Protein liquid preparation and method for producing the 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 protein liquid preparation and a method for producing the same.

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

Background Art

[0003] Granulocyte colony-stimulating factor (G-CSF) is a cytokine that instructs 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. The molecular weight is 18,000 to 19,000 daltons, and it is a glycoprotein with an isoelectric point (pI) of 6.1 (the pI value is 5.5 to 6.1 depending on the degree of glycosylation).

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

[0005] Furthermore, in terms of the commercial viability and efficiency of pharmaceutical protein formulations containing proteins such as G-CSF as described above, dosage form stability can also be overcome by incorporating additional molecules into the dosage form. Protein stability can be improved by including excipients that interact with the protein in solution, keeping it stable, soluble, and non-aggregated. For example, salt compounds and other inonic species are additives to protein formulations. They help prevent protein denaturation by binding to the protein 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), when used as formulation additives, have been shown to reduce alteration of the secondary structure of proteins. Other examples of commonly used additives include polyalcohols such as glycerol and sugars, and nonionic (e.g., Tween, Pluronic) surfactants.

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

[0007] Furthermore, pharmacochemical formulations of proteins must be formulated in high concentrations to improve their therapeutic effect. High-concentration protein formulations are advantageous for therapeutic use because they allow for smaller volume sizes and are more economical to package and store. However, the development of high-concentration protein formulations presents numerous challenges, including manufacturing, 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)). Consequently, side effects that do not appear in low-concentration formulations, such as unnatural protein aggregation and microparticle formation, may appear in high-concentration protein formulations even when additives that provided beneficial effects in low-concentration formulations are used. In addition, the high viscosity of high-concentration proteins can interfere with filtration-based manufacturing processes, potentially causing pain and further side effects to the patient during injection, and resulting in lower patient affinity. Therefore, pharmaceutical protein formulations typically require careful balancing of components and concentrations to improve protein stability, patient affinity, and therapeutic requirements while limiting any adverse effects.

[0008] Therefore, there is a need to develop protein formulations containing high concentrations of non-natural proteins with a high tendency to aggregate, which are not only useful for therapeutic use but also advantageous in terms of solubility and stability, and are patient-friendly. [Overview of the project] [Problems that the invention aims to solve]

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

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

[0011] Another embodiment provides a manufactured article containing the liquid formulation. [Means for solving the problem]

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

[0013] In some examples, the liquid formulation has an conductivity of 15 mS / cm or less. In some examples, the residual rate of efrapegrastim is 98% or more.

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

[0015] In some examples, the concentration of the buffer is approximately 5 mM to approximately 100 mM. In some examples, the buffer is citric acid and / or citrate.

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

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

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

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

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

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

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

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

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

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

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

[0027] Another aspect provides a method for manufacturing the liquid preparation.

[0028] Yet another aspect provides a manufactured article comprising the liquid preparation.

[0029] In yet another embodiment, the Disclosure provides a method for preventing, alleviating, or treating neutropenia in a patient with reduced leukocyte production, comprising administering to the patient a therapeutically effective dose of one of the liquid efrapegrastim formulations described herein.

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

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

[0032] In some examples, the second dose of the liquid efrapegrastim formulation is administered 15 to 25 days after the first dose of the liquid efrapegrastim formulation has been administered to the patient.

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

[0034] In some cases, the therapeutically effective dose was 13.2 mg of liquid efrapegrastim in a 0.6 mL dose.

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

[0036] In several cases, the liquid efrapegrastim formulation was administered to the patient within approximately 6 hours, 5 hours, 2 hours, and 1 hour after the completion of chemotherapy. [Effects 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 excellent solubility and stability, and has the effect of providing a liquid formulation that can be administered in a patient-friendly manner, reducing irritation / pain at the administration site or patient discomfort. [Brief explanation of the drawing]

[0038] [Figure 1] This is the result of confirming the change in the remaining rate of efrapegrastim depending on the concentration of the polysorbate-based nonionic surfactant in a liquid formulation according to one example, via reverse-phase high-performance liquid chromatography (RP-HPLC). [Figure 2] This is the result of confirming the change in the remaining rate of efrapegrastim depending on the concentration of the polysorbate-based nonionic surfactant in a liquid formulation according to one example, via size exclusion chromatography (SE-HPLC). [Modes for carrying out the invention]

[0039] The embodiments illustrated in the accompanying drawings will be referred to in detail, where similar reference numerals refer to similar elements throughout. In this regard, these embodiments may take different forms and should not be construed as being limited to the description herein. Accordingly, these embodiments will be described below with reference only to the drawings in order to illustrate aspects of this specification. As used herein, the term "and / or" includes any and all combinations of one or more of the enumerated items relating to it. When preceding a list of elements, expressions such as "at least one" modify the entire list of elements and not the individual elements of the list.

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

[0041] Eflapegrastim In this specification, the term "efrapegrastim" is the international common name (INN) for a sustained-release G-CSF conjugate containing recombinant human granulocyte-colony-stimulating factor (hG-CSF) variants (WHO Drug Information Volume 29, 2015). Efrapegrastim is also a conjugate in which a bioactive peptide, granulocyte-colony-stimulating factor, a biodegradable polymer, and an immunoglobulin Fc domain are linked together.

