Liquid formulation of GM-CSF for inhalation

Abstract

Provided herein are liquid formulations comprising recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for inhalation by nebulization.

Description

[Technical field]

[0001] The present invention relates to liquid formulations for inhalation comprising recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). [Background technology]

[0002] Granulocyte-macrophage colony-stimulating factor (GM-CSF) was originally identified as a hematopoietic growth factor. Human GM-CSF stimulates the growth of myeloid and erythroid progenitors in vitro and activates monocytes, macrophages, and granulocytes in several immune and inflammatory processes (Gasson JC et al. Prog Clin Biol Res. 1990; 352: 375-384.; Gasson JC et al. Prog Clin Biol Res. 1990; 338: 27-41; Hart SP et al. Biochem Soc Trans. 1998; 26 (4): 650-652; Rapoport AP et al. Blood Rev. 1992; 6 (1): 43-57.). It is produced by many cell types, including lymphocytes, monocytes, endothelial cells, fibroblasts and some malignant cells (Metcalf D. Blood. 1986; 67(2):257-267; Clark SC, Kamen R. Science. 1987; 236(4806):1229-1237; Hart PH et al. J Immunol. 1988; 141(5):1516-1521; Metcalf D et al. Blood. 1986; 67(1):37-45). In addition to having growth stimulatory and differentiation functions on hematopoietic progenitor cells, GM-CSF has also been found to exert a variety of effects on cells of the immune system that express the GM-CSF receptor (for reviews see Hamilton JA. Trends Immunol. 2002;23(8):403-408; de Groot RP et al. Cell Signal. 1998;10(9):619-628).

[0003] Granulocyte-macrophage colony-stimulating factor inhalation therapy has been disclosed for treating patients suffering from autoimmune pulmonary alveolar proteinosis (aPAP) (Tazawa R et al. N Engl J Med. 2019; 381(10): 923-932), nontuberculous mycobacteria (NTM) infection (Scott JP et al. Eur Respir J. 2018; 51(4): 1702-127), acute radiation syndrome (Singh VK et al. Cytokine. 2015; 71(1): 22-37), acute respiratory distress syndrome (Herold S et al. Am J Respir Crit Care Med. 2014; 189(5): 609-611) and cystic fibrosis (Heslet L et al. J Inflamm Res. 2012; 5: 19-27.) by nebulization.

[0004] Inhalation therapy offers the best possibility of optimal delivery to the lungs while reducing systemic side effects. Inhalation treatment targets the airways, lowering the amount of drug required for optimal effectiveness. In addition, the rapid onset of action of drugs is usually accompanied by a low incidence of systemic side effects. Nebulizers are a very suitable treatment method for fungal or bacterial lung infections, as they fill the air with a very fine mist of respirable droplets that can be deposited in all parts of the lungs.

[0005] However, formulation of proteins as liquids for inhalation therapy is a very complicated process, as it requires a product that can be added to a nebulizer and remain stable during aerosol generation. The formulation should remain stable during long-term storage, minimize the effect of nebulization on protein integrity, and have appropriate tonicity for pulmonary delivery. Thus, a delicate balance between stability, pulmonary tolerability, including potential toxicity, and isotonicity is the key to achieving an effective protein formulation for inhalation therapy.

[0006] When proteins are formulated as lyophilized products for resuspension, the same challenges do not arise since long-term stability is ensured such that isotonicity can be achieved upon resuspension, and therefore the same excipient composition is not necessarily applicable for the two different formulations.

[0007] A liquid formulation of GM-CSF (LEUKINE®) has previously been disclosed for intravenous administration (US2002141970A1), which suggests the addition of a chelating agent as an excipient to increase the stability of the compound.

[0008] Formulation of drug solutions for nebulization aims to optimize drug solubility and stability, and small changes in the formulation can also affect inhaled volume, particle size distribution, and treatment time.

[0009] Nebulization has inherent risk of protein instability.The main cause of protein instability during nebulization is unfolding and aggregation at the air-liquid interface, so the use of GM-CSF for nebulization requires a stable liquid formulation that is ready for inhalation and does not undergo significant changes during nebulization. Summary of the Invention

[0010] The present invention discloses a stable liquid formulation of rhGM-CSF that is ready to use for inhalation and does not exhibit undue instability upon nebulization.

