EP0975335A1

EP0975335A1 - Stable pharmaceutical administration forms of peptides, proteins and nucleic acids

Info

Publication number: EP0975335A1
Application number: EP98925470A
Authority: EP
Inventors: Wolfgang Rödel; Markus Mattern; Gerhard Winter
Original assignee: Roche Diagnostics GmbH; Boehringer Mannheim GmbH
Current assignee: Roche Diagnostics GmbH
Priority date: 1997-04-18
Filing date: 1998-04-11
Publication date: 2000-02-02
Also published as: AU7758598A; CA2288649A1; AU744777B2; KR20010006562A; TR199902575T2; CN1261271A; WO1998047490A1; DE19716154A1; JP2001524090A; BR9809103A

Abstract

The invention relates to storage-stable lyophilized pharmaceutical preparations of biomolecules, wherein said biomolecules are selected from the group comprising proteins, peptides, nucleic acids and carbohydrates and further contain one or more basic D or L amino acids in addition to one or more aminodicarboxylic acids, hydroxycarboxylic acids or dicarboxylic acids or their physiologically compatible salts thereof. The auxiliary substances are available in the lyophilized culture partially or fully in amorphous form.

Description

Stable pharmaceutical dosage form for peptides, proteins and nucleic acids

The invention relates to stable lyophilized compositions for pharmaceutical or diagnostic purposes which contain a protein, a peptide, a nucleic acid or a polysaccharide, the auxiliaries being selected such that the lyophilisates are in amorphous or partially amorphous form

The advances in biotechnology in the past 20 years have led to an increasing number of biomolecules that are available in large quantities. A particularly active field for these products is their use in pharmaceutical therapies. For example, certain proteins are used to regulate individual substances Cell types used, nucleic acids to regulate gene expression, polysaccharides for vaccination. In practice, it is advantageous if the preparations can be stored at room temperature, since the storage space for cooling is often limited

In the case of temperature-sensitive preparations, precise control of the storage period between removal from the refrigerator and therapeutic administration (e.g. injection) is necessary, since by-products can form by-products that can falsify the spectrum of activity, particularly in the routine in the hospital and when administering Medicinal products, for example in human clinical studies, it is difficult to ensure continuous control of the storage conditions of the preparations

All biomolecules can hydrolyze more or less easily. Hydrolysis is part of natural metabolism and is necessary, for example, to prevent the accumulation of high-molecular toxic substances in the body The literature also describes a large number of other degradation reactions that apply to varying degrees to biomolecules. In the case of peptides or proteins, such degradation reactions take place through aggregation, denaturation, isomerization or redox processes. In the case of nucleic acids, deamination or addition of a nucleophile, for example, lead to the degradation of Nucleic acids.

In the development of stable lyophilisates, such as pharmaceutical or diagnostic preparations of peptides or proteins, there are still no proven causal methods that have made it possible to reliably select those auxiliary agents from the multitude of possible auxiliary agents and additives that are stable Ensuring the dosage form of the respective active ingredient The selection of suitable excipients to achieve a sufficiently stable dosage form, which, for example, ensure a sufficiently long storage stability or which delay or prevent the above-mentioned degradation reactions, is usually done empirically

It is known that the storage stability of many protein preparations is increased by removing the water. Suitable methods for this are freeze drying and vacuum drying. However, the use of such technical processes can also cause degradation reactions, e.g. a freeze phase is necessary in freeze drying. However, many proteins are not sufficiently stable against freezing processes When the aqueous solution of a biopolymer cools down, the majority of the water crystallizes out, while the biopolymer remains in an amorphous state.This can lead to a change in the molecular environment of the biopolymer, which can also change the spatial structure or conformation of the biopolymer itself. This in turn can promote degradation reactions, for example by increasing the reactivity of individual functional groups or aggregating unfolded chain segments of neighboring polymer molecules. Likewise, in the drying phase, the withdrawal of the Pro in the surrounding hydration shell cause chemical reactions such as oxidation in the protein chain. By adding suitable additives, these degradation reactions can be avoided or at least reduced in extent In freeze drying or vacuum drying, an essential function of the auxiliaries is to create a stabilizing amorphous environment for the biopolymer, which solidifies in the glass state on further cooling. The transition occurs suddenly within a very narrow temperature interval and can be characterized by the glass temperature Tg ' Below this temperature, the molecular mobility and thus also the reactivity is greatly reduced. In a formulation that is well suited for freeze-drying, Tg 'is as high as possible, typically above -40 ° C. The presence of amorphous structures can be determined, for example, by differential scanning calorimetry (DSC) X-ray diffraction studies or by optical and electron microscopic examinations