[0042] Furthermore, in this specification, useful immunoglobulin Fc has a sequence of human immunoglobulin Fc or its closely related analogues, and may be of animal origin such as cattle, goats, pigs, mice, rabbits, hamsters, rats, or guinea pigs. The immunoglobulin Fc region may also be derived from IgG, IgA, IgD, IgE, or IgM, or a combination thereof, or a hybrid thereof. Specifically, the immunoglobulin Fc region may be derived from IgG or IgM, which are most abundant in human blood, and more specifically, from IgG, which is known to improve the half-life of ligand-binding proteins. The immunoglobulin Fc can be produced by treating natural IgG with specific proteolytic enzymes, and can also be produced from cells transformed using recombinant technology. Specifically, the immunoglobulin Fc is recombinant human immunoglobulin Fc produced from E. coli (E. coli) transformants.

[0043] Furthermore, IgG can be divided into subclasses IgG1, IgG2, IgG3, and IgG4, and in the present invention, combinations thereof or hybrids thereof are also possible. Specifically, these are the IgG2 subclass and the IgG4 subclass, and more specifically, the Fc region of IgG4 which has little to no effector function, such as complement-dependent cytotoxicity (CDC). In other words, the immunoglobulin Fc region for drug carriers described herein is a non-glycosylated Fc region derived from human IgG4. Human-derived Fc regions are preferable to non-human-derived Fc regions which can act on antigens in the human body and cause undesirable immune responses, such as generating new antibodies against them.

[0044] The efrapegrastim used herein is produced by conjugating the hG-CSF variant with an immunoglobulin Fc region. The conjugation method used involves cross-linking the hG-CSF variant and the immunoglobulin Fc region using a non-peptide polymer, or by using recombinant technology to produce a fusion protein in which the hG-CSF variant and the immunoglobulin Fc region are linked. The non-peptide polymer used in cross-linking is selected from a group consisting of biodegradable polymers such as polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysacaryl phosphates, dextran, polyvinyl ethyl ether, polylactic acid (PLA) and polylactic-glycolic acid (PLGA), lipid polymers, chitins, hyaluronic acid, and combinations thereof. Derivatives already known in the art, and derivatives that can be readily produced at the level of the art, are also included in the scope of this specification.

[0045] The hG-CSF mutants described herein may be extracted from mammals or chemically synthesized. They may also be obtained from prokaryotes or eukaryotes transconditioned with DNA encoding the hG-CSF mutant using genetic engineering techniques, but the host can be Escherichia coli (e.g., E. coli), yeast (e.g., budding yeast (S. cerevisiae)), or mammalian cells (e.g., Chinese hamster ovary cells, monkey cells). Depending on the host used, the hG-CSF mutant expression product may or may not be glycosylated with mammalian or other eukaryotic carbohydrates. When expressed in prokaryotes, the hG-CSF mutant expression product also contains an initial methionine residue (position 1). hG-CSF mutants suitable for the present invention are also hG-CSF mutants produced using Escherichia coli as the host cell.

[0046] In one specific example, efrapegrastim contains recombinant human granulocyte colony-stimulating factor derivative 17,65 Ser-G-CSF, in which the 17th cysteine ​​and 65th proline residues of the natural G-CSF are substituted with serine and the 1st threonine residue is deleted. As mentioned above, the non-natural protein of efrapegrastim may offer further protein aggregation potential and any side effects compared to the natural protein or 17Ser-G-CSF. Such protein aggregation is a common problem in protein solutions and leads to an increase in protein concentration or viscosity. This specification provides means for achieving high-concentration, low-aggregation protein formulations. The formulations of this specification can achieve stable high concentrations of protein in solution and are also advantageous for therapeutic purposes.

[0047] Furthermore, the function and physiological activity of proteins such as polypeptides are determined by the protein's three-dimensional structure. If the functional part of the protein's three-dimensional structure differs, it will be unable to perform its intended specific function. For example, it is a well-known fact that even a single difference in the amino acid sequence can affect the protein's function if that amino acid corresponds to a functional site in the protein's three-dimensional structure and alters the structure of that site. In addition, in the field of pharmaceutical formulations, the most common discussion regarding protein formulations and peptide formulations concerns the physicochemical stability of the drug. In fact, the characteristics of the drug are crucial in determining the appropriate formulation for successful supply and stability. The initial stage in the development of protein-drug formulations involves complete characterization of the drug's properties and stability within different formulations, and for a typical technician in the art, this begins with considering the physicochemical properties of the protein, such as its isoelectric point, molecular weight, and overall amino acid composition (Jeffrery L. et al., 1994). In other words, for protein-stabilized formulations that exhibit different physicochemical properties from the natural protein, even if only a single amino acid sequence difference is present in the natural protein, as well as in different protein-drugs (e.g., IL-1β), a unique solution method for stability approaches is required.

[0048] The liquid formulation of the present invention has a technical feature in that it can exhibit high stability even under high concentration conditions with respect to efrapegrastim containing 17,65 Ser-G-CSF, which has further amino acid modifications compared to natural hG-CSF or 17Ser-G-CSF.

[0049] In this specification, the term "high concentration" means a dose that can increase the therapeutically advantageous effect. For example, high concentrations of efrapegrastim may also be present in formulations at concentrations of 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, or 20 mg / mL or higher. Specifically, high concentrations of efrapegrastim include 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, and 11 mg / mL. It may also be contained in the preparation at concentrations of 66 mg / mL or more, 12 mg / mL or more than 66 mg / mL, 13 mg / mL or more than 66 mg / mL, 14 mg / mL or more than 66 mg / mL, 15 mg / mL or more than 66 mg / mL, 16 mg / mL or more than 66 mg / mL, 17 mg / mL or more than 66 mg / mL, 18 mg / mL or more than 66 mg / mL, 19 mg / mL or more than 66 mg / mL, or 20 mg / mL or more than 66 mg / mL.