[0011] In one embodiment of the invention, the combination of a sugar alcohol or sugar, albumin, and polyethylene glycol (PEG) with water provides a highly stable liquid formulation of rhGM-CSF.

[0012] In one embodiment, the liquid formulation described above is one that is used as a medicament for nebulization.

[0013] In one embodiment, the liquid formulation of rhGM-CSF is for use in treating aPAP or NTM infection.

[0014] In one embodiment, a method for pulmonary delivery of recombinant rhGM-CSF is disclosed that includes nebulizing rhGM-CSF, a sugar alcohol, albumin and PEG.

[0015] In a further embodiment, a nebulizer containing a liquid formulation of rhGM-CSF is disclosed for use in treating aPAP or NTM infections. [Brief description of the drawings]

[0016] [Figure 1] Figure 1. Effect of mannitol on % rhGM-CSF remaining after 3 months at 40°C. The effect of mannitol concentration on the stability of liquid formulations of rhGM-CSF at accelerated conditions over a period of 3 months was evaluated. The concentration of mannitol was found to affect drug stability. Higher concentrations of mannitol (50 mg / mL) improved the stability of rhGM-CSF. Y-axis is % rhGM-CSF content remaining compared to T0. [Diagram 2] Figure 2. Effect of rhGM-CSF levels within mannitol concentration groups on remaining GM-CSF after 3 months at 40°C. The effect of rhGM-CSF concentration on the stability of liquid formulations at accelerated conditions over 3 months was also evaluated. The concentration of rhGM-CSF was found to affect drug stability. Higher concentrations of rhGM-CSF (400 μg / mL) at a given mannitol concentration improved the stability of rhGM-CSF in liquid formulations. Y-axis is % rhGM-CSF content remaining compared to TO. [Diagram 3]Figure 3. Effect of rHA and PEG4000 on rhGM-CSF impurity formation. Formulations containing rHA and PEG4000 as stabilizers formed less rhGM-CSF impurities over 6 months at refrigerated and accelerated conditions. The simplified formulation (open circles) does not contain rHA or PEG4000. The complex formulation (closed circles) contains 1.0 mg / mL rHA and 0.1 mg / mL PEG4000. [Figure 4A] Figure 4A shows the stability of performance of the complexed rhGM-CSF formulation over 18 months. The stability of performance of the complexed rhGM-CSF formulation upon nebulization was evaluated up to 18 months of storage at 5°C and 25°C. Three parameters were evaluated: DD = Simulated Respiratory Delivered Dose (USP <1601> , by adults), FPF = fine particle fraction (droplets less than 5 μm) and MMAD = mass median aerodynamic diameter were assessed. Little or no variation in performance stability was found upon nebulization up to 18 months. [Figure 4B] FIG. 4B: 18-month performance stability of rhGM-CSF complexed formulations. The performance stability of complexed rhGM-CSF formulations upon nebulization was evaluated up to 18 months of storage at 5° C. and 25° C. Three parameters were evaluated: DD=simulated breath delivered dose (USP <1601> , by adult), FPF=fine particle fraction (droplets less than 5 μm) and MMAD=mass median aerodynamic diameter were assessed. Upon nebulization, little or no variation in performance stability was found at time points up to 18 months. Figures 4A and 4B show two separate batches. [Diagram 5] Figure 5. Three-month stability of low rHA formulations. A conjugated version of rhGM-CSF with low rHA concentration (0.2 mg / mL) was evaluated and showed improved stability over a simplified formulation that did not contain rHA or PEG4000. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention relates to a liquid formulation of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) for use in administration by inhalation, wherein the liquid formulation comprises rhGM-CSF, a sugar alcohol or sugar, albumin and water.

[0018] The combination of specific excipients ensures high stability of rhGM-CSF in a liquid formulation ready for use in inhalation therapy.

[0019] In one embodiment, the rhGM-CSF is molgramostim (SEQ ID NO:1), sargramostim (SEQ ID NO:2), or regaramostim.

[0020] The amino acid sequence of molgramostim is identical to that of endogenous human GM-CSF. The amino acid sequence of sargramostim differs from native GM-CSF by a leucine substitution at position 23. In a preferred embodiment, the rhGM-CSF is molgramostim (SEQ ID NO: 1). Molgramostim is non-glycosylated rhGM-CSF expressed in E. coli.

