WO2013041542A1 - Formulations pharmaceutiques comprenant des sphérolyophilisats de molécules biologiques - Google Patents

Formulations pharmaceutiques comprenant des sphérolyophilisats de molécules biologiques Download PDF

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Publication number
WO2013041542A1
WO2013041542A1 PCT/EP2012/068384 EP2012068384W WO2013041542A1 WO 2013041542 A1 WO2013041542 A1 WO 2013041542A1 EP 2012068384 W EP2012068384 W EP 2012068384W WO 2013041542 A1 WO2013041542 A1 WO 2013041542A1
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spherolyophilisates
pharmaceutical formulation
μιη
cryoprotectant
range
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PCT/EP2012/068384
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English (en)
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Alf Lamprecht
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Alf Lamprecht
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1816—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/24—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • A61K9/1623—Sugars or sugar alcohols, e.g. lactose; Derivatives thereof; Homeopathic globules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1635—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1658—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions

Definitions

  • compositions comprising spherolyophilisates of biological molecules
  • the present invention relates to pharmaceutical formulations comprising
  • pharmaceutically active biological molecules such as proteins, enzymes or antibodies are administered by injection, meaning that they must be provided in the form of a liquid, such as aqueous solutions, emulsions or suspensions.
  • Lyophilisation however, also poses challenges during manufacturing. Lyophilisation itself is a comparatively costly and time-consuming process. Moreover, under standard lyophilisation processes, one frequently obtains a porous solid "cake", which e.g. may not have good flow properties. This e.g. makes filling the
  • the present invention in one aspect relates to a pharmaceutical formulation comprising spherolyophilisates, wherein the spherolyophilisates comprise a mixture of at least one pharmaceutically active agent, at least one cryoprotectant and at least one stabilising agent.
  • the pharmaceutically active agent may be a biological molecule selected from the group comprising proteins, polypeptides, oligopeptides, peptides, DNA, R A and the like.
  • a particularly preferred embodiment of such biological molecules relates to enzymes, hormones, antibodies and the like.
  • Cryoprotectants may be polyhydroxylated carbons such as alcohols and sugars.
  • Alcohols may be preferably polyalcohols such as mannitol, sorbitol, glycol and the like.
  • Sugars may be selected from sucrose, trehalose and the like.
  • the stabilising agent which confers mechanical strength to the spherolyophilisates, may be selected from excipients such as BSA, HSA, recombinant Serum Albumin, dextran, polydextrose, polyvinylpyrollidone (PVP) and the like.
  • the use of such ternary mixtures in a spray freeze drying process allows lyophilisate powders to be obtained, the particles of which show a substantially uniform size distribution and spheroidal or ball-shaped appearance. Moreover, the particles are porous, which allows for quick and efficient reconstitution.
  • the lyophilisate powders of the present invention provide good flow properties being advantageous during manufacturing of storage stable formulations of biological molecules.
  • such lyophilisate powders are thus called spherolyophilisates and in view of the uniform size distribution they may also be considered to be monodisperse spherolyophilisates.
  • the resulting spherolyophilisates may have a diameter in the range of about 1 to about 50 ⁇ , preferably in the range of about 5 to a about 40 ⁇ and more preferably in the range of about 10 to about 30 ⁇ , making them suitable for pulmonary administration.
  • spherolyophilisates with a diameter in the range of about 50 to about 750 ⁇ , preferably of about 100 to about 500 ⁇ , and more preferably in the range of about 200 to about 350 ⁇ .
  • Such spherolyophilisates are suitable for nasal, oral, buccal, sublingual or ophthalmic administration.
  • the present invention also relates to methods of producing formulations comprising spherolyophilisates of biological molecules such as proteins, polypeptides, oligopeptides, peptides and the like by using the aforementioned ternary mixture in a spray freeze drying process.
