WO2013041542A1

WO2013041542A1 - Pharmaceutical formulations comprising spherolyophilisates of biological molecules

Info

Publication number: WO2013041542A1
Application number: PCT/EP2012/068384
Authority: WO
Inventors: Alf Lamprecht
Original assignee: Individual
Current assignee: Individual
Priority date: 2011-09-19
Filing date: 2012-09-19
Publication date: 2013-03-28
Anticipated expiration: 2014-03-19

Abstract

The present invention relates to pharmaceutical formulations comprising monodisperse spherolyophilisates of biological molecules, to methods for making such formulations and uses thereof.

Description

Pharmaceutical formulations comprising spherolyophilisates of biological molecules

FIELD OF THE INVENTION

The present invention relates to pharmaceutical formulations comprising

spherolyophilisates of biological molecules, to methods for making such

formulations and uses thereof. BACKGROUND OF THE INVENTION

The storage of pharmaceutical formulations comprising biological molecules as pharmaceutically active agents for use in therapy poses various technical problems. Typically, 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.

However, storage of liquids comprising proteins, enzymes, antibodies and the like over prolonged periods of time leads to degradation, denaturation and aggregation, which may ultimately render the pharmaceutical product comprising the biological molecule useless. Even though reactions such as degradation, denaturation and aggregation may be slowed down by storage at low temperatures such as 4°C, there has been an interest in formulations that would allow for prolonged storage of such products under common ambient conditions. In a lot of cases, lyophilisation is considered the primary choice for storage of biological molecules such as proteins, enzymes and antibodies as the pharmaceutical products may then be stored over prolonged periods of time and only immediately before use be reconstituted with a solution such a sterile salt solutions that might be provided in parallel in sterile form.

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

lyophilisates into containers difficult.

Therefore, there is a continued interest in improved methods for lyophilisation of biological molecules used for pharmaceutical purposes.

SUMMARY OF THE INVENTION

It is one objective of the present invention to provide pharmaceutical formulations for biological molecules such as proteins, polypeptides, peptides, DNA, RNA and the like which can be stored over prolonged periods of time in a lyophilised form. It is another objective of the present invention to provide lyophilised formulations of biological molecules such as proteins, polypeptides, peptides, and the like which due to the structure and the properties of the lyophilised powders show good flow properties which e.g. make them suitable for oral, nasal, buccal, sublingual or ophthalmic administration.

It is another objective of the present invention to provide lyophilised formulations of biological molecules such as proteins, polypeptides, peptides and the like, which can be used e.g. for pulmonary or oral administration. These and other objectives as they will become apparent from the ensuing description are solved by the subject matter of the independent claims. Some of the preferred embodiments of the invention can be found in the dependent claims. 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.

As will be explained hereinafter, 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. In view of their substantially round shape and uniform size distribution, the lyophilisate powders of the present invention provide good flow properties being advantageous during manufacturing of storage stable formulations of biological molecules. For the purposes of the present invention such lyophilisate powders are thus called spherolyophilisates and in view of the uniform size distribution they may also be considered to be monodisperse spherolyophilisates.

As will be explained hereinafter, using such ternary mixtures in common spray- freeze drying processes allows obtaining of spherolyophilisates with a diameter in the range of about 1 μιη to about 1 mm.

Depending on the process parameters and equipment of the spray freeze drying process, the resulting spherolyophilisates may have a diameter in the range of about 1 to about 50 μιη, preferably in the range of about 5 to a about 40 μιη and more preferably in the range of about 10 to about 30 μιη, making them suitable for pulmonary administration.

By adjusting the process parameters and equipment of the spray freeze drying process, one may also obtain spherolyophilisates with a diameter in the range of about 50 to about 750 μιη, preferably of about 100 to about 500 μιη, and more preferably in the range of about 200 to about 350 μιη. 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

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. 3 a) Size distribution of particles with BSA as stabilizer in 3 concentrations [wt-%];

b) Size distribution of particles with DEX as stabilizer in 3 concentrations [wt-%];

c) Size distribution of particles with PVP as stabilizer in 3 concentrations [wt- %].