[0021] In one embodiment, the liquid formulation of rhGM-CSF comprises a sugar, wherein said sugar is selected from the group consisting of maltose, trehalose, sucrose, mannose, lactose or galactose.

[0022] In another embodiment of the invention, the liquid formulation of rhGM-CSF comprises a sugar alcohol selected from the group consisting of mannitol or sorbitol. The addition of a polyol sugar to the liquid formulation of rhGM-CSF increases the stability. Furthermore, a polyol sugar is used as an isotonicity agent to ensure that the formulation is isotonic with human plasma. In a preferred embodiment, the sugar alcohol is mannitol.

[0023] Albumin is a family of globular proteins, the most common being serum albumin. Human serum albumin (HSA) is the most common protein found in human plasma. The addition of HSA contributes to the stability of rhGM-CSF liquid formulations. Also, the abundance and natural self-tolerance to albumin can minimize the possibility that HSA will induce an immune response in some patient populations. Thus, in one embodiment of the present invention, the liquid formulation of rhGM-CSF contains albumin. In a preferred embodiment, said albumin is HSA. In another embodiment of the present invention, the albumin is recombinant human albumin (rHA).

[0024] In one embodiment of the invention, the liquid formulation of rhGM-CSF further comprises PEG. Such PEGs include, but are not limited to, PEG1000, PEG1550, PEG2000, PEG3000, PEG3350, PEG4000, or PEG8000. Thus, in one embodiment, the liquid formulation of rhGM-CSF further comprises a PEG selected from the group consisting of PEG1000, PEG1550, PEG2000, PEG3000, PEG3350, PEG4000, or PEG8000. In a preferred embodiment, the PEG is PEG-4000.

[0025] The concentration of the protein is crucial to ensure a stable and ready-to-use formulation. Increasing the concentration of rhGM-CSF can increase the stability of liquid formulations of rhGM-CSF. In one embodiment of the invention, the concentration of rhGM-CSF is at least 150 μg / mL. In a further embodiment of the invention, the concentration of rhGM-CSF in the disclosed liquid formulations is in the range of at least 150-500 μg / mL. In another embodiment of the invention, the concentration of rhGM-CSF in the disclosed liquid formulations is in the range of 200-300 μg / mL. The present disclosure shows that increasing the concentration of rhGM-CSF in the presence of the sugar alcohol mannitol stabilizes the liquid formulation (Figure 2).

[0026] To ensure a balance between isotonicity and stability of a liquid formulation of rhGM-CSF for inhalation, an embodiment of the present invention discloses a liquid formulation of rhGM-CSF, wherein the concentration of the sugar alcohol is at least 25 mg / mL. In a further embodiment of the present invention, the concentration of the sugar alcohol is in the range of 45-100 mg / mL. In another embodiment of the present invention, the concentration of the sugar alcohol is in the range of 45-55 mg / mL. As can be seen in FIG. 1 of the present disclosure, increasing the concentration of the sugar alcohol mannitol improves the stability of rhGM-CSF. The stabilizing effect of mannitol is confirmed at a sugar alcohol concentration of 25 mg / mL. This effect is significantly increased at a concentration of 50 mg / mL.

[0027] In one embodiment of the present invention, the liquid formulation of rhGM-CSF comprises albumin at a concentration of at least 0.2 mg / mL. In a further embodiment, the liquid formulation of rhGM-CSF comprises albumin in a concentration range of 0.2-10 mg / mL, e.g., 0.3-5 mg / mL, e.g., 0.5-1.5 mg / mL. Figure 3 of the present disclosure shows that impurities with little rhGM-CSF are formed in rhGM-CSF liquid formulations (complex formulations) containing rHA and PEG4000 as stabilizers both in refrigerated and accelerated conditions. Furthermore, Figure 5 shows that the stabilizing effect of albumin can already be seen at a concentration of 0.2 mg / mL, and the stabilizing effect increases with increasing concentration.

[0028] In one embodiment of the invention, the liquid formulation of rhGM-CSF comprises PEG, e.g., the concentration of PEG is up to 50, e.g., up to 40, e.g., up to 30, e.g., up to 20, e.g., up to 10, e.g., up to 1 mg / mL. In another embodiment of the invention, the concentration of PEG is in the concentration range of, e.g., 0.05-0.5 mg / mL, e.g., 0.05-0.3 mg / mL, e.g., 0.05-0.2 mg / mL. In a further embodiment of the invention, the liquid formulation of rhGM-CSF comprises PEG in the concentration range of 0.05-0.15 mg / mL.

