KR20070057086A - 1,4 o-linked saccharose derivatives for stabilizing antibodies or antibody derivatives - Google Patents

Abstract

The invention relates to compositions, preferably powdered compositions, for example spray-dried or lyophilized powders that contain an antibody or an antibody derivative and one or more 1,4 O-linked saccharose derivatives selected from the compounds: 1,4 O-linked D-Gal saccharose (lactosucrose), 1,4 O-linked D-Glu saccharose (glucosyl sucrose), or 1,4 O-linked Glu-Glu saccharose (maltosyl sucrose). Preferred combinations contain lactosucrose or a combination of glucosyl and maltosyl sucrose.

Description

1,4 O-linked saccharose derivatives for stabilizing antibodies or antibody derivatives}

The present invention relates to the use of the novel oligosaccharide / oligosaccharide mixtures for the preparation and stabilization of pharmaceutical compositions, mainly powders containing antibodies or antibody derivatives as pharmaceutically active substances. The preparation of the powder is preferably carried out by spray drying or freeze drying. The invention relates in particular to the corresponding antibody containing powders and methods for their preparation.

Active substances / active substance formulations formulated in aqueous solutions become somewhat unstable, which can reduce efficacy or bioactivity and increase toxicity or incompatibility. This applies to both conventional pharmaceuticals and peptide containing or protein containing active substances. The stability of the pharmaceutically active substance can be substantially influenced by modification of the structure (internal) or by the addition of a suitable adjuvant (external).

Conventional methods for external stabilization of pharmaceutically active substances are the use of suitable adjuvants. Adjuvants for stabilizing the active substance can be roughly classified into the following categories: sugars and polyols, amino acids, amines, salts, polymers and tensides.

Sugars and polyols are often used as nonspecific stabilizers. Its stabilizing effect on biologically active substances is mainly due to "preferential exclusion" (Xie and Timasheff, 1997, Biophysical Chemistry, 64 (1-3), 25-43; Xie and Timasheff, 1997, Protein Science, 6 (1), 211-221; Timasheff, 1998, Advances in protein chemistry, 51, 355-432]. When sugars are selected, reducing sugars are generally avoided for biologically active substances. Saccharose and trehalose are preferably used as non-reducing sugars. Other examples of suitable adjuvants include glucose, sorbitol, glycerol [Boctor and Mehta, 1992, Journal of Pharmacy and Pharmacology, 44 (7), 600-3; Timasheff, 1993, Annual review of biophysics and biomolecular structure, 22, 67-97; Chang et al., 1993, Pharmaceutical Research, 10 (10), 1478-83] and mannitol (Hermann et al., 1996, Pharmaceutical Biotechnology, 9 (Formulation, Characterization, and Stability of Protein Drugs), 303-328 ; Chan et al., 1996, Pharmaceutical Research, 13 (5), 756-761. It is also known that various polymers have a stabilizing effect on pharmaceutically active substances, mainly proteins such as antibodies. Conventional frequently used human serum albumin (HAS) has very good stabilization and aggregation inhibition properties, but has been found to be inadequate due to the possibility of contamination by “blood-bearing” pathogens. Among the previously known polymers, hydroxypropyl-β-cyclodextrin (HP-β-CD) is particularly suitable because it can be administered parenterally in a completely safe manner. Further examples include high molecular weight dextran (18-82 kD), PVP, heparin, type A and B gelatin and hydroxyethyl starch (HES), heparin, dextran sulfate, polyphosphoric acid, poly-L-glutamic acid, poly- L-lysine.

In addition to sugars and polyols, amino acids can also be used for stabilizing roles or in combination with other adjuvants. Amino acids are mainly used to stabilize proteins. For example, the addition of histidine, glycine, sodium aspartate (Na-Asp), glutamate and lysine hydrochloride (Lys-HCl) may be achieved by rhKGF (recombinant human) in 10 mM sodium phosphate buffer (pH 7.0) with 5% mannitol. Keratinocyte growth factor) (Zhang et al., 1995, Biochemistry, 34 (27), 8631-41). The combination of amino acids and propylene glycol improves the structural stability of rhCNTFs (recombinant human ciliary nerve factor) (Dix et al., 1995, Pharmaceutical Research (Supplement), 12, S97). Lysine and arginine enhance the thermostability (Tm-increase) of IL-1R, while glycine and alanine have a destabilizing effect. Remmele et al., 1998, Pharmaceutical Research, 15 (2), 200-208 ].

In addition, the stability of the pharmaceutically active substance is improved by various drying processes. However, drying is mainly carried out in the presence of an adjuvant in order to maintain the stability of the active substance and to improve the properties of the dry powder. The decisive factor on stabilization by drying is the immobilization of the active substance in the amorphous matrix. The amorphous state has a high viscosity with low molecular mobility and low reactivity. Thus, an advantageous adjuvant should be able to form an amorphous matrix as high as possible with a glass transition temperature into which the active material is introduced. Therefore, the choice of adjuvant depends in particular on its stabilization performance. Other factors, such as the pharmaceutically acceptable and the influence of the adjuvant on particle formation, dispersibility and flowability, play an important role, in particular in the spray drying process.

Spray drying is a particularly suitable process for improving the chemical and physical stability of peptide / protein containing pharmaceutically active substances (Maa et al., 1998, Pharmaceutical Research, 15 (5), 768-775). Spray drying has been described in the field of lung disease treatment; see US Patent No. 5,626,874; US Patent No. 5,972,388; Broadhead et al., 1994, J. Pharm. Pharmacol., 46 (6), 458-467, which is particularly incremental, while at the same time use in inhalers is a viable alternative in the treatment of systemic diseases (see International Publication No. 99/07340). The prerequisite is that the average particle size should be 1-10 μm, preferably 1-7.5 μm, so that the particles can reach deeper into the lungs and enter the bloodstream. For example, JP-A-179 22 07 describes a process for producing the corresponding spray dried particles. On the other hand, a number of processes have been described for the preparation of the corresponding powders; see Publication No. 95/31479; Published Patent Publication No. 96/09814; Published Patent Publication No. 96/32096; Published Patent Publication No. 96/32149; Published Patent Publication No. 97/41833; Published Patent Publication No. 97/44013; Published Patent Publication No. 98/16205; Published Patent Publication No. 98/31346; Published Patent Publication No. 99/66903; Published Patent Publication No. 00/10541; Published Patent Publication No. 01/13893; Maa et al., 1998, supra; Vidgren et al., 1987, Int. J. Pharmaceutics, 35, 139-144; Niven et al., 1994, Pharmaceutical Research, 11 (8), 1101-1109.

In addition, sugars and their alcohols (eg trehalose, lactose, saccharose or mannitol) and various polymers are suitable as adjuvants (Maa et al., 1997, Pharm. Development and Technology, 2 (3), 213-223; Maa et al., 1998, supra; Dissertation Adler, 1998, Universitat Erlangen; Costantino, et al., 1998, J. of Pharm. Sciences, 87 (11), 1406-1411. However, mainly used adjuvants have various drawbacks. For example, the addition of trehalose and mannitol impairs the fluidity of spray dried formulations (C. Bosquillon et al., 2001 Journal of Controlled Release, 70 (3), 329-339). In addition, at concentrations above 20% by weight, mannitol tends to be further recrystallized (Costantino et al., 1998, supra), whereby the stabilizing effect is significantly lowered. A frequently used adjuvant is lactose, which improves the fluidity of spray dried formulations (see C. Bosquillon et al., 2001, supra), while lactose destabilizes Maillard reactants with peptides / proteins due to their reducing properties. This may cause particular problems during the formation of the peptide / protein containing active substance.

During spray drying of antibodies without the addition of stabilizers, dehydration, heating, and shearing cause unfolding of the secondary structure inherently, resulting in significant loss of bioactivity. The prior inwardly hydrophobic portion of the antibody returns to the outwarding state. This occurs gradually at the hydrophobic interface between water droplets and the caution air that occur during spray drying. In addition, antibodies in the aqueous phase accumulate to form dimers or higher aggregates. These aggregates are often irreversible. In addition, the high temperature at which the protein is sprayed is an important variable. When high energy is consumed, destabilization of peptide bonds and denaturation of antibodies occur. In addition, aggregation of the spray dried antibody occurs during storage of the powder. The residual water content in the powder then has a particularly negative effect. Protein aggregates are characterized by reduced or absent biological activity and enhanced antigenicity.

Complex sugars and lactosucrose, described as coupling sugars (oligosaccharides) with the main components of maltosyl sucrose and glucosyl sucrose, are used in the food field. They are used together with sweetening agents such as aspartame, as fillers and dispersants, as a mild sweetening component in chewing gums, to stabilize trehalose syrups from crystallization or as so-called NDO (non-digestible oligosaccharides). The sweetening properties of asparagyl peptides and the improvement and stabilization of sweet-to-sour ratios in ballast and sweetener-containing beverages are known. See US Patent Publication No. 2003/0059511, EP 1 223 175, German Patent Publication No. 199 53 727]. U.S. Patent 5,489,577 and EP 630 651 further describe the use of oligosaccharides to stabilize suspensions prepared from therapeutic proteins and fat or oil bases. It has been described that their activity may be lost during blending and kneading with hydrophobic semisolid materials without premixing the proteins with oligosaccharides. The possibility of stabilization for storage in hydrophobic mixtures or powders is not mentioned in any way.

It is an object of the present invention to provide novel adjuvants for the preparation of pharmaceutical preparations. Corresponding formulations should be distinguished, among other things, by good long term stability.

It is a further object of the present invention to provide a novel adjuvant for preparing a dried pharmaceutical formulation. Corresponding powdered formulations should be distinguished for good long term stability and possibly for inhalation.

It is a further object of the present invention to provide novel adjuvants for preparing peptide / protein containing pharmaceutical formulations, in particular formulations produced by spray drying. Corresponding peptide / protein containing formulations should be distinguished for good long term stability and possibly inhalation.

It is a further object of the present invention to provide novel adjuvants for preparing therapeutic antibodies or antibody derivatives, especially those produced by spray drying. Corresponding antibody containing formulations should be distinguished by good long term stability and possibly inhalation.

A further object of the present invention is to provide a corresponding pharmaceutical formulation for use in an inhaler, regardless of the form of dry powder, propellant containing metered dose aerosol or propellant containing no propellant.

The object on which the present invention is based is solved by the subject / process described in the following description and claims.

Summary of the Invention

The present invention relates to one antibody or one antibody derivative and 1,4 O-linked D-Gal-sacrose (lactosucrose), 1,4 O-linked D-Glu-sacrose (glucosyl sucrose) And at least one 1,4 O-linked saccharose derivative selected from compounds consisting of 1,4 O-linked Glu-Glu-saccharose (maltosyl sucrose).

In addition, lactosucrose means a molecule having the formula:

In addition, glycosyl sucrose in the sense of the present invention means a molecule having the formula:

In addition, maltosyl sucrose means a molecule having the formula:

According to a further aspect of the invention, the corresponding composition, together with the antibody or antibody derivative and the 1,4 O-linked saccharose derivative, further contains one or more monosaccharides, disaccharides and / or polysaccharides, monosaccharides and / or Or disaccharides. In addition, compositions prepared from glucosyl and maltosyl sucrose have been found to be combined with one antibody or one antibody derivative, preferably in combination with monosaccharides, disaccharides and / or polysaccharides, according to the invention.

Thus, in particular the compositions according to the invention comprise a sugar mixture (a) 25 to 99.99% (w / w), preferably containing at least one O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative Preferably from 80 to 90% (w / w) to the dry weight of the composition and one antibody or one antibody derivative (b) preferably from 0.01 to 75% (w / w) (to the dry weight of the composition). Composition with a fraction of) and the sum of the weight percent of the sugar / sugar mixture and the antibody or antibody derivative corresponds to a maximum of 100% (w / w).

The composition may be an aqueous composition, a solid or semisolid composition. Particular preference is given to solid compositions, preferably powdered compositions. Surprisingly, it has been found that the corresponding powdered antibody-containing compositions (i) form an amorphous structure, (ii) have a glass transition temperature above 40 ° C., and (iii) have a low propensity to recrystallize.

Compositions according to the invention, in particular powdered compositions, together with one sugar mixture containing at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative, are further adjuvant And may contain, for example, amino acids, peptides, proteins or other sugars. One or more amino acids, one peptide, together with one sugar mixture and one antibody or antibody derivative containing one or more 1,4 O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives Particularly advantageous are compositions containing dipeptides or tripeptides and / or one salt. According to a preferred embodiment, the present invention, based on dry weight, comprises one sugar mixture containing at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative (a ) 25 to 90% (w / w), 1 to 39.99% (w / w) one or more amino acids and / or one or more peptides (b), one antibody or antibody derivative (c) 0.01% (w) as additional adjuvant / w) or more, to a sprayed composition, such as a spray dried powder. Further auxiliaries preferably mean amino acid isoleucine or peptides, preferably dipeptides or tripeptides having one or more isoleucine groups. According to a particular embodiment, the present invention relates to a sugar mixture containing at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative, based on dry weight (a ) Approximately 60 to 80% (w / w), one or more amino acids, preferably isoleucine (b) approximately 10 to 19.99% (w / w), and one antibody or antibody derivative (c) approximately 0.01 to 30% A composition containing (w / w), for example a powder dried powder. According to yet another aspect, the present invention provides a sugar mixture containing one or more 1,4 O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives, based on dry weight. (a) approximately 60 to 90% (w / w), peptide, preferably isoleucine containing peptide, particularly preferably isoleucine containing dipfetide or tripeptide, more preferably triisoleucine (b) approximately 1 to A composition containing 19.99% (w / w) and approximately 0.01 to 39% (w / w) of one antibody or antibody derivative (c), preferably a powdered composition, for example spray dried powders. will be. After mixing isoleucine or isoleucine containing tripeptides, in particular the powder with the corresponding composition shows very good flowability. In addition, the corresponding powder dried powder has a very high proportion of inhalable particles. In addition, the corresponding powders are very good in processing and storage stability.

According to a further aspect, the present invention provides a mixture containing one or more 1,4 O-linked saccharose derivative (s) or one or more 1,4 O-linked saccharose derivatives, formulated as described above. To a corresponding powdered composition, preferably spray dried powder, containing a sugar mixture (a) and at least one antibody or one antibody derivative (b), whose glass transition temperature is above 40 ° C., preferably It is more than 45 degreeC, More preferably, it is more than 50 degreeC, More especially preferably, it is more than 55 degreeC, Especially preferably, it is more than 60 degreeC. In general, corresponding powders according to the invention have a maximum glass transition temperature of approximately 96 to 110 ° C. In each case, however, the value can be much higher. The fraction of adjuvant added, in particular the fraction of 1,4 O-linked saccharose derivatives, or the mixture of derivatives in the powder, is mainly due to the corresponding glass transition temperature.

According to a further aspect, the present invention relates to a pharmaceutical composition comprising an antibody or antibody derivative for use as an inhaler, comprising and consisting of one powdered composition according to the invention described herein. In this connection, pharmaceutical compositions containing the powders according to the invention as propellant containing metered dose aerosols or solutions for inhalation free of propellants are preferred. Powders according to the invention used to prepare pharmaceutical compositions, preferably powders prepared by spray drying, have a weight median aerodynamic diameter (MMAD) of less than 10 μm, preferably 0.5 to 7.5 μm, according to a further embodiment. More preferably 0.5 to 5.5 μm, particularly preferably 0.5 to 5.0 μm.

In addition, the present invention provides a process for preparing a corresponding composition, preferably a powdered composition according to the invention, for example a process for preparing spray dried powder, the process comprising one or more 1,4 O- Preparation of an aqueous composition containing one sugar mixture (a) containing bound saccharose derivative (s) or one or more 1,4 O-linked saccharose derivatives and one or more antibodies or one antibody derivative (b) And, in the case of powdered compositions, it is dried, for example sprayed, under suitable conditions.

Description of the Drawings

All percentage data specified in the detailed description is for the concentration (w / w) of solids in solution. All symbols in the figures described below refer to the percentage composition (w / w) of the powder obtained by spray drying and freeze drying while grinding. The sign further specifies the total solid concentration (total solid = TS) (% (w / w)) of the solution. In the sign of FIG. 8, the coupling sugar is abbreviated as CS. In the symbols of FIGS. 18, 19, 20 and 21, tri-isoleucine is abbreviated as Ile3. 15, 16, 17, 18, 19, 20 and 21 further specify the spray rate (AAF = spray air flow) set during the spray drying process in Buchi B-290. Subsequently, sign 40 corresponds to a real volumetric flow of about 0.67 m ³ / h, sign 50 corresponds to a real volumetric flow of about 1.05 m ³ / h, and sign 60 corresponds to a real volumetric flow of about 1.74 m ³ / h Corresponds. In all other figures, a spray rate of 40 corresponds to an actual volumetric flow of about 0.67 m ³ / h each.

1 shows the aggregate content after lyophilization, milling and reconstitution by 75% relative humidity and open storage (force storage stability) for 1 week at 40 ° C. (a) 4.5% LS55P fraction and 0.5% IgG fraction, (b) 4.5% coupling sugar fraction and 0.5% IgG fraction, (c) 5.0% IgG fraction and (d) 4.5% mannitol fraction and 0.5% IgG fraction The aqueous solution to be lyophilized. Both LS55P- and coupling sugar containing powders are distinguished by a low aggregate fraction.

Figure 2 shows the aggregate content after lyophilization, pulverization and reconstitution by dry storage (equivalent storage stability) at 40 ° C. for 4 weeks. (a) 4.5% LS55P fraction and 0.5% IgG fraction, (b) 4.5% coupling sugar fraction and 0.5% IgG fraction, (c) 5.0% IgG fraction and (d) 4.5% mannitol fraction and 0.5% IgG fraction The aqueous solution to be lyophilized. Both LS55P- and coupling sugar containing powders are distinguished by a low aggregate fraction.

FIG. 3 shows the aggregate content after lyophilization, milling, vacuum drying and dry storage at 40 ° C. for 4 weeks (vacuum dry storage stability) to reconstitution. (a) 4.5% LS55P fraction and 0.5% IgG fraction, (b) 4.5% coupling sugar fraction and 0.5% IgG fraction, (c) 5.0% IgG fraction and (d) 4.5% mannitol fraction and 0.5% IgG fraction The aqueous solution to be lyophilized. Both LS55P- and coupling sugar containing powders are distinguished by a low aggregate fraction.

4 shows the aggregate content after lyophilization and reconstitution by storage (forced storage stability) for one week at 75% relative humidity and 40 ° C. FIG. (a) 9% LS55P fraction and 1% IgG fraction, (b) 9% coupling sugar fraction and 1% IgG fraction, (c) 9% coupling sugar S fraction and 1% IgG fraction, (d) 9% tre The aqueous solution comprising the haloose fraction and 1% IgG fraction and (e) 10% IgG fraction is spray dried. Both LS55P- as well as coupling sugars and coupling sugar S containing powders are distinguished by low aggregate fractions.

Figure 5 shows the aggregate content after lyophilization and reconstitution by storage (forced storage stability) for one week at 75% relative humidity and 40 ° C. (a) 8% LS55P fraction, 1% isoleucine fraction and 1% IgG fraction, (b) 8% coupling sugar fraction, 1% isoleucine fraction and 1% IgG fraction, (c) 8% coupling sugar S fraction An aqueous solution comprising 1% isoleucine fraction and 1% IgG fraction, (d) 8% trehalose fraction, 1% isoleucine fraction and 1% IgG fraction and (e) 10% IgG fraction is spray dried. Both LS55P- as well as coupling sugars and coupling sugar S containing powders are distinguished by low aggregate fractions.

Figure 6 shows the aggregate content after lyophilization and reconstitution by storage (forced storage stability) for one week at 75% relative humidity and 40 ° C. (a) 8% LS55P fraction, 1% isoleucine fraction and 1% IgG fraction, (b) 8% coupling sugar fraction, 1% isoleucine fraction and 1% IgG fraction, (c) 8% coupling sugar S fraction An aqueous solution comprising 1% isoleucine fraction and 1% IgG fraction, (d) 8% trehalose fraction, 1% isoleucine fraction and 1% IgG fraction and (e) 10% IgG fraction is spray dried. Both LS55P- as well as coupling sugars and coupling sugar-containing powders are distinguished by low aggregate fractions.

FIG. 7 shows the aggregate content after lyophilization and reconstitution by 75% relative humidity and open storage (force storage stability) for 1 week at 40 ° C. FIG. (a) 9.9% LS55P fraction and 0.1% IgG fraction, (b) 9% LS55P fraction and 1% IgG fraction, (c) 6% LS55P fraction and 4% IgG fraction, (d) 4% LS55P fraction and 6% IgG Fraction, (e) 2.5% LS55P fraction and 7.5% IgG fraction, (f) 9% LS55P fraction and 1% IgG fraction, (g) 0.5% LS55P fraction and 9.5% IgG fraction and (h) 10% IgG fraction The aqueous solution to spray-dry. LS55P containing powders are distinguished by a low aggregate fraction.

FIG. 8 shows the aggregate content after lyophilization and reconstitution by 75% relative humidity and open storage (force storage stability) for 1 week at 40 ° C. (a) 9.9% coupling sugar fraction and 0.1% IgG fraction, (b) 9% coupling sugar fraction and 1% IgG fraction, (c) 6% coupling sugar fraction and 4% IgG fraction, (d) 4% Coupling sugar fraction and 6% IgG fraction, (e) 2.5% coupling sugar fraction and 7.5% IgG fraction, (f) 1% coupling sugar fraction and 9% IgG fraction and (g) 10% IgG fraction The aqueous solution is spray dried. LS55P containing powders are distinguished by a low aggregate fraction.

9 shows the aggregate content after spray drying and reconstitution by 75% relative humidity and open storage (force storage stability) for 1 week at 40 ° C. (a) 3.00% LS55P fraction and 0.33% IgG fraction, (b) 2.9166% LS55P fraction, 0.0833% triisoleucine fraction and 0.33% IgG fraction, (c) 2.833% LS55P fraction, 0.166% triisoleucine fraction and 0.33% The aqueous solution containing the IgG fraction and (d) 2.66% LS55P fraction, 0.33% triisoleucine fraction and 0.33% IgG fraction is spray dried. LS55P containing powders are distinguished by a low aggregate fraction. Protein aggregation is significantly reduced by increasing the tri-isoleucine fraction from 0% to 10% relative to the total solids content of the LS55P containing powder.

10 shows the aggregate content after spray drying and reconstitution by 75% relative humidity and open storage (force storage stability) for 1 week at 40 ° C. (a) 3.00% LS90P fraction and 0.33% IgG fraction, (b) 2.9166% LS90P fraction, 0.0833% triisoleucine fraction and 0.33% IgG fraction, (c) 2.833% LS90P fraction, 0.166% triisoleucine fraction and 0.33% An aqueous solution comprising the IgG fraction and (d) 2.66% LS90P fraction, 0.33% triisoleucine fraction and 0.33% IgG fraction is spray dried. LS90P containing powders are distinguished by a low aggregate fraction.

FIG. 11 shows the aggregate content after spray drying and reconstitution by 75% relative humidity and open storage (force storage stability) for 1 week at 40 ° C. FIG. (a) 2.66% LS90P fraction, 0.33% tri-isoleucine fraction and 0.33% IgG fraction, (b) 2.66% LS55P fraction, 0.33% triisoleucine fraction and 0.33% IgG fraction, (c) 2.66% saccharose fraction, 0.33% triisorucin fraction and 0.33% IgG fraction, (d) 2.00% saccharose fraction, 0.66% lactose fraction, 0.33% triisorucin fraction and 0.33% IgG fraction, (e) 2.66% raffinose fraction, 0.33% Triisoleucine fraction and 0.33% IgG fraction, (f) 2.66% hydroxyethyl starch (HES) fraction, 0.33% triisoleucine fraction and 0.33% IgG fraction and (g) 2.66% trehalose fraction, 0.33% triiso An aqueous solution containing the leucine fraction and the 0.33% IgG fraction is spray dried. LS90P- and LS55P- containing powders are distinguished by a low aggregate fraction, in particular by a low aggregate fraction compared to raffinose and hydroxyethyl starch (HES) as specified in the prior art.