  • FIGURE LEGENDS spherolyophilisates with a diameter in the range of about 50 to about 750 ⁇ , preferably of about 100 to about 500 ⁇ , and more preferably in the range of about 200 to about 350 ⁇ .
  • the present invention also relates to methods of producing formulations comprising spherolyophilisates of biological molecules such as proteins, polypeptides, oligopeptid
  • Fig. 1 Set-up for freeze spray freezing used in the examples.
  • Fig. 2 shows HiSIS 2002 ccd camera picture of jet of water droplets (1cm underneath the nozzle).
  • Fig. 5 X-Ray graphs of mannitol, lysozyme, BSA, DEX, PVP, Spray freeze dried (SFD) BSA 0.1%, SFD DEX 0.1%, SFD PVP 0.1%, SFD BSA 10%, SFD DEX 10%, SFD PVP 10%, SFD BSA 10% after 6 months storage, SFD DEX 10%o after 6 months storage, SFD PVP 10%> after 6 months storage.
  • the present invention is based on the finding that, if a pharmaceutically active agent, and in particular biological molecules such as proteins, polypeptides, oligopeptides, peptides and the like are combined with a cryoprotectant and a stabilising agent and subjected to a spray freeze drying process, one can obtain lyophilisate powders of essentially spherical form and uniform size distribution, the latter properties conferring beneficial properties to the lyophilisate powders which may be designated as spherolyophilisates. Moreover, the spherolyophilisates are porous. Without wanting to be bound to a scientific theory, it is believed that the stabilising agent confers mechanical strength to the lyophilisate powder particles, which are formed during the spray freeze drying process. In comparison to known spray freeze drying processes, which do not make use of such ternary combinations, the resulting lyophilisate powders thus show a reduced degree of cracking, abrasion, etc. leading to an intact round structure of substantially uniform size distribution.
  • the terms “about” and “approximately” denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question.
  • the term typically indicates deviation from the indicated numerical value of ⁇ 20%, preferably ⁇ 15%, more preferably ⁇ 10% and even more preferably ⁇ 5%.
  • spray freeze drying is also used in its common sense.
  • the spray freeze drying process as far as it relates to the devices used for performing the method and the method parameters is preferably performed in accordance with the teaching of German patent DE 10 2008 017 461. If in the context of the present invention, specific process parameters have been used, these are indicated in the examples.
  • spherolyophilisates refers to powders with monodisperse, spherical and porous particles produced by freeze drying.
  • the term "monodisperse” means that particles have substantially a uniform size and typically show a span value of less than 10, preferably less than 5, more preferably less than 3, even more preferably less than 2 and most preferably less than 1 or even most preferably less than 0.5.
  • the "span” of size distribution is calculated as (X90 - X10): X50 wherein X10, X50 and X90 are the limit diameters for 10%, 50% and 90% of particles (see also experimental section). A lower value indicates a narrower size distribution.
  • the pharmaceutical formulations in accordance with the invention are obtained by subjecting a ternary mixture comprising at least a pharmaceutically active agent, a cryoprotectant and a stabilising agent to a spray freeze drying process in order to obtain spherolyophilisates.
  • the pharmaceutically active agent may preferably be a biological molecule such as proteins, polypeptides, oligopeptides, peptides, DNA, RNA and the like.
  • peptide designates a molecule where up to and including 10 amino acids are linked by a peptide bond.
  • oligopeptide denotes a molecule where up to 40 amino acids are linked by a peptide bond.
  • peptide denotes a molecule with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids being linked by peptide bonds
  • oligopeptide denotes molecules where 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, e.g. 25, e.g. 30, e.g. 35 and up to and including 40 amino acids are linked by a peptide bond.
  • polypeptide comprises any type of molecules where more than 40 amino acids are linked by a peptide bond.
  • protein typically refers to polypeptide molecules that can be found in nature. The term thus includes protein representatives such as hormones, enzymes, or antibodies.