[0029] The selection of the optimal combination and concentration of excipients and biotherapeutics is a very specific and complex process, specific to each individual protein formulation. The present invention discloses one embodiment of a liquid formulation of rhGM-CSF, where rhGM-CSF is molgramostim (SEQ ID NO: 1), the sugar alcohol is mannitol, the albumin is recombinant human albumin, and the PEG is PEG-4000.

[0030] In one embodiment, the concentration of rhGM-CSF is at least 150 μg / mL, the concentration of mannitol is at least 25 mg / mL, the concentration of recombinant human albumin is at least 0.2 mg / mL, and the concentration of PEG-4000 is up to 1 mg / mL.

[0031] In another embodiment of the invention, the concentration of rhGM-CSF is 150-500 μg / mL, the concentration of mannitol is 45-100 mg / mL, the concentration of recombinant human albumin is 0.5-1.5 mg / mL, and the concentration of PEG-4000 is 0.05-0.15 mg / mL.

[0032] In a preferred embodiment, the concentration of rhGM-CSF is in the range of 200-300 μg / mL, the concentration of mannitol is 50 mg / mL, the concentration of recombinant human albumin is 1 mg / mL, and the concentration of PEG-4000 is 0.1 mg / mL.

[0033] Buffers were added to the formulation to adjust and stabilize pH, and to optimize drug solubility and stability. For drugs intended for inhalation, it is desirable for the pH of the product to be close to physiological pH. In one embodiment of the invention, the liquid formulation of rhGM-CSF further comprises a buffer. In a preferred embodiment, the buffer is citric acid monohydrate or Na 2 HPO 4 Includes.

[0034] The osmolality of a liquid formulation is an important parameter for the suitability of the liquid formulation for inhalation. Preferably, the liquid formulation is essentially isotonic. Thus, in one embodiment of the present invention, the osmolality of the formulation is 250-375 mOsm / L, preferably 325-335 mOsm / L.

[0035] In one embodiment of the invention, a liquid formulation of rhGM-CSF is disclosed that has a % of rhGM-CSF remaining greater than 60% after 3 months storage at 40° C. In a further embodiment of the invention, a liquid formulation of rhGM-CSF is disclosed that has a level of impurity formation less than 5% after 6 months storage at 25° C.

[0036] The invention further discloses, in one embodiment, a method of delivering rhGM-CSF to the lungs, comprising nebulization of rhGM-CSF, a sugar alcohol, albumin and PEG.

[0037] The liquid formulation of rhGM-CSF presented herein is particularly well suited for nebulization. The data of the present disclosure demonstrate that the potency of molgramostim for a liquid formulation containing rhGM-CSF, sugar alcohol or sugar, albumin, PEG and water retains 99.4% of the biological potency of rhGM-CSF upon nebulization. Furthermore, after nebulization of this liquid formulation of rhGM-CSF, it was found that rhGM-CSF remains largely intact after the nebulization process. Finally, little or no change in performance stability is observed when the liquid formulation presented herein is nebulized at up to 18 months.

[0038] In one embodiment of the invention, at least 90%, such as at least 95%, such as at least 98%, such as at least 99% of the biological potency of rhGM-CSF is retained following nebulization.

[0039] In a further embodiment of the invention, the formulation is storage stable at 25°C for at least 18 months.

[0040] In another embodiment of the present invention, the liquid formulation is for use in pulmonary administration. To facilitate pulmonary administration, the formulation must be aerosolized into small, respirable droplets, the majority of which are less than 5 μm in diameter. For biological preparations, the formulation must remain stable during the aerosolization process, which can cause protein denaturation and / or aggregation due to the high levels of mechanical energy and increased air-liquid interface area. The formulation must also contain excipients that are safe for pulmonary administration and have a suitable range of pH and isotonicity.

[0041] In another embodiment of the invention, the liquid formulation of rhGM-CSF is for use in the treatment of pulmonary infections.