In order to be able to produce sufficiently stable lyophilized pharmaceutical dosage forms, it is necessary to select such auxiliaries which do not crystallize, or at most partially, crystallize out during freezing. Such auxiliaries which protect the biomolecule during the introduction process are also referred to as 'cryoprotectants'. In the main drying phase, the ice crystals sublimate , while in the post-drying phase part of the water bound in the amorphous phase and in the biopolymer is drawn off, which as a rule requires more drastic conditions (higher temperature or stronger vacuum) With the decrease in the water content in the freeze-dried material, Tg 'increases the time for the drying process to shorten, the temperature of the plates of the freeze-drying chamber is gradually increased, but the temperature in the freeze-dried material must never exceed Tg '

In the drying phase, the auxiliaries maintain the glass state in which the polymer is embedded. In the post-drying phase, free valences for hydrogen bonds are also created by the removal of water molecules in the biopolymer. This increases the reactivity of the biopolymer by adding suitable stabilizing additives the formation of hydrogen bridges to create an environment for water replacement for the biopolymer. The term 'lyoprotectants' was coined for this purpose The upper limit for the temperature load during storage is determined by the glass transition temperature Tg, above which the molecular mobility increases significantly. As a result, crystallization processes (described by the crystallization temperature Tk) or chemical reactions on macroscopic view often result in the so-called collapse of the lyophilisate cake ( described by the collapse temperature Tc), since the auxiliary molecules associate with one another, which is accompanied by a reduction in the specific surface area of the auxiliary interference matrix

The water in the lyophilisate lowers Tg, in a good recipe the residual moisture after freeze-drying is less than 3%. In the course of longer storage, however, it can increase slightly. In order to have a sufficient safety margin, the storage temperature of the lyophilisate in the glass state should be a maximum of 20 ° C below from Tg.

WO 93/00807 describes a two-component system consisting of a cryoprotectant (such as polyethylene glycol, PVP or starch) and a lyoprotectant (such as sugar, polyhydroxy alcohol or amino acid) for stabilization during the lyophilization

As is known, the tendency towards glass formation increases with the molecular weight. Polymers such as PVP, proteins (in particular serum albumin) or polysaccharides (dextran) are therefore used to form stable glass matrices

However, it is known that protective proteins such as serum albumin can be disadvantageous after injection, since they can cause the formation of antibodies, which impairs the use for parenteral preparations. Furthermore, differences in the raw material batches of the protective proteins can lead to uncertainties, since this increases the process capability and the Quality of the resulting product batches can be adversely affected

The polysaccharides used as auxiliaries can also have a pyrogenic effect in the bloodstream. In addition, polysaccharides have the disadvantage that they often require swelling, so that they rapidly form a when reconstituting a lyophilizate hinder clear solution In addition, the material usually consists of a fraction with different chain lengths, which complicates the batch consistency. The latter also applies to synthetic polymers such as PVP (polyvinylpyrrolidone)

So far, almost exclusively polyhydroxy compounds such as saccharides (sucrose, trehalose, glucose) or sugar alcohols (mannitol) have been used as low-molecular substances for glass formation in lyophilizates for biomolecules. However, the addition of mannitol only results in metastable glass states that can crystallize after storage

Reducing sugars such as glucose or maltose can cause radical or redox reactions, but also form Amadori products with primary amino groups (e.g. in proteins). In addition, the preparation can turn brown due to the Maillard reaction. Non-reducing di- or trisaccharides can hydrolyze, whereby on the one hand can form reducing sugars, on the other hand the physical properties of the excipient matrix may be impaired. Sugar alcohols such as mannitol are known to catalyze hydrolysis reactions in the presence of acetate, for example. They also tend to crystallize out.However, the combination of mannitol / glycine / (possibly phosphate , Detergent) is used as an auxiliary matrix for the lyophilization of proteins (cf. EP 0 597 101, WO 89/09614)