Fig. 4 Electron microscopy pictures

a) DEX 0.1%) particle overview, magnification (mag.) 50x;

b) DEX 10%) particle overview, mag. 5 Ox;

c) BSA 0.1%) single particle, mag. 350x;

d) BSA 10%) single particle, mag. 300x;

e) DEX 0.1%) single particle, mag. 300x;

f) DEX 10% single particle, mag. 300x;

g) PVP 10%o single particle, mag. 300x;

h) DEX 10%o particle surface, mag. lOOOx;

i) BSA 0.1%) particle cross section, mag. 350x;

j) PVP 0.1%) particle cross section, mag. 350x;

k) DEX 0.1%) particle cross section, mag. 350x;

1) DEX 10% particle cross section, mag. 280x;

m) PVP l%o, particle overview, mag. 1500x;

n) PVP l%o, single particle, mag. lOOx. 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. Fig. 6 Lysozyme stability of spray freeze dried formulations containing lysozyme 1 wt-%, Mannitol 5 wt-%, stablilizing agents BSA, DEX and PVP in various concentrations (wt-%), the addition 6M indicates batches stored for 6 months. DETAILED DESCRIPTION OF THE INVENTION

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.

Although the present invention will be described with respect to particular embodiments, this description is not to be construed in a limiting sense. Prior to describing exemplary embodiments of the present invention in detail, definitions important to the comprehension of the present invention are provided. As used in this specification and the appended claims, the singular forms of "a" and "an" also include the respective plurals unless the context clearly dictates otherwise.

In the context of the present invention, 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%.

If in the context of the present invention a range is provided for a specific parameter and the term "between" is used, this means to include the lower and upper limits of the indicated range.

It is to be understood that the term "comprising" is not limiting. For the purposes of the present invention, the term "consisting of is considered to be a preferred embodiment of the term "comprising of. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant also to encompass a group, which preferably consists of these embodiments only. It is to be understood that this invention is not limited to the particular methodology, protocols, reagents, etc. described herein as these may vary. It is also to be understood that the terminology as used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention that will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

The term "lyophilisation" or its synonym "freeze drying" are used in their common sense.

The term "spray freeze drying" is also used in its common sense. In the context of the present invention, 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. The term "spherolyophilisates" refers to powders with monodisperse, spherical and porous particles produced by freeze drying. For the purpose of the present invention, 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.

As mentioned, 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. For the purposes of the present invention, the term "peptide" designates a molecule where up to and including 10 amino acids are linked by a peptide bond. The term

"oligopeptide" denotes a molecule where up to 40 amino acids are linked by a peptide bond. Thus, the term "peptide" denotes a molecule with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids being linked by peptide bonds, while the term "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. The term "polypeptide" comprises any type of molecules where more than 40 amino acids are linked by a peptide bond.

The term "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. For example, 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. Similarly, 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" .

In a preferred embodiment, the pharmaceutical formulations are made from a ternary mixture comprising e.g. a protein such as an enzyme or a hormone.

In another preferred embodiment, an antibody or binding fragment thereof is used as a biological molecule. Again, the person skilled in the art will understand that the term "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. Examples of 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.

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. The term "small molecule compound" is used as common in the art. It thus does not include e.g. proteins, peptides and nucleic acids. Typically, the skilled person in the field of pharmacology and biochemistry will understand the term "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. a nucleic acid, a protein or a polysaccharide). 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

polysaccharide and that alters the activity or function of said biopolymer

The term cryoprotectanf refers to substances as they are commonly used in formulations of biological molecules, which are subjected to lyophilisation.

Such cryoprotectants may for example be alcohols and preferably polyalcohols such as glycol, glycerol, erythritol, treithol, arabitol, xylitol, rebitol, mannitol, sorbitol, dulcitol, iditol, isomalt, maltitol, lactitol, inositol, etc.

The cryoprotectant may also be a sugar selected from mono-, di- and

oligosaccharides; Such sugars include trehalose, mannose, lactose, galactose, maltose, glucose, raffmose, maltotriose, sucrose and the like.