12 shows the aggregate content after reconstitution by spray drying, vacuum drying and then dry storage (vacuum dry storage stability) for 4 weeks at 40 ° C. (a) 9% coupling sugar fraction and 1% IgG fraction, (b) 8% coupling sugar fraction, 1% (w / w) isoleucine fraction and 1% IgG fraction, (c) 3% coupling sugar fraction Spray dried an aqueous solution comprising 6% citrulline fraction and 1% IgG fraction and (d) 10% IgG fraction. Coupling sugar containing powders are distinguished by a low aggregate fraction.

Figure 13 shows the aggregate content after reconstitution by spray drying, vacuum drying and then dry storage (vacuum dry storage stability) for 4 weeks at 40 ° C. (a) 9.9% LS55P fraction and 0.1% IgG fraction, (b) 9% LS55P fraction and 1% IgG fraction, (c) 2.5% LS55P fraction and 7.5% IgG fraction, (d) 1% LS55P fraction and 9% IgG An aqueous solution comprising the fraction, (e) 10% IgG fraction, f) 8% LS55P fraction, 1% isoleucine fraction and 1% IgG fraction and (g) 3% LS55P fraction, 6% citrulline fraction and 1% IgG fraction. Spray dry. LS55P containing powders are distinguished by a low aggregate fraction.

Figure 14 (a + b) shows the aggregate content after reconstitution by spray drying, vacuum drying and 1 or 3 months dry storage (1 month or 3 months stability) at 2-8 ° C, 25 ° C and 40 ° C. . (a) 3.00% LS90P fraction and 0.33% IgG fraction, (b) 2.66% LS90P fraction, 0.33% isoleucine fraction and 0.33% IgG fraction and (c) 2.66% LS90P fraction, 0.33% triisoleucine fraction and 0.33% IgG The aqueous solution containing fractions is spray dried. Both LS90P and LS55P containing powders are distinguished by a particularly low aggregate fraction after three months of storage.

Figure 15 (a + b) is reconstructed by spray drying, vacuum drying, open 1 month or 3 months dry storage (open 1 month or 3 months stability) at 25 ° C. at 29% relative humidity and 43% relative humidity, respectively. The aggregate content after one is shown. (a) 2.9166% LS90P fraction, 0.0833% triisoleucine fraction and 0.33% IgG fraction in AAF of 40, (b) 2.833% LS90P fraction, 0.166% triisoleucine fraction and 0.33% IgG fraction in AAF of 40, (c ) 2.66% LS90P fraction, 0.33% triisoleucine fraction and 0.33% IgG fraction at AFF of 40, (d) 1.60% LS90P fraction, 0.20% triisoruscin fraction and 0.33% IgG fraction at AAF of 40, (e) 50 2.66% LS90P fraction, 0.33% triisoleucine fraction and 0.33% IgG fraction in AAF of (f) 2.66% LS90P fraction, 0.33% triisoleucine fraction and 0.33% IgG fraction in AAF of 60 and (g) AFF of 40 Spray-dry an aqueous solution containing 3.33% IgG fractions. LS90P containing powders are distinguished by a particularly low aggregate fraction after three months of storage.

16 shows fine particle fractions (FPF) with a cut diameter of less than 5 μm for various powders. The powder is prepared by spray drying an aqueous solution containing LS55P and IgG1 or LS55P, isoleucine and IgG1. The solution is prepared and sprayed as described in the examples. The isoleucine containing powder has about 35% FPF and the isoleucine free powder alone has about 16% FPF.

17 shows fine particle fractions (FPF) with a cut diameter of less than 5 μm for various powders. The powder is prepared by spray drying an aqueous solution containing LS90P and IgG1 or LS90P, isoleucine and IgG1. The solution is prepared and sprayed as described in the examples. The isoleucine containing powder has about 28% FPF and the isoleucine free powder alone has about 23% FPF.

18 shows fine particle fractions (FPF) with a cut diameter of less than 5 μm for various powders. The powder is prepared by spray drying an aqueous solution containing LS55P and IgG1 or LS55P, triisoleucine and IgG1. The solution is prepared and sprayed as described in the examples. The triisoleucine containing powder has an FPF greater than 50 or 58% and the triisoleucine free powder alone has about 16% FPF.

20 shows the weight median aerodynamic diameter (MMAD) and the weight median diameter (MMD) for various powders. The powder is prepared by spray drying an aqueous solution containing LS55P and IgG1 or LS55P, triisoleucine and IgG1. The solution is prepared and sprayed as described in the examples. All powders have an MMAD of less than 5 μm and an MMD of less than 3.5 μm. The diagram shows the effect of the triisoleucine fraction on MMAD and MMD at constant total solid concentration and spray parameters. The 10% triisoleucine fraction associated with the total sphere content of the formulation significantly reduces MMAD.

20 shows fine particle fractions (FPF) with a cut diameter of less than 5 μm for various powders. The powder is prepared by spray drying an aqueous solution containing LS90P and IgGl or LS90P, triisoleucine and IgG1. The solution is prepared and sprayed as described in the examples. The triisoleucine containing powder has about 40% to about 59% FPF and the triisoleucine free powder alone has about 24% FPF.

21 shows the weight median diameter (MMD) and the weight median aerodynamic diameter (MMAD) of the various powders. The powder is prepared by spray drying an aqueous solution containing LS90P and IgG1 or LS90P, triisoleucine and IgG1. The solution is prepared and sprayed as described in the examples. All powders have an MMAD of less than 6.5 μm and an MMD of less than 5 μm. The diagram shows the effect of the triisoleucine fraction on MMAD and MMD at constant total solid concentration and spray parameters. The 10% triisoleucine fraction associated with the total solids content of this formulation significantly reduces MMAD. However, low solids content (eg TS "2%) and high spray pressure (AAF of 50 or 60) significantly reduce MMD.

22 shows the residual monomer content after spray drying, forced storage and reconstitution. (a) 3.33% (w / w) lysozyme fraction, (b) 0.33% (w / w) lysozyme fraction and 3.0% (w / w) LS90P fraction, (c) 0.33% (w / w) lysozyme fraction, 0.33 % (w / w) isoleucine fraction and 2.66% (w / w) LS90P fraction and (d) 0.33% (w / w) lysozyme fraction, 0.33% (w / w) triisoleucine fraction and 2.66% (w / w) w) Spray an aqueous solution containing LS90P fractions. LS90P containing powders are distinguished by low residual monomer content.

Figure 23 shows the aggregate content after reconstitution by spray drying, vacuum drying and then 3 months dry storage (3 months stability) at 2-8 ° C, 25 ° C and 40 ° C. (a) 3.33% (w / w) calcitonin fraction, (b) 0.166% (w / w) calcitonin fraction and 3.166% (w / w) LS90P fraction, (c) 0.166% (w / w) calcitonin fraction, 0.33 % (w / w) isoleucine fraction and 2.833% (w / w) fraction and (d) 0.166% (w / w) calcitonin fraction, 0.33% (w / w) triisorucin fraction and 2.833% (w / w) ) Spray an aqueous solution containing the fractions. LS90P containing powders are distinguished by their low aggregate content.

24 shows an inhaler for applying a dry powder formulation.

Justice

The terms and names used within the scope of the invention have the meanings described below. By weight and weight percent expression is meant the dry weight of the composition or the solids content of the solution / suspension, unless stated otherwise. General aspects "containing" and "containing" include more specific aspects "consisting of." In addition, "singular" and "plurality" are used without limitation.

“Sugar mixtures containing 1,4 O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives” are (i) 1,4 O-linked having one formula described in this patent specification. Saccharose derivatives, (ii) mixtures thereof, preferably mixtures of maltosyl and glycosyl sucrose, (iii) one or more 1,4 O-linked saccharose derivatives having one of the formulas described above, and Mixtures with sugars, preferably lactosucrose, lactose and saccharose or glycosyl and / or maltosyl sucrose, saccharose, fructose and glucose, (iv) lactosucrose 55% ( w / w) or more, a mixture of up to 25% (w / w) lactose and up to 10% (w / w) saccharose, (v) more than 88% (w / w) lactoserose, up to 10% lactose (w / w) and a mixture of saccharose (vi) 25% (w / w) of glucosyl and / or maltosyl sucrose, 48% to 56% (w / w) and saccharose, respectively; A mixture of glucose and fructose up to 10% (w / w), (vii) glucosyl and maltosyl sucrose 18% (w / w), saccharose 11% to 15% (w / w) and glucose respectively 5 mixtures% to 9% (w / w), (viii) a nyuka made from manufactured by at Shoji whether or step down a break located at a J-up ^® LS40L (abbreviated as LS40L), nyuka-up ^® LS55L (abbreviated as LS55L) , Nica-Oligo ^® LS55P (abbreviated LS55P), Nica-Oligo ^® LS-90P (abbreviated LS90P), coupling sugar ^® or coupling sugar S ^® .

"Composition" means a liquid, semisolid or solid mixture of one or more starting materials.

"Pharmaceutically acceptable adjuvant" means an adjuvant that can optionally be contained in a formulation within the scope of the invention. Adjuvants may be administered intrapulmonally, for example, without significant toxic side effects to the test subject or the lungs of the test subject.

"Pharmaceutically acceptable salts" include, but are not limited to, salts of inorganic acids, such as salts of chlorides, sulfates, phosphates, diphosphates, bromide and nitrates. In addition, salts of organic acids such as maleate, maleate, fumarate, tartrate, succinate, ethyl succinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para Toluolsulfonates, palmoates, salicylates and stearates, as well as estoleates, gluceptates and lactobionate salts.

"Pharmaceutically acceptable cations" include, but are not limited to, for example, lithium, sodium, potassium, calcium, aluminum, and ammonium (including substituted ammonium).

"Amino acid" means a compound containing at least one amino group and at least one carboxyl group. The amino group is generally in the α-position relative to the carboxyl group, but any other arrangement in the molecule is also conceivable. Amino acids may also contain other additional functional groups such as amino, carboxamide, carboxyl, imidazole, thio, and other groups. Natural or synthetic amino acids having racemic or optical (D or L form) activity can be used, including different stereoisomeric fractions. For example, the term includes isoleucine and D-isoleucine, L-isoleucine, racemic isoleucine and two enantiomers of different fractions.

"Biological macromolecule" means peptides, proteins, fats, fatty acids or nucleic acids and the like.

"Peptide" or "polypeptide" means a polymer of amino acids consisting of 2 to 100 amino acid groups. Peptides or polypeptides are used as nicknames and include both homopeptides and heteropeptides. That is, polymers of amino acids are composed of the same or different amino acid groups. Thus, a "dipeptide" consists of two peptide bound amino acids and a "tripeptide" consists of three peptide bound amino acids. As used herein, a "protein" is a polymer of amino acids having at least 100 amino acid groups.

"Homolog" means a peptide or protein in which a single or plural amino acids are substituted, removed (e.g., fragment), addition (e.g., C- or N-terminal extended derivative) or other modified peptide with respect to a native (wild-type) sequence. Also, for example, derivatives of natural proteins with sugars, polyethylene glycols, and the like can be produced. The homologues have a bioactivity of at least 10, 20, 30 or 40%, preferably at least 50, 60 or 70%, particularly preferably at least 80, 90, 95, 100% or 100% of the natural, nonsynthetic protein. .

"Polysaccharide" or "oligosaccharide" means a complex sugar consisting of three or more monomeric sugar molecules.

"Pure protein formulation" means a powder consisting of one or more antibodies or antibody derivatives and optionally a suitable buffer (typically 0-15% (w / w) by weight of the dry powder). The powder basically contains no other auxiliaries. That is, other adjuvants that may be included are less than 1% by weight of the dry powder.

A "spray dried powder formulation" or "dry powder formulation" generally has a residual moisture of less than approximately 10% (w / w), preferably less than 7% (w / w), particularly preferably 5% (w / powder formulations of less than w), particularly more preferably less than 3% (w / w) When given constant spray, vacuum or lyophilization conditions and the same adjuvant, the residual moisture is largely pharmaceutically active substance in the powder formulation. It depends on the type and fraction of.

"Amorphous" means a powdered formulation having a crystal fraction of less than 10%, preferably less than 7%, more preferably less than 5%, particularly preferably less than 4, 3, 2 or 1%.

"Inhalable" is a powder suitable for use in the lungs. Inhalable powders may be dispersed and inhaled with the aid of an inhaler so that the particles reach the lungs and, if necessary, have a systemic effect through the alveoli. Inhalable particles may, for example, have an average particle size of 0.4 to 10 μm (MMD = weight median diameter), mostly 0.5 to 5 μm, preferably 1 to 3 μm, and / or a weight medium aerodynamic diameter (MMAD = Weight median aerodynamic diameter) is from 0.5 to 10 μm, preferably from 0.5 to 7.5 μm, more preferably from 0.5 to 5.5 μm, even more preferably from 1 to 5 μm, particularly preferably from 1 to 4.5 μm.

"Medium median diameter" or "MMD" is a measure of the average particle size distribution, as the powders described herein are generally polydisperse. The results are expressed as the diameter of the total volume distribution at 50% total throughput. MMD values can be measured, for example, by laser diffraction (see Examples section and method), but any other conventional method may be used (e.g., electron microscopy, centrifugal sedimentation).

"Medium weight aerodynamic diameter (MMAD)" means aerodynamic particle size when 50% of the powder particles generally have a small aerodynamic diameter. The method described in this patent specification (see Examples section and method) is used as a reference method for measuring MMAD when it is not clear.

"Fine particle fraction" means an inhalable portion of a powder consisting of particles having an MMAD of 5 μm or less in particle size. In powders that are well dispersed, the FPF is greater than 20%, preferably greater than 30%, particularly preferably greater than 40%, even more preferably greater than 50%, more preferably greater than 55%. "Cutting diameter" as used in this context means particles that are considered when measuring FPF. A 30% FPF at a cut diameter of ₅ μm (FPF ₅ ) means that at least 30% of all particles in the powder have an average median aerodynamic diameter of less than 5 μm.

"Spray solution" means an aqueous solution or suspension in which the pharmaceutically active substance is dissolved / suspended with one or more adjuvants.

"Flight time" is the name used in standard measurement methods, as described in more detail in the Examples section. MMAD and FPF are measured simultaneously during flight time measurements (see Examples and Methods).

"Surface active" materials can reduce the surface tension of the dissolved solution. Surface activity is measured, for example, by a tension meter proposed by Lecomte du Nouy (Bauer, Fromming, Fuhrer, 6th ed.).

Composition according to the invention

The present invention relates to one antibody or one antibody derivative (a) and a 1,4 O-linked D-Gal-sacrose (lactosucrose), a 1,4 O-linked D-Glu- as a pharmaceutically active substance. Containing at least one 1,4 O-linked saccharose derivative (b) selected from Saccharose (glucosyl sucrose), and a 1,4 O-linked Glu-Glu-Saccharose (maltosyl sucrose) combination It relates to a composition.

According to a further aspect of the invention, the corresponding composition, together with the antibody or antibody derivative and the 1,4 O-linked saccharose derivative, further contains one or more monosaccharides, disaccharides and / or polysaccharides, monosaccharides and / or Or further use of disaccharides is particularly preferred during powder preparation. Furthermore, the present invention consequently comprises a composition having lactosucrose, lactose and saccharose, preferably a powdered composition, for example spray dried powder, and the lactosucrose relative to the total sugar fraction of the composition. The fraction of is at least 40% (w / w), preferably at least 55% (w / w) and also at least 88% (w / w). According to a preferred embodiment, the composition according to the invention, together with one antibody or antibody derivative as a pharmaceutically active substance, as Nyuka-Oligo ^® LS55P (Hayashbara Shoji KK, Japan) (abbreviated as LS55P) Contains the specified sugar mixture, LS55P contains at least 55% lactosucrose, up to 25% lactose (w / w) and up to 10% saccharose (w / w). According to a further preferred embodiment, the composition according to the invention, together with a pharmaceutically active substance, comprises a sugar mixture specified as Nyuka-Oligo ^® LS90P (Hayashbara Shoji, Japan) (abbreviated as LS90P). LS90P contains at least 88% lactosucrose, up to 10% lactose (w / w) and saccharose.

In addition, a composition, preferably a powdered composition, wherein the antibody and antibody derivative are combined with glucosyl and maltosyl sucrose, again preferably in combination with other monomers, dimers and / or multimers, for example Spray dried compositions have been found to be in accordance with the present invention. In addition, the invention consequently comprises the corresponding composition, preferably powdered composition, containing a mixture of glucosyl and maltosyl sucrose, saccharose, glucose and / or fructose together with the antibody or antibody derivative. The fraction of glucosyl and maltosyl-sucrose relative to the total sugar fraction of the composition is preferably at least 25% (w / w). In a further preferred embodiment, each of the glucosyl sucrose fraction and maltosyl sucrose fraction is at least 18% (w / w) of the total sugar fraction of the composition. According to a preferred embodiment, the composition, preferably the powdered composition, according to the invention, together with an antibody or antibody derivative as a pharmaceutically active substance, is specified as Coupling Sugar ^® (Hayashbara Shoji KK, Japan). Containing sugar mixture, Coupling Sugar ^® contains at least 18% (w / w) of glucosyl and maltosyl sucrose, 11-15% (w / w) of saccharose and 5-9% of glucose and fructose, respectively ( w / w). In addition, the present invention provides a pharmaceutical with an antibody or antibody derivative as the active substance, the coupling shoe S ^® (Hayakawa sh manufactured by Shoji whether or views, Japan material) composition containing the mixture per specified as preferably powder It relates to localized compositions, coupling sugar S ^®, glucosyl, and / or the end of tosyl sucrose 25% (w / w) or more, saccharose 48 to 56% (w / w) and the glucose and fructose 10% ( w / w) or less.

Compositions that have been found to be particularly advantageous, preferably powdered compositions, such as spray dried powders, may contain 1,4 O-linked saccharose derivatives or at least one 1,4 O-linked sacca based on the dry weight of the composition. The fraction of the sugar mixture containing the rose derivative is 25 to 99.99% (w / w), preferably 40 to 99% (w / w), more preferably 60 to 99% (w / w), even more preferred. Preferably 60 to 90% (w / w), for example 25, 25.1, 25.2, 25.3,. . . 25.7, 25.8, 25.9 and the like; 26, 27, 28, 29, 30, and the like; 31, 32, 33,. . . 38, 39, 40, and the like; 41, 42, 43,. . . 48, 49, 50, and the like; 51, 52, 53,. . . 58, 59, 60, and the like; 61, 62, 63,. . . 68, 69, 70, and the like; 71, 72, 73,. . . 78, 79, 80, and the like; 81, 82, 83,. . . 88, 89, 90, and the like; 91, 92, 93,. . . 98, 99, and the like; 99.1, 99.2, 99.3,. . . 99.8, 99.9 and the like; 99.91, 99.92, 99.93,. . . 99.98, 99.99% (w / w). In connection with the use of LS55P and also LS90P, a fraction of 60 to 90% (w / w) has been found to be particularly advantageous. When the composition refers to a powdered composition, e.g. spray dried powder, a mixture of sugars containing a 1,4 O-linked saccharose derivative or one or more 1,4 O-linked saccharose derivatives The fraction should be chosen such that the powdered composition is at least partially amorphous, preferably completely amorphous. Fractions of sugar mixtures containing 1,4 O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives can also be reduced below 60% (w / w). The powder is then mixed with the other stabilizing aids in an appropriate amount. Examples of other stabilizing aids are described in this patent specification.

With respect to the dry weight of the composition according to the invention the fraction of the antibody or antibody composition is generally from 0.01 to 75% (w / w), preferably from 0.01 to 50% (w / w), more preferably from 0.33 to 50% (w / w), even more preferably 0.33 to 40% (w / w). According to a further preferred embodiment, for the solids content of the composition according to the invention the fraction of the antibody or antibody derivative corresponds to 0.33 to 35% (w / w), preferably 0.33 to 30% (w / w). For example, the fraction is 0.01, 0.02, 0.03. . . 0.08, 0.09 and the like; 0.1, 0.2, 0.3,. . . 0.8 0.9 and the like; 1, 2, 3,. . . 8, 9, 10 and the like; 21, 22, 23,. . . 28, 29, 30, and the like; 31, 32, 33,. . . 38, 39, 40, and the like; 41, 42, 43,. . . 48, 49, 50, and the like; 51, 52, 53,. . . 58, 59, 60, and the like; 61, 62, 63,. . . 68, 69, 70, and the like; 71, 72, 73, 74, 74.1, 74.2, 74.3,. . . 74.8, 74.9 and the like; 74.91, 74.92, 74.93,. . . 74.98, 74.99, 75% (w / w).

Thus, the compositions according to the invention have a ratio of sugar mixtures containing 1,4 O-linked saccharose derivatives or at least one 1,4 O-linked saccharose derivatives to pharmaceutically active substances, for example 25 /. 75, 26/74, 27/73, 28/72, 29/1, 30/70, 31/69, 32/68, 33/67, 34/66, 35/65, 36/64, 37/63, 38/62, 39/61, 40/60, 41/59, 42/58, 43/57, 44/56, 45/55, 46/54, 47/53, 48/52, 49/51, 50 / 50, 51/49, 52/48, 53/47, 54/46, 55/45, 56/44, 57/43, 58/42, 59/41, 60/40, 61/39, 62/38, 63/37, 64/36, 65/35, 66/34, 67/33, 68/32, 69/31, 70/30, 71/29, 72/28, 73/27, 74/26, 75 / 25, 76/24, 77/23, 78/22, 79/21, 80/20, 81/19, 82/18, 83/17, 84/16, 85/15, 86/14, 87/13, 88/12, 89/11, 90/10, 91/9, 92/8, 93/7, 94/6, 95/5, 96/4, 97/3, 98/2, 99/1, 99.1 / 0.9, 99.2 / 0.8, 99.3 / 0.7, 99.4 / 0.6, 99.5 / 0.5, 99.6 / 0.4, 99.66 / 0.33, 99.7 / 0.3, 99.8 / 0.2, 99.9 / 0.1, 99.99 / 0.01 (w / w). Fraction (a), antibody or antibody of a sugar mixture containing a 1,4 O-linked saccharose derivative or a 1,4 O-linked saccharose derivative if the corresponding composition contains one or more additional excipients The fraction (b) of the derivative, or both (c), is correspondingly reduced and is a 1,4 O-linked saccharose derivative or one or more 1,4 O-linked relative to the dry weight of the composition according to the invention. The fraction of the sugar mixture containing the saccharose derivatives is preferably a value of 80 to 90% (w / w).

In the context of the present invention, the pharmaceutically active substance is an antibody or antibody derivative. Examples thereof include monoclonal, polyclonal, multispecific and single chain antibodies, as well as fragments thereof such as Fab, Fab ', F (ab') ₂ , Fc and Fc 'fragments, light chains ( L) and heavy chain (H) antibodies and their fixed, variant or overvariable regions and Fv and Fd fragments and one or more antibody fractions or fractions of one or more double or single chain antibodies. See Chamov. et al., 1999, Antibody Fusion Proteins, Wiley-Liss Inc.]. The antibody may be of human or non-human origin. Classes known in the following persons include the latter: IgA, IgD, IgE, IgG and IgM, and various subclasses thereof, such as IgA1, IgA2 and IgG1, IgG2, IgG3 and IgG4. In this regard, humanized and chimeric antibodies are also included. Of particular therapeutic importance, the subject of the present invention is therefore a composition comprising a antibody against various surface antigens, eg CD4, CD20 or CD44, or various cytokines such as IL2, IL4 or IL5, preferably Powdered compositions, for example dry sprayed powders, are of particular therapeutic interest and thus form the subject of the present invention. Other examples include antibodies to specific antibody classes (eg anti-IgE antibodies) or viral proteins (eg anti-RSV, anti-CMV antibodies, etc.).

Antigen-binding or Fab fragments consist of variant regions of both chains that are held together by adjacent constant regions. Another antibody fragment is an F (ab ') ₂ fragment that can be prepared by proteolysis with pepsin. In addition, gene cloning can be used to prepare shortened antibody fragments, which consist of only the variant regions of the heavy (VH) and light (VL) chains. These are described as Fv fragments (fragment variation = fragment of the variant portion). Such antibody fragments are also described as single chain Fv fragments (scFv). Examples of scFv antibodies are known and described in Huston et al, 1988, Proc. Natl. Acad. Sci. USA, 16, 5879ff.

