the appropriate range of 3 to 10. Therefore, it was found that in the case of the liquid preparation according to one embodiment, in which the patient affinity value, the main factors of which are osmotic pressure and maximum gliding force, is 3 to 10, it is possible to administer the desired liquid preparation without inducing pain to the patient.

[0224] Test Example 2. Evaluation of the residual rate of liquid preparations depending on the concentration of polysorbate nonionic surfactant For the liquid preparations of Production Examples 1 and 44 to 46, the residual efrapegrastim rate after a 4-week storage test under accelerated conditions (25±2°C / 60±5% RH) was measured using the same method as in Test Example 1 described above.

[0225] 1. Liquid formulation test of Production Example 1 The residual rate under accelerated conditions for the preparation of Preparation Example 1 is shown in Table 15 below.

[0226] [Table 15]

[0227] 2. Liquid formulation test of Manufacturing Example 44 The residual rate under accelerated conditions for the preparation of Preparation Example 44 is shown in Table 16 below.

[0228] [Table 16]

[0229] 3. Liquid formulation test of Manufacturing Example 45 The residual rate under accelerated conditions for the preparation of Preparation Example 45 is shown in Table 17 below.

[0230] [Table 17]

[0231] 4. Liquid formulation test of Manufacturing Example 46 The residual rate under accelerated conditions for the preparation of Preparation Example 46 is shown in Table 18 below.

[0232] [Table 18]

[0233] Figures 1 and 2 show the results of confirming the change in the residual rate of efrapegrastim depending on the concentration of the surfactant in the liquid formulation according to one embodiment. As shown in Figures 1 and 2, the concentration of the surfactant shows a high correlation with the residual rate of the liquid formulation according to one embodiment, and such experimental results indicate that the concentration of the surfactant is a major factor affecting the residual rate of a liquid formulation containing a high concentration of efrapegrastim.

[0234] From the above results, it can be seen that the liquid formulation according to one embodiment has a different ingredient content and manufacturing method from existing liquid formulations, and as a result, while containing a high concentration of active ingredient, it not only has high dosage form stability (e.g., residual rate), but also has a high patient affinity value and can be administered without inducing pain to the patient.

[0235] It should be understood that the embodiments described herein are to be considered in an illustrative sense only and not for the purpose of limitation. The description of a feature or aspect in each embodiment is typically intended to be applicable to other similar features or aspects in other embodiments. Although one or more embodiments have been described with reference to the drawings, those skilled in the art will understand that various changes in form and details may be made therein without departing from the spirit and scope of the present disclosure as defined by the following claims.

Claims

1. A liquid formulation containing efrapegrastim and a buffering agent, It contains efrapegrastim in concentrations of 11 mg / mL to 66 mg / mL. The patient-friendly index (PF) defined by formula 1 below is 10 or less, and formula 1 PF index=Osm (mOsm / kg) / 100+MGF(N) In Equation 1, Osm is the osmotic pressure of the liquid formulation, and MGF is the maximum gliding force when the liquid formulation is administered at a rate of 2.835 mm / s using a 29-cage syringe. The osmotic pressure is between 100 mOsm / kg and 800 mOsm / kg. When the liquid formulation is administered at a speed of 2.835 mm / s using a 29-cage syringe, the maximum sliding force is 5 N or less, or when administered at a speed of 4.725 mm / s, the maximum sliding force is 7 N or less. After storage at 23 to 27°C and 55 to 65% relative humidity for four weeks, the remaining percentage of efrapegrastim measured by reverse-phase high-performance liquid chromatography (RP-HPLC) and size exclusion chromatography (SE-HPLC) was 95% or higher. The concentration of the buffering material is 5 to 100 mM. The buffering substance is citric acid and / or citrate, The aforementioned liquid formulation contains a polysorbate-based nonionic surfactant, The final concentration of the polysorbate-based nonionic surfactant is 0.001 to 5% (w / v) relative to the liquid formulation. Efrapegrastim liquid formulation.