[0042] In a further embodiment, the liquid formulation of rhGM-CSF is for use in treating bacterial, viral, or fungal infections in the lungs. In one embodiment, the bacterial infection is selected from the group consisting of Streptococcus pneumoniae, Haemophilus spp., Staphylococcus aureus, and Mycobacterium tuberculosis. In another embodiment, the viral infection is selected from the group consisting of bronchitis, pneumonia, and bronchiolitis. In yet another embodiment, the fungal infection is selected from the group consisting of Aspergillus, Cryptococcus, Pneumocystis, and endemic fungi.

[0043] In a preferred embodiment, the liquid formulation of rhGM-CSF is for use in the treatment of autoimmune pulmonary alveolar proteinosis (aPAP) or nontuberculous mycobacteria (NTM) infections, in the treatment of tuberculosis, ARDS, influenza infections, coronavirus infections, acute and chronic radiation syndromes, bronchial asthma, COPD, pulmonary fibrosis, lung cancer and inflammatory disorders of the respiratory system. The formulation may also be used prophylactically to stimulate immunological responses and protect against pulmonary infections.

[0044] In an embodiment of the invention, a liquid formulation of rhGM-CSF is disclosed for use as a medicament for nebulization. In another embodiment, a liquid formulation of rhGM-CSF is disclosed for use in treating aPAP or NTM infections.

[0045] In one embodiment, a method of treating a medical condition is provided comprising administering to a subject a liquid formulation of rhGM-CSF by nebulization. In another embodiment, a method of treating aPAP or NTM infection is provided comprising administering to a subject a liquid formulation of rhGM-CSF.

[0046] APAP is a rare autoimmune lung disease. It is the most common form of pulmonary alveolar proteinosis (PAP) (90% of cases). Most cases occur in adults between the ages of 20 and 50. Some people show no symptoms, while others develop progressive dyspnea and shortness of breath on exertion. Other signs and symptoms may include a dry chronic cough, fatigue, weight loss, chest pain, and malaise. In rare cases, hemoptysis, roundness and swelling of the fingertips, and cyanosis may be present. Autoimmune PAP is caused by a malfunction of the immune system due to IgG antibodies that block the effects of GM-CSF. GM-CSF regulates the clearance of surfactant (a mixture of proteins and fats) by alveolar macrophages. Surfactant accumulates in the air sacs of the lungs (alveoli) and eventually makes breathing impossible. The standard treatment is a procedure called lung lavage.

[0047] NTM are mycobacteria that do not cause tuberculosis or leprosy. NTM cause a pulmonary disease that resembles tuberculosis. The most common clinical manifestation of NTM disease is pulmonary disease, although lymphatic, skin / soft tissue, and disseminated disease are also important. Pulmonary disease caused by NTM is most commonly seen in postmenopausal women and in patients with underlying lung disease such as cystic fibrosis, bronchiectasis, and pre-tuberculous disease. It is not uncommon for patients with alpha-1-antitrypsin deficiency, Marfan syndrome, and primary ciliary dyskinesia to develop pulmonary NTM colonization and / or infection. Pulmonary NTM can additionally be found in individuals with AIDS and malignant disease.

[0048] Clinical symptoms vary in scope and intensity but commonly include chronic cough with purulent sputum. Hemoptysis may be present. Constitutional symptoms include malaise, fatigue, and weight loss in advanced disease. The diagnosis of M. abscessus pulmonary infection requires the presence of symptoms, radiological abnormalities, and microbiological cultures.

[0049] An inhaler comprising a liquid formulation of rhGM-CSF according to the present disclosure. In a preferred embodiment, the inhaler is a nebulizer. Types of nebulizers include jet nebulizers, ultrasonic and vibrating mesh. Other embodiments include soft mist inhalers, surface acoustic wave nebulization, and capillary aerosol generators.

[0050] In one embodiment, the present invention discloses a method for delivering rhGM-CSF to the lungs comprising nebulization of a liquid formulation of rhGM-CSF, wherein the liquid formulation comprises rhGM-CSF, a sugar alcohol or sugar, albumin and water, and wherein the method delivers said rhGM-CSF to the lungs.