[0050] In this specification, the term “stable” may mean that a protein substantially retains its physical and / or chemical and / or biological stability during storage. Typically, a formulation is understood to be stable if, for a certain period of time under specific storage conditions, the loss of the active ingredient is less than a certain amount, e.g., less than 10%, less than 7%, less than 5%, less than 4%, or less than 3%.

[0051] Efrapegrastim concentration and remaining percentage As mentioned above, an increase in the concentration of efrapegrastim can negatively affect the survival rate. Furthermore, efrapegrastim, being a further mutant compared to the natural hG-CSF, can have an unpredictable effect on the survival rate. For this reason, the stability of the liquid formulation is also evaluated with protein drug aggregate formation as a major factor. Protein drugs form aggregates when subjected to shear stress or other physical or chemical environments, and such aggregate formation is a major component to consider in the development of liquid formulations because it affects bioavailability, such as reduced efficacy.

[0052] One specific example of a liquid formulation is one in which, when measured by RP-HPLC (reversed-phase high-performance liquid chromatography) or SE-HPLC (size exclusion-high-performance liquid chromatography) at 23 to 27°C and 55 to 65% RH (relative humidity), the remaining protein (e.g., ephrapegrastim) after a 4-week storage test is 95% or higher, 96% or higher, 97% or higher, or 98% or higher. In this case, the remaining percentage refers to the relative ratio of the purity at a specific point in time compared to the initial protein purity, and the n-week remaining percentage of protein (e.g., ephrapegrastim) in the liquid formulation can also be defined by formula 2. Formula 2 Survival rate at week n (%) = Purity value at week n / Initial purity value x 100

[0053] In one specific example, the RP-HPLC measurement was performed on a liquid formulation sample using an appropriate column (for example, a C4 column (particle size 5 μm, interior diameter x length: 4.6 mm x 250 mm)) under conditions of 40 to 80°C. To summarize the HPLC conditions, 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 was set to 214 nm.

[0054] In one specific example, the SE-HPLC measurement was performed on a liquid formulation sample using an appropriate column (for example, a Protein LW-803 column (particle size 5 μm, interior diameter x length: 8.0 mm x 300 mm)). To summarize the HPLC conditions, an isocratic gradient system with a flow rate of 0.3 to 1.2 ml / min (preferably 0.6 ml / min) can be used, and the mobile phase may contain 10 to 10 mM sodium phosphate, 50 to 300 mM sodium chloride, or 1 to 15% isopropyl alcohol. The detector may also be set to 214 nm.

[0055] For example, under the conditions described above, the liquid formulations of this specification maintain 95% or more, 96% or more, 97% or more or 98% or more of the initial purity of the protein drug in monomer form without forming aggregates or degradation products. In other words, under the conditions described above, 5% or less of the initial content of the protein drug in the liquid formulations of this specification, for example, 4% or less or 3% or less, is converted into aggregate form or degradation product form.

[0056] Generally, a formulation is considered stable if, after a 4-week storage test under accelerated conditions (25±2℃ / 60±5% RH), the remaining protein (e.g., efrapegrastim) is maintained at approximately 95%. A formulation is considered to have excellent stability if, after a 4-week storage test under accelerated conditions (25±2℃ / 60±5% RH), the remaining protein is maintained at 97% or higher. A formulation is considered to have very excellent stability if, after a 4-week storage test under accelerated conditions (25±2℃ / 60±5% RH), the remaining protein is maintained at 98% or higher.

[0057] While not bound by any particular theory, the liquid formulation described herein, by containing a high concentration of efrapegrastim, may increase the retention rate of efrapegrastim after long-term storage due to protein-protein interactions. Furthermore, the specific amino acid sequence of the hG-CSF variant may also influence the increase in retention rate, which is thought to be due to electrostatic bonding of charged amino acids and interactions between amino acid products resulting from their chemical structure.

[0058] For the stability of the formulation, in addition to efrapegrastim and buffering agents, other components or substances known in the industry may be selectively included in the liquid formulation, to the extent that they do not impair the effect of the liquid formulation of the present invention.

[0059] Stabilizer In one specific example, the liquid formulation also contains a stabilizer.

[0060] The term “stabilizer” may mean an excipient that improves or enhances stability. Examples of such stabilizers 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, for example, vitamin E acetate, zinc, and any combination thereof. The stabilizer may also be present in the liquid formulation at concentrations 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).

[0061] In one specific example, the stabilizer is a stabilizer that is substantially free of albumin. Human serum albumin, which can be used as a protein stabilizer, is produced from human blood and therefore may be contaminated with pathogenic viruses of human origin. Gelatin and bovine serum albumin may cause disease or, in some patients, induce allergic reactions. The albumin-free stabilizers described herein do not contain human or animal-derived serum albumin or other foreign proteins such as purified gelatin, and therefore do not pose a risk of viral infection.

[0062] In this specification, “substantially does not comprise” means that the substance referred to is present in such an amount 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 present at all.

[0063] While not bound by any particular theory, these substances can play a role in improving the stability of the formulation as described above. Therefore, the remaining amount of efrapegrastim will change due to the altered physical or chemical environment resulting from the use of these substances in specific concentrations.

[0064] surfactant In one specific example, the liquid formulation also contains a surfactant.