2. The efrapegrastim liquid formulation according to claim 1, wherein the conductivity of the liquid formulation is 15 mS / cm or less.

3. The efrapegrastim liquid formulation according to claim 1 or 2, wherein the residual rate is 98% or more.

4. The liquid formulation is the efrapegrastim liquid formulation according to any one of claims 1 to 3, wherein the liquid formulation has a viscosity of 4 cP or less at room temperature of 20°C to 25°C.

5. The liquid formulation of efrapegrastim according to any one of claims 1 to 4, wherein the liquid formulation contains a stabilizer.

6. The efrapegrastim liquid formulation according to claim 5, wherein the stabilizer contains mannitol.

7. The efrapegrastim liquid formulation according to claim 6, wherein the concentration of mannitol is 1 to 20% (w / v) relative to the liquid formulation.

8. The efrapegrastim liquid formulation according to claim 1, wherein the polysorbate-based nonionic surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

9. The efrapegrastim liquid formulation according to claim 8, wherein the final concentration of the polysorbate-based nonionic surfactant is 0.001 to 0.5% (w / v) relative to the liquid formulation.

10. The efrapegrastim liquid formulation according to any one of claims 1 to 9, wherein the pH of the liquid formulation is 4 to 8.

11. An efrapegrastim liquid formulation according to any one of claims 1 to 10, further comprising a tonic modifier.

12. The efrapegrastim liquid formulation according to claim 11, wherein the tonic modifier is sodium chloride.

13. The efrapegrastim liquid formulation according to claim 11, wherein the concentration of the tonic modifier is 5 to 200 mM.

14. The liquid formulation of efrapegrastim according to claim 1, wherein the liquid formulation comprises efrapegrastim in a concentration of 11 to 66 mg / mL, sodium citrate in a concentration of 5 to 100 mM, polysorbate 80 in a concentration of 0.001 to 0.5% (w / v), mannitol in a concentration of 1 to 20% (w / v), and sodium chloride in a concentration of 5 to 200 mM.

15. An efrapegrastim liquid formulation according to any one of claims 1 to 14, used to prevent, alleviate or treat neutropenia in patients with reduced white blood cell production.

16. The efrapegrastim liquid formulation according to claim 15, wherein the neutropenia is severe chronic neutropenia or febrile neutropenia.

17. The efrapegrastim liquid formulation according to claim 15 or 16, wherein the efrapegrastim liquid formulation is administered after the patient has been treated with adjuvant therapy or prior chemotherapy.

18. The liquid efrapegrastim formulation according to any one of claims 15 to 17, wherein the liquid efrapegrastim formulation is administered to the patient within one to five days after treatment with adjuvant therapy or prior chemotherapy.

19. The efrapegrastim liquid formulation according to claim 18, wherein the adjuvant therapy or prior chemotherapy is a combination of docetaxel and cyclophosphamide.

20. The efrapegrastim liquid formulation according to any one of claims 15 to 19, wherein the second dose of the efrapegrastim liquid formulation is administered to the patient between 15 and 25 days after the first dose of the efrapegrastim liquid formulation has been administered.

21. The efrapegrastim liquid formulation according to any one of claims 15 to 20, used in a unit dose form selected from 25 μg / kg, 50 μg / kg, 100 μg / kg, or 200 μg / kg.

22. The liquid formulation of efrapegrastim according to any one of claims 15 to 21, provided as a liquid formulation of 13.2 mg of efrapegrastim in a 0.6 mL dose volume.

23. The efrapegrastim liquid formulation according to any one of claims 15 to 22, wherein the efrapegrastim liquid formulation is used in combination with a second agent.

24. The efrapegrastim liquid formulation according to claim 23, wherein the second activating agent is an anticancer agent.

25. The efrapegrastim liquid formulation according to any one of claims 15 to 24, wherein the efrapegrastim liquid formulation is administered to the patient within 6 hours, 5 hours, 2 hours, or 1 hour after the completion of chemotherapy.

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