[0051] In another embodiment, the present invention discloses nebulizers containing a liquid formulation of rhGM-CSF for use in the treatment of aPAP or NTM infections, including jet nebulizers, ultrasonic and vibrating mesh nebulizers. EXAMPLES

[0052] The effect of different formulations on the stability of rhGM-CSF was studied using HPLC. The content and purity of each formulation was assessed by reversed-phase high-pressure chromatography (RP-HPLC). The determination of higher order structure (aggregates) and degradation products of rhGM-CSF was assessed by size-exclusion high-pressure chromatography (SE-HPLC).

[0053] Example 1 (Figure 1) - Effect of mannitol concentration The three formulation parameters of molgramostim were altered according to the table below (Table 1). 1. Mannitol level (0-50 mg / mL): The buffer concentration was adjusted simultaneously to ensure that the formulation remained isotonic. 2. Stabilizer (one of four options): rHA and PEG, rHA alone, no stabilizer, and Tween 80 alone 3. Molgramostim level (0.1-0.4mg / ml)

[0054] [Table 1]

[0055] Data was collected by RP-HPLC method. Vials were stored at 40°C for 3 months. There were two preparations of each of the 15 formulations in the table above. The main findings of this study were those shown in Figure 1. The level of mannitol had a significant effect on stability at accelerated conditions at 3 months.

[0056] Example 2 (Figure 2) - Effect of rhGM-CSF concentration It was further observed from the data collected as described in Example 1 that increasing the rhGM-CSF concentration also had a significant effect on the stability of the rhGM-CSF formulation at a particular concentration of mannitol.

[0057] Example 3 - Effect of rHA and PEG-4000 (Figure 3) The effect of rHA and PEG-4000 on the stability of molgramostim formulations for nebulization was studied under the following conditions.

[0058] [Table 2]

[0059] Formulation preparation Stability studies were performed using versions of the molgramostim formulation (combined and simplified), as described below.

[0060] Molgramostim combination preparation The composite formulations were prepared on a weight basis as described below. Bacterial disinfection was performed only on the final formulated product. The contents of the final formulated product are as shown in the table below.

[0061] [Table 3]

[0062] To prepare the conjugated rhGM-CSF formulation, a buffer was first prepared according to the following procedure.

[0063] Preparation of citrate / phosphate / HSA buffer (example batch size = 2000 g)

[0064] [Table 4]

[0065] The rHSA was removed from 2-8 °C storage and allowed to equilibrate to room temperature. The required amounts of mannitol, PEG4000, citric acid monohydrate, disodium hydrogen phosphate (anhydrous) and rHSA were pre-weighed into appropriate containers. A clean beaker / bottle and stir bar was placed on a top-loading balance and the weight of the beaker and stir bar was recorded and tared. Approximately 800 g of water for injection (WFI) was added to the beaker. The required amount of citric acid monohydrate, disodium hydrogen phosphate (anhydrous), mannitol and PEG4000 was added to the WFI, the beaker was transferred to a stir plate and the mixture was stirred until all solids were dissolved. The beaker was returned to the top-loading balance and the required amount of rHSA was added to the beaker. The beaker was then transferred to a stir plate and gently stirred for approximately 5 minutes or until the solution is homogenous. Foaming should be avoided. Finally, WFI was added to a target volume of 2000 g and the solution was gently stirred again for approximately 10 min while avoiding foaming. The buffer was labeled and stored at 2-8 °C until further processing. The buffer can be stored at 2-8 °C for up to 100 h.

[0066] Preparation of rhGM-CSF formulated product (example batch size = 1000 g) After preparation of the buffer, the rhGM-CSF complex formulation is prepared as follows. Weight of rhGM-CSF solution (g) = A Weight of the formulated product (g) = A + (10.1507 x A)

[0067] [Table 5]

[0068] The rhGM-CSF was thawed for 24 hours ± 4 hours at 2-8°C. If the bulk material has not thawed within this time, continue thawing by gently swirling the bottle at room temperature. The bottle should not be shaken and foaming should be avoided. The required amount of rhGM-CSF was pre-weighed into an appropriately sized container. A clean bottle and stir bar were placed on a top-loading balance. The weight of the beaker and stir bar were recorded and tared. Approximately 800 g of citrate / phosphate / rHA buffer was added to the beaker, after which the pre-weighed amount of rhGM-CSF was also added to the beaker. The container of rhGM-CSF was rinsed with citrate / phosphate / HSA buffer and the rinse was transferred to the beaker. The beaker was then placed on a stir plate and gently stirred for approximately 5 minutes while avoiding foaming. The mixture was confirmed to be completely homogenous. Citrate / phosphate / HSA buffer was added to a final weight of 1000 g, followed by gentle stirring for approximately 10 minutes while avoiding foaming. The formulated product was filtered using Millipore Stericup filter units (PVDF membrane (Durapore), very low protein binding, 0.22 μm pore size) and stored at 2-8°C protected from light.