In the past, different methods have been developed, for example, to prepare multiple bond scFv derivatives such as dimers, trimers and pentomers. Divalent homodimeric scFv derivatives are referred to as "dimers" to those skilled in the art. If the peptide linker in the scFv molecule is shortened to 5 to 10 amino acids, homodimers are formed by overlapping the VH / VL chains. Dimers can also be stabilized through the introduction of disulfide bridges. Examples of dimers are described in Perisic et al., 1994 (Structure, 2, 1217ff) Bivalent identifying dimeric scFv derivatives are referred to as "minibodies" by one skilled in the art. As a dimeric region, it is a fusion protein containing the CH ₃ region of the antibody, preferably IgG, particularly preferably the CH ₃ region of IgG1, which connects the scFv fragments of the IgG and linker regions through the hinge region. Examples are described in Hu et al., 1996, Cancer Res., 56, 3055ff. Those skilled in the art refer to trivalent co-trimeric scFv derivatives as “tribodies”. Kortt et al., 1997, Protein Engineering, 10, 423ff.] Direct fusion of VH-VL without the use of linker sequences results in the formation of trimers.

In addition, fragments that have a divalent, trivalent or tetravalent structure, called mini-antibodies to those skilled in the art, are derivatives of the scFv fragment. The multimerization can then be carried out via a dimer, trimer or tetramer "twisted coil" structure (Pack, P. et al., 1993, Biotechnology, 11, 1271ff; Lovejoy, B. et al., 1993, Science, 259, 1288ff, Pack, P. et al., 1995, J. Mol. Biol., 246, 28ff.

Particularly preferred embodiments of the invention relate to proteins from the antibody class, more precisely to type 1 antibody G. The antibody is a humanized monoclonal antibody with 95% human and 5% murine antibodies. The antibody has a molecular weight of about 148 kDa and consists of two light chains and two heavy chains and a total of four disulfide bridges.

According to a further aspect, the present invention provides that the dry weight of the corresponding composition is at least 50% (w / w), preferably 50 to 99.99% (w / w), particularly preferably 60 to 90% (w / per sugar) w) and a composition, preferably a powdered composition, such as a spray dried powder, characterized in that it contains at least 40% (w / w) of the antibody or antibody derivative as a pharmaceutically active substance. , The fraction of lactosucrose, maltosyl sucrose and / or glucosyl sucrose is at least 20% (w / w) relative to the dry weight of the composition according to the invention and the sum of the weight percentages is at most 100% (w / corresponds to w). One skilled in the art can also prepare corresponding compositions, preferably powdered compositions, such as spray dried powders. Thus, a person skilled in the art, with respect to the total solid contents of the composition according to the invention, sugar mixtures containing 1,4 O-linked saccharose derivatives or at least one 1,4 O-linked saccharose derivatives It is understood that up to 10% (w / w) of the antibody or antibody derivative can be mixed when the fraction of must be 90%.

Furthermore, the compositions according to the invention, in particular powdered compositions, for example spray dried powders, may contain other auxiliaries such as amino acids, peptides, non-biological or biological polymers and / or one or more sugars. Other conventional high-end aids are, for example, lipids, fatty acids, esters of fatty acids, steroids (such as cholesterol) or chelate binders (such as EDTA) and also various cations (see above). Higher glass transition temperatures, for example, above 40 ° C., preferably above 45 ° C., more preferably above 50 ° C., or preferably 55 when preparing powdered compositions, eg, spray dried powders Adjuvants above < RTI ID = 0.0 > A list of suitable adjuvants is described, for example, in Kippe (Eds.), "Handbook of Pharmaceutical Excipients" 3rd Ed., 2000.

Suitable protein containing adjuvants are, for example, albumin (of human or recombinant origin), gelatin, casein, hemoglobin and the like. The sugars are preferably monosaccharides, disaccharides, oligosaccharides or polysaccharides or combinations thereof. Examples of monosaccharides include fructose, maltose, galactose, glucose, D-mannose, sorbose and the like. Suitable disaccharides in view of the present invention include lactose, saccharose, trehalose, cellobiose and the like. In particular raffinose, melezitose, dextrin, starch and the like may be suitable as complex sugars or oligosaccharides and polysaccharides. Among the sugar alcohols, mannitol, xylitol, maltitol, galactitol, arabinitol, adonitol, lactitol, sorbitol (glucitol), pyranosylsorbitol, inositol, myoinositol and the like are considered as suitable auxiliaries. Suitable amino acids are, for example, alanine, glycine, arginine, histidine, glutamate, asparagine, cysteine, leucine, lysine, isoleucine, valine, tryptophan, methionine, phenylalanine, tyrosine, citrulline, L-aspartyl-L-phenylalanine methyl Esters (= aspartame), trimethylammonioacetate (= betaine) and the like. Amino acids acting as buffers (eg glycine or histidine) and / or dispersants are preferably used. The last mentioned group mainly comprises hydrophobic amino acids such as leucine, valine, isoleucine, tryptophan, alanine, methionine, phenylalanine, tyrosine, histidine or proline.

Within the scope of the present invention, in particular when using amino acids, preferably isoleucine or citrulline, sugar mixtures containing 1,4 O-linked saccharose derivatives or at least one 1,4 O-linked saccharose derivatives are preferred. Preferably from 1 to 19.99% (w / w), particularly preferably from 5 to 19.99% (w / w), particularly preferably from 10 to 19.99% (w / w), even more preferably from 12 to 19.99% ( It has been found advantageous to use together at a concentration of w / w). Such fractions may be obtained by reducing the fraction of the sugar mixture or the fraction of the antibody or antibody derivative containing 1,4 O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives, so that As long as the solids content does not exceed 100% (w / w), it can be increased to a level of 40% (w / w).

Dipeptides, tripeptides, oligopeptides or polypeptides containing one or more of these mainly hydrophobic amino acid groups are particularly advantageous as other adjuvants. 20 or less amino acids, more preferably 15 or less, even more preferably 12 or less, even more preferably 11 or less, even more preferably 10 or less, even more preferably 9 or less Preference is given to peptides that are even more preferably 8 or less, even more preferably 7 or less, and even more preferably 7, 6, 5, 4 or 3 or less. The peptide used for stabilization then does not correspond simultaneously with the pharmaceutically active substance.

Suitable examples of tripeptides are, for example, one or more hypovalent tripeptides: Leu-Leu-Gly, Leu-Leu-Ala, Leu-Leu-Val, Leu-Leu-Leu, Leu-Leu-Met, Leu Leu-Pro, Leu-Leu-Phe, Leu-Leu-Trp, Leu-Leu-Ser, Leu-Leu-Thr, Leu-Leu-Cys, Leu-Leu-Tyr, Leu-Leu-Asp, Leu-Leu -Glu, Leu-Leu-Lys, Leu-Leu-Arg, Leu-Leu-His, Leu-Gly-Leu, Leu-Ala-Leu, Leu-Val-Leu, Leu-Met-Leu, Leu-Pro-Leu , Leu-Phe-Leu, Leu-Trp-Leu, Leu-Ser-Leu, Leu-Thr-Leu, Leu-Cys-Leu, Leu-Try-Leu, Leu-Asp-Leu, Leu-Glu-Leu, Leu -Lys-Leu, Leu-Arg-Leu and Leu-His-Leu. Formula Ile-XX; X-Ile-X; Tripeptides of XX-Ile, wherein X may be one of the following amino acids: alanine, glycine, arginine, histidine, glutamic acid, glutamine, asparagine, asparagine, cysteine, leucine, lysine, isoleucine (Ile), valine, It has been found to be particularly advantageous to use tryptophan, methionine, phenylalanine, proline, serine, threonine, tyrosine, L-aspartyl-L-phenylalanine-methylester (= aspartame), trimethylammonio-acetate. Corresponding tripeptides of formula (Ile) ₂ -X, for example Ile-I0le-X, Ile-X-IIe or X-IIe-Ile, wherein X can be one of the amino acids described above, in particular desirable. For example, the following tripeptides include: Ile-Ile-Gly, Ile-Ile-Ala, Ile-Ile-Val, Ile-Ile-Ile, Ile-Ile-Met, Ile-Ile-Pro, Ile-Ile-Phe, Ile-Ile-Trp, Ile-Ile-Ser, Ile-Ile-Thr, Ile-Ile-Cys, Ile-Ile-Tyr, Ile-Ile-Asp, Ile-Ile-Glu, Ile- Ile-Lys, Ile-Ile-Arg, Ile-Ile-His, Ile-Gly-Ile, Ile-Ala-Ile, Ile-Val-Ile, Ile-Met-Ile, Ile-Pro-Ile, Ile-Phe- Ile, Ile-Trp-Ile, Ile-Ser-Ile, Ile-Thr-Ile, Ile-Cys-Ile, Ile-Try-lie, Ile-Asp-lie, Ile-Glu-Ile, Ile-Lys-Ile, Ile-Arg-Ile, Ile-His-Ile. Particular preference is given to using Ile-Ile-Ile.

Suitable polymers include, for example, those mentioned above as auxiliaries, polyvinylpyrrolidone, derivatized celluloses such as hydroxymethyl, hydroxyethyl or hydroxypropyl ethylcellulose, polymer sugars, for example , Piscol, starch, for example hydroxyethyl- or hydroxypropyl starch, dextrin, for example cyclodextrin (2-hydroxypropyl-β-cyclodextrin, sulfobutylether-β-cyclodextrin), Polyethylene, glycol and / or pentine.

Salts are, for example, inorganic salts, such as chlorides, sulfates, phosphates, diphosphates, hydrobromide and / or nitrates. In addition, the powders according to the invention can be prepared using organic salts, for example maleates, maleates, fumarates, tartrates, succinates, ethyl succinates, citrates, acetates, lactates, methanesulfonates, benzoates , Salts of ascorbate, paratoluenesulfonate, palmoate, salicylate, stearate, estoleate, glutceptate or lactobionate. At the same time, the corresponding salts may contain pharmaceutically acceptable cations such as sodium, potassium, calcium, aluminum, lithium or ammonium. It has been found to be particularly suitable to use the corresponding cations during stabilization of the antibody or antibody derivative.

Accordingly, the present invention also relates to sugar mixtures containing 1,4 O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives and one antibody or one antibody derivative as a pharmaceutically active substance. A composition, preferably a powdered composition, such as a spray dried powder, containing one or more pharmaceutically acceptable adjuvants and / or one or more salts. Adjuvants may be, for example, amino acids, peptides and salts thereof, sugars, polyols, salts of amino acids, and / or the polymers described above.

According to a further aspect, the invention provides a mixture of sugars containing 1,4 O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives and one or one antibody as a pharmaceutically active substance. A composition, preferably a powdered composition, such as a spray dried powder, containing one or more amino acid (s), preferably one amino acid, as an additional adjuvant, together with a derivative. In this regard, the present invention also relates to 25% of sugar mixtures (a) containing 1,4 O-linked saccharose derivatives or at least one 1,4 O-linked saccharose derivatives, on their dry weight ( w / w) or more, preferably 50 to 90% (w / w), particularly preferably 60 to 90% (w / w) and amino acids (b) 1 to 19.99% (w / w) and pharmaceutical activity A composition, preferably a powdered composition, eg, containing an antibody or antibody derivative (c) from 0.01 to 74% (w / w), wherein the sum of the total fractions is 100% (w / w) For example, it relates to a spray dried powder. In a preferred embodiment, a fraction of the sugar mixture containing 1,4 O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives is 60% (w / w) relative to the dry weight of the corresponding composition. ), Preferably 70 to 90% (w / w). In the corresponding formulations, the fraction of amino acids is preferably 1 to 19.99% (w / w) and the fraction of the antibody or antibody derivative is 0.01 to 39% (w / w).

In addition, according to a further aspect of the invention, for example, 80% (w / w) sugar mixtures containing 1,4 O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives Compositions containing 1% (w / w) / antibody or antibody derivative 1% (w / w) (80/19/1), preferably powdered compositions, such as spray dried powders, For example (80/18/2); (80/17/13); (80/16/4); (80/15/5); (80/14/6); (80/13/7); (80/12/8); (80/11/9); (80/10/10); (70/20/10); (70/19/11); (70/18/12); (70/17/13); (70/16/14); (70/15/15); (70/14/16); (70/13/17); (70/12/18); (70/11/19); (70/10/20); (60/20/20); (60/19/21); (60/18/22); (60/17/23); (60/16/24); (60/15/25); (60/14/26); (60/13/27); (60/12/28); It relates to a powder containing or consisting of (60/11/29) or (60/10/30). The fraction of the antibody or antibody derivative is 20% (w / w) up to 0.01% (w / w), for example 9.99,. . . 9.9, 9.8, 9.7. . . 9.3, 9.2, 9.1. . . 9, 8 7, 6, 5, 4, 3, 2, 1,. . . 0.9, 0.8, 0.7,. . . 1,4 O-linked saccharose as long as it is reduced to 0.66, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03 0.02, 0.01% (w / w) The fraction of the sugar mixture containing the derivative or one or more 1,4 O-linked saccharose derivatives is such that the sum of the weight fractions of each component of the composition is 100% (w / w) relative to the dry weight of the composition, eg For example, 80.01,. . . 80.1, 80.2, 80,3. . . 80.8, 80.9, 81, 82, 83, 84, 85, 86, 87, 88, 89,. . . , 89.1, 89.2, 89.3,. . . 89.33,. . . 89.4, 89.5, 89.6, 89.7, 89.8, 89.9,. . . 89.91, 89.92, 89.93,. . . 89.97, 89.98, 89.99% (w / w). By addition of other adjuvants or salts, the fraction of sugar mixtures, amino acids / peptides and / or antibodies or antibody derivatives containing 1,4 O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives The corresponding weight can be adjusted / reduced such that the total weight fraction of each component is 100% (w / w).

Thus, if the added amino acid is isoleucine, according to a further embodiment, a fraction of the sugar mixture (a) containing a 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative This is at least 25% (w / w), preferably 50 to 90% (w / w), particularly preferably 60 to 90% (w / w), and the fraction of isoleucine (b) is 1 to 19.99%. (w / w), wherein the fraction of the antibody or antibody derivative, preferably the peptide / protein (c), as a pharmaceutically active substance is at least 0.01% (w / w), preferably 0.01 to at most 74% (w / w) ), Preferably powdered compositions, such as spray dried powders, are in accordance with the present invention. Preferably the fraction of isoleucine is from 5 to 19.99% (w / w), more preferably from 10 to 19.99% (w / w) of the total solid fraction of the composition according to the invention. In addition, the total weight percentage of each component is at most 100% (w / w). In addition, according to the invention, compositions having the following composition, preferably powdered compositions, for example spray dried powders, conform to the invention: 1,4 O-linked saccharose derivatives or at least one , 80% (w / w) / amino acid or peptide 10% (w / w) / antibody or antibody derivative 10% (w / w) (80/10/10, sugar mixture containing 4 O-linked saccharose derivatives) ); (79/11/10); (78/12/10); (77/13/10); (76/14/10); (75/15/10); (74/16/10); (73/17/10); (72/18/10); (71/19/10); (70/20/10).

Wherein the fraction of antibody or antibody derivative is also 10 to 0.01% (w / w), for example 9.99,. . . 9.9, 9.8, 9.7. . . 9.3, 9.2, 9.1. . . 9, 8, 7, 6, 5, 4, 3, 2, 1,. . . 0.9, 0.8, 0.7,. . . 0.66,. . . 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03 0.02, 0.01% (w / w) and can thus be reduced to 1,4 O-linked saccharose The fraction of the sugar mixture containing the derivative or 1,4 O-linked saccharose derivative is such that the total weight fraction of each component of the composition is up to 100% (w / w) relative to the dry weight of the corresponding composition, For example, 80.01 ,. . . 80.1, 80.2, 80.3. . . 80.8, 80.9, 81, 82, 83, 84, 85, 86, 87, 88, 89,. . . , 89.1, 89.2, 89.3,. . . 89.33,. . . , 89.4, 89.5, 89.6, 89.7, 89.8, 89.9,. . . 89.91, 89.92, 89.93,. . . , 89.97, 89.98, 89.99% (w / w). Thus, compositions having the following composition, preferably powdered compositions, such as spray dried powders, are according to the invention: 1,4 O-linked saccharose derivatives or at least one 1,4 O-linked Sugar mixtures containing saccharose derivatives 80% (w / w) / isoleucine 19% (w / w) / antibody or antibody derivative 1% (w / w) (80/19/1); (80/18/2); (80/17/3); (80/16/4); (80/15/5); (80/14/6); (80/13/7); (80/12/8); (80/11/9); (80/10/10); (70/19/11); (70/18/12); (70/17/13) (70/16/14); (70/15/15); (70/14/16); (70/13/17); (70/12/18); (70/11/19); (70/10/20); (60/19/21); (60/18/22); (60/17/23) (60/16/24); (60/15/25); (60/14/26); (60/13/27); (60/12/28); (60/11/29); (60/10/30). By adding other adjuvants or salts, the sugar mixture, isoleucine and / or the fraction of the antibody or antibody derivative containing 1,4 O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives, Corresponding adjustments can be made such that the total weight fraction of each component is at most 100% (w / w).

A further aspect of the invention relates to stabilizing a composition, preferably a powdered composition, such as a spray dried powder, containing the antibody or antibody derivative, preferably in the form of a peptide, protein or mixture thereof. The use of sugar mixtures and peptides, preferably dipeptides, or tripeptides containing at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative. This patent specification discloses 1,4 O-linked saccharose derivatives or one or more 1,4 O-linkeds for producing a composition according to the invention, preferably a powdered composition, for example spray dried powder. Several tripeptides can be used that can be used with sugar mixtures containing the derived saccharose derivatives. According to a particular embodiment, the peptides, preferably dipeptides or tripeptides, contain at least one isoleucine, preferably two isoleucines, or in particular advantageous embodiments are those consisting of three isoleucines.

In this regard, the fraction of the sugar mixture (a) containing at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative is at least 25% (w / w), preferably Preferably from 60 to 99% (w / w), particularly preferably from 80 to 90% (w / w) and the fraction of the tripeptide, preferably the dipeptide or tripeptide (b) is from 1 to 19.99% (w). / w), and compositions, powdered compositions, such as spray dried powders, wherein the fraction of the antibody or antibody derivative (c) is between 0.01 and up to 74% (w / w) is considered to be in accordance with the present invention. In addition, the sum of each solid may not exceed 100% (w / w). In addition, compositions having the following composition, preferably powdered compositions, such as spray dried powder, are according to the invention: one or more 1,4 O-linked saccharose derivatives or one or more 1,4 Sugar mixture containing O-linked saccharose derivatives 89% (w / w) / peptide, preferably dipeptide or tripeptide, particularly preferably isoleucine containing tripeptide, even more preferably triisoleucine 1 % (w / w) / antibody or antibody derivative 10% (w / w) (89/1/10); (88/2/10); (87/3/10); (86/4/10); (85/5/10); (84/6/10); (83/7/10); (82/8/10); (81/9/10); (80/10/10); (79/11/10), (78/12/10); (77/13/10), (76/14/10); (75/15/10), (74/16/10); (73/17/10); (72/18/10) or (71/19/10). Wherein the fraction of antibody or antibody derivative is 10 to 0.01% (w / w), for example 9.99,. . . 9.9, 9.8, 9.7. . . 9.3, 9.2, 9.1. . . 9, 8, 7, 6, 5, 4, 3, 2, 1,. . . 0.9, 0.8, 0.7,. . . 0.66,. . . 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03 0.02, 0.01% (w / w) and can thus be reduced to 1,4 O-linked saccharose The fraction of the sugar mixture containing the derivative or one or more 1,4 O-linked saccharose derivatives is such that the total weight fraction of each component of the composition is up to 100% (w / w) relative to the dry weight of the composition, For example, 80.01,. . . 80.1, 80.2, 80.3. . . 80.8, 80.9, 81, 82, 83, 84, 85, 86, 87, 88, 89,. . . 89.1, 89.2, 89.3,. . . 89.33,. . . 89.4, 89.5, 89.6, 89.7, 89.8, 89.9,. . . 89.91, 89.92, 89.93,. . . 89.97, 89.98, 89.99% (w / w). Thus, according to the invention of a composition having a composition, preferably a powdered composition, for example spray dried powders: at least one 1,4 O-linked saccharose derivative or at least one O-linked saccharose Sugar mixtures containing derivatives 80% (w / w) / peptides, preferably dipeptides or tripeptides, particularly preferably isoleucine containing peptides, even more preferably triisoleucine 19% (w / w) / 1% (w / w) antibody or antibody conductor material (80/19/1); (80/18/2); (80/17/3); (80/16/4); (80/15/5); (80/14/6); (80/13/7); (80/12/8); (80/11/9); (80/10/10); (70/19/11); (70/18/12); (70/17/13); (70/16/14); (70/15/15); (70/14/16); (70/13/7); (70/12/18); (70/11/19); (70/10/20); (60/20/20); (60/19/21); (60/18/22); (60/17/23); (60/16/24); (60/15/25); (60/14/26); (60/13/27); (60/12/28); (60/11/29); (60/10/30). Wherein the amount of peptide (diisoleucine, triisoleucine, isoleucine or triisoleucine containing peptide, tripeptide or dipeptide) is also in the range of 10 to 1% (w / w), for example 9.99,. . . 9.9, 9.8, 9.7. . . 9.3, 9.2, 9.1. . . 9, 8, 7, 6, 5, 4, 3, 2, 1.9, 1.8, 1.7,. . . 1.66,. . . Can be reduced to 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1% (w / w), so that the amount of antibody or antibody derivative is equal to the total weight fraction of each component of the composition relative to the dry weight of the composition. To be at most 100% (w / w), for example, 30.1, 30.2, 30.3. . . 30.8, 30.9, 31, 32, 33, 34, 35, 36, 37, 38, 38.1, 38.2, 38.3,. . . 38.33,. . . , 38.4, 38.5, 38.6, 38.738.8, 38.9,. . . Can increase to 39% (w / w). If the fraction of peptides (diisoleucine, triisoleucine, isoleucine or triisoleucine containing peptides, tripeptides or dipeptides) is reduced to 10 to 1 (w / w) as described herein, 1,4 The fraction of sugar mixtures containing O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives can also be increased in the powder. In a constant active substance fraction of 10% (w / w) of the antibody or antibody derivative, for example, sugars containing 1,4 O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives The fraction of the mixture is 80.1, 80.2, 80.3. . . 80.8, 80.9, 81, 82, 83, 84, 85, 86, 87, 88, 88.1, 88.2, 88.3,. . . 88.33,. . . , 88.4, 88.5, 88.6, 88.7, 88.8, 88.9 or 89% (w / w) can be prepared.

Fraction 60 to 80% (w / w) of a sugar mixture (a) containing at least one 1,4 O-linked saccharose derivative or at least one 1,4-linked saccharose derivative, peptide, preferably di Peptide or tripeptide, particularly preferably isoleucine containing peptide, more preferably tripeptide, preferably fraction of 10 to 19.99% (w / w) of triisoleucine (b), and antibody or antibody derivative (c) Compositions having a fraction of 0.01 to at most 30% (w / w), preferably powdered compositions, for example spray dried powders, are compositions which have been found to be particularly preferred. In addition, the sum of each solid does not exceed 100% (w / w).

In addition, according to a further aspect according to the invention, the composition, preferably the powdered composition, for example spray dried powder, may contain a surface active material such as Tween 20, 40, 60, 80, bri It may further contain Brij 35, Pluronic F 88 and Pluronic F 127. These are preferably used at concentrations of 0.01 to 0.1% (w / w). 0.01 to 0.1% (w / w) containing a sugar mixture containing at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative Particular preference is given to compositions containing at a concentration of, preferably powdered compositions, for example spray dried powders.

According to a further embodiment, the particles in the powdered composition according to the invention have an MMD of 1 to 10 μm, preferably 1 to 5 μm.

According to a further aspect, the present invention provides a sugar mixture (a) and one antibody or one antibody containing at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative A composition containing a derivative, preferably a powdered composition, such as a spray dried powder, is characterized in that the glass transition temperature exceeds 40 ° C. In general, corresponding powders according to the invention have a maximum glass transition temperature of about 96 to 110 ° C. However, in each case the value can be much higher.

In addition, the present invention relates to pharmaceutical compositions containing one or more compositions described herein.

