METHOD FOR PREPARING STABLE PEPTIDE FORMULATIONS.

Abstract

The present invention relates to an improved method for preparing a powder formulation containing a peptide. The present invention also provides an improved method for preparing a powder formulation containing glucagon or a glucagon analogue, wherein said powder formulation is suitable for nasal administration.

Description

METHOD FOR PREPARING STABLE PEPTIDE FORMULATIONS The present invention relates to the field of medicine. More particularly, the present invention provides an improved method for preparing a powder formulation containing a peptide. The present invention also provides an improved method for preparing a powder formulation containing glucagon or a glucagon analogue, wherein said powder formulation is suitable for nasal administration. Peptides are prone to physical instability, such as aggregation during and after the manufacturing process. Aggregation is a complex process that originates through several different mechanisms. Aggregation can typically be induced by the nucleation of a small number of peptides or proteins, which form small, soluble aggregates; these then serve as nucleation foci for the subsequent growth of larger, insoluble aggregates. The nucleation-growth process can increase with time, temperature, protein concentration, and other parameters. During manufacturing, proteins are purified and concentrated using a variety of methods, such as ultrafiltration, affinity chromatography, selective absorption chromatography, ion-exchange chromatography, lyophilization, dialysis, and precipitation or salting-out.Such concentration processes can produce aggregation (Maggio, BioProcess International 2008, 6(10): 58-65). Removing or solubilizing these aggregates requires additional process steps that can be costly and compromise overall product performance. The effects of aggregation can include material loss, reduced efficacy, altered pharmacokinetics, reduced product stability and shelf life, and induction of unwanted immunogenicity. Aggregation has become an important issue for biopharmaceutical manufacturers, particularly because the current trend toward high-concentration solutions increases the likelihood of protein-protein interactions, which in turn promote aggregation (Maggio, BioProcess International 2008; 6(10): 58-65). Several approaches to limiting peptide aggregation have been studied, including, but not limited to, adjusting pH, buffer conditions, ionic resistance, and / or adding other excipients, such as cyclodextrins. Glucagon is known for its tendency to aggregate in aqueous solutions (Pedersen JS., J Diabetes Sci Technol. 2010; 4(6): 1357-1367; Beaven et al., The European J. Biochem. 1969; 11(1): 3742; Matilainen et al., European J. of Pharmaceutical Sciences 2009; (36): 412-420), which can cause problems during the manufacture of glucagon powder formulations. Previous methods for preparing glucagon powder formulations suitable for nasal administration are disclosed in WO2016 / 133863. There is a need for alternative methods to prepare peptide powder formulations, particularly glucagon or glucagon analogue powder formulations. Specifically, there is a need for methods that reduce or eliminate peptide aggregation in aqueous solution. By reducing or, preferably, eliminating aggregation, the final powder formulation will retain a very high percentage of active peptide, which is highly advantageous. Preferably, the method results in an aqueous solution before drying that is physically and chemically stable for an extended period, for example, up to 24 hours. This extended stability makes the process much more suitable for large-scale manufacturing. Furthermore, there is a need for a method that produces a final powder formulation with a long shelf life, preferably up to approximately 24 months. Accordingly, the present invention provides an improved and cost-effective method for reducing peptide aggregation during the manufacture of a powder formulation. This method incorporates a double filtration step. One of the peptides used in the present invention is glucagon or a glucagon analogue. Powder formulations prepared according to the present method are particularly suitable for nasal administration. According to one aspect of the invention, a method for preparing a peptide powder formulation is provided. This method comprises the following steps: a. form a first mixture of an acid, a phospholipid surfactant and a cyclodextrin in an aqueous carrier; b. subjecting the first mixture to a first filtration stage, wherein the filter comprises a membrane with a pore size of about 0.4 pm to about 0.5 pm; c. adding a peptide to the first filtration product to form a second mixture, and subjecting the second mixture to a second filtration stage, wherein the filter comprises a membrane with a pore size of about 0.4 pm to about 0.5 pm; and d. Dry the second