Further disadvantages or problems in the production of really sufficiently dry, storage-stable sugar preparations are drastically longer drying times, since only a small amount of heat can be added because of the stability of the biological materials used. Long process times are economically unfavorable, there is also an increased process risk, e.g. leaks in the vacuum chamber can occur, the cow unit fails, etc

It has surprisingly been found that certain combinations of auxiliaries are suitable as glass formers in the lyophilization of biomolecules. The present invention therefore relates to lyophilized preparations comprising a) biomolecules selected from the group consisting of proteins, peptides, nucleic acids and carbohydrates and b) one or more basic D- or L-amino acids and c) one or more aminodicarboxylic acids, hydroxycarboxylic acids, hydroxydicarboxylic acids or dicarbonaurs, or their physiologically acceptable salts, the auxiliaries in the lyophilisate being at least partially present in amorphous form. To facilitate drying and to improve the morphological structure of the lyophilisate cake, one or more neutral amino acids can optionally also be added.

The selection of the excipients means that the excipients in the lyophilisates are either completely amorphous or at least in a partially amorphous modification. In contrast to crystalline compositions, such lyophilisates have a glass transition temperature (Tg) which is above the desired storage temperature. Suitable combinations of auxiliaries Mixtures containing at least one substance each from groups (A) and (B), where (A) is a basic D- or L-amino acid, and (B) an aminodicarboxylic acid, especially an acidic D- or L-amino acid, Is aminocarboxylic acid, monocarboxylic acid, dicarboxylic acid or hydroxydicarboxylic acid, or their physiologically tolerable salts. Mixtures of this type are suitable as glass formers in the lyophilization of biomolecules and have the advantage that the lyophilisates prepared in this way are stable over a longer period of time, depending on the sensitivity of the biomolecule used, preferably for at least one year, in particular 1-2 years at refrigerator temperature or room temperature . This makes it possible to reduce or completely avoid the less suitable groups of substances mentioned above, so that the disadvantages when using the groups of substances mentioned in the production of pharmaceutical dosage forms can be largely avoided. Another advantage of the lyophilisates produced according to the invention is a great reduction in drying times, in particular when using a hydrophobic amino acid of less than 30 hours, preferably less than 24 hours, in particular less than 15 hours. This means that instead of drying, which often takes several days, the lyophilisates can be prepared by drying overnight. Pharmaceutically stable lyophilisates can be obtained if the pair of the basic amino acid and the counterion necessary for pH adjustment is selected so that a lyophilization matrix is at least partially amorphous and has a glass transition temperature of more than 50 ° C. preferably more than 65 ° C., in particular more than 80 ° C. For the manufacturing process it is advantageous if the frozen solution has a glass transition temperature of more than -40 ° C.

Physiologically acceptable acids or bases and their salts can also be used to adjust the pH. Suitable acids are inorganic or organic acids, such as phosphoric acid, acetic acid, etc. Free acids or bases are preferably used in order to achieve the lowest possible salt concentration in the lyophilisate With some peptides and proteins in arginine phosphate, the formation of protein aggregates was observed in the preparation of the solution to be lyophilized, provided the phosphate content was higher than 5 mM. Similar behavior was surprisingly found when using arginine citrate (c = 10 mM) the formation of aggregates can be reduced or largely avoided if mono- or dicarboxylic acids are used as counterions instead of the phosphate salt of the amino acid arginine. Lyophilisates with a stable glass state were obtained especially when aminodicarbonaurs (e.g. acidic D- or L-amino acids) or he dicarboxylic acids can optionally be used to fine-tune the pH in the range 5-7 when using a basic amino acid phosphoric acid in concentrations less than 5 mM

In addition, the dosage forms according to the invention have the further advantage that the glass transition temperature and the appearance of the lyophilisate cake have been further improved, in particular if a neutral amino acid has additionally been added, even if it partially crystallizes out. The amount can be varied within wide limits (5 - 50% of the total amount of excipients)

The dosage forms according to the invention have the advantage that they are stable in storage at room temperature over a long period of time. This ensures safe use as a medicament even when the cooling chain is interrupted Suitable additives or auxiliaries in the sense of the present invention are a preferred embodiment of a combination of a basic, an acidic and at least one neutral amino acid. These combinations are physiologically well tolerated, have good freeze-drying properties and also improve the thermal stability of lyophilized biopolymers dissolving the lyophilizate with water quickly leads to a clear solution