Further, such cryprotectants may be amino acids. Examples of 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. The term "stabilising agent" for the purposes of the present invention relates to a pharmaceutically acceptable excipient that is commonly used for protein

formulations such as BSA, HSA or recombinant versions thereof, gelatine, casein, dextran, polydextrose, cyclodextrine, polyvinylpyrollidone, polyvinyl alcohol, poloxamer, poly ethylenegly col, hydroxylethyl cellulose, hyaluronic acid, hydroxyethyl starch and carboxymethyl cellulose. The 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. For example, stabilizing agents include hydrohilic polymers such as dextran, polyvinylpyrollidone, polyvinyl alcohol, poloxamer, poly ethylenegly col, hyaluronic acid, lactoglobulin and the like. The 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. The solution comprising the ternary mixture used for manufacturing the

pharmaceutical formulations 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. Preferably the 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. For example, if 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. Thus, under 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.

As can be taken from Table 2 of Examples, 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.-%. Preferably, 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. In general 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

DE 10 2008 017 461 and the examples mentioned hereinafter.

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 μιη 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 μιη 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 μιη to 1 mm.

Depending on the size of the spherolyophilisates, the formulations in accordance with the present invention may be suitable for different modes of administration. For example, usually, a diameter range of between 1 to 50 μιη and preferably below 50 μιη 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.

For other modes of administration, such as nasal, oral, buccal or sublingual administration it has, however, to be ensured that the powders have good flow properties given that it is the powder that will typically be administered to the patient. In the context of the present invention, it has been shown that for the model substance of lysozyme, one can produce spherolyophilisates of substantially uniform size and form having a diameter in the range of approximaetly 100 μιη to 1000 μιη, preferablly approximately 150μι ίο 700 μιη, more preferably approximately 150 μιη to 500 μηι and evene more preferably approximately 200 μιη to 350 μιη which have excellent flow properties, which are storage stable and which should thus be suitable for nasal, oral, sublingual and buccal administration. For the model substance insulin it has even been shown that such spherolyophilisates indeed provide bioavailability upon nasal administration.

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 μιη, preferably about 5 to about 40 μιη, and more preferably of about 10 to about 30 μιη such as about 20 μιη that can be used in pulmonary administration.

In another embodiment 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 μιη to about 1 mm and preferably in the range of about 150 μιη to about 500 μιη and more preferably in the range of about 200 μιη to about 400 μιη, such as about 250 to about 300 and about 350 μιη. Such 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

manufacturing, 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.

As mentioned above, 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. In brief, the solution is injected into a cooling chamber where the particles immediately freeze. Given that a nozzle is used which is controlled by a piezoelectric element, it is ensured that the drop-size is constantly the same. By gravity 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. As a consequence 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.

In an alternative embodiment 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). In any case, 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. In a preferred embodiment 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.

Moreover, there is a selection of suitable spray systems like ultrasonic nozzles, two- fluid nozzles, four-fluid nozzles, drop on demand nozzles and drop jet nozzles that can be involved in the spray freeze drying process and effects the drop formation and therefore particle characteristics. In addition the stimulation of atomization like mass flow ratio (two-fluid nozzle, four-fluid nozzle) ultrasound (ultrasonic nozzle) or piezoelectric stimulation (drop on demand nozzle, drop jet nozzle) cause differences in generated particles for instance in size, size distribution and specific surface area. The influence of process parameters during spray freeze drying is discussed inter alia in Maa et al., (Pharm. Research, 1999, 16(2)) or Costantino et al. (Pharm. Research, 2000, 17 (11)). In general, spray settings can be seen as fine adjustment with only minor influence on the particle formation. For example by replacing the orifice of a drop jet nozzle by a model with twice the diameter drop size may double in diameter were changing stimulation frequency to double value result in about 10% difference in drop size. 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.

This process thus overall leads to essentially uniform lyophilisate particles, which are therefore considered to be spherolyophilisates. 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.

The invention will now be illustrated with respect to specific embodiments, which however are not to be construed as limiting.