filtration product to form a solid formulation and process the solid formulation to produce a final powder formulation. In one embodiment, the peptide is glucagon or a glucagon analogue. In particular, it is glucagon. In one embodiment, the acid is either citric acid or acetic acid. Specifically, it is acetic acid. More specifically, the acetic acid is at a concentration of 1M. In one embodiment, the surfactant, cyclodextrin, and peptide together constitute between approximately 1.5% and approximately 3% by weight of the second mixture. In a particular embodiment, they constitute approximately 2% by weight of the second mixture. In a further embodiment, they constitute approximately 2.5% by weight of the second mixture. In one embodiment, the surfactant is dodecylphosphocholine (DPC), didecylphosphatidylcholine (DDPC), lisolauroylphosphatidylcholine (LLPC), dioctanoylphosphatidylcholine (D8PC) or dilauroylphosphatidylglycerol (DLPG). In particular, the surfactant is DPC. In one embodiment, cyclodextrin is α-cyclodextrin, β-cyclodextrin, hydroxypropyl β-cyclodextrin, or γ-cyclodextrin. Specifically, cyclodextrin is β-cyclodextrin. In one embodiment, more than 98% of the peptide in the final powder formulation is unaggregated peptide as measured by reversed-phase HPLC. Preferably, more than 99% of the peptide is unaggregated peptide. More preferably, 100% of the peptide is unaggregated peptide. / Ul According to another aspect of the invention, a method is provided for preparing a peptide powder formulation comprising the following steps: a. form a first mixture of a phospholipid surfactant and a cyclodextrin in an aqueous carrier; b. subjecting the first mixture to a first filtration stage, wherein the filter comprises a membrane with a pore size of about 0.4 pm to about 0.5 pm; c. adding a peptide to the first filtration product to form a second mixture, and subjecting the second mixture to a second filtration stage, wherein the filter comprises a membrane with a pore size of about 0.4 pm to about 0.5 pm; and d. Dry the second filtration product to form a solid formulation and process the solid formulation to produce a final powder formulation. In one embodiment, the surfactant, cyclodextrin, and peptide together constitute between approximately 1.5% and approximately 3% by weight of the second mixture. In a particular embodiment, they constitute approximately 2% by weight of the second mixture. In a further embodiment, they constitute approximately 2.5% by weight of the second mixture. In one embodiment, the surfactant is DPC, DDPC, LLPC, D8PC, or DLPG. In particular, the surfactant is DPC. In one embodiment, cyclodextrin is α-cyclodextrin, β-cyclodextrin, hydroxypropyl β-cyclodextrin, or γ-cyclodextrin. Specifically, cyclodextrin is β-cyclodextrin. In one embodiment, more than 98% of the peptide in the final powder formulation is unaggregated peptide as measured by reversed-phase HPLC. Preferably, more than 99% of the peptide is unaggregated peptide. More preferably, 100% of the peptide is unaggregated peptide. According to another aspect of the invention, a method is provided for preparing a glucagon powder formulation comprising the following steps: a. form a first mixture of acetic acid, DPC and β-cyclodextrin in an aqueous carrier; b. subjecting the first mixture to a first filtration stage, wherein the filter comprises a membrane with a pore size of about 0.4 pm to about 0.5 pm; c. adding glucagon to the first filtration product to form a second mixture, and subjecting the second mixture to a second filtration stage, wherein the filter comprises a membrane with a pore size of about 0.4 pm to about 0.5 pm; and d. Dry the second filtration product to form a solid formulation and process the solid formulation to produce a final powder formulation. In one embodiment, glucagon, DPC, and β-cyclodextrin together constitute between approximately 1.5% and approximately 3% by weight of the second mixture. In a particular embodiment, they constitute approximately 2% by weight of the second mixture. In a further embodiment, they constitute approximately 2.5% by weight of the second mixture. / Ul In one embodiment, the acetic acid is at a concentration of 1M. In one embodiment, more than 98% of the glucagon in the final powder formulation is unaggregated glucagon as measured by reversed-phase HPLC. Preferably, more than 99% of the glucagon is unaggregated glucagon. More preferably, 100% of the glucagon is unaggregated glucagon. The present invention further provides a powder formulation prepared according to a method of the invention. In specific embodiments, the drying of the second filtration product can be carried out by freeze drying (lyophilization) or spray drying. In one specific embodiment, the filter membrane in both the first and second filtration stages comprises, among other materials, polyvinylidene difluoride (PVDF), cellulose