For the purposes of the present invention, the basic amino acids are all physiologically compatible amino acids with a basic side group, for example histidine, lysine, arginine, ornithine or citrulline. The neutral amino acids are the physiologically compatible amino acids with hydrophobic or hydrophilic side groups, for example phenylalanine, Glycine, leucine or isoleucm Suitable acids are aminodicarboxylic acids, hydroxycarboxylic acids, hydroxydicarboxylic acids, dicarboxylic acids or their physiologically acceptable salts, for example aspartic or glutamic acid. If these acids have a chiral center, the racemates or the optically active derivatives can be used

The amount of additives according to the invention are preferably selected such that the weight ratio of the acids mentioned in group c) (aminodicarboxylic acids, hydroxycarboxylic acids or dicarboxylic acids) to the basic D- or L-amino acids of group a) in the lyophilisate in the range from 0.01 1 to 2 1 amounts A range from 0.1 1 to 1 1, in particular approximately 0.5 1, is particularly advantageous

For the purposes of the present invention, numerous peptides or proteins are suitable as active ingredients for producing the pharmaceutical dosage forms according to the invention, such as, for example, immunomodulators, lymphokines, monokines, cytokines, enzymes, antibodies, growth factors, growth-inhibiting factors, blood proteins, hormones, vaccines, blood coagulation factors , and corresponding precursor proteins, muteins or fragments thereof. The peptides or proteins have a molecular weight of 0 5 - 500 kD, preferably 2 0-200 kD. The following peptides or proteins are examples called atrial naturetic factor or ANP (see also WO 85/33768), urodilatin or ularitide (see also WO 88/06596, WO 95/33768), cardiodilatin (see also WO 85/02850), BNP (brain natural peptides), auriculin, interferons, Colony stimulating factors, interleukins (IL-1, IL-1α, IL-1β, IL-2, IL-3, IL-4, etc) macrophage activating factors, B-cell factors, urokinase, plasminogen activators, TNF, NGF , Erythropoietin, EGF, hGH, BMP (bone morphogenic proteins), calcitonin, insulin or relaxin

Nucleic acids such as plasmids, DNA fragments or RNA strands are also suitable for the dosage form according to the invention

The lyophilized pharmaceutical dosage forms according to the invention are particularly suitable for parenteral administration in liquid form

In the following examples and comparative examples, the invention is exemplified and explained below. Formulations were found which, as glass formers in the lyophilization of biomolecules, which increase the glass transition temperature drastically, largely prevent aggregation of the biomolecules, improve the appearance of the lyophilizate cake and for thermal stabilization Lyophilized biomolecules are suitable. The formulations shown have shown that only the mixtures according to the invention lead to the desired result, ie they enable stable protein formulations in wholly or partially amorphous structures to be obtained in a short lyophilization process

The concentrations given in the formulations of the following examples relate to the solution before the lyophilization example 1

The pH of the solution is adjusted to pH 7.4 using H, PO ₄

2 g of L-arginine and 1 g of L-aspartate were dissolved in 50 ml of water. 35 mg of G-CSF (dissolved in 30 ml of 10 mM phosphate buffer) were pipetted into this solution and the mixture was stirred for 5 min. Then 100 μl of Tween 80 (as 10% water solution) pipetted in and stirred for a further 20 min. The pH was adjusted to 7.4 by adding phosphoric acid and the volume made up to 100 ml. This solution was membrane-filtered (PVDF filter 0.22 μm) and 1 ml each was filled into glass vials After fitting a suitable stopper, freeze-drying was carried out, the drying taking place over a total period of 40 hours. The vials were then sealed and stored at different temperatures until analysis. DSC determined that the glass transition temperature of the cake was 95 ° C. After 26 weeks, different samples of this lyophilizate had X-ray diffraction spectra recorded.These show that even after storage at a Temperature of + 60 ° C is amorphous Example 2 (comparative example)

The pH of the solution is adjusted to pH 7.4 with NaOH

2 g of L-valine and 2 g of glycine were dissolved in 50 ml of water. 100 μl of Tween 80 (as a 10% aqueous solution) were pipetted in and stirred for 20 min. The pH was then adjusted to 7.4 by adding NaOH. 35 mg of G-CSF (dissolved in 30 ml of 10 mM phosphate buffer) were pipetted in and stirred for 5 min. The pH was checked and the volume made up to 100 ml. After filling into vials, a lyophilizate was prepared as in Example 1. In the DSC of this lyophilizate, no glass transition was evident immediately after production. The X-ray diffraction spectra and images in the Scanning electron microscope showed that the cake is fully crystalline. No amorphous or partially amorphous structures can be detected