Preparation of the composition according to the invention

The composition according to the invention comprises a sugar mixture (a) and one antibody or one antibody derivative (b) containing at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative ), A liquid, semisolid, or solid composition. Particular preference is given to solid powdered compositions, for example compositions formed by freeze drying or spray drying of aqueous solutions or suspensions. Aqueous solutions or suspensions can be obtained by mixing or suspending the corresponding components (eg 1,4 O-linked saccharose derivatives and antibodies) in a suitable solvent. In addition, each substance can be added to the solvent as a dry substance and dissolved or suspended in it. Semi-solid formulations are also suitable. Semi-solid preparations consist of simple or complex bases and, according to the invention, sugar mixtures (a) and antibodies or containing 1,4 O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives or The antibody derivative (b) is dissolved or dispersed. Sugar mixtures and antibody or antibody derivatives containing 1,4 O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives, individually or in the powders prepared by a suitable process described above are described above. As can be added to the base. The formulations may further contain suitable adjuvants, such as preservatives, antioxidants, stabilizers, emulsifiers, thickeners, and penetration enhancers, or other suitable adjuvants. Suitable semisolid bases include, but are not limited to, hydrophobic ointments, absorbent ointments, hydrophilic ointments, lipophilic creams, hydrophilic creams, lipophilic gels, hydrophilic gels, pastes, and enrobing pastes.

Particular preference is given, for example, to solid powdered compositions formed through drying of the previous aqueous solution or suspension. Known drying processes include, among other things, freeze drying, spray drying, vacuum drying, infrared drying, or ultrasonic drying. General descriptions of suitable processes are described, for example, in Willser, Dissertation, 2000, Dr., the contents of which are incorporated herein by reference. Hut Verlag, Munich, Germamy-ISBN 3-89963-027-0). A sugar mixture (a) and one antibody or antibody derivative containing one or more 1,4 O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives, prepared by spray drying or freeze drying ( It has been found that the powdered composition consisting of b) is particularly stable.

Preparation of the dried powder according to the invention

The present invention also provides a process for the preparation of the dried powder, preferably spray dried powder, described in more detail above. Spray drying processes include, for example, an antibody or antibody derivative (a) as a pharmaceutically active substance, and one or more 1,4 O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives. The solution / suspension to be sprayed containing the sugar mixture (b) is below 200/120 ° C. (inlet / outlet temperature), preferably below 186/96 ° C., preferably 186/96 ° C. to 60/40 ° C., for example For example, it is characterized by spraying at 180 to 150/95 to 80 ℃. The method according to the invention is described in more detail on the basis of many embodiments in the Examples section.

Powders according to the invention can be prepared by dissolving the antibody or antibody derivative in an aqueous solution basically depending on the solubility conditions of the antibody or antibody derivative. Buffer solutions with a pH of 3 to 11, preferably 3.5 to 9 are mainly used. During the preparation of inhalable powders, aqueous solutions with a pH of 4 to 7.8 are particularly advantageous. To ensure proper solubility, the pH value of the solution should be less than the pH value of the antibody / antibody derivative. The aqueous solution may optionally contain further water soluble organic solvents such as acetone, alcohols and the like. For example, lower alcohols such as methanol, ethanol or propanol (n-propanol or iso-propanol) are particularly suitable. Such mixed solvent systems generally contain 10-20% (v / v) of water soluble organic solvents. The solid fraction in the solution to be dried generally corresponds to 0.01 to 20% (w / w), preferably 0.05 to 10% (w / w), particularly preferably 0.1 to 5% (w / w). Within the scope of the present invention, spray dried powders are prepared based on an aqueous solution with a solid fraction of 10% (w / w), 3.33% (w / w), or 2.00% (w / w), and lyophilized. Powders are prepared based on an aqueous solution with a solid fraction of 10% (w / w).

For example, the adjuvant or mixture of suitable adjuvant as described above is generally placed in a second container in a suitable buffer solution having an ultrapure water or pH value of 3 to 11, preferably 3.5 to 9, particularly preferably 4.0 to 7.8. It is dissolved and mixed in a second step with a solution of the active substance containing the antibody or antibody derivative. The solution or suspension is then adjusted to the required solids content with a suitable buffer solution having an ultrapure water or pH value of 3 to 11, preferably 3.5 to 9, particularly preferably 4.0 to 7.8.

As a result, the present invention

Dissolving the antibody or antibody derivative in an aqueous solution / suspension (a),

Sugar mixtures containing one or more 1,4 O-linked saccharose derivatives selected from lactosucrose, glucosyl sucrose, or maltosyl sucrose compounds or one or more 1,4 O-linked saccharose derivatives described above Dissolving / suspending in aqueous solution / suspension (b),

A sugar mixture containing an antibody or antibody derivative and a 1,4 O-linked saccharose derivative or one or more 1,4 O-linked saccharose derivatives is dissolved / suspended in different solutions / suspensions and mixed Step (c) and

And (d) drying the solution / suspension containing at least one 1,4 O-linked saccharose derivative (s) and the antibody or antibody derivative.

When the drying process is spray drying, drying under step (d) is carried out by spraying the corresponding solution / suspension at below 200/120 ° C. (inlet / outlet temperature), preferably 60/40 ° C. to 1 86/96 ° C. do.

In addition, the 1,4 O-linked saccharose derivatives may form part of a sugar mixture containing one or more 1,4 O-linked saccharose derivatives. Examples of correspondingly suitable sugar mixtures are described in more detail in, for example, "definitions". The sugar mixture may then further contain one or more monosaccharides, disaccharides and / or polysaccharides, together with 1,4 O-linked saccharose derivatives, in particular adding monosaccharides and / or disaccharides during powder preparation. It is preferable to use as. Thus, within the scope of the present invention, sugar mixtures may be used, for example, with lactosucrose, lactose, and saccharose, with a fraction of lactosucrose relative to the total sugar fraction of the composition being> 40% (w / w), preferably> 55% (w / w), and also> 88% (w / w). The sugar mixture is a sugar mixture, preferably referred to as Nyuka-Oligo ^® LS55P (abbreviated to LS55P) of Hayashibara Shoji Kabushiki Kaisha, Japan, which is at least 55% lactosucrose and up to 25% lactose (w) / w), containing up to 10% saccharose (w / w). According to a further preferred embodiment, the sugar mixture is a sugar mixture, preferably referred to as Nyuka-Oligo ^® LS9OP (abbreviated as LS9OP) of Hayashibara Shoji Kabushiki Kaisha, Japan, which has 88% or more of lactosucrose, It contains up to 10% (w / w) lactose and saccharose. A sugar mixture may be further used in combination with glucosyl and maltosyl sucrose, again preferably in combination with other monosaccharides, disaccharides and / or polysaccharides. Suitable sugar molecules in view of the invention consequently consist of glucosyl and maltosyl sucrose, saccharose, glucose, and / or fructose, the fraction of glucosyl and maltosyl sucrose relative to the total sugar fraction of the composition Preferably it corresponds to 25% (w / w) or more. According to a further preferred embodiment, each of the glucosyl sucrose fraction and maltosyl sucrose fraction corresponds to at least 18% (w / w) total sugar fraction. According to another preferred embodiment, the sugar mixture used is Coupling Sugar ^® of Hayashbara Shoji Kabushiki Kaisha, Japan, which has at least 18% (w / w) of glucosyl and maltosyl sucrose, saccharose, respectively. 11 to 15% (w / w) and 5 to 9% (w / w) of glucose and fructose, respectively. Also in view of the present invention, a sugar mixture described by Coupling Sugar S ^® of Hayashibara Shoji Kabushiki Kaisha, Japan, is suitable, which is at least 25% (w / w) of glucosyl and / or maltosyl sucrose , Saccharose 48-56% (w / w), and glucose and fructose less than 10% (w / w).

The adjuvant content of sugar mixtures containing 1,4 O-linked saccharose derivatives or one or more 1,4 O-linked saccharose derivatives in a solution / suspension to be dried is 25% relative to the solids content of the solution or suspension to be dried. To 99.99% (w / w), preferably 60% to 99% (w / w), particularly preferably 60% to 90% (w / w). The active substance concentration of the antibody or antibody derivative is generally from 0.01 to 75% (w / w), preferably from 0.01 to 40% (w / w), particularly preferably from 0.01 to the solids content of the solution or suspension to be dried. To 30% (w / w). Based on the powder compositions according to the invention described above, the skilled person can prepare solutions or suspensions to be dried and produce the corresponding powder compositions after drying, in particular after spraying.

In addition, the present invention contemplates sugar mixtures having a solids content of the solution / suspension to be dried which contains at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative 25 to 99.99 A process for the preparation of dried powders as described above, characterized in that it contains% (w / w), preferably 60 to 99% (w / w). According to a further preferred embodiment, the present invention provides that the solid fraction of the solution or suspension to be dried comprises 0.01 to 75% (w / w), preferably 0.01 to 30% (w / w) of the antibody or antibody derivative as a pharmaceutically active substance. , Particularly preferably from 0.33 to 30% (w / w).

According to a further aspect, at least 25% (w / w) sugar mixture (a) containing at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative, eg For example, in a solids content of 25 to 99.99% (w / w), and at least 0.01% (w / w), preferably 0.01 to 75% (w / w), of the antibody or antibody derivative (b) as a pharmaceutically active substance. The solution or suspension to be dried is prepared and dried, preferably sprayed, and the sum of the weight percents corresponds to at most 100% (w / w). In a preferred embodiment, at least 60% (w / w) sugar mixture (a) containing at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative Prepare a solution / suspension to be dried to a solids content with 60 to 90% (w / w), and 0.01 or 40% (w / w) of the antibody or antibody derivative (b), dry, preferably spray, and solution Or the sum of the weight percentages of the suspension corresponds to a maximum of 100% (w / w) relative to the solids content of the spray solution / spray suspension.

According to the powder according to the invention as described above, according to a further aspect the solution / suspension to be dried further contains at least one pharmaceutically compatible adjuvant and / or at least one salt. Adjuvants are mainly amino acids, peptides or salts thereof, sugars, polyols, salts of organic acids, and / or polymers.

Together with one antibody or antibody derivative (a) as a pharmaceutically active substance and a sugar mixture (b) containing at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative, The solution or suspension to be dried preferably contains one or more amino acids and / or peptides or protein (c) as other auxiliaries. In addition, the present invention contemplates that a solution or suspension to be dried as a sugar mixture, containing at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative, for its solids content (a) at least 25% (w / w), preferably at least 60 to 90% (w / w), at least one amino acid and / or at least one peptide (b) 1 to 19.99% (w / w), and the antibody Or 0.01 to 74% (w / w) of the antibody derivative (c). Examples of suitable adjuvants comprising pharmaceutically compatible salts, peptides, and amino acids are described herein under "powders according to the invention". One skilled in the art can produce the corresponding powder and adjust the weight fraction such that the sum of the solid fractions corresponds to up to 100% (w / w). A fraction of the antibody or antibody derivative (relative to total solids content), for example, corresponds to 10% (w / w) and at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-binding When the fraction of the sugar mixture containing the extracted saccharose derivatives corresponds to 80% (w / w), the skilled artisan can add up to 10% (w / w) of amino acids to the solution / suspension to be dried. You will be able to recognize it.

According to a further preferred embodiment, the solution or suspension to be dried, together with a sugar mixture containing at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative, adds isoleucine It is further contained as an adjuvant. Solutions or suspensions considered to be advantageous include: 25% (w / w) sugar mixture (a) in which the solid fraction contains at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative ), Preferably at least 60 to 90% (w / w), isoleucine (b) 1 to 19.99% (w / w), and 0.01% (w / w) of the antibody or antibody derivative (c) as a pharmaceutically active substance It contains more than). The fraction of the antibody or antibody derivative is then preferably from 0.01 to at most 74% (w / w) and the sum of the solid fractions corresponds to at most 100% (w / w). One skilled in the art can preferably produce the corresponding powder by spray drying and adjust the weight fraction such that the sum of the solid fractions corresponds to at most 100% (w / w). A fraction of the antibody or antibody derivative (relative to total solids content), for example, corresponds to 10% (w / w) and at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-binding When the fraction of the sugar mixture containing the saccharose derivative is equivalent to 80% (w / w), the skilled person can add up to 10% (w / w) of isoleucine to the solution / suspension to be dried. You will recognize that.

In a further embodiment, the solution or suspension to be dried, together with a sugar mixture containing at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative, comprises at least one tripeptide, Especially preferably, it contains triisoleucine. Solutions or suspensions considered to be advantageous include: 25% (w / w) sugar mixture (a) in which the solid fraction contains at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative ), Preferably 60 to 98.99% (w / w), particularly preferably 60 to 90% (w / w), peptides, preferably dipeptides or tripeptides, particularly preferably isoleucine containing peptides, More preferably 1 to 19.99% (w / w) of triisoleucine (b), and 0.01% (w / w) or more of the antibody or antibody derivative (c) as a pharmaceutically active substance, and the sum of the solid fractions Corresponds to the maximum of 100% (w / w). The fraction of the antibody or antibody derivative then preferably corresponds to 0.01 up to 74% (w / w). Those skilled in the art can produce the corresponding powders and adjust the weight fractions so that the sum of the solid fractions corresponds to at most 100% (w / w). A fraction of the antibody or antibody derivative (relative to total solids content), for example, corresponds to 10% (w / w) and at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-binding When the fraction of the sugar mixture containing the modified saccharose derivatives corresponds to 80% (w / w), the person skilled in the art will know that up to 10% (w / w) peptides, preferably dipeptides or tripeptides, are particularly preferred. It will be appreciated that the isoleucine containing peptide, more preferably triisoleucine, can be added to the solution / suspension to be dried, preferably to be sprayed.

As already mentioned, it is advantageous to prepare and spray a solution to be dried, preferably to be sprayed, having a pH value of 3 to 11, preferably 3.5 to 9, particularly preferably 4.0 to 7.8. Suitable buffer systems are known to those skilled in the art. The use of inorganic salts or organic salt buffer systems has generally been found to be particularly advantageous.

Optimal adjuvant and protein content for each antibody or antibody derivative is generally determined experimentally. In addition, preferred formulations of the present invention may contain additional auxiliaries to improve powder properties such as dispersibility and flowability while retaining good aggregation inhibition.

Spray drying

Spray drying is carried out with conventional spray dryers, for example by instruments manufactured by Niro A / S (Soeborg, DK), Buchi Labortechnik GmbH (Flawil, CH) and the like. Perform. Optimum conditions for spray drying depend on the respective formulation and must be determined experimentally. Air is generally used as a gas. However, other inert gases such as nitrogen or argon are suitable. The spray drying temperature, ie the inlet temperature and the outlet temperature, is determined by the temperature sensitivity of the active substance used and in each case by the stabilizer used. It is common to have an inlet temperature of 50 to 200 ° C, while the outlet temperature is mainly 30 to 150 ° C. Within the scope of the present invention, the process is carried out at an inlet temperature of approximately 170 to 185 ° C and an outlet temperature of 80 to 100 ° C. However, it may also have an inlet temperature of 200 ° C. or less, preferably 60 to 185 ° C., and an outlet temperature of 120 ° C. or less, preferably 40 to 105 ° C., depending on the stabilizer fraction. Spraying is generally carried out at a pressure of approximately 29 to 150 psi, preferably approximately 30 or 40 to 100 psi, for example approximately 30, 40, 50, 60, 70, 80, 90, or 100 psi.

In connection with the Butch Form B-290 spray dryer, the liquid feed rate is generally from 0.1 to 100 ml / min, preferably from 0.1 to 30 ml / min, for example approximately 3 ml / min. In this regard, an aspirator flow rate of 20 to 40 m 3 / h, preferably 30 to 40 m 3 / h, for example 38.3 m 3 / h, and 0.3 to 2.5 m 3 / h, preferably approximately 0.67 m 3 h Atomization flow rates of 1.05 m 3 / h, and 1.74 m 3 / h have been found to be particularly suitable.

Spray dried antibody or antibody derivative formulations may optionally be subjected to a second mild drying (post-drying). The aim is to obtain a standard residual water content in the formulation, preferably less than 2% (w / w), to increase both active material stability and powder properties such as glass transition temperature, flowability, and dispersibility. The conditions of the post-drying process should be chosen so that the aggregation of the antibody or antibody derivative does not significantly increase. Spray dried active substance formulations are preferably prepared, further processed and stored under dry conditions (at low relative air humidity). The post-drying process allows the water content of the powder to be further reduced despite the relatively high initial residual water content after spray drying. Surprisingly, the adjuvant of the invention stabilizes the protein in an excellent manner in the desired formulations even under process and storage conditions that are not optimal.

Freeze drying

Freeze drying of the aqueous solution can be carried out according to the description described in "Lyophilisation" by Essig and Oschmann (Wissenschaftliche Verlagsgesell-schaft, Stuttgart, 1993). The therapeutically active substance and the antibody or antibody derivative are generally lyophilized as an aqueous solution or suspension. Appropriate concentration and pH values should be considered. In a preferred formulation, the antibody or antibody derivative is initially dissolved in an aqueous solution having a suitable buffer system. The pH value of the protein containing solution is generally from 3 to 11, preferably from 3.5 to 9, particularly preferably from 4.0 to 8. The pH value of the solution should be set below or above the isotonic point of the antibody / antibody derivative. In a second container, the adjuvant or a mixture of suitable auxiliaries is dissolved in a suitable buffer solution with ultrapure water or a pH value of 3 to 11, preferably 3.5 to 9, particularly preferably 4.0 to 8.5, and in a second step with the protein solution Mix. Finally, a solution with ultrapure water or a suitable buffer solution with a pH value of 3 to 11, preferably 3.5 to 9, particularly preferably 4.0 to 8.5 is set to the required solids content. Suitable total solids content is 0.1 to 30% (w / w), preferably 0.5 to 20% (w / w), particularly preferably 0.75 to 15.0% (w / w).

The solution is then lyophilized in a conventional commercial freeze dryer, such as Christ LPC-16 / NT Epsilon 2-12 D, manufactured by Martin Christ Gefriertrocknungsanlagen GmbH. Let's do it. The product is a protein containing powder or cake and before further processing, the size is then reduced by a suitable process to obtain a polydisperse powder.

The temperature in the freeze dryer is experimentally optimized and is generally -70 ° C to + 100 ° C, preferably -50 ° C to + 40 ° C. Preferred pressure parameters in the lyophilizer are pressures of lO * e-5 to 1013 mbar. After reducing its size, the lyophilized protein formulation can preferably be subjected to a second mild drying (post-drying). The aim is to obtain a constant residual water content in the formulation, preferably less than 2% (w / w), to increase both antibody stability and powder properties such as glass transition temperature, flowability, and dispersibility. The conditions of the post-drying process should be chosen so that the aggregation of proteins does not increase significantly.

Characteristics of Spray Dried Dry Powder Formulations

The residual water content of the dry protein-powder formulation prepared within the scope of the present invention is less than 15% (w / w), generally less than 10% (w / w), preferably less than 6% (w / w). In addition, the residual water content of the spray dried protein-powder formulation is preferably less than 5% (w / w), particularly preferably less than 3% (w / w), most preferably 0.2 to 2.0% (w / w). )to be. Formulations with a low residual moisture content generally exhibit increased stability during packaging and storage. In addition, the dry protein-powder formulations according to the invention are especially hydroscopic. That is, they tend to absorb moisture from their environment. To avoid this, the powder is generally stored in a container, such as a blister pack, which ensures the exclusion of moisture from the air. Surprisingly, it has been found that in selected formulations of powders according to the invention, even after one month of open storage at 43% relative air humidity, the powder remains stable in terms of both protein stability and inhalation potential.

The stabilizing effect of the adjuvants described herein can protect the protein from excessive filling during spray drying and storage. Pure protein formulations spray dried in the absence of adjuvants form large aggregates. Process dependent factors such as heat, shear stress, and denaturation at the air-water interface cause coagulation during spray drying (up to about 6.6% aggregation) and subsequent post-drying (up to about 5.8% aggregation). During the storage process, the absence of the stabilized hydrate shell of the protein results in large aggregation (approximately 11.8 to approximately 18.9% aggregation).

In contrast to pure protein formulations, both preferred spray dried formulations of the present invention can reduce the formation of aggregates after spray drying and maintain them at very low levels even under various storage conditions. Through spray drying and subsequent vacuum drying, approximately 4.0% aggregates are formed in the pure protein formulation, whereas only approximately 0.5 to approximately 1.8% aggregates are formed in the preferred formulation. Under particularly challenging storage conditions of forced storage stability (40 ° C., 75% relative humidity), the preferred formulations are certainly superior (approximately) to pure protein formulations (approximately 18.2 to 18.9% aggregation) and similar reference formulations with tlehalose as an adjuvant 1.0 to approximately 13.1% aggregation). This advantage is particularly evident when compared to the formulation described in Example 4. The addition of triisoleucine in the spray solution significantly improves the aerodynamic properties of the powder. Surprisingly, only a combination containing at least one 1,4 O-linked saccharose derivative, preferably lactosucrose and triisoleucine, in particular LS55P and triisoleucine and LS9OP and triisoleucine, aggregates the protein (only 0.7 To 4.4% aggregation). The highest trehalose described as raffinose (12.6% flocculation) and hydroxyethyl starch (roughly 18.6% flocculation) or similar stabilizer used in combination with the triisoleucine described in International Publication No. 01/32144 is triiso. It is not possible to protect proteins from aggregates, particularly under challenging conditions combined with leucine. In addition, both the LS55P-triisoleucine formulation and the LS9OP-triisoleucine formulation are clearly advantageous for the saccharose-triisoleucine composition (5.6% aggregation) and the saccharose-lactose-triisoruscin composition (8.8% aggregation). see. In addition, LS55P is even more surprising in that it contains 25% or less of lactose together with saccharose. It is clear that the negative effect exerted by reducing sugar lactose in protein stability is compensated for by LS55P by 1,4 O-linked saccharose derivative lactosucrose. The high fraction of lactosucrose in the sugar fraction of this formulation is even more advantageous in protein stability (see LS9OP formulation).

During relatively short periods of storage, especially under destabilizing conditions (1 week at 40 ° C., 75% relative humidity), formulations that already have a significant storage effect on the incorporated proteins are much milder standard storage conditions (eg 1 year drying at approximately 25 ° C.). Protein is stabilized for a long time.

Following equilibration, after 4 weeks of storage under dry conditions at 40 ° C. (equilibrated storage stability), powder formulations containing LS55P and coupling sugars have a particularly low aggregate content compared to pure protein powders (approximately 11.8% aggregation). (Approximately 1.4 to 3.2% aggregation).

After 4 weeks of storage under vacuum drying followed by drying conditions at 40 ° C. (vacuum dried storage stability), powder formulations containing LS55P and coupling sugars were particularly low compared to pure protein powders (approximately 13.2% aggregation). And aggregate content (approximately 1.1 to 2.1% aggregation).

LS55P (80%), isoleucine (10%), and IgG1 (10%) formulations having approximately 35% fine particle fractions were subjected to vacuum drying followed by drying conditions at 2-8 ° C, 25 ° C, and 40 ° C. Storage for 3 months under nitrogen and then up to 1.9% aggregate content after filling under nitrogen.

LS55P (80%), triisoleucine (10%), and IgG1 (10%) formulations with MMAD and 58.3% fine particle fraction of approximately 3.9 μm after spray drying were dried vacuum followed by 2-8 ° C., and It shows up to 1.9% aggregate content after filling under nitrogen after drying for 3 months at 25C, and up to 2.6% aggregate content under dry storage conditions (3 months stability) at 40 ° C.

After approximately 43% relative air humidity and one month open storage (one month open stability) at 25 ° C., the specific LS55P (80%), triisoleucine (10%), and IgG1 (10%) formulations were further approximately The same low MMAD (approximately 3.8 μm) and the same high fine particle fraction (approximately 59.6%) show low aggregate content (approximately 1.3%).

LS9OP (90%) and IgG1 (10%) formulations having an MMAD of approximately 3.8 μm, approximately 2.8 μm, and approximately 24% fine particle fraction after spray drying, followed by vacuum drying, 2-8 ° C., 25 ° C., and It exhibits aggregate contents of 1.2 and 2.2% or less after filling under nitrogen after 1 month and 3 months of storage (1 month and 3 months of stability) under dry conditions at 40 ° C.