  • the person skilled in the art will understand that the aforementioned molecules may be modified, e.g. by a chemical modification and still exert their original function.
  • a protein, polypeptide, oligopeptide, or a peptide may comprise a non- natural amino acid conferring e.g. stability over the non-modified version of the molecule.
  • proteins, polypeptides, oligopeptides and peptides may be modified by glycosylation, pegylation, hesylation and the like. All these types of common modifications are meant being encompassed by the term “derivative" .
  • the pharmaceutical formulations are made from a ternary mixture comprising e.g. a protein such as an enzyme or a hormone.
  • an antibody or binding fragment thereof is used as a biological molecule.
  • antibody for the purposes of the present invention covers the common type of antibodies and antigen binding molecules or fragments. Binding fragments may thus include portions of an intact full-length antibody, such as an antigen binding or variable region of the complete antibody.
  • antibody fragments include Fab, F(ab') 2 , Fd and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibody fragments such as bispecific, trispecific, and multispecific antibodies (e.g., diabodies, triabodies, tetrabodies); minibodies; chelating recombinant antibodies; tribodies or bibodies; intrabodies; nanobodies; small modular immunopharmaceuticals (SMIP), binding-domain immunoglobulin fusion proteins; camelized antibodies; VHH containing antibodies; and any other polypeptides formed from antibody fragments.
  • SMIP small modular immunopharmaceuticals
  • Typical representatives of biological molecules may be therapeutic antibodies for cancer treatment, known biologies such as EPO, G-CSF, insulin, growth factors, luteinizing hormone, follicle stimulating hormone, lysozyme, etc.
  • a pharmaceutically active agent may, however, also comprise small molecules.
  • small molecule compound is used as common in the art. It thus does not include e.g. proteins, peptides and nucleic acids.
  • small molecule compound also termed "small molecule” as referring to a low molecular weight, preferably organic compound, which is not a biopolymer (such as e.g.
  • the upper molecular weight limit may be regarded as being less than about 5000 Daltons. Preferably the upper molecular weight limit is less than about 2000 Daltons, more preferably less than about 1000 Dalton and even more preferably less than about 800 Dalton. Functionally, the skilled person in the field of pharmacology may understand the term as referring to a molecule that binds with high affinity to a biopolymer such as a protein, a nucleic acid or a
  • cryoprotectanf refers to substances as they are commonly used in formulations of biological molecules, which are subjected to lyophilisation.
  • cryoprotectants may for example be alcohols and preferably polyalcohols such as glycol, glycerol, erythritol, trithol, arabitol, xylitol, rebitol, mannitol, sorbitol, dulcitol, iditol, isomalt, maltitol, lactitol, inositol, etc.
  • polyalcohols such as glycol, glycerol, erythritol, trithol, arabitol, xylitol, rebitol, mannitol, sorbitol, dulcitol, iditol, isomalt, maltitol, lactitol, inositol, etc.
  • the cryoprotectant may also be a sugar selected from mono-, di- and
  • Such sugars include trehalose, mannose, lactose, galactose, maltose, glucose, raffmose, maltotriose, sucrose and the like.
  • cryprotectants may be amino acids.
  • amino acids include gylcin, arginine, glutamic acid and proline.
  • Particularly preferred representatives of alcohols as cryoprotectants are mannitol and sorbitol.
  • Particularly preferred representatives of sugars as cryoprotectants are trehalose and sucrose.
  • a particularly preferred cryoprotectant is mannitol.
  • stabilising agent for the purposes of the present invention relates to a pharmaceutically acceptable excipient that is commonly used for protein
  • stabilising agent may be an excipient, which usually does not crystallize, as this helps preventing re- crystallization of other components. As explained hereinafter, the stabilising agent also contributes to the mechanical stability of the spherolyophilisates. Therefore stabilising agents being pharmaceutically acceptable excipients with a polymeric structure can be preferred.