acetate, cellulose nitrate, polytetrafluoroethylene (PTFE, Teflon), polyvinyl chloride, polyethersulfone, or other filter materials suitable for use in a cGMP manufacturing environment. In a preferred embodiment, the filter membrane comprises PVDF. In one specific embodiment, the filter membrane in both the first and second filtration stages is a mixture with a pore size of approximately 0.45 µm. In a preferred embodiment, the filter membrane is a PVDF membrane with a pore size of 0.45 µm. In one embodiment, the pH of the solution during the method of the present invention is maintained between 2 and 3. In one embodiment, the solution phase of the method of the present invention is carried out at a temperature between 15 and 30°C, preferably between 18 and 25°C, more preferably around 20°C. The methods of the present invention can be used for peptides that tend to aggregate during the manufacture of a powder formulation. In particular, the methods can be used for peptides including, but not limited to, amylin, amylin analogs, recombinant human factor VIII (rfVII), calcitonin gene-related peptide (CGRP), calcitonin, GLP-1 analogs, dual GLP-1-GLP agonists, GIP agonists, recombinant human growth hormone (rhGH), D-ala-T-amide inhibitor CCR5 octapeptide, recombinant human insulin, insulin analogs, cyclic peptide analogs PTH 1-31, interferon-β, interferons β-α and β-1β, interleukin-2 (IL-2), erythropoietin (EPO), pramlintide acetate, and enzymes such as urokinase. In particular, the methods of the present invention can be used to prepare a powdered formulation of glucagon. Glucagon is a highly effective treatment for severe hypoglycemia both in and out of the hospital setting. Glucagon is available as powdered formulations that must be mixed with a diluent immediately before administration by injection. Liquid formulations of glucagon are also known (Pontiroli et al., Br Med J (Clin Res Ed) 1983; 287: 462-463). A glucagon powder for nasal administration for the treatment of severe hypoglycemia, described in WO2016 / 133863, has recently been approved in the United States and Europe under the name Baqsimi™. ivIAzuz i / ui Glucagon or glucagon analogue formulations produced according to the methods of the present invention are particularly suitable for nasal administration. In preferred embodiments, the formulations produced according to the methods of the present invention have one or more of the following: • A low proportion of small particles that could reach the lungs. • Drug content adequate to provide the total drug dose required to achieve the therapeutic effect as a single dose in one nostril. • Drug content suitable to deliver the total dose in a few tens of milligrams, or the maximum allowed by the delivery device. • The appropriate drug content and absorption characteristics are effective despite the presence of nasal congestion that may be associated with allergies or the common cold. • Stability during storage under ambient conditions for a prolonged period, preferably at least 24 months. • Good safety and tolerability profile. As used herein, the term “aggregation” refers to the accumulation, clumping, agglomeration, dimerization, polymerization, or formation of seed nuclei, nucleation foci, fibrils, or gels of small oligomeric precursors, such as peptides. Aggregate size ranges from soluble dimers and other multimers (approximately 5–10 nm in apparent globular diameter) to larger, insoluble species identified as subvisible and visible particles (approximately 20–50 pm in apparent globular diameter). Of the soluble aggregates, the larger ones, such as high-molecular-weight species, may be capable of eliciting immunogenic responses that could have an adverse clinical outcome. As used herein, the expressions “seed nuclei” or “nucleation foci” refer to the smallest aggregate size from which larger aggregates are formed. Reversed-phase HPLC can be used to determine the amount of unadded peptide in the final powder formulation. Standard conditions familiar to mid-level tradespeople can be used, such as those described in the examples below. As used herein, “glucagon” refers to a polypeptide of the sequence His-Ser-GIn-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-GIn-Asp-PheVal-GIn-Trp-Leu-Met-Asn-Thr (SEQ ID NO:1). Glucagon can be chemically synthesized, produced using recombinant DNA technology, or extracted from natural sources. The term “glucagon analog” refers to variants of this sequence that retain the ability to stimulate an increase in blood glucose in vivo. In Chabenne et al., Molecular Metabolism 2014, 3:293-300, examples of glucagon analogs are reported in which one amino acid in the natural sequence is replaced with alanine, as well as analogs with multiple substitutions. An example analog in which three amino acids are modified to produce a glucagon analog with enhanced biological activity is [Lys1718, G1u21]glucagon. Zealand