LS9OP (80%), isoleucine (10%), and IgG1 (10%) formulations having approximately 28% fine particle fraction after spray drying were subjected to vacuum drying at 2-8 ° C, 25 ° C, and 40 ° C. After 1 month and 3 months of storage (1 month and 3 months of stability) under dry conditions, the aggregate content is below 0.9 and 1.1% after filling under nitrogen, respectively.

The LS9OP (80%), triisoleucine (10%), and IgG1 (10%) formulations having a MMAD of approximately 4.8 μm and a fine particle fraction of approximately 53.2% after spray drying were vacuum dried followed by 2-8 ° C., Agglomerates content of 1.0 and 2.3% or less after filling under nitrogen after 1 month and 3 months of storage (1 month and 3 months of stability) under dry conditions at 25 ° C. and 40 ° C., respectively.

After approximately 43% relative air humidity and 1 month and 3 months of open storage (stability of 1 month and 3 months open) at 25 ° C., LS9OP (80%), triisorucin (10%), and IgG1 (10) as described above. %) Changes in formulation further exhibit low aggregate contents of approximately 0.5% and 0.8% or less. After spray drying, the MMAD value is 3.9-3.3 μm and the FPF value is approximately 55.6-58.9%. After approximately 43% relative air humidity and one month open storage at 25 ° C., the formulations described above further exhibit low MMAD values (approximately 4.1 to 3.5 μm) and high fine particle fractions (approximately 62.3 to 67.3%).

Through a change in spray drying conditions, it is possible to produce powders which preferably have an average particle size (MMD) of less than 20 μm, preferably less than 10 μm. According to a particularly preferred embodiment, the average particle size of such particles according to the invention is less than 7.5 μm, preferably less than 5 μm. Particular preference is given to particles having an average particle size of less than 4 μm, more preferably less than 3.5 μm. In addition, particles having an average particle diameter of 0.1 to 5 mu m, preferably 0.2 to 4 mu m, can be produced. In a further embodiment, the corresponding particles are mixed with non-breathable particles such as lactose having a particle size of at least 40 μm. The fraction preferably corresponds to at least 15%, more preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, particularly preferably at least 50 or 60%.

In addition to the mean particle size (MMD), the inhalability is highly dependent on the mean aerodynamic particle diameter (MMAD). The MMAD of the particles according to the invention is preferably less than 10 μm, more preferably less than 7.5 μm. Particularly advantageous are powders consisting of particles having an MMAD of less than 5.5 μm, preferably less than 5 μm, even more preferably less than 4.5 μm. The powders described in the examples can be prepared in corresponding particle sizes by combining optimum spray drying conditions with suitable selection and concentrations of the adjuvants according to the invention. The addition of amino acids and / or tripeptides improves particle performance, in particular while increasing the fraction of inhalable particles with an MMAD of less than 7.5 μm, preferably less than 5.5 μm. Through addition of the prepared isoleucine and triisoleucine, inhalables having an FPF greater than 28%, preferably greater than 40%, more preferably greater than 50%, even more preferably greater than 55% (see examples) Particles can be prepared.

In addition, the powders according to the invention are distinguished by glass transition temperatures of at least 40 ° C, preferably at least 50 ° C, more preferably at least 55 ° C, even more preferably at least 60 ° C. Especially preferred powders have a glass transition temperature of at least 65 ° C. The glass transition temperature of the powders according to the invention generally corresponds to 40 to 110 ° C. The present invention therefore also relates to powders, preferably spray dried powders, containing pharmaceutically active substances and LS9OP, LS55P, and coupling sugars or coupling sugars, the glass transition temperature of which is at least 40 ° C, preferably Corresponds to 45 to 60 ℃ or more. According to a further preferred embodiment, the glass transition temperature corresponds to at least 55 ° C, preferably at least 55 to 60 ° C.

Use of Spray Dried Powder

The powders according to the invention are suitable for the preparation of medical drugs, preferably for the preparation of medical inhalants.

Freeze dried powder

In addition, the powder may be prepared by or freeze-dried to ensure pulverisation (see Examples). In the examples given in this regard, powdering is preformed in the simplest manner imaginable by a spatula in a lyophilisation vial. In addition, lyophilisate can be readily powdered into suitable mills such as friction mills, ball mills, rod mills, mortar mills, air jet mills, or other suitable processes (see Bauer, Fromming, Fuhrer, 6th edition). Can be.

Properties of Freeze Dried Powdered Dry Powder Formulations

The residual water content of the dry protein-powder formulation prepared within the scope of the invention is less than 15% (w / w), generally less than 10% (w / w), preferably less than 5% (w / w). In addition, the residual water content of the lyophilized protein-powder formulation is preferably less than 3% (w / w), particularly preferably less than 2% (w / w), most preferably 0.2-1.5% (w / w). )to be. Formulations with a low residual moisture content generally exhibit improved stability during packaging and storage. In addition, the dry protein-powder formulations according to the invention are especially hydroscopic. That is, they tend to absorb moisture from their environment. To avoid this, the powder is generally stored in a container, such as a blister pack, which ensures the exclusion of moisture from the air.

The stabilizing effect of the adjuvants described herein can protect the protein from extreme stress during lyophilization and storage. Pure protein formulations lyophilized in the absence of adjuvants form large aggregates. Process dependent factors such as heat, shear stress, and denaturation at the air-water interface cause coagulation (aggregation of approximately 2.1% or less) during freeze drying. During the course of storage, the absence of a stabilized hydrate shell of protein leads to large aggregation (20.5% aggregation).

In contrast to pure protein formulations, both preferred lyophilized formulations of the present invention can reduce the formation of aggregates after lyophilization and maintain them at very low levels even under different storage conditions. Under particularly challenging storage conditions of forced storage stability (40 ° C., 75% relative humidity), lyophilized and powdered lyophyllate has mannitol as a pure protein formulation (approximately 14.5% aggregation) and as an adjuvant (approximately 34.0% aggregation). Clearly good (approximately 1.2 to approximately 1.5% aggregation) for similar reference formulations.

During relatively short periods of storage, especially under destabilizing conditions (40 ° C., 1 week at 75% relative humidity), formulations that already have a significant stabilizing effect on the incorporated proteins are much milder than standard storage conditions (eg 1 year drying at approximately 25 ° C.). Protein is stabilized for a long time.

After 4 weeks of storage under dry conditions (equilibrated storage stability) at 40 ° C. following freeze drying, powdering, and equilibration, powder formulations containing LS55P and coupling sugars were particularly pure protein powders (approximately 15.3% aggregation). And low aggregate content (approximately 2.6 and 4.6% aggregation) compared to similar reference formulations with mannitol as adjuvant (approximately 11.6% aggregation).

After 4 weeks of storage under dry conditions (vacuum dried storage stability) at 40 ° C. following freeze drying, powdering, and equilibration, powder formulations containing LS55P and coupling sugars were particularly pure protein powders (approximately 14.5% aggregation). ) And low aggregate content (approximately 1.2 and 1.5% aggregation) compared to similar reference formulations with mannitol as adjuvant (approximately 6.2% aggregation).

In addition, the powders according to the invention are distinguished by glass transition temperatures of at least 40 ° C, preferably at least 50 ° C, more preferably at least 55 ° C. The glass transition temperature of the powders according to the invention generally corresponds to 40 to 110 ° C., but in each case may exceed this value. Accordingly, the present invention also relates to powders containing pharmaceutically active substances and LS9OP, LS55P, and coupling sugars or coupling sugars, preferably lyophilized and powdered powders, the glass transition temperature of which is 40 ° C. Above, Preferably it corresponds to 45-60 degreeC or more. According to a further preferred embodiment, the glass transition temperature corresponds to at least 55 ° C, preferably 55 to 60 ° C or 110 ° C.

Use of Dried Powder

The powders according to the invention are suitable for the preparation of medical drugs, preferably for the preparation of medical inhalants.

Delivery of Powders According to the Invention

Spray dried powder preparations according to the invention can be used directly as dry powders, basically via a so-called dry powder inhaler or the like, or after recombination, in the form of aerosols via so-called atomizers. The inhalable powders according to the invention can then be used as known top inhalers.

Inhalable powders according to the invention can be used as inhalers for delivering a single dose from a storage container to a measuring chamber as described in US Pat. No. 4,570,630 or to other instruments as described in DE 36 25 685. It is available. Inhalable powders according to the invention are preferably filled in capsules (so-called inhalets) for use in inhalers, for example as described in WO 94/28958.

Further suitable inhalers are described, for example, in US Pat. No. 5,458,135, US Pat. No. 5,785,049, or International Publication No. 01/00263. Further suitable inhalers are known from WO 97/41031, US Pat. No. 3,906,950, and US Pat. No. 4,013,075. Further dispersive inhalers for dry powder formulations are described in EP 129 985, EP 472 598, EP 467 172, and US Pat. No. 5,522,385.

Inhalable powders according to the invention can be used, for example, using an inhaler known under the trade name Turbuhaler ^® (manufacturer: AstraZeneca LP), for example in European Patent Publication No. 237 507 It can be used as an inhaler disclosed in the call. Other suitable inhalers are Rotahaler ^® (manufacturer: GlaxoSmithKline Corp.), Discus ^® (manufacturer: GlaxoSmithKline Corporation), Spiros ^TM inhaler [manufacturer: Dura Pharma Dura Phrmaceuticals and Spinhaler ^® (Fiscon).

A particularly preferred inhaler for administering a pharmaceutical combination according to the invention in the form of an inhalet is shown in FIG. 24. The inhaler (handheld) for inhaling the powdered pharmaceutical composition from the capsule comprises two windows (2), a deck (3) provided with a fixed sieve (5) with an air inlet opening and located through the sieve housing (4). Housing for adjusting the flow resistance and suction chamber 6 connected with a deck 3 provided with a trigger 9 movable with a spring 8 with two sharp needles 7. ), A deck 3, and a cap 11, and a mouthpiece 12 hinged through a shaft 10 having an air hole 13.

When the inhalable powder according to the invention is filled into capsules (inhalets) in connection with the preferred use described above, a filling amount of 1 to 30 mg per capsule is suitable.

Further inhalable powders according to the invention can be used as inhalable aerosols containing propellants or inhalable aerosols containing no propellants. For this purpose, the powders according to the invention are suspended in pressure liquefiable solvents or solvent mixtures or recombined in aqueous solutions. Suitable suspensions or solutions are known in the art. For example, recombination is advantageous in physiological solutions with a pH of 3-11, preferably 4-9. Recombination in aqueous solutions with a pH of 5.5 to 7.8 is particularly advantageous. Propellant-containing suspensions or solutions for the recombination of powders according to the invention may further contain stabilizers, emulsifiers, surfactants, or other auxiliaries in the form of water-soluble organic solvents. Suitable materials are described, for example, in Bauer, Lehrbuch der Pharmazeutischen Technologie, Wissenschaftl. Verlagsgesellschaft mbH, Stuttgart, 178-184; Adler, 1998, Journal of Pharmaceutical Sciences, 88 (2), 199-208, are known to those skilled in the art. In addition, the corresponding inhalable aerosols produced by suspension or recombination of the powders according to the invention constitute the object of the invention.

In addition, propellants used to prepare inhalable aerosols according to the invention are known in the art. Suitable propellants are chlorolated and fluorinated hydrocarbons such as e-propane, e-butane, or isobutane, and halogenated hydrocarbons, for example, methane, ethane, propane, butane, cyclopropane, or cyclobutane It is selected from the group consisting of derivatives. The propellants described above can be used alone or as mixtures thereof. Particularly preferred propellants are TG11, TG12, TG134 (1,1,1,2--tetrafluoroethane), TG227 (1,1,12,3,3,3-heptafluoropropane), and mixtures thereof Halogenated alkane derivatives selected from among which propellants TG134, TG227, and mixtures thereof are preferred.

Propellant-containing inhalable aerosols according to the invention may contain up to 5% (w / w) of active substance. The aerosols according to the invention, for example, contain 0.002 to 5% (w / w), 0.01 to 3% (w / w), 0.015 to 2% (w / w), 0.1 to 2% ( w / w), 0.5 to 2% (w / w), or 0.5 to 1% (w / w). Inhalable aerosols having the corresponding active substance concentrations can be prepared and achieved by recombination of the powders according to the invention in an appropriate amount of solvent.

In addition, the propellant-containing inhalable aerosols according to the invention described above can be used with known state-of-the-art inhalers (MDI = metered dose inhalers). In this connection, Ben tolrin (Ventolin) ^®: reference can be made to the inhaler described in the manufacturer's Ben tolrin Pharma City (Ventolin Pharmacy)] or United States Patent No. 5,32,094 No. or U.S. Patent No. 5,672,581 calls. Thus, a further aspect of the invention relates to a medical drug in the form of a propellant containing aerosol as described above in combination with one or more inhalers suitable for delivery of the aerosol. The invention further relates to an inhaler characterized by containing a propellant containing aerosol according to the invention described above.

In addition, the present invention relates to a cartridge containing a propellant-containing inhalable aerosol according to the invention described above, which can be used by mounting a suitable valve in a suitable inhaler. Suitable cartridges and processes for filling the cartridges with propellant containing inhalable aerosols according to the invention are known in the art.

The powders according to the invention can further be recombined into inhalable solutions or suspensions that do not contain propellants. Inhalable solutions that do not contain the corresponding propellant contain, for example, an aqueous or alcoholic, preferably ethanol solvent, if necessary, ethanol solvent mixed with an aqueous solvent. In the case of an aqueous / ethanol solvent mixture, the relative fraction of ethanol relative to water is not limited, but the maximum limit is preferably below 70% (v / v), in particular below 60% (v / v) of ethanol. . Residual volume fraction is filled with water. Inhalable solutions containing no propellant according to the invention may be mixed with cosolvents and / or other auxiliaries described above. For example, hydroxyl groups or other polar groups such as alcohols, in particular isopropyl alcohol, glycols, in particular propylene glycol, polyethylene glycol, polypropylene glycol, glycol ethers, glycerol, polyoxyethylene alcohol, and polyoxyethylene Cosolvents containing fatty acids can be used. In this regard, auxiliaries and additives are not active substances but are meant pharmacologically compatible substances which can be formulated with a pharmacologically suitable solvent together with the active substance (s) to ensure the quality of the active substance formulation. It is understood that. Such substances preferably have no pharmacological effect, or have no apparent or at least undesired pharmacological effect with respect to the desired treatment. Auxiliaries and additives include surfactants such as soya lecithin, olefinic acids, sorbitan esters such as polysorbates, polyvinylpyrrolidone, other stabilizers, complexing agents, antioxidants, and / or final drugs. Antiseptics, vitamins, and / or other additives known in the art, together with preservatives to ensure or prolong the shelf life of the formulation. Additives also include pharmacologically harmless salts, for example sodium chloride as an isotonic agent. Preferred adjuvants include antioxidants such as ascorbic acid, vitamin A, vitamin E, tocopherol, and similar vitamins or provitamins present in humans, if not already used to adjust pH values. Preservatives can be used to protect the formulation from contamination by bacteria. Suitable preservatives known in the art include in particular cetyl pyridinium chloride, benzalkonium chloride, or benzoic acid or benzoate, for example sodium benzoate, at concentrations known in the art. The preservative described above is preferably contained at a concentration of up to 50 mg / 100 ml, particularly preferably from 5 to 20 mg / 100 ml. The invention therefore also relates to inhalable aerosols which do not contain propellants prepared by the recombination of the powders according to the invention.

Inhalers capable of atomizing within a few seconds at therapeutically necessary dosages with suitable therapeutic aerosols for use in small amounts of liquid formulations are particularly suitable for the field of inhalable solutions containing no propellant according to the invention. A mean particle size of the active substance solution in an amount of less than 100 μl, preferably less than 50 μl, particularly preferably 10 to 30 μl, is atomized at one stroke such that the inhalable fraction of the aerosol corresponds to a therapeutically effective amount. Atomizers capable of forming aerosols of size less than 20 μm, preferably less than 10 μm are preferred within the scope of the present invention.

Devices of the kind for the delivery of a liquid drug in a dose-free form for use in an inhaler are described in detail, for example, in WO 91/14468 and WO 97/12687 (in particular FIG. 6a and 6b).

Within the scope of the present invention, reference is made explicitly to the corresponding figures 6a and 6b of WO 97/12687, including the relevant parts used in the description. The atomizer (apparatus) described in this publication is also known under the trade name Respimat ^® (manufacturer: Boehringer Ingelheim Pharma). Because of its nearly cylindrical shape and small and convenient size of less than 9 to 15 cm in length and 2 to 4 cm in width, the device can always be carried by the patient. The atomizer sprays a specific volume of drug formulation through a small nozzle under high pressure to produce an inhalable aerosol.

Preferred atomizers are basically

A pump housing which is closed to the upper housing part and carries a nozzle body with a nozzle or nozzle arrangement at one end,

Hollow piston with valve body,

A power take-off flange flange, with the hollow piston closed and located in the upper housing part,

-Locking tensioning mechanism located in the upper housing part,

A spring housing in which the spring is located, mounted on the upper housing part by a rotating bearing and rotating;

An upper housing part, a pump housing, a nozzle, a locking tensioning mechanism, a spring housing, a spring, and a storage container, characterized by a lower housing part inserted in the spring housing in the axial direction.

The hollow piston with the valve body corresponds to the device described in WO 97/12687. It can partially fire the cylinder of the pump housing and move axially between the cylinders. Within the scope of the present invention, reference is made to FIGS. 1 to 4, in particular FIG. 3 and related parts used in the detailed description. In spring operation, the hollow piston with the valve body exerts a pressure of 5 to 60 MPa (about 50 to 600 bar), preferably 10 to 60 MPa (about 100 to 600 bar) on a metered amount of active material solution that is a fluid. A volume of 10-50 μl is preferred. Particularly preferred is a volume of 10-20 μl, in particular 15 μl per stroke.

The valve body is preferably arranged at the end of the hollow piston facing the valve body.

The nozzle of the nozzle body is preferably manufactured by microstructure, ie by microtechnology. Microstructured nozzle bodies are described, for example, in WO 94/07607, the disclosure of which specifically refers to FIG. 1 and the detailed description thereof. The nozzle body consists of, for example, two tightly joined plates of glass and / or silicon, one or more of which comprise one or more microstructured channels connecting the nozzle inlet side to the nozzle outlet side. do. At the nozzle outlet side is located one or more rounded or unrounded openings with a depth of 2 to 10 μm, a width of 5 to 15 μm, preferably a depth of 4.5 to 6.5 μm and a length of 7 to 9 μm. When there are a plurality of nozzle openings, preferably two, the nozzle spraying directions in the nozzle body tend to extend in parallel to one another or incline each other in the nozzle opening direction. In a nozzle body with two or more nozzle openings on the outlet side, it may tend to be from 20 to 160 degrees, preferably from 60 to 150 degrees, in particular from 80 to 100 degrees from each other, the angle in the spray direction. The nozzle openings are preferably arranged at a distance of 10 to 200 μm, more preferably 10 to 100 μm, particularly preferably 30 to 70 μm. Most preferred is a distance of 50 μm.

Thus, the spray direction will meet near the nozzle opening.

The liquid drug formulation engages the nozzle body at an inlet pressure of 600 bar or less, preferably 200 to 300 bar, and atomizes to form an inhalable aerosol through the nozzle opening. The preferred particle or droplet size of the aerosol is 20 μm or less, preferably 3 to 10 μm.

The locking tensioning mechanism comprises a spring, preferably a cylindrical spiral compression spring, for the accumulation of mechanical energy. The spring moves on the takeoff flange as actuation member and this movement is measured by the position of the locking member. The path of the takeoff flange is precisely limited by the upper and lower interruptions. The spring is preferably bent through a power transmission gear, for example a helical thrust gear, by an external rotational force generated while the upper housing portion rotates with respect to the spring housing of the lower housing portion. In this case, the upper housing portion and the takeoff flange comprise single or multiple V-shaped gears.

The locking member attached to the locking surface is arranged annularly around the takeoff flange. This consists, for example, of an essentially radially elastically deformable plastic or metal ring. The rings are arranged in a plane perpendicular to the atomizer axis. After the spring is bent, the locking face of the locking member moves in the path of the takeoff flange and prevents the spring from loosening. The lock member is operated by a button. The actuation button is connected or coupled with the locking member. To activate the locking tensioning mechanism, the actuation button is moved parallel to the round plane, preferably parallel to the atomizer, to deform the deformable ring in the round plane. Detailed description of the design of the locking tensioning mechanism is given in International Publication No. 97/20590.

The lower housing portion moves axially relative to the spring housing and covers the bearings , spindle drive and fluid storage container.

When the atomizer is in operation, the upper housing portion rotates relative to the lower housing portion and the lower housing portion takes on a spring housing. The spring is then compressed and bent by the helical thrust gear and the locking mechanism works automatically. The rotation angle is preferably a numerical fraction of 360 °, for example 180 °. At the same time as the spring is bent, the takeoff portion of the upper housing portion moves in a predetermined path, the hollow piston is drawn into the cylinder of the pump housing and a portion of the fluid is sucked from the storage container into the high pressure chamber in front of the nozzle.

Multiple exchangeable storage containers containing the fluid to be atomized may be continuously pushed by the atomizer and used continuously if necessary. The storage container contains an aqueous aerosol according to the invention.

The atomizing process is activated by pressing the operation button badly. The locking mechanism then obstructs the path for the drive portion. The bent spring moves the piston into the cylinder of the pump housing. The fluid emerges from the atomizer nozzles in atomized form.

Further details of the design are known from PCT International Publication No. 97/12683 and International Publication No. 97/20590, the contents of which are incorporated herein by reference.

The atomizer member is made of a functionally suitable material. The atomizer housing is made of plastic, made of, for example, injection molding, as well as other components, as long as operation is permitted. For medical purposes, physiologically safe substances are used.

6A and 6B of International Publication No. 97/12687 (including detailed description thereof), which are hereby incorporated by reference in their entirety, refer to the corresponding atomizer (Resmatmat ^® ). Such atomizers are particularly suitable for use with inhalable aerosols which do not contain propellants according to the invention.

FIG. 6A of WO 97/12687 shows a longitudinal section of a spring-tensioned atomizer and FIG. 6B of WO 97/12687 shows a longitudinal section of a spring relaxed atomizer. The upper housing portion 51 comprises a pump housing 52 with a holder 53 for the atomizer nozzle arranged at the distal end. The nozzle body 54 and the filter 55 are located in the holder. The hollow piston 57, which is fixed to the take-off flange 56 of the locking tensioning mechanism, partially projects into the cylinder of the pump housing. At its end, the hollow piston carries the valve body 58. The hollow piston is sealed by the seal 59. Inside the upper housing portion there is a distal stop 60 where the takeoff flange stops when the spring relaxes. A distal stop 61, where the spring stops taking off the flange, is located in the takeoff flange. After spring tension, the locking member 62 slides between the end stop 61 and the support 63 of the upper housing portion. The actuation button 64 is connected with the locking member. The upper housing portion is terminated in the mouthpiece 65 and sealed with an insertable protective cap 66. The spring housing 67 with the compression spring 68 is mounted on the upper housing portion by the snap projection 69 and the rotating bearing to rotate. The lower housing portion 70 moves into the spring housing. A replaceable storage container 71 for the fluid 72 to be atomized is located inside the spring housing. The storage container is sealed by the stopper 73 by the hollow piston protruding into the storage container and the end thereof submerged in the fluid (supply of the active substance solution). The spindle 74 for the mechanical counter is arranged in a spring housing mold. The drive pinion 75 is located at the distal end of the spindle opposite the upper housing portion. Slider 76 is located on the spindle.

When the formulations according to the invention are atomized with the technique described above (Respimat ^® ), the weight delivered during at least 97%, preferably at least 98% of the total inhaler operation (stroke) is less than 25% in the tolerance range. , Preferably 20%. The formulations are delivered as specific weights, from 5 to 30 mg, particularly preferably from 5 to 20 mg, per stroke.

However, formulations according to the invention can also be atomized using inhalers other than those described above, for example jet-stream inhalers or other stationary atomizers.