  • stabilizing agents include hydrohilic polymers such as dextran, polyvinylpyrollidone, polyvinyl alcohol, poloxamer, poly ethylenegly col, hyaluronic acid, lactoglobulin and the like.
  • hydrophilic polymers may be selected dependin on the route of administration such as oral, buccal or nasal administration. The skilled person is aware which polymers may be particularky suitable for a certain route of administration.
  • Preferred stabilising agents are BSA, HSA, recombinant versions thereof, dextran and PVP.
  • a particularly preferred stabilising agent is PVP.
  • compositions in accordance with the invention by a spray freeze drying process will typically comprise between about 0.05 wt.-% to 20 wt.-% of the stabilising agent.
  • solution comprising the ternary mixture will comprise between 0.1 wt.-% to 10 wt.-% of the stabilising agent. It has been found that an increasing amount of stabilising agent increases the mechanical strength of the lyophilisate powders obtained in the spray freeze drying process.
  • the amount of stabilising agents such as BSA, dextran or PVP is increased from 0.1 wt.-% to 1 wt.-% and even 10 wt.-% in the starting solution, the mechanical properties of the resulting spherolyophilisates clearly improve as can be detected in electron microscopy.
  • the surface of particles having higher concentrations of these stabilising agents appears smoother, has fewer cracks and is more uniform.
  • the beneficial influence of the stabilising agent on the mechanical properties of the resulting lyophilisates can also be deduced by that the span for the size distribution of the resulting lyophilisates is reduced with increasing amounts of stabilising agent.
  • an increasing amount of stabilising agent irrespective of the chemical nature of the stabilising agent, leads to a reduction in the span value, meaning that the particles become less polydisperse, i.e. having a more uniform size distribution. This is explainable by the assumption that the increased mechanical strength of the particles prevents breakage during the spray freeze drying process.
  • the cryoprotectant will usually be comprised in the solution comprising the ternary mixture used for the spray freeze drying process in amounts of 1% to 20% such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 wt.-%.
  • the cryoprotectant is used in an amount of about 3 to 10 wt.-% such as 5 wt.-%.
  • the average size i.e. the average diameter of the spherolyophilisates which are obtained by using the ternary mixture described herein in a spray freeze drying process can be adjusted by the process parameters for the spray freeze drying process.
  • the droplets and thus the resulting particles may be smaller if a nozzle with a smaller diameter is used. More information as regards the performance of a spray freeze drying process, the process parameters etc. can be found in
  • the desired size of the spherolyophilisates will to some extent pose restrictions on the stabilising agent that can be used in the ternary mixture. For example if spherolyophilisates of a small diameter such as 1 to 20 ⁇ are to be manufactured, the stabilising agent will have to have a lower viscosity than if spherolyophilisates with a diameter in the range of about 500 ⁇ are to be produced. For the purposes of the present invention, it has been found that stabilising agents such as BSA, dextran and PVP are suitable to produce spherolyophilisates across a diameter range of 1 ⁇ to 1 mm.
  • the formulations in accordance with the present invention may be suitable for different modes of administration.
  • a diameter range of between 1 to 50 ⁇ and preferably below 50 ⁇ is considered to be suitable in the case of spherolyophilisates for pulmonary applications. If spherolyophilisates with such small diameters are produced in accordance with the present invention, it has been observed that flow properties may be affected as the resulting lyophilisate powders, despite their round curvature, tend to aggregate. For pulmonary application this should however be less of a problem given that the actual administration will occur by a nebuliser, which will ensure a homogenous distribution of the powder particles.
  • the present invention therefore also pertains to pharmaceutical formulations having spherolyophilisates being made from the aforementioned ternary mixtures having a diameter in the range between about 1 to about 50 ⁇ , preferably about 5 to about 40 ⁇ , and more preferably of about 10 to about 30 ⁇ such as about 20 ⁇ that can be used in pulmonary administration.