EXAMPLES MATERIALS AND METHODS

Chemicals Lysozyme (LYS) also named muramidase from hen egg white was used as a model enzyme for activity measurements and purchased from Roche Diagnostics,

Mannheim, Germany. Insulin was purchased from Sigma Immunoglobulin G (IgG, Gamunex ®) was purchased from Sigma. Bovine serum albumin (BSA) [albumin fraction V, purity >98%, M-69.000] was purchased from Carl Roth, Karlsruhe, Germany. Maltodextrin (DEX) [Roquette LAB 2509, dextrose equivalent of DE=19] was a gift from Roquette Freres, Lestrem Cedex, France. Polylvinylpyrollidone (PVP) [Kollidon 12 PF, K-value range = 10.2 - 13.8] was obtained from BASF, Ludwigshafen, Germany. D(-)-Mannitol [Ph. Eur.] was purchased from VWR International, Amsterdam, The Netherlands. Ultrapure water was made with a MiliQ, Millipore Corp., Billerica, MA, USA. Liquid nitrogen was purchased from Linde, Pullach, Germany.

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 0.45μιη cellulose acetate filter before the spray process.

Spray Freeze Drying

The Spray Freeze Drying (SFD) process consisted of three steps: Droplet formation, freezing followed by freeze drying. For droplet formation a drop jet nozzle (MJK- 104 Dispenser, Microdrop Technologies GmbH, Norderstedt, Germany) with a nozzle diameter of 50μιη was installed at the top of the spray tower. 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. Hence 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). In operation 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, Density and Surface Tension Measurements of Solutions

Viscosity was measured with a "Haake RheoStress 1" rheometer (Haake GmbH, Karlsruhe, Germany). A cone-plate tool (a= 1°, 0=5Omm) was utilized and results were recorded at a shear rate of 50/s at 20°C. The density of the solutions was measured with a Mohr balance (Gottlieb Kern & 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.

Scanning Electron Microscopy 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.

Density and Porosity of Particles

Samples of particles were distributed on black adhesive tape, weighed and counted with image analysis software(ImageJ 1.37a, Wayne Rasband National Institutes of Health, Betheda, MD, USA, http://rsb.info.nih.gov/ij/) to calculate the average weight per particle. Then the mean diameter of particles was taken from laser diffraction measurements to calculate the mean particle volume where particles were considered as perfect spheres. Particle volume was used in combination with the weight to calculate apparent density. The particle density of pure substances was analyzed with a helium pycnometer (Quantachrome Ultrapycnometer 1000, Boynton Beach, FL, USA) and density of formulations was calculated. Finally, the porosity was calculated from the ratio of apparent density to particle density.

Gas Sorption Analysis (BET)

The specific surface area was measured with a Quantachrome Nova 3200 high speed gas sorption analyser, Version 6.11, (Quantachrome, Boynton Beach, FL, USA). The used adsorbate was nitrogen. X-Ray Diffraction

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 °2Θ to 45 °2Θ, Generator was set to 40mA; 45 kV. Evaluation of the data was carried out with PANalytical X'Pert HighScore Plus, Version 2.2.c.

Lysozyme Assay

To investigate the integrity of lysozyme tertiary structure upon freezing and drying lysozyme activity assay was performed according to the method described by Shugar et al. (Biochimica et Biophysica, 1952 (8)). The pH-value was regulated with phosphate buffer solution to pH 7.0. The change of absorbance was detected over 6 minutes at wavelength of 450nm with a UV7VIS Spectrometer Lambda 12

(Bodenseewerk Perkin-Elmer GmbH, Uberlingen, Germany) which is proportional related to the catalysis of 1,4-beta- linkages between N-acetylmuramic acid and N- acetyl-D-glucosamine residues in the peptidoglycan skeleton of the bacterial cell walls of Micrococcus lysodeicticus. Immunoglobulin G stability:

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.

To determine the degradation of the IgG caused by freezing and drying, the spherolyophilisates were dissolved in water and subjected to size exclusion chromatography in order to determine the percentage of aggregates The

determination of degradation was done in triplicates for each batch.

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 μιη cellulose acetate filter before the spray process. All further steps were according to the procedure described under Spray freeze drying.