Thus, a further aspect of the present invention relates to a medical drug in the form of an inhalable solution or suspension containing no propellant as described above, in connection with a device suitable for the delivery of these formulations, preferably Respimat ^® . . The invention preferably relates to inhalable solutions or suspensions which contain one of the powders according to the invention and which do not contain a propellant in connection with a device known under the tradename Respimat ^® . The invention further relates to, characterized by containing the inhaled solution or suspension not containing the above-described propellant, the inhalation device of the above-mentioned bar, preferably less blood mat ^®.

Inhalable solutions containing one of the powders according to the invention as described in this regard in a unique commercially available form are preferably consistent with the invention.

Inhaled a solution containing no propellants according to the invention or suspensions, less blood for use in a mat ^® with solutions and suspensions as described above bar, concentrate or ready-to-use may be used as a possible sterile inhaled solution or suspension have. By adding isotonic saline solutions, ready-to-use formulations can be prepared, for example, from concentrated solutions. Ready-to-use sterile formulations can be used as power-driven stop atomizers or portable atomizers that produce inhalable aerosols using ultrasonic or compressed air by the Venturi principle or other principles.

Thus, a further aspect of the invention is in the form of inhalable solutions or suspensions containing no propellant as described above, using concentrated or ready-to-use sterile formulations in connection with suitable devices for the delivery of these solutions. It relates to a medical drug, characterized in that the device is a power-driven stop atomizer or a portable atomizer that produces inhalable aerosols using ultrasonic or compressed air by the Venturi principle or other principles.

Other suitable atomizers for the use of recombinant aerosols in inhalers are AERx ^™ (manufacturer: Aradigm), Ultravent ^® [manufacturer: Mallinkrodt] and AconII. ) ^® [Manufacturer: Do Quest Medical Products (Maquest Medical Products)] it is.

Aspect Example

Substances and Methods

matter

Humanized monoclonal antibodies of approximately 148 kDa molecular weight from Boehringer Ingelheim, Germany are used as IgG1. The antibody is derived from a murine antibody in which the complementary determining region of the murine antibody is implanted with human immunoglobulin. This produced a chemotherapy antibody with 95% human and 5% murine fractions. The antibody is expressed by a murine myeloma cell line. Cells are removed by tangential flow microfiltration, and solutions without cells are purified by various chromatographic methods. Additional steps include nuclease treatment, low pH treatment and nanofiltration. The antibody containing bulk solution contains histidine 25 mM and glycine 1.6 mM as buffer and is concentrated to approximately 100 mg / ml by diafiltration for the preparation of spray dried solutions. The bulk for the preparation of the solution to be sprayed contains 0.4 to 0.8% of aggregates. The final drug can be stored at 2-8 ° C. for at least 2 years. New Car-Oligo ^® LS55P, New Car-Oligo ^® LS90P, Coupling Sugar ^® and Coupling Sugar S ^® were purchased from Hayashibara Shoji Kabushi Kaisha, Japan. Saccharose, lactose, mannitol, raffinose, hydroxyethyl starch and L-isoleucine were purchased from Sigma-Aldrich Chemie GmbH, Germany. Trehalose was purchased from Georg Breuer GmbH, Germany. Triisoleucine was purchased from Iris Biotech GmbH, Germany. Hen's egg albumin lysozyme (lysozyme) (135500 U / mg) was purchased from SERVA Electrophoresis GmbH, Germany. Synthetic salmon calcitonin (calcitonin) was purchased from Biotrend Chemikalien GmbH, Germany.

Spray drying by Butch B-290

Spray drying was carried out using a B-290 Buchi Mini Spray Dryer from Buchi Labortechnik GmbH. Spray drying of the formulations was basically performed as described in "Spray Drying Handbook", 5th edition, K. Masters, John Wiley and Sons, Inc., NY, NY (1991).

The spray dryer consists of a heating system, a filter, an aspirator, a drying cylinder, a cyclone, a temperature sensor for measuring inlet and outlet temperatures and a collection vessel. The solution to be sprayed is pumped to a two-fluid nozzle using a peristaltic pump. Here, compressed air is used to atomize the solution into small droplets. Drying proceeds in the spray tower with warm air drawn through the drying tower with an aspirator during the continuous flow process. The product is collected in a collection vessel after passing through a cyclone.

Two kinds of cyclones were used.

Cyclone I: Butch Cyclone (Product No .: 4189)

Cyclone II: Butch high performance cyclone (product no .: 46369)

The solids content of the sprayed solution is 10% (w / v), 3.3% (w / v) and 2.00% (w / v) in 50-600 ml of solution. The inlet temperature is about 170 to 158 ° C., the liquid feed rate is about 3 to 3.33 ml / min, the aspirator flow rate is about 36.8 to 38.3 m 3 / h, and the atomizing air flow (AAF) is 0.67 M 3 / h, 1.05 m 3 / h and 1.74 m 3 / h, with the result that the outlet temperature was 80 to 95 ° C.

Freeze drying

Freeze drying was carried out using a Christ LPC-16 / NT epsilon 2-12 D freeze dryer manufactured by Martin Christ Gepretrokkesan ragen GmbH. The freeze dryer consists of a drying chamber, a sublimated solvent separation condenser, a vacuum generating pump and an electric device. Drying is controlled by control of the surface temperature and drying of the drying chamber.

The solid fraction of the lyophilized solution was 5% (w / v). The solution was dispensed 0.5 ml each into 2R vials and placed in a freeze dryer with a flow through freeze-dryer stopper. First, the solution was frozen to −40 ° C. within 30 minutes. It was then subjected to major drying at 0.11 mbar in three stages. First 30 hours at −40 ° C., then 8 hours at −30 ° C., and finally 8 hours at −16 ° C. were performed. In the next step, post-drying was carried out at 20 ° C., 0.001 mbar for 20 hours. In the last step, the vial was automatically sealed with a freeze-drying stopper that was only used initially. The lyophyllate obtained thus obtained was powdered in vials with spatula.

X-ray Diffraction [Wide-angle X-ray scattering (WAXS)]

To determine the crystallinity of the dried samples, the samples were tested at a controlled temperature of 22 ° C. with a Seifert X-ray diffractometer XRD 3000 TT (manufactured by Seifert, Arensburg, Germany). . An X-ray tube (λ = 0.15418 mm) (Ni primary filter) using a Cu cathode, Cu-Kα ray, was operated at a cathode voltage of 40 kV and a current of 30 mA. After the sample plate was placed in the instrument, the samples were measured for 5-40 ° at scan speed 2θ = 0.05 ° with a measurement time of 2 seconds at each angle.

Powder diffraction patterns were recorded on a SC 1000 V detector with ScanX-Rayflex instrument (version 3.07), device XRD 3000 (scan) or Rayflex (version 2.1) (analysis).

Size Exclusion Chromatography (SEC-HPLC)

a) IgG1 protein aggregates

In order to quantify IgG1 protein aggregates in recombinable powders, size exclusion chromatography (SEC-HPLC) was performed. SEC-HPLC was performed with HP1090 units from Agilent. A TSK3000SWXL column (300 × 7.8 mm) from Tosoh Biosep (Tosoh Bioscience, Stuttgart, Germany) was used for separation. As mobile phase, a buffer consisting of 0.1 M disodium hydrogen phosphate and 0.1 M sodium sulfate was dehydrogenated and the pH was adjusted to 6.8 with 85% orthophosphoric acid. The amount of sample filled was 25 μl at a protein concentration of 2-10 mg / ml. Protein detection was performed at 280 nm with a diode array detector from Agilent. Chromatograms were evaluated using HP Chemstation software from Agilent.

b) calcitonin protein aggregates

SEC-HPLC was performed to quantify calcitonin protein calcitonin aggregates in recombinant powders. SEC-HPLC was performed using an HP1100 unit from Agilent. A TSK3000SWXL column (300 × 7.8 mm) from Tosoh Biocep [Tosoh Bioscience, Stuttgart, Germany] was used for separation. As mobile phase, a buffer of pH about 6 consisting of 0.25M sodium sulfate was used (Windisch et al., 1997). Alternatively, a dehydrogenation buffer consisting of 0.1 M disodium hydrogen phosphate dihydrate and 0.1 M sodium sulfate adjusted to pH 6.8 with 85% orthophosphoric acid can be used. The amount of sample filled was 20 μl at a protein concentration of 0.5 to 2 mg / ml. Protein detection was performed at 210 nm with an Agilent UV detector. Chromatograms were evaluated using HP ChemStation Software from Agilent.

c) Lysozyme residual monomer content

To quantify the lysozyme residual monomer content in the recombinant lysozyme formulations, a modified SEC-HPLC method (van de Weert, 2000) was performed. SEC-HPLC was performed using an HP1100 unit from Agilent. A TSK3000SWXL column (300 × 7.8 mm) from Tosoh Biocep [Tosoh Bioscience, Stuttgart, Germany] was used for separation. As mobile phase, a buffer consisting of 0.05 M disodium hydrogen phosphate dihydrate and 0.2 M sodium chloride was adjusted to pH 7.0 with 85% orthophosphoric acid. The amount of sample filled was 25 μl at a protein concentration of 2-10 mg / ml. Protein detection was performed at 280 nm with an Agilent UV detector. Chromatograms were evaluated using HP ChemStation Software from Agilent.

To evaluate the formulation, the remaining soluble monomers were quantified by the following method. First, a calibration line was shown using standard solutions of lysozyme at concentrations of 2.5 mg / ml, 5.0 mg / ml and 10 mg / ml. The AUC \ of the monomer peak was then taken into account for the corresponding lysozyme concentration of the standard solution tested.

Residual monomer content of each lysozyme formulation tested was calculated on the basis of a calculation line. If the residual monomer content of the formulation is high, the protein stability is good.

Particle Size Measurement (MMD)

Mass average diameter (MMD) or average particle size was measured with a Sympatech Helos unit manufactured by Sympatech GmbH, Klaustal-Cellerfeld, Germany. This measurement principle is based on laser diffraction and uses a helium-neon laser. Some 1-3 mg of powder was dispersed at 2 bar of air pressure and placed in front of the Fourier lens (50 mm) by a parallel laser beam. The Fraunhofer model evaluates the particle size distribution. Two measurements were taken per powder.

Mass Average Aerodynamic Diameter (MMAD) and Fine Particle Fraction (FPF)

A few powders of 12-18 mg each were filled into hard gelatin capsules (size: 3), introduced into a handicap (inhaler manufactured by Boehringer Ingelheim) and used for measurement. The adapter was used to contact the handheld with the USP / EP throat of the impactor inlet of the measuring device. The power was discharged at a rate of 39.0 L / min with a suction time of 6.15 seconds. An external control board was used to control the air flow rate. Three or more capsules were measured for each power.

APS 3321 units from TSI Inc., Minnesota, USA, were used simultaneously in combination with the 3306 impactor inlet to measure aerodynamic particle size (MMAD) using a “flight time” measurement and to measure single stage impactors ( Fine particle fraction (FPF) was measured with a single-stage impactor (effective cut off diameter at 39 L / min: 5.0 μm). After draining through the EP / USP passage or sample inlet, the powder was passed through a thin capillary and 0.2% of the powder was sampled under constant velocity conditions for flight time measurements. After the capillary passes two laser beams, the flight time is measured and the flight time is measured for a specific distance as a light shield. The result is a counted distribution, which subsequently converts into a mass distribution and into a mass mean aerodynamic diameter (MMAD).

The remaining 99.8% of the powder group passed through the capillary was separated through a single stage impactor. Fractions larger than 5.0 μm are separated as a result of mass inertia on the impact plate in the impactor. Fine particle fractions (FPF) follow the air stream and are finally separated in a depth filter. Fine particle fractions are measured by weight. The fine particle fraction is calculated from the fraction of powder separated in the filter for the total amount of powder used, ie the powder weighed per capsule.

Residual water content

Residual moisture content of the dried product was measured by a coulometric titration (Metrohm 737 KF coulometer made in Germany, with a type 703 titration standard). Dissolved or dispersed in [Hydranal-Methanol dry, (VWR / Merck Eurolab)]. (Hydranal-Coulomat solution), VWR / Merck Eurolab] was conditioned at the beginning of the bottom of the bottom, ie the measurement solution was adjusted to a water content of 0. Samples were measured by injecting into titration cells.

Stability measurement

Powders or proteins contained in powders were tested for different stability after spray drying. The percentage of protein aggregates was set as a measure of formulation stability. Pure protein formulations, similar trehalose formulations, similar raffinose formulations, similar saccharose formulations, similar saccharose-lactose formulations or similar hydroxyethyl starch formulations are partially compared with the innovative aids described herein as reference materials. Aggregates were analyzed by validated size exclusion chromatography (SEC-HPLC) with UV detection (DAD). For this purpose, the pretreated powder was first recombined in ultrapure water (pH 6-8).

Forced Storage Stability: The selected formulations were tested for stability after open storage for one week at about 40 ° C. and about 75% relative humidity of air (40 ° C., 75% RH) in open glass vials.

Equilibrated Storage Stability: After spray drying, the selected formulations were stored for 1 day at about 22 ° C. and 50-55% relative humidity of air in an open glass vial (equilibration). The glass vial was subsequently sealed, beaded under the conditions described above, and stored for 4 weeks at about 40 ° C., after which stability was tested.

Vacuum dried storage stability: After spray drying, the selected formulations were stored for 1 day at about 30 ° C. and about 0.15 mbar in open glass vials in a vacuum drying cabinet from Memmert, Germany (vacuum drying). ). The glass vial was subsequently taken out of the vacuum drying cabinet, sealed, beaded, and stored for 4 weeks at about 40 ° C., after which stability was tested.

Stability for 3 months: After spray drying, the selected formulations were vacuum dried in an open glass vial (see description above). The glass vial was sealed under nitrogen, beaded and stored at three different temperatures: 2-8 ° C, 25 ° C and 40 ° C. After 1 month, the stability of the powder was tested.

Open Stability for 3 Months: After spray drying, the selected formulations were stored at 25 ° C. and about 29% and / or 43% relative humidity of air in open glass vials, respectively. After 1 month and 3 months, the stability of the powder was tested. In selected formulations, the aerodynamic performance of the powder was also tested by time of flight measurement (see description above).

Example 1

Spray drying of 10% (w / v) IgG1 formulation

Dilute the formulated concentration of about 109 mg / ml of pure IgG1 in glycine-histidine buffer at pH 6 to 100 mg / ml with demineralized water (pH about 7.5), optionally Cyclone I in the absence of other auxiliaries was spray dried as described above at atomization air flow rate of about 0.67 m 3 / h. The volume of this solution was 50 ml. Aggregate content was investigated as described above.

After 1 week of open storage at 75% relative humidity of air and 40 ° C. (forced storage stability), the solution prepared from the recombinant powder contained about 18.9% and 18.2% aggregates, respectively.

After equilibration at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombinant powder contained about 11.8% aggregates.

After vacuum drying at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombinant powder contained about 13.2% aggregates.

Spray drying of 3.33% (w / v) IgG1 formulation

Dilute pure IgG1 at a concentration of about 102.8 mg / mL formulated in glycine-histidine buffer at pH 6 (see "Substances") with demineralized water (pH about 7.5) to a content of 33 mg / mL, and any other auxiliaries. Using cyclone I in the absence of was spray dried as described above at atomized air flow rate of about 0.67 m 3 / h. The volume of this solution was 150 ml. Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 16.3% of aggregates.

After storage for 1 month and 3 months (stabilization for 3 months) at 2-8 ° C., the solution prepared from the recombinant powder contained about 4.5% and 4.4% aggregates, respectively.

After storage for 1 month and 3 months (stabilization for 3 months) at 25 ° C., the solution prepared from the recombined powder contained about 7.4% and 7.1% aggregates, respectively.

After 1 month and 3 months storage at 40 ° C. (stabilization for 3 months), the solution prepared from the recombined powder contained about 13.3% and 18.1% aggregates, respectively.

After storage for 1 month and 3 months at about 29% relative humidity of air and 25 ° C., the solution prepared from the recombined powder contained about 5.5% and 6.6% aggregates, respectively.

After storage for 1 month and 3 months at about 43% relative humidity of air and 25 ° C., the solution prepared from the recombined powder contained about 5.6% and 7.0% aggregates, respectively.

Spray drying of 9% (w / v) trehalose and 1% (w / v) IgG1 formulation

4.5 g of trehalose was dissolved in about 40 mL of demineralized water (pH about 7.5). As a next step, add about 4.6 mL of pure IgG1 at a concentration of about 109 mg / mL formulated in glycine-histidine buffer at pH 6 (see "Material" description), and with demineralized water (pH about 7.5) to a volume of 50 mL. Diluted. The resulting solution contained about 9% (w / v) adjuvant or matrix and 1% (w / v) protein, using cyclone I as described above at atomized air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 12.6% of aggregates.

Spray drying of 3.00% (w / v) LS90P and 0.33% (w / v) IgG1 formulations

4.5 g of LS90P was dissolved in about 140 mL of demineralized water (pH about 7.5). As a next step, add about 4.864 mL of pure IgG1 at a concentration of about 102.8 mg / mL formulated in glycine-histidine buffer at pH 6 (see "Material" description) and demineralized water (pH about 7.5) to a volume of 150 mL. Diluted with. The resulting solution contained about 3.00% (w / v) adjuvant or matrix and 0.33% (w / v) protein, using cyclone I as described above at atomizing air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 1.0% of aggregates.

After storage for 1 month and 3 months (stabilization for 3 months) at 2-8 ° C., the solution prepared from the recombinant powder contained about 0.6% and 0.9% aggregates, respectively.

After 1 month and 3 months storage at 25 ° C. (stabilization for 3 months), solutions prepared from the recombined powder contained about 0.8% and 1.3% aggregates, respectively.

After 1 month and 3 months of storage at 40 ° C. (stabilization for 3 months), the solution prepared from the recombined powder contained about 1.1% and 2.2% aggregates, respectively.

The MMD of the powder was measured as described above.

MMD of the powder after spray drying was 2.8 μm.

MMAD and FPF of the powders were measured as described above.

MMAD of the powder after spray drying was 3.8 μm, and FPF was 23.6% by weight of the powder capsule.

Spray drying of 9.9% (w / v) LS55P and 0.1% (w / v) IgG1 formulations

4.950 g of LS55P was dissolved in about 40 mL of demineralized water (pH about 7.5). As a next step, add about 0.518 ml of pure IgG1 at a concentration of about 96.55 mg / ml in glycine-histidine buffer (see "Material" description) at pH 6 and demineralized water (pH about 7.5) to a volume of 50 ml. Diluted with. The resulting solution contained about 9.9% (w / v) adjuvant or matrix and 0.1% (w / v) protein, using cyclone I as described above at atomizing air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombined powder contained about 5.7% aggregates.

After vacuum drying for 1 day at 40 ° C. and dry storage for 4 weeks, the solution prepared from the recombined powder contained about 4.7% aggregates.

Spray drying of 9% (w / v) LS55P and 1% (w / v) IgG1 formulations

4.5 g of LS55P was dissolved in about 40 mL of demineralized water (pH about 7.5). As a next step, add about 4.6 mL of pure IgG1 at a concentration of about 109 mg / mL formulated in glycine-histidine buffer at pH 6 (see "Material" description), and with demineralized water (pH about 7.5) to a volume of 50 mL. Diluted. The resulting solution contained about 9% (w / v) adjuvant or matrix and 1% (w / v) protein, using cyclone I as described above at atomized air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 2.3% of aggregates.

After equilibration at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombined powder contained about 1.8% aggregates.

After vacuum drying at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombinant powder contained about 1.4% aggregates.

Spray drying of 6% (w / v) LS55P and 4% (w / v) IgG1 formulations

3.0 g of LS55P was dissolved in about 15 mL of demineralized water (pH about 7.5). As a next step, add about 19.45 ml of pure IgG1 at a concentration of about 102.8 mg / ml formulated in glycine-histidine buffer at pH 6 (see "Material" description) and demineralized water (pH about 7.5) to a volume of 50 ml. Diluted with. The resulting solution contained about 6% (w / v) adjuvant or matrix and 4% (w / v) protein, using cyclone I as described above at atomized air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 4.0% of aggregates.

Spray drying of 4% (w / v) LS55P and 6% (w / v) IgG1 formulations

2.0 g of LS55P was dissolved in about 15 mL of demineralized water (pH about 7.5). As a next step, add about 29.18 ml of pure IgG1 at a concentration of about 102.8 mg / ml formulated in glycine-histidine buffer at pH 6 (see "Material" description) and demineralized water (pH about 7.5) to a volume of 50 ml. Diluted with. The resulting solution contained about 4% (w / v) adjuvant or matrix and 6% (w / v) protein, using cyclone I as described above at atomizing air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

 In view of storage stability, the following aggregate contents were obtained.

After a week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombined powder contained about 6.9% aggregates.

Spray drying of 2.5% (w / v) LS55P and 7.5% (w / v) IgG1 formulations

1.25 g of LS55P was dissolved in about 10 mL of demineralized water (pH about 7.5). As a next step, add about 38.84 mL of pure IgG1 at a concentration of about 96.55 mg / mL in glycine-histidine buffer (see "Material" description) at pH 6 and demineralized water (pH about 7.5) to a volume of 50 mL. Diluted with. The resulting solution contained about 2.5% (w / v) adjuvant or matrix and 7.5% (w / v) protein, using cyclone I as described above at atomized air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombined powder contained about 5.9% aggregates.

After vacuum drying at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombinant powder contained about 6.1% aggregates.

Spray drying of 1.0% (w / v) LS55P and 9.0% (w / v) IgG1 formulations

0.50 g of LS55P was dissolved in about 5 mL of demineralized water (pH about 7.5). As a next step, add about 41.43 ml of pure IgG1 at a concentration of about 96.55 mg / ml, formulated in glycine-histidine buffer at pH 6 (see "Material" description), and demineralized water (pH about 7.5) to a volume of 50 ml. Diluted with. The resulting solution contained about 1.0% (w / v) adjuvant or matrix and 9.0% (w / v) protein, using cyclone I as described above at atomizing air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 10.8% of aggregates.

After vacuum drying at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombinant powder contained about 8.0% aggregates.

Spray drying of 0.5% (w / v) LS55P and 9.5% (w / v) IgG1 formulations

0.25 g of LS55P was dissolved in about 2.5 mL of demineralized water (pH about 7.5). As a next step, add about 46.21 ml of pure IgG1 at a concentration of about 102.8 mg / ml formulated in glycine-histidine buffer at pH 6 (see "Material" description) and demineralized water (pH about 7.5) to a volume of 50 ml. Diluted with. The resulting solution contained about 0.5% (w / v) adjuvant or matrix and 9.5% (w / v) protein, using cyclone I as described above at atomizing air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

 In view of storage stability, the following aggregate contents were obtained.

After a week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombined powder contained about 13.7% of aggregates.

Spray drying of 3.00% (w / v) LS55P and 0.33% (w / v) IgG1 formulations

9.0 g of LS55P was dissolved in about 280 mL of demineralized water (pH about 7.5). As a next step, add about 9.73 ml of pure IgG1 at a concentration of about 102.8 mg / ml formulated in glycine-histidine buffer at pH 6 (see "Material" description) and demineralized water (pH about 7.5) to a volume of 300 ml. Diluted with. The resulting solution contained about 3.0% (w / v) adjuvant or matrix and 0.33% (w / v) protein, using cyclone II as described above at atomization air flow rate of about 0.67 m 3 / h Spray dried. Aggregate content was investigated as described above.

 In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 5.0% of aggregates.

The MMD of the powder was measured as described above.

MMD of the powder after spray drying was 2.9 μm.

MMAD and FPF of the powders were measured as described above.

MMAD of the powder after spray drying was 4.3 μm, and FPF was 15.9% by weight of the powder capsule.

Spray drying of 9.9% (w / v) coupling sugar and 0.1% (w / v) IgG1 formulation

6.290 g of coupling sugar containing syrup (corresponding to 4.950 g of coupling sugar) were dissolved in about 40 mL of demineralized water (pH about 7.5). As a next step, add about 0.518 ml of pure IgG1 at a concentration of about 96.55 mg / ml in glycine-histidine buffer (see "Material" description) at pH 6 and demineralized water (pH about 7.5) to a volume of 50 ml. Diluted with. The resulting solution contained about 9.9% (w / v) adjuvant or matrix and 0.1% (w / v) protein, using cyclone I as described above at atomizing air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above. In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 14.9% of aggregates.