Pharma has disclosed a number of glucagon analogues, for example, in US patent publications 20140080757, 2014001733, 20130316941, 20130157935, 20130157929, 20120178670, 20110293586, 20110286982, 20110286981, and 20100204105. These analogues are reported to have a higher binding affinity for the GLP receptor than the glucagon receptor, but despite this, they retain glucagon activity. Zealand Pharma has also begun clinical trials of a glucagon analogue for the treatment of hypoglycemia, designated ZP4207.US patent publication 20130053310 discloses other glucagon analogues useful in the treatment of hypoglycemia. Phospholipid surfactants are ubiquitous components of biological membranes found in cells and tissues throughout the human body, including the nasal mucosa. The most prevalent phospholipid surfactants in cells are phosphatidylcholines and phosphocholines (PCs), although phosphatidylglycerols (PGs) are also significant components of biological membranes. Lysophospholipids derived from a diacyl PC or PG can also be used by removing one of the acyl groups. Example phospholipid surfactants that can be used in the present invention are dodecylphosphocholine (DPC), didecylphosphatidylcholine (DDPC or 1,2-didecyl-sn-glycero-3-phosphocholine), lysolauroylphosphatidylcholine (LLPC or 1-didecanyl-sn-glycero-3-phosphocholine), dioctanoylphosphatidylcholine (D8PC or 1,2-dioctanoyl-sn-glycero-3-phosphocholine) and dilauroylphosphatidylglycerol (DLPG or 1,2-dilauroyl-sn-glycero-3-phospho(T-rac-glycerol)). The preferred phospholipid surfactants are those that form micelles, rather than bilayers, at the concentration used during powder formulation manufacturing. These include DPC, DDPC, LLPC, and D8PC, but not DLPG. DPC is the most preferred. In specific embodiments of the invention, a single type of phospholipid surfactant is used. In other embodiments, the phospholipid surfactant component may be composed of mixtures of phospholipid surfactants, including, for example, a combination of any two, three, or four of the surfactants identified above. As used herein, the term “cyclodextrin” refers to a cyclodextrin containing six, seven, or eight glucose residues in the ring, creating a cone shape, namely: • α (alpha)-cyclodextrin: 6-membered sugar ring molecule • β (beta)-cyclodextrin: 7-membered sugar ring molecule • γ (gamma)-cyclodextrin: 8-membered sugar ring molecule α-CD was used in the powder formulation (HypoGon® Nasal) by Novo Nordisk in clinical trials (Stenniger et al., Diabetologia 1993; 36: 931-935; Rosenfalck AM, et al., Diabetes Res Clin Pract 1992; 17: 43-50). The aqueous solubility of α-CD is reported to be around 5% by weight. Two other cyclodextrins, one with less aqueous solubility than α-CD (β-CD, 1.85% by weight) and another with greater aqueous solubility than α-CD (HR-β-CD), are also suitable for use in the invention, as is γ-cyclodextrin which is freely soluble in water. Cyclodextrins in the formulations act as a bulking agent and also adhere to the surface of the nasal mucosa, aiding in glucagon absorption. After administration into the nasal cavity, the main ingredient (90% to 70% by weight), namely cyclodextrin, helps the powder adhere to the mucosal surface. Cyclodextrins can be used individually or as mixtures of any two or more cyclodextrins. In one particular embodiment, the glucagon powder formulation prepared according to the present method comprises glucagon, DPC, and β-cyclodextrin. Preferably, the powder formulation comprises glucagon, DPC, and β-cyclodextrin in a weight ratio of 10:10:80 (glucagon:DPC:cyclodextrin). Preferably, the glucagon is present in a therapeutic amount that is effective when administered as a single dose into one nostril. In one embodiment, the dose of glucagon is approximately 3 mg. Mixing can be achieved through methods including static and dynamic mixing. Dynamic mixing can be performed using a blade inserted into the liquid, attached to a shaft and rotated by a motor. Static mixing can be achieved by flowing the liquid through a tortuous path within a static mixer. The presence of an air-water interface during high-speed mixing can lead to foaming. High-speed mixing can also, in turn, result in protein destabilization due to shear stress. To minimize, and preferably eliminate, foaming, low-speed mixing conditions are preferred. In the case of dynamic mixing, the speed is determined by the revolutions per minute (rpm) of the agitator.The preferred rpm values ​​are between 50 and 300, with greater preference between 50 and 250, and even greater preference between 50 and 100. The second filtrate is dried to remove the solvent and leave a solid product. Drying can be carried out by freeze-drying, spray-drying, tray-drying, or other techniques. The macroscopic physical characteristics of the product will vary depending on the drying technique, and it may be in the form of a flaky solid (due