[0069] Molgramostim simplified preparation The simplified formulation of rhGM-CSF was prepared on a volumetric basis because the density of the buffer for the simplified formulation was unknown. The component concentrations and amounts per vial of the final formulated product are shown in the table below.

[0070] [Table 6]

[0071] Preparation of rhGM-CSF Formulated Product (Example Batch Size = 1000 mL)

[0072] [Table 7]

[0073] Required amounts of mannitol, citric acid monohydrate, disodium hydrogen phosphate (anhydrous) were pre-weighed into suitable containers. Approximately 800 mL of water for injection (WFI) was added to a beaker equipped with a stir bar. Citric acid monohydrate, disodium hydrogen phosphate (anhydrous) and mannitol were added to the WFI. The mixture was stirred until all solids were dissolved. Required amount of rhGM-CSF was added to the beaker, the container of rhGM-CSF was rinsed with WFI and the rinse was transferred to the beaker. The beaker was placed on a stir plate and the mixture was gently stirred for approximately 5 minutes while avoiding foaming. Check for complete homogeneity. Contents of the beaker were transferred to a 1 L volumetric flask. The beaker was rinsed with WFI and the rinse was transferred to the volumetric flask. Care should be taken to avoid foaming during the transfer. WFI was added to the volumetric flask to QS up to 1 L. Contents were also mixed by gently inverting the flask. The formulated product was filtered using a Millipore Stericup filter unit (PVDF membrane (Durapore), very low protein binding, pore size 0.22 μm). The final formulated product was stored at 2-8° C. protected from light.

[0074] The main findings of this study are shown in Figure 2. The formulation containing rHA and PEG4000 as a stabilizer (combined formulation) resulted in less formation of impurities in rhGM-CSF under both refrigerated and accelerated conditions. Furthermore, the table below shows that the combined formulation has a significantly higher purity when stored at 40°C for 12 days compared to the simplified formulation.

[0075] [Table 8]

[0076] [Table 9]

[0077] Example 4 - Stability after nebulization The stability of the rhGM-CSF complex formulation after nebulization was evaluated by comparing the impurities and protein aggregates detected in non-nebulized and concentrated nebulized formulations. The aerosol generated by a vibrating mesh was collected immediately after nebulization by collecting it in a polypropylene tube placed against the mesh. Impurities and protein aggregates were detected by high performance liquid chromatography (HPLC) and size exclusion chromatography (SEC), respectively. The formulation evaluated in this study was identical to the rhGM-CSF complex formulation described above, except that the rhGM-CSF concentration was 300 μg / mL instead of 250 μg / mL. Higher concentrations of molgramostim are more likely to exhibit instability and represent a worst-case scenario.

[0078] The detection of unknown impurities determined by HPLC is tabulated in Table 2 below. The relative retention times (RRT) of these peaks relative to the main peak were 0.87, 0.96, and 1.07. After nebulization using the Head type 30 device, a slight increase in RRT of 0.87 (0.26%) was detected in the nebulized sample, and residues in the reservoir (0.32%). These results demonstrate that rhGM-CSF remains largely intact after the nebulization process.

[0079] [Table 10]

[0080] The generation of impurities and aggregates due to nebulization was initially investigated by collecting and analyzing aerosols produced by a vibrating mesh nebulizer (PARI eFlow). Assessment of aggregation by SEC revealed observable differences in albumin-related aggregates, designated HMW1 and HMW2. This data suggests that some aggregation of albumin occurs during the nebulization process, but the majority of the protein in the formulation remains unaggregated.

[0081] [Table 11]

[0082] [Table 12]

[0083] The data collected in Table 4 are the rhGM-CSF concentrations in the combined formulation before and after nebulization with a vibrating mesh nebulizer. The content was measured by RP-HPLC.

[0084] Finally, the potency of molgramostim was also evaluated before and after nebulization, and it was found that the combined formulation retained 99.4% of the biological potency of GM-CSF upon nebulization. Biological potency was measured using a TF-1 cell line assay.