Spray drying of 9% (w / v) coupling sugar and 1% (w / v) IgG1 formulation

5.71 g of coupling sugar containing syrup (corresponding to 4.5 g of coupling sugar) was dissolved in about 40 ml of demineralized water (pH about 7.5). As a next step, add about 4.6 mL of pure IgG1 at a concentration of about 109 mg / mL formulated in glycine-histidine buffer at pH 6 (see "Material" description), and with demineralized water (pH about 7.5) to a volume of 50 mL. Diluted. The resulting solution contained about 9% (w / v) adjuvant or matrix and 1% (w / v) protein, using cyclone I as described above at atomized air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 4.9% of aggregates.

After equilibration at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombined powder contained about 3.2% aggregates.

After vacuum drying at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombinant powder contained about 2.1% aggregates.

Spray drying of 6% (w / v) coupling sugar and 4% (w / v) IgG1 formulation

3.81 g of coupling sugar containing syrup (corresponding to 3.0 g of coupling sugar) was dissolved in about 25 ml of demineralized water (pH about 7.5). As a next step, add about 19.45 ml of pure IgG1 at a concentration of about 102.8 mg / ml formulated in glycine-histidine buffer at pH 6 (see "Material" description) and demineralized water (pH about 7.5) to a volume of 50 ml. Diluted with. The resulting solution contained about 6% (w / v) adjuvant or matrix and 4% (w / v) protein, using cyclone I as described above at atomized air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 5.0% of aggregates.

Spray drying of 4% (w / v) coupling sugar and 6% (w / v) IgG1 formulation

2.54 g of coupling sugar-containing syrup (corresponding to 2.0 g of coupling sugar) was dissolved in about 15 mL of demineralized water (pH about 7.5). As a next step, add about 29.18 ml of pure IgG1 at a concentration of about x mg / ml formulated in glycine-histidine buffer at pH 6 (see "Material" description) and demineralized water (pH about 7.5) to a volume of 50 ml. Diluted with. The resulting solution contained about 4% (w / v) adjuvant or matrix and 6% (w / v) protein, using cyclone I as described above at atomizing air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above. In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombined powder contained about 9.9% aggregates.

Spray drying of 2.5% (w / v) coupling sugar and 7.5% (w / v) IgG1 formulation

1.59 g of coupling sugar containing syrup (corresponding to 1.250 g of coupling sugar) was dissolved in about 8 ml of demineralized water (pH about 7.5). As a next step, add about 38.84 mL of pure IgG1 at a concentration of about 96.56 mg / mL in glycine-histidine buffer (see "Material" description) at pH 6 and demineralized water (pH about 7.5) to a volume of 50 mL. Diluted with. The resulting solution contained about 2.5% (w / v) adjuvant or matrix and 7.5% (w / v) protein, using cyclone I as described above at atomized air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 10.8% of aggregates.

Spray drying of 1.0% (w / v) coupling sugar and 9.0% (w / v) IgG1 formulation

0.653 g of coupling sugar-containing syrup (corresponding to 0.50 g of coupling sugar) was dissolved in about 5 ml of demineralized water (pH about 7.5). As a next step, add about 41.43 ml of pure IgG1 at a concentration of about 96.56 mg / ml in glycine-histidine buffer (see "Material" description) at pH 6 and demineralized water (pH about 7.5) to a volume of 50 ml. Diluted with. The resulting solution contained about 1.0% (w / v) adjuvant or matrix and 9.0% (w / v) protein, using cyclone I as described above at atomizing air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above. In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 13.1% aggregates.

Spray drying of 9% (w / v) coupling sugar S and 1% (w / v) IgG1 formulation

5.86 g of coupling sugar S containing syrup (corresponding to 4.5 g of coupling sugar S) was dissolved in about 40 mL of demineralized water (pH about 7.5). As a next step, add about 4.6 mL of pure IgG1 at a concentration of about 109 mg / mL formulated in glycine-histidine buffer at pH 6 (see "Material" description), and with demineralized water (pH about 7.5) to a volume of 50 mL. Diluted. The resulting solution contained about 9% (w / v) adjuvant or matrix and 1% (w / v) protein, using cyclone I as described above at atomized air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombined powder contained about 5.4% aggregates.

Example 2

Spray drying of 8% (w / v) trehalose, 1% (w / v) L-isoleucine and 1% (w / v) IgG1 formulation

In the ultrasonic bath, 4.0 g of trehalose and 0.5 g of L-isoleucine were dissolved in about 40 mL of demineralized water (pH about 7.5). As a next step, add about 4.6 mL of pure IgG1 at a concentration of about 109 mg / mL formulated in pH 6 glycine-histidine buffer (see "Material" description) and dilute with demineralized water (pH about 7.5) to a volume of 50 mL. I was. The resulting solution contained about 9% (w / v) adjuvant or matrix and 1% (w / v) protein, using cyclone I as described above at atomized air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 22.2% of aggregates.

Spray drying of 2.66% (w / v) LS90P, 0.33% (w / v) L-isoleucine and 0.33% (w / v) IgG1 formulations

In an ultrasonic bath, 4.0 g of LS90P and 0.50 g of L-isoleucine were dissolved in about 140 mL of demineralized water (pH about 7.5). As a next step, add about 4.864 ml of pure IgG1 at a concentration of about 102.8 mg / ml formulated in pH 6 glycine-histidine buffer (see "Material" description), and with demineralized water (pH about 7.5) to a volume of 150 ml. Diluted. The resulting solution contained about 3.00% (w / v) adjuvant or matrix and 0.33% (w / v) protein, using cyclone II as described above at atomization air flow rate of about 0.67 m 3 / h Spray dried. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombined powder contained about 0.7% aggregates.

After storage for 1 month and 3 months (stabilization for 3 months) at 2-8 ° C., the solution prepared from the recombinant powder contained about 0.7% and 1.0% aggregates, respectively.

After storage for 1 month and 3 months (stabilization for 3 months) at 25 ° C., the solution prepared from the recombined powder contained about 0.8% and 1.1% aggregates, respectively.

After 1 month and 3 months storage at 40 ° C. (stabilization for 3 months), the solution prepared from the recombined powder contained about 0.6% and 1.1% aggregates, respectively.

MMAD and FPF of the powders were measured as described above.

MMAD of the powder after spray drying was 7.3 μm, and FPF was 28.1% by weight of the powder capsule.

Spray drying of 8% (w / v) LS55P, 1% (w / v) L-isoleucine and 1% (w / v) IgG1 formulations

In the ultrasonic bath, 4.00 g of LS90P and 0.50 g of L-isoleucine were dissolved in about 40 mL of demineralized water (pH about 7.5). As a next step, add about 4.60 ml of pure IgG1 at a concentration of about 109 mg / ml in glycine-histidine buffer (see "Substances" description) at pH 6, and with demineralized water (pH about 7.5) to a volume of 50 ml. Diluted. The resulting solution contained about 9% (w / v) adjuvant or matrix and 1% (w / v) protein, using cyclone I as described above at atomized air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 5.5% of aggregates.

After equilibration at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombined powder contained about 1.8% aggregates.

After vacuum drying at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombined powder contained about 1.8% aggregates.

Spray drying of 2.66% (w / v) LS55P, 0.33% (w / v) L-isoleucine and 0.33% (w / v) IgG1 formulations

In an ultrasonic bath, 8.0 g of LS55P and 1 g of L-isoleucine were dissolved in about 280 mL of demineralized water (pH about 7.5). As a next step, add about 9.7 mL of pure IgG1 at a concentration of about 102.8 mg / mL formulated in pH 6 glycine-histidine buffer (see "Material" description) and add demineralized water (pH about 7.5) to a volume of 300 mL. Diluted. The resulting solution contained about 3% (w / v) adjuvant or matrix and 0.33% (w / v) protein, using cyclone II as described above at atomization air flow rate of about 0.67 m 3 / h Spray dried. Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombined powder contained about 5.9% aggregates.

After storage for 1 month and 3 months (stabilization for 3 months) at 2-8 ° C., the solution prepared from the recombinant powder contained about 1.6% of aggregates, respectively.

After 1 month and 3 months storage at 25 ° C. (stabilization for 3 months), solutions prepared from the recombined powder contained about 1.6% and 1.8% aggregates, respectively.

After 1 month and 3 months storage at 40 ° C. (stabilization for 3 months), the solution prepared from the recombined powder contained about 1.6% and 1.8% aggregates, respectively.

MMAD and FPF of the powders were measured as described above.

MMAD of the powder after spray drying was 4.9 μm, and FPF was 34.7% by weight of the powder capsule.

Spray drying of 8% (w / v) coupling sugar, 1% (w / v) L-isoleucine and 1% (w / v) IgG1 formulation

In an ultrasonic bath, 5.08 g of coupling sugar-containing syrup (corresponding to 4.0 g of coupling sugar) and 0.50 g of L-isoleucine were dissolved in about 40 mL of demineralized water (pH about 7.5). As a next step, add about 4.60 ml of pure IgG1 at a concentration of about 109 mg / ml in glycine-histidine buffer (see "Substances" description) at pH 6, and with demineralized water (pH about 7.5) to a volume of 50 ml. Diluted. The resulting solution contained about 9% (w / v) adjuvant or matrix and 1% (w / v) protein, using cyclone I as described above at atomized air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombined powder contained about 7.1% aggregates.

After equilibration at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombinant powder contained about 2.5% aggregates.

After vacuum drying at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombined powder contained about 1.1% aggregates.

Spray drying of 8% (w / v) coupling sugar S, 1% (w / v) L-isoleucine and 1% (w / v) IgG1 formulation

In the ultrasonic bath, 5.21 g of coupling sugar S-containing syrup (corresponding to 4.0 g of coupling sugar S) and 0.50 g of L-isoleucine were dissolved in about 40 mL of demineralized water (pH about 7.5). As a next step, add about 4.60 ml of pure IgG1 at a concentration of about 109 mg / ml in glycine-histidine buffer (see "Substances" description) at pH 6, and with demineralized water (pH about 7.5) to a volume of 50 ml. Diluted. The resulting solution contained about 9% (w / v) adjuvant or matrix and 1% (w / v) protein, using cyclone I as described above at atomized air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 6.8% of aggregates.

Example 3

Spray drying of 3% (w / v) trehalose, 6% (w / v) L-citrulline and 1% (w / v) IgG1 formulation

In the ultrasonic bath, 1.50 g of trehalose and 3.00 g of L-citrulline were dissolved in about 40 mL of demineralized water (pH about 7.5). As a next step, add about 4.6 mL of pure IgG1 at a concentration of about 109 mg / mL formulated in pH 6 glycine-histidine buffer (see "Material" description) and dilute with demineralized water (pH about 7.5) to a volume of 50 mL. I was. The resulting solution contained about 9% (w / v) adjuvant or matrix and 1% (w / v) protein, using cyclone I as described above at atomized air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombined powder contained about 5.9% aggregates.

Spray drying of 3% (w / v) LS55P, 6% (w / v) L-citrulline and 1% (w / v) IgG1 formulation

In the ultrasonic bath, 1.50 g of LS55P and 3.00 g of L-citrulline were dissolved in about 40 mL of demineralized water (pH about 7.5). As a next step, add about 4.60 ml of pure IgG1 at a concentration of about 109 mg / ml in glycine-histidine buffer (see "Substances" description) at pH 6, and with demineralized water (pH about 7.5) to a volume of 50 ml. Diluted. The resulting solution contained about 9% (w / v) adjuvant or matrix and 1% (w / v) protein, using cyclone I as described above at atomized air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 2.9% aggregates.

After equilibration at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombined powder contained about 1.8% aggregates.

After vacuum drying at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombinant powder contained about 1.3% of aggregates.

Spray drying of 3% (w / v) coupling sugar, 6% L-citrulline and 1% (w / v) IgG1 formulation

In the ultrasonic bath, 1.91 g of coupling sugar containing syrup (corresponding to 1.5 g of coupling sugar) and 3.00 g of L-citrulline were dissolved in about 40 ml of demineralized water (pH about 7.5). As a next step, add about 4.60 ml of pure IgG1 at a concentration of about 109 mg / ml in glycine-histidine buffer (see "Substances" description) at pH 6, and with demineralized water (pH about 7.5) to a volume of 50 ml. Diluted. The resulting solution contained about 9% (w / v) adjuvant or matrix and 1% (w / v) protein, using cyclone I as described above at atomized air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombined powder contained about 3.9% aggregates.

After equilibration at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombinant powder contained about 1.4% aggregates.

After vacuum drying at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombinant powder contained about 1.3% of aggregates.

Spray drying of 3% (w / v) coupling sugar S, 6% L-citrulline and 1% (w / v) IgG1 formulation

In the ultrasonic bath, 1.95 g of coupling sugar S-containing syrup (corresponding to 1.5 g of coupling sugar S) and 3.00 g of L-citrulline were dissolved in about 40 mL of demineralized water (pH about 7.5). As a next step, add about 4.60 ml of pure IgG1 at a concentration of about 109 mg / ml in glycine-histidine buffer (see "Substances" description) at pH 6, and with demineralized water (pH about 7.5) to a volume of 50 ml. Diluted. The resulting solution contained about 9% (w / v) adjuvant or matrix and 1% (w / v) protein, using cyclone I as described above at atomized air flow rate of about 0.67 m 3 / h. Spray dried. Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 3.1% of aggregates.

Example 4

Spray drying of 2.66% (w / v) trehalose, 0.33% (w / v) tri-isoleucine 0.33% (w / v) IgG1 formulation

8.0 g trehalose and 1 g tri-isoleucine were dissolved in about 280 ml of demineralized water (pH about 7.5) in an ultrasonic bath. As a next step, formulated in pure IgG1 pH 6 glycine-histidine buffer (see "Material" description) at about 12.30 mL concentration of about 96.55 mg / mL, diluted with demineralized water (pH about 7.5) to a volume of 300 mL. I was. The resulting solution contained about 3% (w / v) adjuvant or matrix and 0.33% (w / v) protein, using cyclone II as described above at atomization air flow rate of about 0.67 m 3 / h Spray dried. Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 26.7% of aggregates.

Spray drying of 2.66% (w / v) raffinose, 0.33% (w / v) tri-isoleucine and 0.33% (w / v) IgG1 formulations

In the ultrasonic bath, 8.0 g of raffinose and 1 g of tri-isoleucine were dissolved in about 140 mL of demineralized water (pH about 7.5). As a next step, add about 4.87 ml of pure IgG1 at a concentration of about 102.8 mg / ml formulated in pH 6 glycine-histidine buffer (see "Material" description), and with demineralized water (pH about 7.5) to a volume of 300 ml. Diluted. The resulting solution contained about 3% (w / v) adjuvant or matrix and 0.33% (w / v) protein, using cyclone II as described above at atomization air flow rate of about 0.67 m 3 / h Spray dried. Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 12.6% of aggregates.

Spray drying of 2.66% (w / v) hydroxyethyl starch (HES), 0.33% (w / v) tri-isoleucine and 0.33% (w / v) IgG1 formulations

While stirring at about 80 ° C. in an ultrasonic bath, 8.0 g of HES and 1 g of tri-isoleucine were dissolved in about 140 mL of demineralized water (pH about 7.5). As a next step, about 4.87 ml of pure IgG1 at a concentration of about 102.8 mg / ml formulated in glycine-histidine buffer (see "Substances" description) at pH 6 is added to the previously frozen turbid solution to a volume of 150 ml. Diluted with demineralized water (pH ca. 7.5). The resulting solution contained about 3% (w / v) adjuvant or matrix and 0.33% (w / v) protein, using cyclone II as described above at atomization air flow rate of about 0.67 m 3 / h Spray dried. Aggregate content was investigated as described above.

After one week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombined powder contained about 18.6% aggregates.

After 1 month and 3 months of open storage at about 43% relative humidity and 25 ° C. of air (open stabilization for 3 months), the solution prepared from the recombinant powder contained about 11.9% and 15.4% aggregates, respectively.

Spray drying of 2.00% (w / v) saccharose, 0.66% (w / v) lactose, 0.33% (w / v) tri-isoleucine and 0.33% (w / v) IgG1 formulations

In an ultrasonic bath, 6.0 g of saccharose, 2.0 g of lactose and 1 g of tri-isoleucine were dissolved in about 280 ml of demineralized water (pH about 7.5). As a next step, add about 9.73 ml of pure IgG1 at a concentration of about 102.8 mg / ml formulated in glycine-histidine buffer at pH 6 (see "Material" description) and demineralized water (pH about 7.5) to a volume of 300 ml. Diluted with. The resulting solution contained about 3% (w / v) adjuvant or matrix and 0.33% (w / v) protein, using cyclone II as described above at atomization air flow rate of about 0.67 m 3 / h Spray dried. Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 8.8% of aggregates.

Spray drying of 2.66% (w / v) saccharose, 0.33% (w / v) tri-isoleucine and 0.33% (w / v) IgG1 formulations

In the ultrasonic bath, 8.0 g saccharose and 1 g tri-isoleucine were dissolved in about 280 mL of demineralized water (pH about 7.5). As a next step, add about 9.73 ml of pure IgG1 at a concentration of about 102.8 mg / ml formulated in pH 6 glycine-histidine buffer (see "Material" description), and with demineralized water (pH about 7.5) to a volume of 300 ml. Diluted. The resulting solution contained about 3% (w / v) adjuvant or matrix and 0.33% (w / v) protein, using cyclone II as described above at atomization air flow rate of about 0.67 m 3 / h Spray dried. Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombined powder contained about 5.6% aggregates.

Spray drying of 2.66% (w / v) LS90P, 0.33% (w / v) tri-isoleucine and 0.33% (w / v) IgG1 formulations

4.0 g of LS90P and 0.50 g of tri-isoleucine were dissolved in about 140 mL of demineralized water (pH about 7.5). As a next step, add about 4.864 mL of pure IgG1 at a concentration of about 102.8 mg / mL formulated in glycine-histidine buffer at pH 6 (see "Material" description) and demineralized water (pH about 7.5) to a volume of 150 mL. Diluted with. The resulting solution contained about 3.00% (w / v) adjuvant or matrix and 0.33% (w / v) protein, using cyclone II as described above at atomization air flow rate of about 0.67 m 3 / h Spray dried. Aggregate content was investigated as described above. In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 2.3% of aggregates.

After storage for 1 month and 3 months (stabilization for 3 months) at 2-8 ° C., the solution prepared from the recombinant powder contained about 0.7% and 1.0% aggregates, respectively.

After storage for 1 month and 3 months (stabilization for 3 months) at 25 ° C., the solution prepared from the recombinant powder contained about 0.8% and 1.4% aggregates, respectively.

After storage for 1 month and 3 months (stabilization for 3 months) at 40 ° C., the solution prepared from the recombined powder contained about 0.9% and 2.2% aggregates, respectively.

After 1 month and 3 months of open storage at about 29% relative humidity of air and 25 ° C., the solution prepared from the recombinant powder contained about 0.4% and 0.7% aggregates, respectively.

After 1 month and 3 months of open storage at about 43% relative humidity of air and 25 ° C., the solution prepared from the recombinant powder contained about 0.5% and 0.6% aggregates, respectively.

The MMD of the powder was measured as described above.

MMD of the powder after spray drying was 4.7 μm.

MMAD and FPF of the powders were measured as described above.

MMAD of the powder after spray drying was 4.8 μm, and FPF was 53.2% based on the powder capsule weight.

Spray drying of 2.66% (w / v) LS90P, 0.33% (w / v) tri-isoleucine and 0.33% (w / v) IgG1 formulations

4.0 g of LS90P and 0.50 g of tri-isoleucine were dissolved in about 140 mL of demineralized water (pH about 7.5). As a next step, add about 4.864 mL of pure IgG1 at a concentration of about 102.8 mg / mL formulated in glycine-histidine buffer at pH 6 (see "Material" description) and demineralized water (pH about 7.5) to a volume of 150 mL. Diluted with. The resulting solution contained about 3.00% (w / v) adjuvant or matrix and 0.33% (w / v) protein and spray dried as described above at about 1.05 m 3 / h of air flow using Cyclone II. I was. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 2.2% of aggregates.

After storage for 1 month and 3 months (open stabilization for 3 months) at about 29% relative humidity and 25 ° C. of air, the solution prepared from the recombinant powder contained about 0.5% and 0.6% aggregates, respectively.

After storage for 1 month and 3 months (relative stabilization for 3 months) at about 43% relative humidity of air and 25 ° C., the solution prepared from the recombinant powder contained about 0.5% and 0.7% aggregates, respectively.

The MMD of the powder was measured as described above.

MMD of the powder after spray drying was 2.7 μm.

MMAD and FPF of the powders were measured as described above.

MMAD of the powder after spray drying was 3.6 탆 and FPF was 58.0% by weight of the powder capsule.

Spray drying of 2.66% (w / v) LS90P, 0.33% (w / v) tri-isoleucine and 0.33% (w / v) IgG1 formulations

4.0 g of LS90P and 0.50 g of tri-isoleucine were dissolved in about 140 mL of demineralized water (pH about 7.5). As a next step, add about 4.864 mL of pure IgG1 at a concentration of about 102.8 mg / mL formulated in glycine-histidine buffer at pH 6 (see "Material" description) and demineralized water (pH about 7.5) to a volume of 150 mL. Diluted with. The resulting solution contained about 3.00% (w / v) adjuvant or matrix and 0.33% (w / v) protein and spray dried as described above at about 1.74 m 3 / h of air flow using Cyclone II. I was. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about ??% aggregates.

After storage for 1 month and 3 months (open stabilization for 3 months) at about 29% relative humidity and 25 ° C. of air, the solution prepared from the recombinant powder contained about 0.5% and 0.6% aggregates, respectively.

After storage for 1 month and 3 months (open stabilization for 3 months) at about 43% relative humidity of air and 25 ° C., the solution prepared from the recombinant powder contained about 0.5% and 0.8% aggregates, respectively.

The MMD of the powder was measured as described above.

MMD of the powder after spray drying was 2.6 탆.

MMAD and FPF of the powders were measured as described above.

MMAD of the powder after spray drying was 3.3 μm and FPF was 58.9% based on the powder capsule weight.

Spray drying of 1.60% (w / v) LS90P, 0.20% (w / v) tri-isoleucine and 0.20% (w / v) IgG1 formulations

4.0 g of LS90P and 0.50 g of tri-isoleucine were dissolved in about 220 mL of demineralized water (pH about 7.5). As a next step, add about 4.864 mL of pure IgG1 at a concentration of about 102.8 mg / mL formulated in glycine-histidine buffer at pH 6 (see "Material" description) and demineralized water (pH about 7.5) to a volume of 250 mL. Diluted with. The resulting solution contained about 1.80% (w / v) adjuvant or matrix and 0.20% (w / v) protein, using cyclone II as described above at atomization air flow rate of about 0.67 m 3 / h Spray dried. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 2.2% of aggregates.

After storage for 1 month and 3 months (open stabilization for 3 months) at about 29% relative humidity and 25 ° C. of air, the solution prepared from the recombinant powder contained about 0.5% and 0.5% aggregates, respectively.

After storage for 1 month and 3 months (open stabilization for 3 months) at about 43% relative humidity of air and 25 ° C., the solution prepared from the recombinant powder contained about 0.7% and 0.7% aggregates, respectively.

The MMD of the powder was measured as described above.

MMD of the powder after spray drying was 3.2 μm.

MMAD and FPF of the powders were measured as described above.

MMAD of the powder after spray drying was 3.9 μm and FPF was 55.6% by weight of the powder capsule.

Spray drying of 2.833% (w / v) LS90P, 0.166% (w / v) tri-isoleucine and 0.33% (w / v) IgG1 formulations

4.25 g of LS90P and 0.25 g of tri-isoleucine were dissolved in about 140 mL of demineralized water (pH about 7.5). As a next step, add about 4.864 mL of pure IgG1 at a concentration of about 102.8 mg / mL formulated in glycine-histidine buffer at pH 6 (see "Material" description) and demineralized water (pH about 7.5) to a volume of 150 mL. Diluted with. The resulting solution contained about 3.00% (w / v) adjuvant or matrix and 0.33% (w / v) protein, using cyclone II as described above at atomization air flow rate of about 0.67 m 3 / h Spray dried. Aggregate content was investigated as described above. In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombined powder contained about 1.5% of aggregates.