Example 3 (comparative example

The pH of the solution is adjusted to pH 7.4 with NaOH. 1 g of L-valine and 2 g of glycine were dissolved in 70 ml of water, 100 μl of Tween 80 (as a 10% aqueous solution) were added and the mixture was stirred for 20 minutes pH adjusted to 7.0 by adding NaOH. 30 mg (15 kU) of LDH (from pig muscle, dissolved in 20 ml of 20 mM phosphate buffer) were pipetted into this solution and the mixture was stirred for 5 minutes. The pH was checked and the volume to 100 ml Filled with this solution, after filling into vials, a lyophilizate was prepared as in Example 1. This recipe is also fully crystalline. No amorphous structures can be detected

Example 4

The pH of the solution is adjusted to pH 7.4 using H ₃ PO ₄

2 g of L-arginine, 1 g of aspartic acid and 1 g of L-phenylalanine were dissolved in 70 ml of water, 100 μl of Tween 80 were added (as a 10% aqueous solution) and the mixture was stirred for 20 minutes. The pH was then brought to 7.4 by adding phosphoric acid 50 mg (15 kU) of LDH (from pork muscle, dissolved in 30 ml of 100 mM phosphate buffer) were pipetted into this solution and the mixture was stirred for 5 minutes. The pH was checked and the volume was made up to 100 ml. After filling into vials a lyophilizate as prepared in example 1. The analysis showed that part of the phenylalanine is in crystalline form, that is to say the cake is partly crystalline and partly amorphous. The crystalline fraction remained constant during storage Example 5

After storage of the vials at room temperature (RT), the content of unchanged G-CSF of formulations 1 and 2 was determined using RP-HPLC

After storage of the vials at RT (after 5 and 13 weeks, respectively), the enzymatic activity of formulations 3 and 4 was determined in the coupled optical test.

Example 6 Lyophilisates were prepared analogously to formulation 4, but arginine was replaced by the same molar amount of other basic aminocarboxylic acids. The enzyme activity of LDH in the lyophilisate was determined after 5 weeks after storage at room temperature (RT)

Example 7

The pH of the solution is adjusted to pH 6.3 using acid (s u)

In each case 2 g of L-arginine were dissolved in 50 ml of water and a pH of 6.3 was adjusted by adding an acid. 1 g of isoleucine and 1 mg of rhNGF were added and made up to a volume of 100 ml with water. This solution was membrane-filtered (PVDF filter 0.22 μm) and 1 ml each filled into glass vials. After fitting a suitable stopper, freeze-drying was carried out after a total drying time of about 40 hours. The lyophilisates were then measured with DSC Result

Example 8 (comparative example)

The pH of the solution is adjusted to pH 6.0 with H ₃ PO ₄ .

2 g of L-arginine were dissolved in 50 ml of water and the pH was adjusted to 6.0 by adding phosphoric acid. 100 mg of ularitides (dissolved in 30 ml of H ₂ O) were pipetted into this solution and 10 min. touched. After 60 minutes the solution became cloudy and the protein flocculated. The experiment was then stopped Example 9 (comparative example)

The pH of the solution is adjusted to 6.0 with citric acid

2 g of L-arginine were dissolved in 50 ml of water and the pH was adjusted to 6.0 by adding citric acid. 100 mg of ularitide (dissolved in 30 ml of H ₂ O) were added with a pipette and the mixture was stirred for 10 min. After 2 hours, the solution became cloudy and the protein flocculated. The experiment was then stopped

Example 10

Lyophilisates with the active ingredient ularitide were produced according to the procedure of Example 7 with the following composition per vial

a) Recipe 19 (comparative example) 1 mg ularitis

10 mg mannitol with acetic acid ad pH 6.3

Analysis in the X-ray diffraction pattern shows that the lyophilisate has a fully crystalline structure. No amorphous or partially amorphous structures are detectable b) Recipe 20

1 mg ularitide 20 mg L-arginine 10 mg L-isoleucine with aspartic acid ad pH 6.3

Assessment in the DSC.