[0085] Example 5 - Performance stability of rhGM-CSF complex formulations. The performance stability of rhGM-CSF complex formulations upon nebulization was evaluated up to 18 months storage at 5° C. and 25° C. Three parameters were evaluated: DD = Simulated Respiratory Dose (USP) <1601> , adults), FPF = fine particle fraction (droplets less than 5 μm), and MMAD = mass median aerodynamic diameter. Little or no change in performance stability with nebulization was observed at time points up to 18 months. Performance data is based on USP <601> Produced by pharmaceutical cascade impaction in accordance with.

[0086] Example 6 - Stability of low concentration rHA formulations A version of conjugated rhGM-CSF containing a low concentration (0.2 mg / mL) of rHA was evaluated and showed improved stability over the simplified formulation without rHA or PEG 4000. The rhGM-CSF content was measured by RP-HPLC.

Claims

1. a. rhGM-CSF at a concentration of 150-500 μg / mL; b. recombinant human albumin (rHA) at a concentration of 0.5-1.50 mg / mL; c. polyethylene glycol-4000 (PEG-4000) at a concentration of 0.05-0.15 mg / mL; d. Mannitol at a concentration of 45-100 mg / mL; A stable liquid formulation of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF), comprising:

2. The concentration of the rhGM-CSF is 200 to 300 μg / mL, The concentration of the rHA is 0.5 to 1.50 mg / mL; the concentration of the PEG-4000 is 0.05-0.15 mg / mL; and 2. The liquid formulation of claim 1, wherein the concentration of mannitol is 45-100 mg / mL.

3. The liquid formulation of claim 1, wherein the rhGM-CSF is molgramostim (SEQ ID NO: 1), sargramostim (SEQ ID NO: 2), or regramostim.

4. The liquid formulation of claim 3, wherein the rhGM-CSF is molgramostim (SEQ ID NO: 1).

5. The liquid formulation of claim 1, wherein less than 5% impurities are formed in the stable liquid formulation after storage at 25°C for 6 months.

6. The liquid formulation of claim 5, wherein the impurities are determined using HPLC.

7. 3. The liquid formulation of claim 2, further comprising a buffer.

8. The liquid formulation of claim 7, wherein the buffer is citric acid monohydrate or Na2HPO4.

9. The rhGM-CSF is molgramostim (SEQ ID NO: 1), wherein the liquid formulation contains Tween 80, Na ２ H.P.O. ４ and citric acid monohydrate, The Tween 80, the Na ２ H.P.O. ４ and one or more of the citric acid monohydrates are a concentration of Tween 80 from 0 mg / mL to 0.1 mg / mL; 2.6mg / mL to 15.5mg / mL Na ２ H.P.O. ４ and / or comprising a concentration of citric acid monohydrate from 0.28 mg / mL to 1.7 mg / mL; 9. The liquid formulation of claim 8.

10. 2. The liquid formulation of claim 1, wherein the liquid formulation has an osmolality of 250-375 mOsm / L.

11. 2. The liquid formulation of claim 1, wherein the % of rhGM-CSF remaining after 3 months of storage is greater than 60% and / or less than 5% of impurities are formed in the stable liquid formulation after 6 months of storage at 25°C.

12. 12. The liquid formulation of claim 11, wherein the impurities are determined using HPLC.

13. The liquid formulation of any one of claims 1 to 12, wherein the liquid formulation is nebulized.

14. The liquid formulation of claim 13, wherein at least 90% of the biological potency of the rhGM-CSF is retained after nebulization.

15. 13. An inhaler comprising the liquid formulation of claim 1.

16. The inhaler of claim 15, wherein the liquid formulation is nebulized.

17. The liquid formulation of claim 1 for pulmonary administration.

18. The liquid formulation of claim 1 for administration to the lungs.

19. A liquid formulation as described in claim 1 for inhalation.

20. The liquid formulation of claim 1 for the treatment of a pulmonary infection or a bacterial, viral or fungal infection in the lungs.

21. The liquid formulation of claim 1 for the treatment of autoimmune pulmonary alveolar proteinosis (aPAP) infection.

22. The liquid formulation of claim 1 for the treatment of nontuberculous mycobacterial (NTM) infections.

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