  • the present invention relates to spherolyophilisates being made from the ternary mixtures in a spray freeze drying process having a diameter in the range of about 100 ⁇ to about 1 mm and preferably in the range of about 150 ⁇ to about 500 ⁇ and more preferably in the range of about 200 ⁇ to about 400 ⁇ , such as about 250 to about 300 and about 350 ⁇ .
  • spherolyophilisates due to their spherical shape, smooth surface, mechanical stability and narrow size distribution have excellent flow properties. Such excellent flow properties provide a significant improvement in handling of such lyophilisate powders during
  • such formulations thus may be used as lyophilisate powders and reconstituted in solution immediately prior to administration, exemplarily prior to parenteral administration. They may also be directly used in a powder form for nasal, oral, sublingual or buccal administration or reconstituted before use.
  • the present invention uses the common spray freeze dry method known from e.g. DE 10 2008 017 461 for a ternary mixture as mentioned above to obtain spherolyophilisates for preferably biological molecules.
  • the solution is injected into a cooling chamber where the particles immediately freeze.
  • a nozzle which is controlled by a piezoelectric element, it is ensured that the drop-size is constantly the same.
  • the frozen droplets then enter a drying chamber in which an upstream gas stream as well as the velocity of the liquid stream dispersed into droplets initiates the drying process.
  • the frozen droplets will release humidity into the gas stream.
  • the frozen droplets will have decreased density and thus move up the drying chamber.
  • At the upper end of the drying chamber they will then be transported with the drying gas and can subsequently be separated from the drying gas.
  • the ternary solution will be introduced in a device as described above to produce frozen droplets of uniform size and form. These frozen droplets may then be transferred into a common lyophilisation device to initiate the drying step (see Fig. 1).
  • the ternary mixtures of the present invention ensure that the frozen droplets, which are formed when the solution enters the cooling chamber, will have a uniform size and form and will not change the size and form, e.g. due to collision, cracking etc. whilst being in the cooling and/or drying chamber when being moved by the gas stream.
  • the spray freeze drying process uses simply cold air for the freezing step.
  • the avoidance of using liquid nitrogen has the advantage that no physical deformation of the particles occurs upon contacting the nitrogen. Further, agglomeration of particles during the process of production is reduced if the particles are not directly contacted with liquid nitrogen.
  • spray settings can be seen as fine adjustment with only minor influence on the particle formation.
  • spray settings can be seen as fine adjustment with only minor influence on the particle formation.
  • the freezing temperature impacts the particle morphology and is especially a valuable option for manipulation of porosity and specific surface area.
  • the porosity of particles depends to some extent on the solid concentration of the initial solution. Therefore the porosity can be adjusted by the composition of the initial solution.
  • a further parameter to control porosity is the freezing rate.
  • a fast freezing rate in spray freeze also facilitates to achieve amorphous particles.
  • the spherolyophilisates of the present invention which are spheroidal particles of substantially uniform size and shape, are characterised by a high porosity and low density. In view of their smooth surface and their substantially round form, their flow properties are excellent.
  • the high porosity of the sponge like structure lead to a low density structure, which is easily deformable and can be quickly dissolved in aqueous solutions. They are thus suitable for storage and quick reconstitution of biological molecules.
  • Chemicals Lysozyme also named muramidase from hen egg white was used as a model enzyme for activity measurements and purchased from Roche Diagnostics,
  • Solution Preparation Solutions consist of 5g D(-)-Mannitol in 100ml and BSA, DEX or PVP in following concentrations: O.lg in 100ml, lg in 100ml or lOg in 100ml (see table no. 1). All solutions are also prepared with an additional lg in 100ml of LYS (see table no. 2). Solutions were filtrated through a 0.45 ⁇ cellulose acetate filter before the spray process.
  • the Spray Freeze Drying (SFD) process consisted of three steps: Droplet formation, freezing followed by freeze drying.