Recipe 19 no glass transition recognizable, is fully crystalline recipe 20 'Tg = 85, 1 ° C, partially amorphous structure

Both formulations 19 and 20 were stored for 1 year at room temperature. The subsequent gel electrophoresis gave the following results Rec. 19 dimers> 1% and soluble aggregates Rec. 20 100% monomer,

Example 11

The active ingredient Ularitide was used to produce lyophilisates with the following recipe compositions per vial using the procedure from Example 7:

a) Rec. 21 (comparative example) 1 mg ularitide

50 mg sucrose

10 mg glycine

6 mg polyethylene glycol 6000 b) Rez 22

1 mg ularitide 20 mg L-arginine 10 mg L-isoleucine with aspartic acid ad pH 6.3

c) Rez 23

4 mg ularitide 20 mg L-arginine

10 mg L-isoleucine with aspartic acid ad pH 6.3

d) Rez 24 1 mg ularitide

20 mg L-arginine 10 mg L-leucine with aspartic acid ad pH 6.3

e) Rez 25 (comparative example) 1 mg ularitide 25 mg sucrose 20 mg glycine

All formulations 21-25 were stored in a stress test at different temperatures and then the content was determined using RP-HPLC

The results of Examples 10 and 11 show that the formulations according to the invention stabilize the peptide better against thermal stress than the formulations based on sucrose or sucrose / PEG corresponding to the prior art. The mannitol used in many formulations does not lead to a sufficiently stable formulation

Examples 8 and 9 show that the acids often used in buffer systems lead to flocculation in certain peptides. This was not observed in the acids used in the recipes according to the invention (preferably aspartic acid and glutamic acid)

Example 12

a) Rez 26 (comparative example)

1 mg ularitide 15 mg glycine

2 mg L-isoleucine 10 mg urea 0.5 mg polysorbate with Na acetate buffer ad pH 6.8, completely crystalline b) Rez 27

1 mg ularitide 20 mg D-arginine 10 mg D-isoleucine with D-aspartic acid ad pH 6.8

c) Rez 28

1 mg ularitide 20 mg L-arginine 10 mg L-isoleucine with L-aspartic acid ad pH 6.8

d) Rez 29 (comparative example) 1 mg ularitide 20 mg L-threonine

10 mg L-isoleucine

e) Rez 30 (comparative example) 1 mg ularitide 15 mg polyethylene glycol 6000

5 mg phenylalanine

The formulations were stored in a stress test at different temperatures and then the amount of the main breakdown product was determined using RP-HPLC ('Peak XI)

In the formulations according to the invention, significantly less degradation product of the peptide Formulation 29 is produced, although it results in an amorphous lyophilisate cake, which, however, is not sufficiently suitable for stabilizing the protein

EXAMPLE 13 Lyophilisates with the active ingredient ularitide were produced using the procedure from example 7, with the following composition per vial

a Rez 31

1 mg ularitide

70 mg sucrose

10 mg L-phenylalanine b) Rez 32

1 mg ularitide

85 mg sucrose

c) Rez 33

1 mg ularitide

46 mg raffinose

10 mg L-phenylalanine d) Rez 34

1 mg ularitide 20 mg L-arginine 5 mg L-phenylalanine with L-aspartic acid ad pH 6.3

All formulations 31-34 were stored in a stress test at different temperatures, then the lyophilizate was dissolved in 1 ml of water and the turbidity of the reconstituted solution after 1 hour in a nephelometer (Hach brand) determines turbidity values above 1.0 are considered questionable Excipients are readily soluble, the cause of the clouding observed in some formulations is an aggregation of the peptide

Results

KS = refrigerator temperature -8 ° C RT = room temperature 20-22 ° C

Only the formulations according to the invention do not show that the turbidity limit of 1.0 is exceeded after exposure to temperature

Example 14

Ularitide was reconstituted in various formulations after storage as in Example 11 and the solution was examined for particles after 3 hours using a light scattering meter (PMS) Results (given are mean values from the payment of 5 vials)

The formulations according to the invention show no critical increase in the number of particles after storage at elevated temperature