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

[0066] When the concentration of polysorbate-based nonionic surfactants exceeds 5% (w / v) relative to the total solution, it significantly affects the stability of liquid formulations containing efrapegrastim, and the remaining percentage can be applied as a stability evaluation measure. For example, if the protein remaining percentage is maintained at 97% or higher under accelerated conditions (25±2℃ / 60±5% RH) after a 4-week storage test, such a formulation is understood to have excellent stability.

[0067] Although polysorbate-based nonionic surfactants have been widely used as additives in the pharmaceutical and cosmetic fields since their initial approval in Europe, some recent reports indicate that they may have negative effects on the human body. For example, there are reports that injecting polysorbate 80 into the human body can induce anaphylaxis (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 induce patient discomfort upon injection. Furthermore, it is technically necessary to prevent the concentration of polysorbate-based nonionic surfactants from inevitably increasing during the liquid formulation manufacturing process.

[0068] In one specific example, the polysorbate-based nonionic surfactant is used in the following final concentrations relative to the total solution: 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), and 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) ) can be included in, 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 also includes components at concentrations of 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 this specification, the term "final concentration" is a concept distinct from the stated concentration and refers to the actual concentration substantially contained within the liquid formulation. For example, in the manufacturing of a liquid formulation, in the process of concentrating efrapegrastim to the target concentration after the exchange of buffering material, the final concentration of the surfactant in the solution may be higher than the stated concentration. For example, in examples where 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 0.005% (w / v) by at least 1,000 times. For example, the final concentration (i.e., actual concentration) of 0.005% (w / v) or 0.01% (w / v) polysorbate 80 described in the examples of the Registered Patent Publication of the Republic of Korea No. 10-1340710 (published 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). Conversely, in this specification, when the final concentration of the polysorbate nonionic surfactant is indicated as 0.005% (w / v), it means that the formulation was obtained by replacing the polysorbate nonionic surfactant with a buffer substance substantially free of it, for example, by using diafiltration, then concentrating the buffer substance to the target efrapegrastim concentration, and finally spiking the polysorbate nonionic surfactant to 0.005% (w / v) until a concentration of 0.005% (w / v) was achieved. Accordingly, the liquid formulation according to one specific example may be pre-treated using a purification column, or the pre-treated liquid formulation may be concentrated after replacing it with a buffer substance substantially free of the polysorbate nonionic surfactant. Thus, without being limited to a specific theory, the liquid formulations described herein may offer significantly greater formulation stability and improved properties compared to conventional liquid formulations containing highly aggregated non-natural proteins, even at high concentrations.

[0070] Tonic modifier In one specific example, the liquid formulation also contains a tonic modifier.

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

[0072] The tonic modifier is one or more selected from the group consisting of pharmaceutically acceptable salts, sugars, and amino acids. Specifically, the tonic modifier is 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, it is sodium chloride, etc. Furthermore, the tonic modifier is 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. Furthermore, the tonic modifier is 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 tonic modifier for stabilizing the formulations according to this specification is also an amount that can maintain or adjust the osmotic pressure range, for example, 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] The tonic modifier may be added in an amount sufficient to provide the appropriate osmotic pressure, as described below.

[0075] Buffer substances and pH In this specification, the term “buffering agent” may mean one or more components that can protect a solution from changes in pH when added to an aqueous solution, when an acid or alkali is added, or when diluted in a solvent. The buffering agent is any substance that can bring the pH of a composition for stabilizing a formulation into a specific range, for example, an organic acid buffer or an inorganic acid buffer, for example, an organic acid, an inorganic acid, or a salt of an organic or inorganic acid. More specifically, the buffering agent is 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, succinates, acetates, citrates, phosphates, lactates, etc. of the organic or inorganic acids. More specifically, examples of buffering 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 pharmaceutically acceptable pH buffering substances known to the art, and mixtures thereof may also be used.

[0076] In one specific example, the concentration of the buffering material may be 5 to 100 mM, 5 to 80 mM, 10 to 80 mM, 10 to 60 mM, 10 to 50 mM, or 15 to 25 mM.

[0077] In one specific example, the specific pH range of the liquid formulation is 4 to 8, 5 to 8, 5 to 7, or 5 to 6, and in the specific example, it is also 5.5.

[0078] The buffering material can be dissolved in a liquid medium such as water and used in solution with a pH of 4 to 8, 5 to 8, 5 to 7, or 5 to 6.

[0079] Osmotic pressure Based on a combination of various literatures, the osmotic pressure of drugs can be adjusted to 300 ± 30 mOsm / kg, but due to the need for various excipients, they are also manufactured as hypertonic solutions for technological and industrial purposes. For intravenous or intravascular administration, the upper limit of osmotic pressure is generally suggested to not exceed 1,000 mOsm / kg, and since the osmotic pressure of serum is approximately 285 mOsm / L, the lower limit of osmotic pressure is generally suggested to exceed 100 mOsm / L or 200 mOsm / L. Therefore, it is generally expected that patients can tolerate osmotic pressures of 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 specific example, the osmotic pressure of a liquid formulation exhibiting excellent stabilization effects is also 400 to 800 mOsm / kg.

[0080] Osmotic pressure can be measured using measurement methods and instruments known in the relevant technical field.

[0081] While not bound by any particular theory, it is presumed that the osmotic pressure of the liquid formulation described herein is affected by the concentration of efrapegrastim or by the concentration of stabilizers or tonic modifiers, which may be added to the formulation. The stability of the liquid formulation described herein is increased by efrapegrastim within a certain concentration range and / or by hG-CSF variants having a specific amino acid sequence, allowing for more flexible adjustment of the composition of the liquid formulation that may affect stability compared to the composition of conventional liquid formulations. This allows for more flexible adjustment of the composition necessary to achieve the osmotic pressure of the liquid formulation in order to realize the effects of the invention. For example, the osmotic pressure can be adjusted by relatively lowering the concentration of stabilizers or tonic modifiers, which may also be added to the liquid formulation for stability.