These spherolyophilisates were administered nasally to Wistar rats at a dose of 20IU/kg and compared to control groups receiving either insulin- free

spherolyophilisates or insulin subcutaneously at a dose of HU/kg. Blood samples were taken a predetermined time points over 5 hours, analysed for their glucose level which were expressed as percentage of the initial value before the administration. Typical group size was 2 rats. RESULTS

Solution properties and Particle formation 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. The diameter of droplets formed by a laminar liquid jet of low viscosity is described with following approximation according to Walzel et al (Chem.-Ing.-Tech., 1996, 62 S. 983-994). d ~ dj * 1.9 = 95μπι

dj ^ D

d = diameter of droplets; dj = diameter of liquid jet; D= diameter of the capillary (50 μιη)

The consistent drop break-up of the drop jet nozzle was analysed by photographs (see Fig. 2). It was visible that the measured droplet diameter of approximately ΙΟΟμιη from image fits to the calculated value.

The properties of solutions can be summarized with the dimensionless Ohnesorge number, which relates viscosity, density and surface tension of a liquid.

V

Oh = 7 r

V⁷ P ^dj)

Oh= Ohnesorge number; η= viscosity; σ= surface tension; p= density

Measured results of the physicochemical properties as well as the calculated Ohnesorg numbers are compiled in table no. 1. The influences on particles generation appear in the change of particle size (table no. 2), where the increase of Ohnesorge numbers complies with the increase in mean diameter of corresponding particles. Nevertheless, the average resulting particle diameter is about 2 to 4 times larger after SFD than the droplet diameter, independent from frequency, pressure or solution properties.

Particle Properties

Results from laser diffraction spectrometry show that the volume median diameters range from 230 - 310 μιη 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).

xS G [μιη]

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.

On the particle surface both smooth and rough porous sections can be found. While the appearance of particles with low quantity of stabilizing agents is mainly influenced by mannitol (Fig. 4c, 4e), higher concentrations of stabilizing agents indicate the influence of the different components: Where mannitol is the dominant component porous sections are interrupted with smooth sections that build a thin layer on the surface and cover the pores, though this thin layer seems to break of very easily. While particles with 10 wt-% PVP form coarse diffuse structures (Fig. 4g), 10 wt-% DEX (Fig. 4f) and 10 wt-% BSA (Fig. 4d) particles develop surface structures with more systematic arrangements. Possibly, in some particles with 10 wt-% DEX nucleation areas can be recognized as little section centers. Starting from these section centers a star-shaped structure is created that ends in more concentrated areas representing borders to areas that were affected by adjacent nucleation points. In particles containing 10 wt-% BSA often a small number of little holes, which could be perhaps also considered as nucleation areas, are found affecting a large proportion of the particle surface. Taking a closer look at the at surface structures it becomes visible that high freezing rates in SFD cause forming of many thin walls by freeze concentration which represents the glassy phase that is separated by a very fine pore structure (Fig. 4h). BSA and PVP are molecules that can accumulate at interfaces hence a higher proportion of them could be expected on the surface. However, the fraction of interfacial active molecules that finds their way to the surface within the spray process depends on concentration and freezing rate. In case rapid freezing which can be achieved by SFD, diffusivity of macromolecules becomes very low as the quick increase of viscosity restricts their mobility within the cooling drop in very short terms and they are immobilized before they can accumulate at the surface. Reducing the freezing rate by lowering the temperature in the spray tower leads to a higher solid load at the surface where most surface pores are closed (Fig. 4g). Cross sections show that the interior of the particles is highly porous (Fig. 4i-41), where obviously more coarse and stable structures are created with an increasing fraction of macro molecular stabilizers within the formulation. Adaptation of the parameters of spray freeze drying enables the production of spherolyophilisates with a diameter in the range of about 1 μιη to about 1 mm. For instance, smaller particles (Fig. 4m) or larger particles (Fig. 4n) can be produced byadapting the size of the noozle, Spray freeze drying leads to highly porous structures that facilitate disintegration. Formulations with low stabilising agent proportion and thus with low solid content (6.1% and 7%) show values over 90% porosity. The formulations with higher solid content (16.0%) have porosity values between 70%> and 85%. Due to the fast freezing step pore sizes are very small in SFD particles (see table no. 2).