  • a drop jet nozzle MJK- 104 Dispenser, Microdrop Technologies GmbH, Norderstedt, Germany
  • the nozzle is capable of producing a jet of monodispersed droplets in the dimension of nano liter volume, and its throughput is approximately 1ml per minute.
  • an amount of 3 to 10 g dried particles per hour can be produced depending on spray settings and the solid concentration of the initial solution.
  • the jet produced has a velocity of approximately 10 m/s and undergoes a constant Rayleigh drop dispersion around 3 cm after leaving the nozzle which is stimulated by a piezoelectric pulse that generates the monodispersed droplets.
  • the batches were produced either with a pressure of lOOkPa and a frequency of 40kHz or with a pressure of 150kPa and a frequency of 57kHz (all batches containing Lysozyme).
  • Drop generation was visualized with a high speed camera (HiSIS 2000, KSV Instruments) before each spray process, and frequencies were adjusted where necessary to assure a consistent generation of droplets.
  • the initially monodispersed droplet stream breaks up into a narrow spray cone due to collisions, aerodynamic disintegration and perturbation.
  • the freezing process was performed within a cooled, stainless steel spray tower where direct spraying into the liquid nitrogen was avoided by design.
  • the spray tower was encased by a cooling jacket, with liquid nitrogen distributed from an attached storage container.
  • the cooling chamber was strongly insulated heavy to avoid a huge cold loss (heat transfer coefficient of isolation ⁇ 0.4 W/m2K).
  • the system was cooled to temperatures below -90°C. Particles froze rapidly within the tower and were collected in a cooled and isolated beaker at the bottom of the spray tower. (Fig. 1).
  • the frozen particles were transferred into a freeze dryer (STERIS Lyovac GT2, Hurth, Germany) and subsequently dried under vacuum for at least 36 hours. Particles were stored in a desiccator at 21°C / 10%rH.
  • Viscosity was measured with a "Haake RheoStress 1" rheometer (Haake GmbH, Düsseldorf, Germany).
  • the density of the solutions was measured with a Mohr balance (Gott Kunststoff & Sohn, Ebingen Wurttemberg, Germany).
  • the surface tension was measured with the drop volume method by a "Kruss FM40 Easy Drop” tensiometer (Kruss GmbH, Hamburg, Germany). Size Distribution of Particles
  • the particle size distributions were measured by laser diffraction spectrometry (LD) using a Sympatec Helos LF instrument in combination with a Sympatec Rodos SR dispersing module (both Sympatec GmbH, Clausthal-Zellerfeld, Germany) at lOOkPa to distribute the particles in front of the laser. Diffraction spectra were evaluated using the Fraunhofer theory option of Windox 3.4 software.
  • the particles produced were sputter coated (Polaron SC7640 Sputter Coater, Quorum Technologies Ltd., Newhaven, UK) for 4 - 8 minutes with gold and imaged with a Hitachi S-2460N (Hitachi High Tech. Corp., Tokyo, Japan) scanning electron microscope (SEM) to examine surface appearance, while cross sections were used to visualize the porous structure of the particles.
  • X-ray diffraction was measured to specify the crystalline fraction of SFD particles. Measurements were made with a X'Pert x-ray diffractometer (Philips Analytical B.V., Almelo, The Netherlands). Configuration was set to Transmission-Reflection- Spinner, scan axis angle was measured from 5 °2 ⁇ to 45 °2 ⁇ , Generator was set to 40mA; 45 kV. Evaluation of the data was carried out with PANalytical X'Pert HighScore Plus, Version 2.2.c.
  • Spray freeze dried spherolyophilisates were prepared from a solution that consists of 5% (w/v) Mannitol, 1% (w/v) PVP, and 0.5 % IgG in glycine buffer. All further steps of the spherolyophilisates preparation were according to the procedure described under Spray freeze drying.
  • the spherolyophilisates were dissolved in water and subjected to size exclusion chromatography in order to determine the percentage of aggregates.