to freeze-drying) or a dry solid cake. Powders with excessive moisture content can be sticky and clumpy, resulting in a powder that is difficult to handle for filling a delivery device. It is important to note that the level of residual water content has a direct impact on stability. Residual moisture content levels above 5% in bulk powder result in reduced stability compared to powder with a residual water content below 5%. Therefore, in a particular embodiment, powder formulations prepared according to the present invention preferably have a residual water content of less than 5%. In one particular embodiment, the amount of acid in the powder formulations prepared according to the present invention is less than 10% w / w, preferably less than 6% w / w. Powders suitable for nasal administration require physical characteristics that allow for adequate flowability so they can be filled into a nasal delivery device. Flowability is determined by several parameters, including particle size, shape, density, surface texture, surface area, cohesion, adhesion, elasticity, porosity, hygroscopicity, and friability. Powders with the desired particle size and flow characteristics can be produced by processing bulk powder to remove particles that are too small or too large. Methods for processing bulk powder to remove particles that are too small or too large may include milling the bulk powder to break up larger particles and sieving to isolate particles within the desired particle size range. Several sieving methods can be performed, including vertical-motion (throw-action) vibratory sieving, horizontal sieving, tapping, supersonic sieving, and circular air jet sieving. Sieves may be used as individual sieves with a fixed nominal opening, or the bulk powder may be processed through a series of sieves with progressively smaller openings to obtain the desired particle size distribution.The sieves can be woven wire mesh sieves with nominal openings ranging from 25 to 1000 pm. Examples Example 1 - Preparation of the glucagon powder formulation - Double filtration stage DPC is dissolved in a 1 M acetic acid solution by stirring. β-Cyclodextrin is then added to the DPC solution and stirred until dissolved to form a first solution. This first solution undergoes a first filtration stage through a 0.45 µm PVDF filter. The filtrate (excipient solution) is collected in a new, clean tank, and the tank temperature is set to 20°C ± 2°C to ensure the solubility of the materials in the solution. Once the target temperature is reached in the tank, glucagon or a glucagon analogue is added to the tank while the solution is stirred. As soon as the glucagon appears to be dissolved (by visual confirmation), stirring is stopped immediately. The glucagon solution is then filtered through a second 0.45 µm PVDF filter, and the filter material is collected in a second clean tank.This second filter material (second filtration product) contains 97.5% w / w 1 M aqueous acetic acid, 0.25% w / w DPC, 2% w / w β-cyclodextrin, and 0.25% w / w glucagon (total 2.5% w / w solids by weight). The material is then lyophilized and subjected to a densification step to produce the final glucagon powder formulation. Comparative example - Preparation of the glucagon powder formulation - Simple filtration stage DPC is dissolved in 8 liters of 1 M acetic acid solution by stirring. Glucagon is added while stirring the solution. As soon as the glucagon appears to be dissolved (by visual confirmation), β-cyclodextrin is added while stirring. Once all added solids appear to have dissolved, the solution is filtered through a 0.45 µm PVDF filter. The use of multiple filters may be necessary if a single filter membrane becomes clogged or fouled. The filtrate contains 0.3% w / w DPC, 2.4% w / w β-cyclodextrin, and 0.3% w / w glucagon (total 3% w / w solids). The filtrate is collected and lyophilized. Stability of the excipient solution after the first filtration The excipient solution (acetic acid, DPC, and β-cyclodextrin) was prepared essentially as described in Example 1 at a concentration of 2.5% w / w solids. The solution was maintained at 25°C during the study. The data are summarized in Table 1. There were no significant changes in content over a period of 22 hours, and the mass balance was confirmed. Table 1 / U1 Sample time in hours DPC content (% w / w) β-Cyclodextrin content (% w / w) 0 0.26 2.02 1 0.26 2.00 2.7 0.26 2.05 3.2 0.26 1.97 7.4 0.25 1.96 22 0.25 1.98 Stability of the aqueous solution containing glucagon Solution test The glucagon solution is prepared essentially as described in Example 1 (second filtration product). After preparation, the glucagon solution is allowed to stand without stirring. Samples of the solution are taken at predetermined times and passed through a 0.45 µm filter before the assay. Any glucagon that has formed aggregates is removed by this filtration step; therefore, this assay provides an estimate of the degree of aggregation. Fluorescence assay The basis of the fluorescence method is the utilization of the change in emission wavelength of the single tryptophan residue in the glucagon molecule (Pedersen JS., J Diabetes Sci Technol. 