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). Further 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).

Further, the stability of Immunoglobulin G in spherolyophilisates was tested. As shown in Table 3 up to 99% of undegraded IgG could be recovered after reconstitution with water. The results in Table 3 are shown as mean values with standard deviation.

Additionally to the stability of pharmaceutically active agents in spherolyophilisates, the bioavailability of such agents was tested exemplarily for Insulin. Therefore,

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

spherolyophilisates.

Table 1

c (M) c (BSA) c (DEX) c (PVP)

η [mPas] σ [mN/m] P [g/cc ] Oh

[wt-%] [wt-%] [wt-%] [wt-%]

5.0% 0.1% 1.31 65.9 1.015 0.0227

5.0% 1.0% 1.41 65.3 1.019 0.0244

5.0% 10.0% 2.41 54.4 1.042 0.0453

5.0% 0.1% 1.42 73.5 1.016 0.0232

5.0% 1.0% 1.49 73.5 1.019 0.0243

5.0% 10.0% 2.22 73.7 1.054 0.0356

5.0% 0.1% 1.34 71.2 1.016 0.0223

5.0% 1.0% 1.42 70.3 1.018 0.0237

5.0% 10.0% 2.24 67.3 1.036 0.0379 concentration c, viscosity η, surface tension σ, density p, Ohnesorge number Oh

Table 2

c BSA [wt-%] c DEX [wt-%] c PVP [wt-%]

Dimension

0.10% 1.00% 10.00% 0.10% 1.00% 10.00% 0.10% 1.00% 10.00%

0

x50 [μπι] 252,2 283,2 300,2 237,3 253,5 309,8 231,3 234,5 272, 1 particle

(x90 - xl0)

Span 1 ,35 1 ,29 0,62 1 ,24 1 ,23 0,65 1 ,43 1 ,43 0,71 x50

Ssa [sq. m g] 21,9733 19,4059 9,5171 20,4025 19,7802 : 9,3417 20,873 20,6396 5,6935

V

[ml] 0,0084 0,01 19 0,0142 0,007 0,0085 0,0156 0,0065 0,0068 0,0105 particle

w

[ §] 0,7344 0,7292 3,625 0,7417 0,9178 3,7937 0,8121 0,8764 3,7762 particle

P

[g/cc] 0,0874 0,0613 0,2559 0,106 0,1076 0,2437 0,1253 0,1298 0,358 apparent

p true [g/cc] 1 ,4698 1 ,4428 1 ,315 1 ,4835 1 ,484 1 ,5017 1 ,4968 1 ,5034 1 ,3381

Φ [%] 94,051 95,75 80,54 92,854 92,749 83,773 91 ,626 91 ,366 73,247 concentration c, average particle diameter 0 particle, span = (x90 - xlO) / x50, ssa = specific surface area, average particle volume V particle, average particle weight w particle, apparent density papparent, true density ptrue, porosity Φ Table 3

%IgG degraded SD

Batch 1 0.66 0.15

Batch 2 1.27 0.53

Batch 3 2.31 0.35

Table 4

Some embodiments of the invention relate to:

1. 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.

2. A pharmaceutical formulation of 1 , wherein the pharmaceutically active agent is a biological molecule.

3. 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. A pharmaceutical composition of any of 1 , 2, 3 or 4, wherein the

cryoprotectant is a polyhydroxylated carbon.

6. A pharmaceutical composition of any of 1 , 2, 3, 4 or 5, wherein the

cryoprotectant is a polyhydroxylated carbon selected from the group comprising alcohols and sugars.

7. A pharmaceutical formulation of 6, wherein the cryoprotectant is a polyalcohol selected form the group comprising mannitol, sorbitol and glycol. 8. A pharmaceutical formulation of 7, wherein the cryoprotectant is mannitol.

9. A pharmaceutical formulation of 6, wherein the sugar is selected from the group comprising sucrose and trehalose. 10. A pharmaceutical formulation of any of 1, 2, 3, 4, 5, 6, 7, 8 and 9, wherein 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.