  • Insulin Bioavailability Insulin loaded spray freeze dried spherolyophilisates were prepared from a solution that consists of 5% (w/v) Mannitol, 1% (w/v) PVP, and 1.3% (w/v) Insulin in distilled water. The pH of the solution was adjusted to pH 3 to dissolve Insulin. The usual volume of the solution was 10ml which was filtered through a 0.45 ⁇ cellulose acetate filter before the spray process. All further steps were according to the procedure described under Spray freeze drying.
  • Droplets of uniform size are generated by Rayleigh disintegration of coherent liquid jet exiting from the nozzle at a velocity of about 10 m/s. Single droplets are visible approximately 3 mm below the orifice.
  • the optimal frequency for the drop break-up depends on viscosity, surface tension and density of the fluid and the frequency of piezoelectric excitation has to be adjusted accordingly.
  • d diameter of droplets
  • dj diameter of liquid jet
  • D diameter of the capillary (50 ⁇ )
  • results from laser diffraction spectrometry show that the volume median diameters range from 230 - 310 ⁇ over all measured batches. Comparing the distribution of initially formed droplet sizes which can be regarded as monodisperse with the distribution after droplet fusion and crust formation, a wider but still narrow distribution is observed. The span- value is used to compare size distributions where small span-values indicate narrow distributions (Table 2 and Fig. 3a, 3b, 3c).
  • the value xlO is used to describe the size of a particle that is larger than 10% (V/V) of all particles in the sample and the same applies to x50 and x90.
  • Populations of small particles detected at low concentrations of stabilizer can be considered as artefacts of the measurement due to small particle fragments. This causes a significant decrease in xlO values of these batches and therefore also an increase of the span- values.
  • the SEM images (Fig. 4a to 4n) show the spherical shape of particles, which was formed by drop creation. Distinct single particles are visible.
  • Lysozyme was entrapped to control the process stability, which includes drop formation and disintegration, spray- freezing and freeze drying.
  • An effect of specific surface area (ssa) on lysozyme stability is not recognizable (see table no. 2).
  • Ssa is higher in particles with a low solid content in the formulation independent from used stabilizing agent. But in all formulations more than 90% of the lysozyme activity was kept.
  • the standard deviation includes the 100% value in 7 of 9 formulations and according to Kruskal Wallis test there is no significant difference in the results. Also the activity after 6 months of storage of 3 batches containing each 10% [wt-%] stabilizer at 21°C, 10%rH conditions remains unchanged (Fig. 6).
  • the structure of the particles was analyzed by X-ray diffraction, to obtain data on the degree of crystallinity 4 weeks after production and after 6 months of storage, since crystalline structures, that are already present from the beginning or occur during storage caused by the incipient re-crystallization of the contained mannitol, can influence the dissolution rate.
  • the structure of the particles was mainly amorphous. In formulations with 10% content of polymeric stabilizers, less crystalline structure was found. Among them formulations containing Bovine serum albumin showed the lowest crystallization effect. In retained samples that were stored for 6 months at room temperature under dry conditions (conventional for freeze dried products), re- crystallisation could be observed in a fraction of about 1 - 3%. Therefore it can be predicted that an effect on dissolution will be very low (see Fig. 5).
  • Insulin loaded spherolyophilisates were administered nasally to rats. The results are shown as mean values with standard deviation in Table 4. After subcutaneous injection (at HU/kg) blood glucose level decreases to 67% of the initial value after one hour while it immediately drops to 58% after 30min when spherolyophilisates are given (at 20IU/kg) nasally. No effect was found with drug-free
  • a pharmaceutical formulation comprising spherolyophilisates, wherein the spherolyophilisates comprise a mixture of at least one pharmaceutically active agent, at least one cryoprotectant and at least one stabilizing agent.
  • a pharmaceutical formulation of 1 wherein the pharmaceutically active agent is a biological molecule.