Example 15 Lyophilisates of the following recipes were produced with the rhNGF protein:

a) Rec. 35

0.025 mg rhNGF 20 mg L-arginine 10 mg L-aspartic acid with acetic acid ad pH 6.3

b) Rec. 36

0.025 mg rhNGF 20 mg L-arginine

10 mg L-aspartic acid 10 mg L-isoleucine with acetic acid ad pH 6.3 c Rez 37 (comparative example)

0.025 mg rhNGF 20 mg L-arginine 30 mg sucrose with acetic acid ad pH 6.3

d) Rez 38 (comparative example ^" )

0.025 mg rhNGF 20 mg L-arginine 30 mg raffinose with acetic acid ad pH 6.3

The program for lyophilization was greatly shortened compared to the usual programs in total 15 hours instead of usually 40-50 hours. The residual moisture in the lyophilisate was then determined in two independent determinations

Result

Recipe 35 4.7% / 5.0%

Recipe 36 l, 0% / 0.9%

Recipe 37 5.9% / 6.6%

Recipe 38 5.6% / 5.6%

The example shows that a shortening of the lyophilization time is only possible with the formulations according to the invention, in particular when using a hydrophobic amino acid as a 3 component Example 16

With the rhNGF protein, lyophilisates of the following recipes were produced

a) Rez 39

0.025 mg rhNGF 20 mg L-arginine 10 mg ß-alanine 10 mg L-aspartic acid with acetic acid ad pH 6.3

b) Rez 40

0.025 mg rhNGF 20 mg L-arginine 8 mg L-aspartic acid

10 mg L-isoleucine with acetic acid ad pH 8.7

c) Rez 41 0.025 mg rhNGF

20 mg L-arginine 12 mg L-aspartic acid 10 mg L-isoleucine with malic acid ad pH 4.5

It was lyophilized with the same program as specified in Example 15 and then the residual moisture was determined

Result

Recipe 39 1.2% / 1.4%

Recipe 40 1.6% / 1.9%

Recipe 41 0.8% / 0.9%

Claims

claims

Lyophilized preparations containing a) biomolecules selected from the group consisting of proteins, peptides,

Nucleic acids and carbohydrates, b) one or more basic D- or L-amino acid and c) one or more aminodicarboxylic acid, hydroxycarboxylic acid, hydroxydicarboxylic acid or dicarbonaure, or their physiologically acceptable salts, the auxiliaries in the lyophilisate being wholly or partly in amorphous form

Lyophilized preparations according to claim 1, characterized in that the weight ratio of the additives mentioned in c) to the additives mentioned in b) is in the range from 0.01 1 to 2 1

Lyophilized preparations according to one of claims 1 to 2, characterized in that they contain as auxiliary substances polymers (compounds with a molecular weight> 1000 Da) in an amount of less than 10% by weight of the total auxiliary substance mass

Lyophilized preparations according to one of claims 1-3, characterized in that they contain sugar as auxiliary substances in an amount of less than 10% by weight of the total auxiliary substance mass

Lyophilized preparations according to one of claims 1-4, characterized in that they have a glass transition temperature of> 50 ° C

Lyophilized preparations according to any one of claims 1-5, characterized in that the frozen solution has a glass transition temperature above -40 ° C before lyophilization Lyophilized preparation according to one of claims 1-6, characterized in that the aminocarboxylic acid is aspartic acid or glutamic acid

Lyophilized preparations according to any one of claims 1-7, characterized in that they additionally contain one or more amino acids with a hydrophobic residue

Lyophilized preparations according to claim 8 containing leucine, isoleucine, valine or phenylalanine, in particular isoleucine or phenylalanine

Lyophilized preparations according to any one of claims 1-9, characterized in that the solution reconstituted with water has a pH between about 3 and 9

Lyophilized preparations according to any one of claims 1-10, characterized in that the biomolecule to excipient mass ratio is less than 1-10

Lyophilized preparation according to one of claims 1-11, characterized in that the biomolecule is a peptide from the class of the atrials

Natπureti see peptides (ANP) is

A process for the preparation of lyophilized preparations according to any one of claims 1-12, characterized in that a solution or suspension of the biomolecule is prepared in a physiologically acceptable solvent and a) one or more basic D- or L-amino acids, and b) at least one or more aminodicarboxylic acids or organic or inorganic acids are added, and then the solution is lyophilized A method according to claim 13, characterized in that the lyophilization is carried out starting from an aqueous solution with a phosphate content of less than 5mM