After storage for 1 month and 3 months (open stabilization for 3 months) at about 29% relative humidity and 25 ° C. of air, the solution prepared from the recombinant powder contained about 0.5% and 0.5% aggregates, respectively.

After storage for 1 month and 3 months (open stabilization for 3 months) at about 43% relative humidity of air and 25 ° C., the solution prepared from the recombinant powder contained about 0.5% and 0.6% aggregates, respectively.

The MMD of the powder was measured as described above.

MMD of the powder after spray drying was 4.8 μm.

MMAD and FPF of the powders were measured as described above.

MMAD of the powder after spray drying was 5.2 μm and FPF was 45.7% by weight of the powder capsule.

Spray drying of 2.9166% (w / v) LS90P, 0.0833% (w / v) tri-isoleucine and 0.33% (w / v) IgG1 formulations

4.375 g of LS90P and 0.125 g of tri-isoleucine were dissolved in about 140 mL of demineralized water (pH about 7.5). As a next step, add about 4.864 mL of pure IgG1 at a concentration of about 102.8 mg / mL formulated in glycine-histidine buffer at pH 6 (see "Material" description) and demineralized water (pH about 7.5) to a volume of 150 mL. Diluted with. The resulting solution contained about 3.00% (w / v) adjuvant or matrix and 0.33% (w / v) protein, using cyclone II as described above at atomization air flow rate of about 0.67 m 3 / h Spray dried. Aggregate content was investigated as described above. In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 1.2% of aggregates.

After storage for 1 month and 3 months (relative stabilization for 3 months) at about 29% relative humidity and 25 ° C. of air, the solution prepared from the recombinant powder contained about 0.4% and 0.5% aggregates, respectively.

After storage for 1 month and 3 months (open stabilization for 3 months) at about 43% relative humidity of air and 25 ° C., the solution prepared from the recombinant powder contained about 0.5% and 0.6% aggregates, respectively.

The MMD of the powder was measured as described above.

MMD of the powder after spray drying was 4.2 탆.

MMAD and FPF of the powders were measured as described above.

MMAD of the powder after spray drying was 6.1 μm and FPF was 39.6% by weight of the powder capsule.

Spray drying of 2.66% (w / v) LS55P, 0.33% (w / v) tri-isoleucine and 0.33% (w / v) IgG1 formulations

8.0 g of LS55P and 1 g of tri-isoleucine were dissolved in about 280 mL of demineralized water (pH about 7.5) in an ultrasonic bath. As a next step, add about 9.73 ml of pure IgG1 at a concentration of about 102.8 mg / ml formulated in pH 6 glycine-histidine buffer (see "Material" description), and with demineralized water (pH about 7.5) to a volume of 300 ml. Diluted. The resulting solution contained about 3% (w / v) adjuvant or matrix and 0.33% (w / v) protein, using cyclone II as described above at atomization air flow rate of about 0.67 m 3 / h Spray dried. Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 2.1% aggregates.

After storage for 1 month and 3 months (stabilization for 3 months) at 2-8 ° C., the solution prepared from the recombinant powder contained about 0.8% and 1.5% aggregates, respectively.

After storage for 1 month and 3 months (stabilization for 3 months) at 25 ° C., the solution prepared from the recombined powder contained about 0.9% and 1.5% aggregates, respectively.

After 1 month and 3 months storage at 40 ° C. (stabilization for 3 months), the solution prepared from the recombined powder contained about 1.3% and 2.6% aggregates, respectively.

After storage for 1 month and 3 months (open stabilization for 3 months) at about 43% relative humidity of air and 25 ° C., the solution prepared from the recombinant powder contained about 1.0% and 1.0% aggregates, respectively.

The MMD of the powder was measured as described above.

MMD of the powder after spray drying was 3.4 μm.

MMAD and FPF of the powders were measured as described above.

MMAD of the powder after spray drying was 3.9 μm, and FPF was 58.3% based on the powder capsule weight.

After 1 month of storage at about 43% relative humidity of air and 25 ° C. (open stabilization over 3 months), MMAD was 3.8 μm and FPF was 59.6% by weight of powder capsule.

Spray drying of 2.833% (w / v) LS55P, 0.166% (w / v) tri-isoleucine and 0.33% (w / v) IgG1 formulations

8.5 g of LS90P and 0.5 g of tri-isoleucine were dissolved in about 280 mL of demineralized water (pH about 7.5) in an ultrasonic bath. As a next step, add about 9.73 ml of pure IgG1 at a concentration of about 102.8 mg / ml formulated in pH 6 glycine-histidine buffer (see "Material" description), and with demineralized water (pH about 7.5) to a volume of 300 ml. Diluted. The resulting solution contained about 3% (w / v) adjuvant or matrix and 0.33% (w / v) protein, using cyclone II as described above at atomization air flow rate of about 0.67 m 3 / h Spray dried. Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 3.4% of aggregates.

After 1 month and 3 months of storage at about 43% relative humidity and 25 ° C. of air (open stabilization for 3 months), the solution prepared from the recombinant powder contained about 1.3% and 1.5% aggregates, respectively.

The MMD of the powder was measured as described above.

MMD of the powder after spray drying was 2.9 μm.

MMAD and FPF of the powders were measured as described above.

MMAD of the powder after spray drying was 4.4 μm and FPF was 58.6% by weight of the powder capsule.

Spray drying of 2.9166% (w / v) LS55P, 0.0833% (w / v) tri-isoleucine and 0.33% (w / v) IgG1 formulations

8.75 g of LS90P and 0.25 g of tri-isoleucine were dissolved in about 280 mL of demineralized water (pH about 7.5). As a next step, add about 9.73 ml of pure IgG1 at a concentration of about 102.8 mg / ml formulated in glycine-histidine buffer at pH 6 (see "Material" description) and demineralized water (pH about 7.5) to a volume of 300 ml. Diluted with. The resulting solution contained about 3% (w / v) adjuvant or matrix and 0.33% (w / v) protein, using cyclone II as described above at atomization air flow rate of about 0.67 m 3 / h Spray dried. Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 4.4% of aggregates.

After storage for 1 month and 3 months (open stabilization for 3 months) at about 43% relative humidity of air and 25 ° C., the solution prepared from the recombinant powder contained about 0.7% and 0.8% aggregates, respectively.

The MMD of the powder was measured as described above.

MMD of the powder after spray drying was 2.9 μm.

MMAD and FPF of the powders were measured as described above.

MMAD of the powder after spray drying was 4.4 μm and FPF was 58.6% by weight of the powder capsule.

Example 5

Preparation of further powders according to the invention

Spray Drying of a 3.33% (w / v) Lysozyme Formulation

5 g of lysozyme was dissolved in about 140 mL of demineralized water (pH about 7.5) and diluted with demineralized water (pH about 7.5) to a volume of 150 mL. The resulting solution was spray dried using cyclone II as described above.

Residual monomer content was measured as described above. After forced storage, the solution prepared from the recombinant powder had a residual monomer content of 35.3%. The MMD of the powder was measured as described above. The MMD of the powder was 3.2 μm. MMAD and FPF of the powders were measured as described above. MMAD was 4.0 μm and FPF was 70.4% by weight of the powder capsule.

Spray drying of 3.00% (w / v) LS90P and 0.33% (w / v) lysozyme formulations

In an ultrasonic bath, 9.0 g of LS90P was dissolved in about 280 ml of demineralized water (pH about 7.5). As a next step, 1 g of lysozyme was added and diluted with demineralized water (pH about 7.5) to a volume of 300 ml. The resulting solution was spray dried using cyclone II as described above.

Residual monomer content was measured as described above. After forced storage, the solution prepared from the recombinant powder had a residual monomer content of 62.1%. The MMD of the powder was measured as described above. The MMD of the powder was 4.0 μm. MMAD and FPF of the powders were measured as described above. MMAD was 3.7 μm and FPF was 24.7% by weight of the powder capsule.

Spray drying of 2.66% (w / v) LS90P, 0.33% (w / v) isoleucine and 0.33% (w / v) lysozyme formulations

In the ultrasonic bath, 8.0 g of LS90P and 1 g of isoleucine were dissolved in about 280 mL of demineralized water (pH about 7.5). As a next step, 1 g lysozyme was added and diluted with demineralized water (pH about 7.5) to a volume of 300 ml. The resulting solution was spray dried using cyclone II as described above.

Residual monomer content was measured as described above. After forced storage, the solution prepared from the recombinant powder had a residual monomer content of 47.9%. The MMD of the powder was measured as described above. The MMD of the powder was 3.9 μm. MMAD and FPF of the powders were measured as described above. MMAD was 4.1 μm and FPF was 29.0% by weight of the powder capsule.

Spray drying of 2.66% (w / v) LS90P, 0.33% (w / v) tri-isoleucine and 0.33% (w / v) lysozyme formulations

In the ultrasonic bath, 8.0 g of LS90P and 1 g of tri-isoleucine were dissolved in about 280 mL of demineralized water (pH about 7.5). As a next step, 1 g of lysozyme was added and diluted with demineralized water (pH about 7.5) to a volume of 300 ml. The resulting solution was spray dried using cyclone II as described above.

Residual monomer content was measured as described above. After forced storage, the solution prepared from the recombinant powder had a residual monomer content of 47.9%. The MMD of the powder was measured as described above. The MMD of the powder was 2.7 μm. MMAD and FPF of the powders were measured as described above. MMAD was 3.6 μm and FPF was 58.6% by weight of the powder capsule.

Spray Drying of a 3.33% (w / v) Calcitonin Formulation

1 g of calcitonin was dissolved in about 25 ml of demineralized water (pH about 7.5) and diluted with demineralized water (pH about 7.5) to a volume of 30 ml. The resulting solution was spray dried using cyclone II as described above.

Aggregate content was investigated as described above.

After 3 months storage at 2-8 ° C. (stabilization for 3 months), solutions prepared from the recombined powder contained about 4.1% of aggregates each.

After 3 months storage at 25 ° C. (stabilization for 3 months), solutions prepared from the recombined powder contained about 4.9% of aggregates respectively.

After 3 months storage at 40 ° C. (stabilization for 3 months), solutions prepared from the recombinant powders contained about 7.4% of aggregates each.

MMAD and FPF of the powders were measured as described above. MMAD was 3.9 μm and FPF was 59.0% by weight of the powder capsule.

Spray Drying of 3.166% (w / v) LS90P and 0.166% (w / v) Calcitonin Formulations

4.750 g of LS90P was dissolved in about 140 mL of demineralized water (pH 5 about 7.5). As a next step, 0.250 g of calcitonin was added and diluted with demineralized water (pH about 7.5) to a volume of 150 ml. The resulting solution was spray dried using cyclone II as described above.

Aggregate content was investigated as described above.

After storage for 3 months (stabilization for 3 months) at 2-8 ° C., solutions prepared from the recombined powder contained about 3.6% of aggregates each.

After 3 months storage at 25 ° C. (stabilization for 3 months), the solutions prepared from the recombined powder contained about 3.9% of aggregates each.

After 3 months storage at 40 ° C. (stabilization for 3 months), the solutions prepared from the recombined powder contained about 4.6% of aggregates respectively.

The MMD of the powder was measured as described above. The MMD of the powder was 2.6 μm. MMAD and FPF of the powders were measured as described above. MMAD was 4.3 μm and FPF was 47.3% by weight of the powder capsule.

Spray drying of 2.833% (w / v) LS90P, 0.33% (w / v) isoleucine and 0.166% (w / v) calcitonin formulation

In the ultrasonic bath, 4.250 g of LS90P and 0.50 g of isoleucine were dissolved in about 140 mL of demineralized water (pH about 7.5). As a next step, 0.250 g of calcitonin was added and diluted with demineralized water (pH about 7.5) to a volume of 150 ml. The resulting solution was spray dried using a cyclone II roll as described above.

Aggregate content was investigated as described above.

After 3 months storage at 2-8 ° C. (stabilization for 3 months), solutions prepared from the recombined powder contained about 3.3% of aggregates each.

After 3 months storage at 25 ° C. (stabilization for 3 months), solutions prepared from the recombined powder contained about 3.6% of aggregates each.

After 3 months storage at 40 ° C. (stabilization for 3 months), solutions prepared from the recombinant powders contained about 3.6% of aggregates each.

The MMD of the powder was measured as described above. MMD of the powder was 2.8 μm. MMAD and FPF of the powders were measured as described above. MMAD was 4.4 μm and FPF was 49.2% by weight of the powder capsule.

Spray Drying of 2.866% (w / v) LS90P, 0.33% (w / v) Tri-Isoleucine and 0.166% (w / v) Calcitonin Formulations

In the ultrasonic bath, 4.250 g of LS90P and 0.50 g of tri-isoleucine were dissolved in about 140 mL of demineralized water (pH about 7.5). As a next step, 0.250 g of calcitonin was added and diluted with demineralized water (pH about 7.5) to a volume of 150 ml. The resulting solution was spray dried using cyclone II as described above.

Aggregate content was investigated as described above.

After 3 months storage at 2-8 ° C. (stabilization for 3 months), solutions prepared from the recombined powder contained about 3.3% of aggregates each.

After 3 months storage at 25 ° C. (stabilization for 3 months), solutions prepared from the recombined powder contained about 3.6% of aggregates each.

After 3 months storage at 40 ° C. (stabilization for 3 months), solutions prepared from the recombined powder contained about 3.9% aggregates each.

The MMD of the powder was measured as described above. The MMD of the powder was 2.5 μm. MMAD and FPFF of the powders were measured as described above. MMAD was 3.5 μm and FPF was 60.4% by weight of the powder capsule.

Example 6

Freeze drying of 5% (w / v) IgG1 formulation

In a pH 6 glycine-histidine buffer (see "Material" description), formulated concentrations of about 109 mg / ml of pure IgG1 are diluted with demineralized water (pH about 7.5) to a content of 50 mg / ml and the absence of any other auxiliaries. Under freeze drying. The volume of this solution was 50 ml and distributed in commercially available 2R vials before lyophilization. Lyophilisate was lyophilized in 2R vials using Spatura and further treated as described above.

Aggregate content was investigated as described above.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 20.5% of aggregates.

After equilibration at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombinant powder contained about 15.3% of aggregates.

After vacuum drying at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombined powder contained about 12.6% aggregates.

Freeze drying of 4.5% (w / v) mannitol and 0.5% (w / v) IgG1 formulation

2.25 g of mannitol was dissolved in about 40 mL of demineralized water (pH about 7.5). As a next step, 2.3 ml of pure IgG1 at a concentration of about 109 mg / ml formulated in glycine-histidine buffer (see "Material" description) at pH 6 was diluted with demineralized water (pH about 7.5) to a content of 50 mg / ml. . The resulting solution contained about 4.5% (w / v) adjuvant or matrix and 0.5% (w / v) protein and was distributed in commercial 2R vials and lyophilized as described above. Lyophilisate was lyophilized in 2R vials using spatula and further treated as described above. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 34.0% of aggregates.

After equilibration at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombinant powder contained about 11.6% of aggregates.

After vacuum drying at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombinant powder contained about 6.2% aggregates.

Freeze drying of 4.5% (w / v) LS55P and 0.5% (w / v) IgG1 formulations

2.25 g of LS55P was dissolved in about 40 mL of demineralized water (pH 5 about 7.5). As a next step, 2.3 ml of pure IgG1 at a concentration of about 109 mg / ml formulated in glycine-histidine buffer (see "Material" description) at pH 6 was diluted with demineralized water (pH about 7.5) to a content of 50 mg / ml. . The resulting solution contained about 4.5% (w / v) adjuvant or matrix and 0.5% (w / v) protein and was distributed in commercial 2R vials and lyophilized as described above. Lyophilisate was lyophilized in 2R vials using spatula and further treated as described above. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombined powder contained about 2.5% of aggregates.

After equilibration at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombined powder contained about 2.6% aggregates.

After vacuum drying at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombinant powder contained about 1.2% of aggregates.

Freeze drying of 4.5% (w / v) coupling sugar and 0.5% (w / v) IgG1 formulation

2.25 g of coupling sugar was dissolved in about 40 mL of demineralized water (pH about 7.5). As a next step, 2.3 ml of pure IgG1 at a concentration of about 109 mg / ml formulated in glycine-histidine buffer (see "Material" description) at pH 6 was diluted with demineralized water (pH about 7.5) to a content of 50 mg / ml. . The resulting solution contained about 4.5% (w / v) adjuvant or matrix and 0.5% (w / v) protein and was distributed in commercial 2R vials and lyophilized as described above. Lyophilisate was lyophilized in 2R vials using spatula and further treated as described above. Aggregate content was investigated as described above.

In view of storage stability, the following aggregate contents were obtained.

After 1 week of open storage (forced storage stability) at 75% relative humidity of air and 40 ° C., the solution prepared from the recombinant powder contained about 5.5% of aggregates.

After equilibration at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombinant powder contained about 4.6% aggregates.

After vacuum drying at 40 ° C. for 1 day and dry storage for 4 weeks, the solution prepared from the recombinant powder contained about 1.5% aggregates.

Claims (52)

One or more antibodies or one derivative thereof and 1,4 O-linked D-Gal-sacrose (lactosucrose), 1,4 O-linked D-Glu-sacrose (glucosyl sucrose), and 1 A composition comprising one or more 1,4 O-linked saccharose derivatives selected from compounds consisting of 4 O-linked Glu-Glu-sacrose (maltosyl sucrose). The composition of claim 1, wherein the composition is a pharmaceutical composition. The composition of claim 1, wherein the antibody is a therapeutic antibody or derivative thereof. The composition of any one of claims 1 to 3, wherein the antibody is a monoclonal antibody or derivative thereof. The composition according to any one of claims 1 to 4, characterized in that it contains lactosucrose as 1,4 O-linked saccharose derivatives. 6. The composition of claim 5, further comprising one or more monosaccharides, disaccharides and / or polysaccharides. 7. The composition of claim 6, further comprising lactose and saccharose. 8. The composition of claim 5, wherein the fraction of lactosucrose is at least 55% (w / w) based on the sugar fraction contained in the composition. 9. The composition according to any one of claims 1 to 4, characterized by containing a mixture of glucosyl sucrose and maltosyl sucrose. 10. The composition of claim 9, further comprising one or more monosaccharides, disaccharides and / or polysaccharides. The composition of claim 10, further comprising fructose, glucose, and / or saccharose. The method according to any one of claims 9 to 11, characterized in that the total fraction of glucosyl sucrose and maltosyl sucrose is at least 25% (w / w) based on the sugar fraction contained in the composition, Composition. The composition of claim 12, wherein each of the glucosyl sucrose fraction and the maltosyl sucrose fraction is at least 18% (w / w) based on the sugar fraction contained in the composition. The sugar fraction according to any one of claims 1 to 13, wherein the sugar fraction containing one or more 1,4 O-linked saccharose derivatives is 25 to 99.99% (w / w) of the dry weight of the composition. , Composition. The method of claim 1, wherein the fraction of the antibody or antibody derivative is 0.1 to 75% (w / w) of the dry weight of the composition, and the sum of the weight percentages is at most 100% (w / w). Characterized in that, the composition. The composition of claim 1, wherein the dry weight of the composition comprises 60 to 90% (w / w) per sugar and 40% (w / w) or less of the antibody or antibody derivative, Wherein the fraction of maltosyl sucrose, and / or glucosyl sucrose is at least 20% (w / w) of the dry weight of the composition, and the sum of the weight percents is at most 100% (w / w). The composition of claim 1, which contains at least one pharmaceutically compatible adjuvant and / or at least one salt. The composition of claim 17, wherein the adjuvant is an amino acid or a peptide. The composition of claim 18, wherein the amino acid is isoleucine. The composition of claim 18, wherein the peptide is a dipeptide or tripeptide. The composition of claim 18, wherein the peptide is an isoleucine containing peptide. The composition of claim 20 or 21, wherein the peptide is diisoleucine or triisoleucine. The sugar mixture of claim 19, wherein the dry weight of the composition is from 60 to 99% (w / w) sugar mixture containing at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative And 1 to 40% (w / w) of isoleucine. The sugar mixture according to any one of claims 20 to 23, wherein the dry weight of the composition contains at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative. 60 to 99% (w / w), and 1 to 40% (w / w) peptide. The composition of claim 1, wherein the composition is an aqueous solution, a semisolid formulation, or a powder. The powder of claim 25, wherein the MMD of the particles in the powder is 1 to 10 μm. 27. The powder of claim 25 or 26, wherein the MMAD of the particles in the powder is between 1 and 5 microns. The powder of claim 25 or 26, wherein the MMAD of the particles in the powder is less than 10 μm. 27. The powder of claim 25 or 26, wherein the MMAD of the particles in the powder is from 0.5 to 10 mu m. Dissolving / suspending the antibody or antibody derivative in an aqueous solution / suspension (a), Aqueous solutions of one or more 1,4 O-linked saccharose derivatives selected from lactosucrose, glucosyl sucrose, or maltosyl sucrose or sugar mixtures, combinations containing one or more 1,4 O-linked saccharose derivatives Dissolving / suspending in suspension (b), A sugar mixture containing an antibody or antibody derivative and a 1,4 O-linked saccharose derivative or one or more 1,4 O-linked saccharose derivatives is dissolved / suspended in different solutions / suspensions and mixed Step (c) and (D) drying the solution / suspension containing the antibody or antibody derivative and 1,4 O-linked saccharose derivative (s), 30. A method for producing a powder according to any one of claims 25 to 29. 31. The method of claim 30, wherein the drying process is freeze or spray drying. 32. The method of claim 30 or 31, wherein the 1,4 O-linked saccharose derivative is lactosucrose. 33. The method of claim 32, wherein the solution or suspension further contains one or more monosaccharides, disaccharides or polysaccharides. 34. The method of claim 33, wherein the solution to be dried further contains lactose and saccharose. 35. The method of any one of claims 32 to 34, wherein the fraction of lactosucrose is at least 55% (w / w) of the sugar fraction present in the solution to be dried. 32. The method of claim 30 or 31, wherein the 1,4 O-linked saccharose derivative is a mixture of glucosyl sucrose and maltosyl sucrose. 37. The method of claim 36, wherein the solution to be dried further contains one or more monosaccharides, disaccharides or polysaccharides. 38. The method of claim 36 or 37, wherein the solution to be dried contains fructose, saccharose, and / or glucose. 39. The method of any of claims 36 to 38, wherein the total fraction of glucosyl sucrose and maltosyl sucrose is at least 25% (w / w) of the fraction present in the solution to be dried. Manufacturing method. 40. The method of claim 39, wherein each of the glucosyl sucrose fraction and the maltosyl sucrose fraction is at least 18% (w / w) of the fraction present in the solution to be dried. 41. The sugar fraction according to any one of claims 30 to 40 wherein the sugar fraction containing one or more 1,4 O-linked saccharose derivatives is 25 to 99.99% (w / w) of the dry weight of the solution to be dried. The manufacturing method of powder. The method of claim 30, wherein the fraction of the antibody or antibody derivative is from 0.1 to 75% (w / w) of the dry weight of the solution to be dried and the sum of the weight percentages is at most 100% (40). w / w), characterized in that the manufacturing method of the powder. 43. The method of claim 30, wherein the solution to be dried contains one or more pharmaceutically compatible adjuvants and / or one or more salts. The method of claim 43, wherein the adjuvant is an amino acid or a peptide. 45. The method of claim 44, wherein the amino acid is isoleucine. 45. The method of claim 44, wherein the peptide is a dipeptide or tripeptide. 45. The method of claim 44, wherein the peptide is an isoleucine containing peptide. 48. The method of claim 44, 46 or 47, wherein the peptide is triisoleucine. 43. A sugar mixture according to claim 41 or 42, wherein the dry weight of the solution to be dried is a sugar mixture containing at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative A method for producing a powder, characterized in that it contains% (w / w) and 1 to 19.99% (w / w) amino acids and the sum of the weight% is up to 100% (w / w). 43. A sugar mixture according to claim 41 or 42, wherein the dry weight of the solution to be dried is a sugar mixture containing at least one 1,4 O-linked saccharose derivative or at least one 1,4 O-linked saccharose derivative % (w / w), and 1 to 19.99% of the peptide. Use of a powder according to any one of claims 25 to 29 for the manufacture of a medical drug. Use of a spray dried powder according to any one of claims 26 to 29 for the manufacture of a medical inhalant.

Images