[0082] conductivity In other specific examples, the formulations of this specification also 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. An intermediate range of the aforementioned cited values, for example, 1 to 20 mS / cm, is intended to be included in the present invention. For example, a numerical range using a combination of the aforementioned cited values ​​as an upper and / or lower limit is intended to be included. Also included in this specification are numerical values ​​that are included in the cited values, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mS / cm, etc.

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

[0084] Maximum gliding force and viscosity In this specification, the term "maximum gliding force (MGF)" refers to the maximum force applied when administering a drug using a syringe, and is influenced by the viscosity of the formulation, the injection speed, and the characteristics of the syringe. Furthermore, the fluid properties of protein formulations are affected by the protein concentration. Additionally, needles thinner than 29G substantially increase the maximum gliding force. Ultimately, aggregation due to high protein concentrations and the resulting increase in viscosity lead to an increase in the maximum gliding force, sometimes requiring administration with needles smaller than 29G to reduce the maximum gliding force, which can cause discomfort to the patient. Generally, patients are reported to be able to tolerate a maximum gliding force of around 15N to 20N (Development of Syringeability Guide for Subcutaneous Protein Formulations, L. Joseph et al., Pfizer Global Research & Development, 2010).

[0085] In one specific example, when the liquid formulation is administered at a rate of 2.835 mm / s using a 29-cage syringe, even while containing a sufficiently high concentration of epilapegrastim for therapeutic effect, the maximum gliding force is 7N, 6N, 5N, 4N, or 3N or less. Furthermore, when the liquid formulation is administered at a rate of 4.725 mm / s using a 29-cage syringe, the maximum gliding force is 10N, 9N, 8N, 7N, 6N, 5N, 4N, 3.5N, or 3N or less. The maximum gliding force can be measured using a gliding force measuring device known in the art, for example, a rheometer sold by DAEGO TRADING CO. (Seoul, South Korea). In one specific example, the liquid formulations of this specification have a viscosity of 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 or less at room temperature of 20°C to 25°C. Viscosity can be measured using measurement methods and instruments known in the art.

[0086] As mentioned above, the maximum sliding force is affected by the viscosity of the formulation, and viscosity, i.e., the fluid properties of the protein formulation, is affected by its concentration. Therefore, the maximum sliding force is affected by the remaining percentage, concentration, or buffering agent of the protein drug (efrapegrastim), and may also be affected by any additional surfactants, stabilizers, or tonic modifiers included.

[0087] Patient-friendly formulations In the case of protein-based drugs, high concentrations are required for therapeutic effect, but generally, problems such as aggregation, insolubility, and degradation are amplified as the protein concentration increases. Therefore, balancing the component and concentration in pharmacologic protein formulations to improve stability and therapeutic requirements while limiting any side effects is one of the technical challenges that must be solved in this industry. To this end, this specification has made it possible to produce formulations that contain high concentrations of active ingredients while maintaining high stability and solubility. In addition to the remarkable advances in such protein formulations for therapeutic use described herein, when such liquid formulations of protein drugs are administered to an individual, irritation / pain and discomfort at the administration site remain challenges that must be addressed.

[0088] Osmotic pressure, and injection site irritation / pain Osmotic changes in tissues and cells are perceived as dangerous signals in the human body, activating dendritic cells and stimulating immune and inflammatory responses (Gallo and Gallucci, 2013). Hypertonicity in the gastrointestinal tract of human infants has been reported to cause necrotizing colitis (Atakent et al., 1984). It is already well known that the presence of pain receptors is responsible for the sensation of pain induced by various events, including injections. The sensation of peripheral pain is mediated through afferent fibers (sensory nerve fibers) called pain receptors (Brazeau et al., 1998). Functionally, these pain receptors are classified into two main types: polymodal nociceptors that respond to chemicals, and mechanothermal nociceptors that 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 induce water absorption into cells), activating compression (or stretch)-sensitive channels and causing pain.

[0089] Maximum gliding force and viscosity, as well as patient discomfort. Higher-concentration protein formulations are attracting attention due to their ability to reduce injection volume and secure storage space. However, the development of high-concentration protein formulations presents several practical challenges in terms of stability, manufacturing, and supply, stemming from the tendency of proteins to aggregate at high concentrations. The physical properties of high-concentration protein formulations affect their ability to be easily transferred; such high-concentration solutions sometimes exhibit high viscosity, preventing the solution from passing through the syringe needle. Therefore, there is a correlation between material viscosity and protein concentration; higher concentrations of protein formulations result in higher viscosity and cause discomfort to patients. Consequently, developing formulations that enhance therapeutic efficacy while further improving patient tolerance through increased concentration and decreased viscosity represents a significant advance in protein formulation design.

[0090] In order to reduce maximum gliding force and enhance therapeutic effect by administering protein preparations with even finer needles, the preparation must contain a high concentration of protein, exhibit minimal aggregation, and have low viscosity. However, these conflicting requirements (high protein concentration and low viscosity) must be resolved.