2010; 4(6): 1357-1367). As the glucagon molecule changes its conformation from a random coil or alpha helix to aggregated forms, the local environment of the tryptophan molecule changes, resulting in a blue shift in the emission spectrum. Therefore, by monitoring the change in the wavelength of the glucagon emission fluorescence signal over time with a fiber-optic-coupled backscatter fluorescence probe, the ratio of emission peaks from non-aggregated glucagon to aggregated forms of the molecule can be used as a tool for real-time aggregation monitoring. The glucagon solution is prepared essentially as described in Example 1 (second filtration product). A fluorescence probe is used to monitor changes in the emission spectra over time. The solution is not stirred and is monitored at room temperature for 24 hours. In a smaller-scale experiment (100 mL), no change in the glucagon fluorescence ratio was observed over this 24-hour period. In further experiments, the glucagon solution is prepared essentially as described in Example 1 (second filtration product) and held at different temperatures. For comparison, this is compared to a glucagon solution that has not undergone the second filtration stage. The results are summarized in Table 2. Table 2 Glucagon solution filtration studies and holding temperature Experiments Filtration Holding temperature, time Agitation Results A Yes 20°C, 24 hours No No change in glucagon solution assay B Yes 5°C, 24 hours No No change in glucagon solution assay C No 20°C, 24 hours No Loss in glucagon solution assay; change in peak fluorescence emission ratio The study results show that when the glucagon solution undergoes the second filtration stage and is kept undisturbed at 5°C or 20°C, no glucagon is lost through aggregation from the system. However, when the solution is not filtered, it loses approximately 8% of its glucagon content within 24 hours. The chemical stability of the glucagon solution prepared essentially as set out in Example 1 can also be evaluated by reversed-phase HPLC essentially as set out below. In preparations made essentially as described above with double filtration in quantities as large as 100 liters (with 2.5% w / w solids), surprisingly, the solution collected after the second filtration stage proved to be physically and chemically stable for up to 24 hours without any detectable aggregation (as determined by one or more of the methods described above). In contrast, a glucagon solution subjected to a single filtration stage (comparative example - 8 liters and 3% w / w solids) showed visible aggregation within approximately 15 minutes of the addition of glucagon. Chemical stability analysis by HPLC of the nasal glucagon powder formulation The stability of the nasal glucagon powder formulation prepared according to Example 1, relative to well-defined external reference standards, is determined using routine RP-HPLC techniques. Briefly, a C18 reversed-phase HPLC column, 3.0 mm id x 150 mm, 2.6 µm particle size, is used with a mobile phase of potassium phosphate:acetonitrile buffer with a UV detection wavelength of 214 nm. The gradient mobile phase composition starts with a 3-minute retention of 54%, 80:20, 150 mM potassium phosphate:acetonitrile buffer and ends with a 70% potassium phosphate:acetonitrile buffer, 60:40, over the course of 8 minutes. In experiments conducted essentially as described above, as shown in Table 3, representative samples from three different batches of the nasal glucagon powder formulation prepared according to Example 1 (100 I) retained about 100% of the glucagon activity within experimental precision. Bioassay of the potency of the nasal glucagon powder formulation A genetically engineered embryonic kidney cell line, HEK293, stably expressing both a cell-surface receptor for glucagon and a CRE-luciferase reporter gene, is used to determine the relative potency of the final nasal glucagon formulation. In this cell-based assay, luciferase transcription from the CRE promoter is regulated by triggering a response along the endogenous cyclic AMP (cAMP) signaling pathway. Thus, glucagon binding to the cell-surface receptor induces cAMP production. This leads to phosphorylation and activation of the cAMP-responsive element-binding protein (CREB), resulting in luciferase expression via the CRE-luciferase reporter gene. Luciferase production is determined by adding a luciferin substrate to the reaction mixture and quantifying luciferin oxidation using a luminometer.The luminescence signal is proportional to the amount of luciferase present, which is directly proportional to the amount of glucagon used