11. A pharmaceutical formulation of any of l, 2, 3, 4, 5, 6, 7, 8, 9 and 10, wherein the spherolyophilisates are producible by a spray freeze drying process using a solution comprising a mixture of at least a pharmaceutically active agent, a cryoprotectant and a stabilizing agent. A pharmaceutical formulation of 11, wherein the solution comprises the stabilizing agent in the range of about 0.05 wt-% to about 20 wt-%.

A pharmaceutical formulation of 12, wherein the formulation comprises the stabilizing agent in the range of about 0.1 wt-% to about 10 wt-%.

A pharmaceutical formulation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13, wherein the spherolyophilisates have a diameter in the range between about 1 μιη to about 50 μιη.

A pharmaceutical formulation of 14, wherein the spherolyophilisates have a diameter in the range of about 5 μιη to about 40 μιη, preferably in the range of about 10 μι ίο about 30 μιη.

A pharmaceutical formulation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13, wherein the spherolyophilisates have a diameter in the range of about 50 μιη to about 1 mm. A pharmaceutical formulation of 16, wherein the spherolyophilisates have a diameter in the range of about 100 μιη to about 700 μιη, preferably in the range of about 150 μιη to about 500 μιη and more preferably in the range of about 200 μι ίο about 350 μιη. A pharmaceutical formulation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 for use in pulmonary administration.

A pharmaceutical formulation of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 16 and 17 for use in nasal, oral, buccal, sublingual or ophthalmic

administration. 20. 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.

21. 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-%.

Claims

1. 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, wherein said pharmaceutically active agent is selected from the group comprising proteins, polypeptides, oligopeptides, peptides or derivatives thereof and said spherolyophilisates have a diameter in the range between about 1 μιη to about 1 mm.

2. A pharmaceutical composition of claim 2, wherein the protein is an antibody or binding fragment thereof.

A pharmaceutical composition of any of claims 1 or 2, wherein the cryoprotectant is a polyhydroxylated carbon selected from the group comprising alcohols and sugars.

A pharmaceutical formulation of claim 3, wherein the cryoprotectant is selected form the group comprising mannitol, sorbitol and glycol.

A pharmaceutical formulation of claim 4, wherein the cryoprotectant is mannitol.

A pharmaceutical formulation of claim 3, wherein the sugar is selected from the group comprising sucrose and trehalose.

A pharmaceutical formulation of any of claims 1, 2, 3, 4, 5 and 6, wherein the stabilizing agent confers mechanical stability to the spherolyophilisates and is selected from the group comprising BSA, HSA, recombinant SA, gelatine casein, dextran,cyclodextrin, polydextrose, polyvinylpyrrolidone, polyvinyl alcohol, poloxamer, polyethyleneglycol, hydroxyethyl cellulose, hyaluronic acid, hydroxyethyl starch and carboxymethyl cellulose.

8. A pharmaceutical formulation of any of claims 1, 2, 3, 4, 5, 6 and 7, wherein the spherolyophilisates are producible by a spray freeze drying process using a solution comprising a mixture of at least a pharmaceutically active agent, a cryoprotectant and a stabilizing agent.

9. A pharmaceutical formulation of claim 8, wherein the solution comprises the stabilizing agent in the range of about 0.05 wt-% to about 20 wt-%.

10. A pharmaceutical formulation of any of claims 1, 2, 3, 4, 5, 6, 7, 8 and 9,

wherein the spherolyophilisates have a diameter in the range between about 1 μιη to about 50 μιη.

11. A pharmaceutical formulation of any of claims 1, 2, 3, 4, 5, 6, 7, 8 and 9,

wherein the spherolyophilisates have a diameter in the range of about 50 μιη to about 1 mm.

12. A pharmaceutical formulation of any of claims 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 for use in pulmonary administration.

13. A pharmaceutical formulation of any of claims 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 for use in nasal, oral, buccal, sublingual or ophthalmic administration.

14. Method of producing a pharmaceutical formulation of any of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13, 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.

15. Method of claim 14, wherein the solution comprises the stabilizing agent in the range of about 0.05 wt-% to about 20 wt-%.