[0091] Patient friendly index (PF) As mentioned above, the production of high-concentration protein formulations involves opacity, aggregation, and precipitation, leading to considerable problems. Besides the possibility of non-natural protein aggregation and microparticle formation, reversible self-binding occurs, resulting in increased viscosity and other properties that complicate injection-based supply. High viscosity also complicates the production of high-concentration proteins by filtration. Therefore, developing formulations with high stability and patient affinity requires careful and comprehensive consideration of various factors. Specifically, the residual rate, a factor related to formulation stability, is affected by stabilizers and surfactants, which in turn affect the viscosity of the substance and thus the maximum sliding force. Furthermore, osmotic pressure is affected by tonic modifiers and buffering substances, which can influence conductivity.

[0092] In one specific example, this specification provides a liquid formulation that satisfies the 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 Equation 1, Osm is the osmolarity 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.

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

[0094] Specifically, the patient affinity index for liquid formulations is 3 to 10, 5 to 10, 6 to 10, or 6 to 9. Considering that the maximum sliding force, which is affected by viscosity, is almost always at least 1N, if the osmotic pressure is 1,000 mOsm / kg, the patient affinity index exceeds 10, making patient-friendly infusion difficult. Also, considering that the maximum sliding force is almost always 1N or more, if the osmotic pressure is less than 200 mOsm / kg, the patient affinity index does not exceed 3, making patient-friendly infusion difficult.

[0095] Therefore, liquid formulations that fall within the range of the patient affinity index have a low viscosity, low gliding force, and appropriate osmotic pressure range that takes into account high-concentration protein formulations, and by solving the technical challenges mentioned above, they can be administered with patient affinity while having high dosage form stability.

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

[0097] The injection volume of the liquid formulation of the present invention can be appropriately adjusted 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] Another embodiment provides a manufactured article containing the liquid formulation.

[0099] In other embodiments of this specification, a manufactured article is provided which includes a drug product and provides instructions relating to its use. The manufactured article includes a container. Suitable containers may 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 for illustrative purposes only, and the scope of the present invention is not limited to these examples.

[0101] Example 1. Analysis of a patient-friendly injectable liquid formulation. In this embodiment, various variables of the manufactured formulation that affect the patient during administration are utilized to derive a patient-friendly injectable formulation of the final liquid formulation. To this end, as described above, the appropriate value for osmotic pressure was determined to be 100 to 1,000 mOsm / kg, or preferably 200 to 1,000 mOsm / kg, based on the descriptions 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 appropriate value for maximum gliding force was calculated to be 5N or less, referring to the description in "Development of Syringeability Guide for Subcutaneous Protein Formulations, L. Joseph et al., Pfizer Global Research & Development, 2010".

[0102] Considering the complementary nature of these values, a mediating variable was introduced and shown in Equation 1 below. When Equation 1 is satisfied, it is possible to manufacture a liquid formulation with high patient affinity, and the result 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 formulations Maximum gliding force when administering the liquid formulation at a speed of 2.835 mm / s using an MGF:29 cage syringe.

[0104] The aforementioned patient affinity index was determined to be between 3 and 10, taking into account the appropriate osmotic pressure and appropriate maximum gliding force values. Considering that the maximum gliding force is almost always 1N or more, if the osmotic pressure of the liquid formulation exceeds 1,000 mOsm / kg, the patient affinity index will exceed 10, making patient-friendly injection difficult. Also, considering that the maximum gliding force is almost always 1N or more, if the osmotic pressure is less than 200 mOsm / kg, the patient affinity index will not exceed 3, making patient-friendly injection difficult. In other words, if the patient affinity index is between 3 and 10, it is considered to be an excellent patient-friendly injectable formulation.

[0105] Therefore, liquid formulations that fall within the range of the patient affinity index are considered to have low viscosity, low gliding force, and an appropriate osmotic pressure range for high-concentration protein formulations. By resolving the technical challenges mentioned above, it is possible to administer them with patient affinity while maintaining high formulation stability.

[0106] Example 2. Stability analysis of liquid formulation The stability of high-concentration protein formulations was measured by the remaining percentage after a 4-week storage test. Specifically, to measure the remaining percentage of liquid formulations, the remaining percentage of efrapegrastim was measured by RP-HPLC and SE-HPLC after a 4-week storage test under accelerated conditions (25±2℃ / 60±5% RH). The n-week remaining percentage of efrapegrastim in the liquid formulation was calculated using Equation 2. Formula 2 Survival rate at week n (%) = Purity value at week n / Initial purity value x 100

[0107] Purity is the relative ratio of the main peaks obtained by HPLC.

[0108] HPLC was performed using an Agilent 1200 series analyzer, and RP-HPLC was performed using a PhenomenexJ upiter C4 column at 60°C. A 2-eluent linear gradient system with a flow rate of 1.0 mL / min was used. Mobile phase A was 20% acetonitrile containing 0.1% trifluoroacetic acid, and mobile phase B was 80% acetonitrile containing 0.1% trifluoroacetic acid. After a minimum of 1 hour of initial stabilization with 76% mobile phase A and 24% mobile phase B, measurements were taken using a linear gradient system with 24-60% mobile phase B for 0-15 minutes, 60-73% mobile phase B for 15-48 minutes, and 73-100% mobile phase B for 48-75 minutes. Re-equilibrium was then performed with 24% mobile phase B for 75-85 minutes. The sample injection volume was set to 20 μg, the detector wavelength was set to 214 nm, and the entire process was coordinated using 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, measurements were taken for 60 minutes. The sample injection volume was set to 20 μg, the detector wavelength was set to 214 nm, and the entire process was coordinated using Agilent Chemstation software.