to induce the cells. The relative potency of a test sample is determined by comparing an 8-point typical dose-response curve of the reference standard with that of the sample. The response data are fitted to a 4-parameter logistic model to determine the ECs of the reference standard and the ECs of the sample, where the ratio between these ECs represents the relative potency of the test material. HEK293 cells are placed in 96-well cell culture plates in growth medium (10% fetal bovine serum (FBS) in Dulbecco's Modified Eagle Medium (DMEM) with 1.0 mg / mL Genetecin® and 125 pg / mL hygromycin B. Penicillin and streptomycin may be added to a final concentration of 100 units / mL penicillin and 100 pg / mL streptomycin) and allowed to adhere for 30 minutes to 2.5 hours at 37°C. The growth media are washed and replaced with assay media consisting of 0.25% FBS in DMEM with 0.5% bovine serum albumin, 1 x penicillin / streptomycin, and glucagon at concentrations ranging from 0.00032 ng / mL to 25 ng / mL. The plates are incubated for 4.5 hours at 37°C. 100 pL of SteadyGlo® are added per cavity, and then the cavities are shaken continuously for 30 minutes at room temperature. The plates are read on a luminometer. In the experiments conducted essentially as described above, as shown in Table 3, the relative potency percentage of glucagon measured via the cell-based assay was found to be between 94% and 102%, demonstrating that no aggregation occurred during the preparation of the formulation according to Example 1 (100 L). These results were comparable to the results of the chemical-based assay of glucagon using the same reference standard. Analysis of impurities in the nasal glucagon powder formulation The identification and quantification of potential impurities in the nasal glucagon powder formulation is performed using routine RP-HPLC techniques. Impurities may arise due to the manufacturing process or chemical decomposition of the materials in the final formulation. The method is based on the conditions described in USP41-NF36. This analysis provides an indication of the stability of the glucagon powder formulation. In experiments conducted essentially as described above, as shown in Table 3, the total impurity level in the batch release ranged from approximately 0.4% to approximately 0.56%. Furthermore, the proposed shelf-life specification analysis for the nasal glucagon powder formulation prepared according to Example 1 shows a total impurity level of approximately 20% (a / a) or lower for up to approximately 24 months. Remarkably, the nasal glucagon powder formulation has a total impurity level that is significantly lower than that recommended for current glucagon emergency kits on the market, for which the current USP monograph (USP41-NF36) specifies a limit of no more than 31% (a / a) of total impurities and related compounds. Table 3 Chemical stability analysis, bioassay and impurities of the nasal glucagon powder formulation iviAazuz i / ui Lot No. Glucagon Chemical Assay (%) Glucagon Bioassay (% Relative Potency) Total Impurities (%) 1 103.1 102 0.40 2 101.1 94 0.39 3 102.1 97 0.56 The above data is for batches of powder formulation that were loaded into a nasal delivery device and then discharged. Clinical efficacy of the nasal glucagon powder formulation The clinical efficacy of a single-batch, quality-controlled nasal glucagon powder formulation, manufactured using the two-stage filtration process of Example 1, was analyzed in clinical trial study NCT03339453 (Suico et al., EASD-2008; abstract 150). Briefly, the efficacy and safety of the nasal (NG) glucagon powder formulation were compared to intramuscular (IMG) glucagon in adult patients with type 1 diabetes mellitus during insulin-controlled hypoglycemia. The nasal glucagon powder formulation is packaged in a device for administration into one nostril at a dose of 3.0 mg. The results shown in Table 4 demonstrate that 100% of patients were successfully treated with either NG or IMG, and that the activity of NG was comparable to the activity of IMG in this study. Table 4 Primary Efficacy Analysis IGBI (TID) (N=66)a 3 mg NG 1 mg IMG Treatment Success - n (%) 66(100%) 66(100%) Treatment Difference (95% two-sided confidence limit)b 0% (-1.5%, 1.55)c Glucagon criterion met - n (5) (i) >70 mg / dl (3.9 mmol / l) (ii) Increase of 20 mg / dl (1.1 mmol / l) from nadir Both (i) and (ii) 66(100%) 66(100%) 66(100%) 66(100%) 66(100%) 66(100%) The efficacy analysis populations consisted of all patients who received both doses of the study drug with eligible glucose concentrations. bDifference calculated as (percentage of success in IMG) - (percentage of success in NG), non-inferiorityc95% two-sided confidence interval (Cl) of the Wald method with continuity adjustment Sequences (SEQ ID NO:1) His-Ser-GIn-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-GIn-Asp-Phe-Val-GInTrp-Leu-Met-Asn-Thr