  • a pharmaceutical formulation of 2 wherein the biological molecule is selected from the group comprising proteins, polypeptides, oligopeptides, peptides, DNA, R A or derivatives thereof. 4.
  • a pharmaceutical composition of 3 wherein the protein is an antibody or binding fragment thereof. 5.
  • cryoprotectant is a polyhydroxylated carbon.
  • cryoprotectant is a polyhydroxylated carbon selected from the group comprising alcohols and sugars.
  • cryoprotectant is a polyalcohol selected form the group comprising mannitol, sorbitol and glycol.
  • cryoprotectant is mannitol.
  • the stabilizing agent confers mechanical stability to the spherolyophilisates and is selected from the group comprising BSA, HSA, recombinant SA, dextran, polyvinylpyrrolidone, polyvinyl alcohol, poloxamer, poly ethylenegly col, hydroxyethylcellulose, hyaluronic acid.
  • Method of producing a pharmaceutical formulation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 and 17, comprising at least the step of subjecting a solution comprising a mixture of at least one pharmaceutically active agent, at least one cryoprotectant and at least one stabilizing agent to a spray freeze drying step in order to obtain spherolyophilisates.
  • Method of 20 wherein the solution comprises the stabilizing agent in the range of about 0.05 wt-% to about 20 wt-%. 22. Method of 20, wherein the solution comprises the stabilizing agent in the range of about 0.05 wt-% to about 20 wt-%.

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Abstract

La présente invention concerne des formulations pharmaceutiques comprenant des sphérolyophilisats de molécules biologiques, des procédés pour fabriquer de telles formulations et des utilisations de celles-ci.
PCT/EP2012/068384 2011-09-19 2012-09-19 Formulations pharmaceutiques comprenant des sphérolyophilisats de molécules biologiques WO2013041542A1 (fr)

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DE102015105375B4 (de) * 2015-01-07 2016-11-03 Richard Süverkrüp Vorrichtung und Verfahren zur Erzeugung monodisperser gefrorener Tropfen
DE102019133243A1 (de) * 2019-12-05 2021-06-10 Rheinische Friedrich-Wilhelms-Universität Bonn Verfahren zur Herstellung sprühgefriergetrockneter Partikel und entsprechend hergestellte Partikel
CN113576944A (zh) * 2021-08-09 2021-11-02 中山中研化妆品有限公司 冻干球及其制备方法、护肤品
CN113908254A (zh) * 2021-10-19 2022-01-11 山西锦波生物医药股份有限公司 一种干粉吸入剂及其制备方法和用途
CN114159555A (zh) * 2021-12-08 2022-03-11 江西赛基生物技术有限公司 抗原蛋白冻干小球及其制备方法

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Publication number Priority date Publication date Assignee Title
DE102015105375B4 (de) * 2015-01-07 2016-11-03 Richard Süverkrüp Vorrichtung und Verfahren zur Erzeugung monodisperser gefrorener Tropfen
DE102019133243A1 (de) * 2019-12-05 2021-06-10 Rheinische Friedrich-Wilhelms-Universität Bonn Verfahren zur Herstellung sprühgefriergetrockneter Partikel und entsprechend hergestellte Partikel
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CN113576944A (zh) * 2021-08-09 2021-11-02 中山中研化妆品有限公司 冻干球及其制备方法、护肤品
CN113576944B (zh) * 2021-08-09 2023-07-07 中山中研化妆品有限公司 冻干球及其制备方法、护肤品
CN113908254A (zh) * 2021-10-19 2022-01-11 山西锦波生物医药股份有限公司 一种干粉吸入剂及其制备方法和用途
CN113908254B (zh) * 2021-10-19 2024-05-28 山西锦波生物医药股份有限公司 一种干粉吸入剂及其制备方法和用途
CN114159555A (zh) * 2021-12-08 2022-03-11 江西赛基生物技术有限公司 抗原蛋白冻干小球及其制备方法

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