[0110] Furthermore, considering the physicochemical properties of efrapegrastim and general knowledge in the field of protein formulations, it was understood that efrapegrastim is stable if its residual rate is maintained as follows under accelerated conditions (25±2℃ / 60±5% RH) after a 4-week storage test: Maintaining over 95%: Stable Maintaining over 97%: Excellent stability. Maintaining over 98%: Possesses extremely excellent stability.

[0111] Manufacturing Example 1-46: Manufacturing of a liquid formulation containing efrapegrastim We designed a liquid formulation that contains a high concentration of efrapegrastim while exhibiting patient-friendly properties and dosage form stability as presented in Examples 1 and 2.

[0112] Based on simulation predictions, liquid formulations from Production Examples 1 to 36 were manufactured that were deemed to have dosage form stability and allow for patient-friendly infusion. Furthermore, liquid formulations from Production Examples 37 to 39 were manufactured to determine the dosage form stability when high concentrations of polysorbate-based nonionic surfactants are included using conventional manufacturing methods. In addition, liquid formulations from Production Examples 40 to 43 were manufactured that were expected to cause patient discomfort or have problems in terms of dosage form stability. Finally, liquid formulations from Production Examples 44 to 46 were manufactured to confirm how the concentration of polysorbate-based nonionic surfactants affects the residual rate of efrapegrastim, which is a major factor in dosage form stability.

[0113] 1. Manufacturing Examples 1 to 36 The liquid formulation containing efrapegrastim was manufactured as follows:

[0114] First, a liquid formulation was prepared with the following composition: 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, polysorbate 80 was removed from the prepared liquid formulation using an SQ purification column (Source 15Q, GE Healthcare). Afterward, only the most important fraction of the purified profile was recovered. Next, buffer exchange was performed on the pretreated liquid formulation using filtration. Specifically, using a buffer without polysorbate 80, a VivaSpin 20 (Sartorius) was used to perform a total of five buffer exchanges at 3,700 rpm for 1 hour. Finally, the buffer-exchanged liquid formulation was concentrated to approximately 2 to 3 times the target concentration. Considering the final volume and target concentration, a buffer without polysorbate 80 was added to the concentrated liquid formulation. Using a polysorbate 80 stock concentrated 100 times above the target concentration, the liquid formulation was spiked to achieve a final polysorbate concentration (actual concentration) of 0.005% (w / v), thereby producing a liquid formulation containing 0.005% (w / v) polysorbate 80. Furthermore, in Production Examples 2 to 37, liquid formulations with different compositions from those of Production Example 1 were produced using the same method as in Production Example 1.

[0115] In other words, the compositions of the liquid formulations in Manufacturing Examples 1 to 36 are as follows:

[0116] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing Example 9] 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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. Manufacturing Examples 37 to 39 The liquid formulation of Production Example 37 was manufactured with reference to Registered Korean Patent No. 10-1340710, however, efrapegrastim containing an hG-CSF variant having a different amino acid sequence than the hG-CSF variant amino acid sequence described in Registered Korean Patent No. 10-1340710 was used as the active ingredient, and the concentration was also different.

[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) polysorbate 80 (concentration before concentration). Subsequently, buffer exchange was immediately performed using filtration without sample pretreatment. Specifically, buffer exchange was performed a total of 5 times in a buffer containing polysorbate 80 using a VivaSpin 20 (Sartorius) at 3,700 rpm for 1 hour. The buffer-exchanged liquid formulation was then concentrated to approximately twice the target concentration. Considering 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 aforementioned manufacturing example 37, the concentration of polysorbate 80 is the concentration before the concentration process. In the test example below, the concentration process resulted in the application of polysorbate 80 exceeding a minimum of 5% (w / v (final concentration)).

[0154] In other words, the composition of the liquid formulation in manufacturing example 37 is as follows:

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

[0156] Furthermore, in manufacturing examples 38 and 39, liquid formulations were produced using the same method as in manufacturing example 37, but with different compositions from the formulation in manufacturing example 37, as described below.

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

[0159] 3. Manufacturing Examples 40 to 43 A liquid formulation containing efrapegrastim was produced using the same method as in Production Example 1, but with a different composition from the formulation described above.

[0160] In other words, the compositions of the liquid formulations in manufacturing examples 40 to 43 are as follows:

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

[0162] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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. Manufacturing Examples 44 to 46 A liquid formulation containing efrapegrastim was produced using the same method as in Production Example 1, but with a different composition from the formulation described above.

[0166] In other words, the composition of the liquid formulations of manufacturing examples 44 to 46 is as follows:

[0167] [Manufacturing 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] [Manufacturing 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] [Manufacturing 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 the remaining rate and patient affinity index of liquid formulations. For the liquid formulations of Production Examples 1 to 6 and Production Examples 37 to 43, osmotic pressure measurements, conductivity measurements, viscosity measurements, and maximum sliding force measurements were performed. Subsequently, the remaining percentage of efrapegrastim after a 4-week storage test under accelerated conditions (25±2℃ / 60±5% RH) was measured.

[0171] 1. Liquid formulation test of manufacturing example 1 The osmotic pressure of the liquid formulation in Production Example 1 was measured using an automated osmometer (Gonotec, OSMOMAT auto) and was 645 mOsm / kg.

[0172] Furthermore, the conductivity of the liquid formulation in Production Example 1 was measured at room temperature using a conductivity meter (Compact Conductivity Meter EC33, LAQUAtwin, Horiba) as described by the manufacturer. As a result, the conductivity of the liquid formulation in Production Example 1 was 14.37 mS / cm.