Claims

1. A method for preparing a peptide powder formulation comprising the following steps: a. forming a first mixture of an acid, a phospholipid surfactant, and a cyclodextrin in an aqueous carrier; b. subjecting the first mixture to a first filtration step, wherein the filter comprises a membrane with a pore size of about 0.4 pm to about 0.5 pm; c. adding a peptide to the first filtration product to form a second mixture, and subjecting the second mixture to a second filtration step, wherein the filter comprises a membrane with a pore size of about 0.4 pm to about 0.5 pm; and d. drying the second filtration product to form a solid formulation and processing the solid formulation to produce a final powder formulation.

2. The method according to claim 1, wherein the peptide is glucagon or a glucagon analogue.

3. The method according to claim 2, wherein the peptide is glucagon.

4. The method according to any one of claims 1 to 3, wherein the surfactant, cyclodextrin and peptide together constitute between about 1.5% and about 3% by weight of the second mixture.

5. The method according to claim 4, wherein the surfactant, cyclodextrin and peptide together constitute about 2% by weight of the second mixture.

6. The method according to claim 4, wherein the surfactant, cyclodextrin and peptide together constitute about 2.5% by weight of the second mixture.

7. The method according to any of claims 1 to 6, wherein the membrane, in both the first and second filtration stage, comprises a polyvinylidene difluoride (PVDF) membrane.

8. The method according to any of claims 1 to 7, wherein the membrane, in both the first and second filtration stage, comprises a pore size of about 0.45 pm.

9. The method according to any of claims 1 to 8, wherein the acid is citric acid or acetic acid.

10. The method according to claim 9, wherein the acid is acetic acid.

11. The method according to claim 10, wherein the acetic acid is at a concentration of 1M.

12. The method according to any of claims 1 to 11, wherein the surfactant is dodecylphosphocholine, didecylphosphatidylcholine, lysolauroylphosphatidylcholine, dioctanoylphosphatidylcholine or dilauroylphosphatidylglycerol.

13. The method according to claim 12, wherein the surfactant is dodecylphosphocholine. / Ul 14. The method according to any of claims 1 to 13, wherein the cyclodextrin is a-cyclodextrin, β-cyclodextrin, hydroxypropyl β-cyclodextrin or y-cyclodextrin.

15. The method according to claim 14, wherein the cyclodextrin is β-cyclodextrin.

16. A method for preparing a peptide powder formulation comprising the following steps: a. forming a first mixture of a phospholipid surfactant and a cyclodextrin in an aqueous carrier; b. subjecting the first mixture to a first filtration step, wherein the filter comprises a membrane with a pore size of about 0.4 pm to about 0.5 pm; c. adding a peptide to the first filtration product to form a second mixture, and subjecting the second mixture to a second filtration step, wherein the filter comprises a membrane with a pore size of about 0.4 pm to about 0.5 pm; and d. drying the second filtration product to form a solid formulation and processing the solid formulation to produce a final powder formulation.

17. The method according to any of claims 1 to 16, wherein more than 98% of the peptide in the final powder formulation is a non-aggregated peptide, as measured by reversed-phase HPLC.

18. A powder formulation prepared by the method according to any of claims 1 to 17.

19. A method for preparing a glucagon powder formulation comprising the following steps: a. forming a first mixture of acetic acid, dodecylphosphocholine, and β-cyclodextrin in an aqueous carrier; b. subjecting the first mixture to a first filtration step, wherein the filter comprises a membrane with a pore size of about 0.4 µm to about 0.5 µm; c. adding glucagon to the first filtrate product to form a second mixture, and subjecting the second mixture to a second filtration step, wherein the filter comprises a membrane with a pore size of about 0.4 µm to about 0.5 µm; and d. drying the second filtrate product to form a solid formulation and processing the solid formulation to produce a final powder formulation.

20. The method according to claim 19, wherein the surfactant, cyclodextrin and peptide together constitute about 2.5% by weight of the second mixture.

21. The method according to claim 19 or claim 20, wherein the membrane, in both the first and second filtration stage, comprises a PVDF membrane.

22. The method according to any of claims 19 to 21, wherein the membrane, in both the first and second filtration stage, comprises a pore size of / Ul around 0.45 μπi.

23. The method according to any of claims 19 to 22, wherein the acetic acid is at a concentration of 1M.

24. The method according to any of claims 19 to 23, wherein more than 98% of the glucagon in the final powder formulation is unadded glucagon, as measured by reverse-phase HPLC.

25. A powder formulation prepared by the method according to any one of claims 19 to 24.

26. A powder formulation according to claim 25, wherein more than 98% of the glucagon in the final powder formulation is unaggregated glucagon, as measured by reverse-phase HPLC.