[0173] Furthermore, the viscosity of the liquid formulation in Production Example 1 was measured at room temperature (20 to 25°C) using a Vibration Viscometer ((A&D, SV-1A)), and the viscosity value was 1.86 cP.

[0174] Furthermore, the maximum sliding force of the liquid formulation in Manufacturing Example 1 was measured using a Rheo Meter (Sun Scientific, Compac-100) with a 29-cage syringe (based on 400 μL, length 22.68 mm) when the liquid formulation was administered at speeds 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 results were 3.099 N and 2.099 N, respectively.

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

[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 Manufacturing Example 2 In the same manner as in Test Example 1 described above, the osmotic pressure, conductivity, viscosity, maximum sliding force, and residual rate of the formulation were measured.

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

[0180] Furthermore, the residual rate of the formulation from manufacturing example 2 under accelerated conditions (25±2℃ / 60±5% RH) is shown in Table 2 below.

[0181] [Table 2]

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

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

[0184] Furthermore, the residual rate of the formulation from manufacturing example 3 under accelerated conditions (25±2℃ / 60±5% RH) is shown in Table 3 below.

[0185] [Table 3]

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

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

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

[0189] [Table 4]

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

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

[0192] Furthermore, the residual rate of the formulation of manufacturing example 5 under accelerated conditions (25±2℃ / 60±5% RH) is shown in Table 5 below.

[0193] [Table 5]

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

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

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

[0197] [Table 6]

[0198] 7. Liquid Formulation Test of Manufacturing Example 37 Similar to Test Example 1 mentioned above, the residual rates of the formulation from Manufacturing Example 37 under accelerated conditions (25±2℃ / 60±5% RH) are shown in Table 7 below.

[0199] [Table 7]

[0200] 8. Liquid Formulation Test of Manufacturing Example 38 Similar to Test Example 1 mentioned above, the residual rates of the formulation from Manufacturing Example 38 under accelerated conditions (25±2℃ / 60±5% RH) are shown in Table 8 below.

[0201] [Table 8]

[0202] 9. Liquid Formulation Test of Manufacturing Example 39 Similar to Test Example 1 mentioned above, the residual rates of the formulation from Manufacturing Example 39 under accelerated conditions (25±2℃ / 60±5% RH) are 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 sliding force, and residual rate of the formulation were measured.

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

[0206] Furthermore, the residual rates of the formulation of manufacturing example 40 under accelerated conditions (25±2℃ / 60±5% RH) are 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 sliding force, and residual rate of the formulation were measured.

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

[0210] Furthermore, the residual rates of the formulation from manufacturing example 41 under accelerated conditions (25±2℃ / 60±5% RH) are 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 sliding force, and residual rate of the formulation were measured.

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

[0214] Furthermore, the residual rates of the formulation of manufacturing example 42 under accelerated conditions (25±2℃ / 60±5% RH) are 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 sliding force, and residual rate of the formulation were measured.

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

[0218] Furthermore, the residual rates of the formulation from manufacturing example 43 under accelerated conditions (25±2℃ / 60±5% RH) are shown in Table 13 below.

[0219] [Table 13]

[0220] 14. Evaluation of Patient Affinity Index To confirm whether the aforementioned manufactured liquid formulations satisfy Formula 1, the patient affinity index for 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 manufacturing examples 37 to 39, which contain high concentrations of polysorbate-based nonionic surfactants, some data shows problems with the residual rate. Furthermore, the liquid formulation in manufacturing example 43 shows a serious residual rate problem.

[0223] Furthermore, as shown in Table 14 above, in the case of manufacturing example 43, although the osmotic pressure was within the appropriate range, the maximum gliding force was high and still induced patient discomfort, and in the case of manufacturing example 41, although the maximum gliding force was within the appropriate range, the high osmotic pressure could still induced patient discomfort. In contrast, it was found that the liquid formulation according to one specific example fell within the appropriate osmotic pressure range, the maximum gliding force range was 5N or less, and the patient affinity index was within the appropriate range of 3 to 10. Therefore, it was found that in the case of the liquid formulation according to one specific example, where the patient affinity value, with osmotic pressure and maximum gliding force as the main factors, is between 3 and 10, it is possible to administer the desired liquid formulation without inducing patient discomfort.

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

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

[0226] [Table 15]

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

[0228] [Table 16]

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

[0230] [Table 17]

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

[0232] [Table 18]

[0233] Figures 1 and 2 are the results of confirming the change in the residual rate of efrapeglastim depending on the concentration of the surfactant in a liquid preparation according to an embodiment. As shown in Figures 1 and 2 described above, the concentration of the surfactant shows a high correlation with the residual rate of the liquid preparation according to an embodiment, and such experimental results indicate that the concentration of the surfactant is the main factor affecting the residual rate of the liquid preparation containing a high concentration of efrapeglastim.

[0234] From these results, it can be seen that the liquid formulation described in this example differs from existing liquid formulations in terms of ingredient content and manufacturing method. As a result, it not only contains a high concentration of active ingredients but also exhibits high dosage form stability (e.g., residual rate), high patient affinity, and can be administered without causing discomfort to the patient.

[0235] It should be understood that the examples described herein are for illustrative purposes only and are not intended to be limiting. Descriptions of features or embodiments within each example are typically applicable to other similar features or embodiments in other examples. Even if one or more examples are described with reference to the drawings, it will be understood by those skilled in the art that various modifications in form and detail may be made therein without departing from the spirit and scope of this 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 efrapegrastim liquid formulation according to any one of claims 15 to 17, wherein the efrapegrastim liquid 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.

Images