AU738891B2 - Stable transglutaminase preparations and process for producing them - Google Patents

Description

Regulation 3.2(2) Regulation 3.2(2)

AUSTRALIA

Patents Act 1990 a baa 4 C C C a.

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Application Number: Lodged: Invention Title: STABLE TRANSGLUTAMINASE PREPARATIONS AND PROCESSES FOR PRODUCING THEM The following statement is a full description of this invention, including the best method of performing it known to us Stable transglutaminase preparations and processes for producing them The present invention relates to stable transglutaminase preparations, in particular stable factor XIII preparations, and processes for producing them.

Factor XIII (F XIII, fibrin-stabilizing factor), which is a transglutaminase which is present as a proenzyme in plasma, platelets and monocytes/macrophages, is of importance, inter alia, for ensuring efficient blood coagulation and wound healing. F XIII, in the form of fresh frozen plasma, isolated from placenta or plasma, 15 has already been successfully employed therapeutically for many years for the therapy of factor XIII deficiency.

In the meantime, it has also become possible to prepare factor XIII recombinantly (rF XIII).

The commercially available, purified or parti- 20 ally purified transglutaminase or F XIII preparations contain added stabilizers, such as human serum albumin (HSA), which is, however, a disadvantage for protein preparations in view of the concomitant decrease in the specific activity, the additional protein load and/or potential immunogenicity and also the adverse effect on assessment of purity. Particularly in the case of highly pure proteins (such as recombinant proteins), it is desirable not to reduce once again the purity which has been achieved by adding foreign proteins. Furthermore, there is the potential for contamination with viral antigens when albumin, for example, is added.

T.he composition and activity of protein preparations which can be employed therapeutically generally musb be stable over a relatively long period of time. Since it 2is only rarely possible to achieve this in solution, such products are frequently marketed in the dry state. Mild freeze-drying is the method of choice for drying such products. However, even when this method is used, stable preparations, which fulfill the requirements for integrity and durability, are only obtained under certain conditions.

Since freeze-drying unstabilized transglutaminase solutions, for example, leads to a marked drop in activity and to considerable turbidity, formulations based on albumin and containing relatively high concentrations of salts have, for example, been previously described for use with purified F XIII preparations (DE-C-2063 070, JP Soo. 53/59018). However, these formulations suffer from the 15 previously described disadvantage of the addition of foreign protein, with all the problems attached thereto.

The freeze-drying of rF XIII in the presence of glycine or arginine and non-reducing sugars has also been described (WO 93/03147) In this case, however, no 20 statements were made with regard to the stability and solubility or clarity of the reconstituted lyophilisate.

Furthermore, the resulting product was stored at -20 0

C,

presumably to keep the material stable owing to inadequate stability at 4°C.

The underlying object of the present invention is, therefore, to obtain, by means of a suitable formulation for transglutaminases, in particular for F XIII, a nonperishable product, as a protein which can be administered locally topically as well) or parenterally, which is stable at 2-8 0 C (or higher) and which does not require the addition of, for example, HSA. Furthermore, the lyophilisate should be readily soluble and, after having been dissolved, should yield a clear solution which lacks turbidity and which, in addition, should also be sufficiently stable.

3 This objective was achieved by the provision of stable preparation forms for transglutaminase preparations, obtained by using defined stabilizers or mixtures thereof, as described in more detail below, and by processes for producing them. The invention consequently relates to a stable preparation form of a transglutaminase which, after lyophilization, is readily soluble without turbidity and which comprises the purified transglutaminase and also Dand/or L-amino acids, apart from glycine, their salts, derivatives and homologs, or dimers or oligomers thereof or mixtures thereof and/or sugars or sugar alcohols, where appropriate in combination with surface-active agents and/or reducing agents.

15 While the exemplary embodiments demonstrate this with 15 reference to recombinant factor XIII or F XIII which is isolated from placenta or plasma, they are not restricted thereto. In a preferred embodiment, the present invention n consequently relates to stable preparation forms of factor XII, and to biologically active fragments, 20 t derivatives or muteins thereof.

o In a particularly preferred embodiment, the stable :preparation forms are preparation forms which comprise F 25 XIII from plasma, placenta, thrombocytes or macrophages/ monocytes, or comprise recombinant F XIII.

The novel investigations can essentially be divided into two areas: investigations on the freeze drying itself, and on subsequent stability during storage.

rF XIII (Metzner et al., in J. McDonagh, R. Seitz, R. Egbring: "Factor XIII", 87 93, Schattauer (1993)) and also placental and plasma F XIII (Karges and Rapp, in J. McDonagh, R. Seitz, R. Egbring: "Factor XIII", 66 76, Schattauer (1993)) were employed in the investigations.

S- 4 The freeze-drying experiments were carried out in commercially available units using small glass bottles with and without a siliconized surface.

Freeze-drying: In a comparison of different additives, surprisincly it was found, that the activity and solubility of the F XIII can be preserved very well during freeze-drying in some cases when certain additives from the group of the D- and/or L-amino acids, their salts, derivatives or homologs are used (Table I).

In a further preferred embodiment, the present invention relates, therefore, to stable preparation forms of a transglutaminase which comprise the amino acids His, Glu, Met, Thr, Lys, Ala, Ile or Cys, their salts, derivatives or homologs, dimers or oligomers thereof, or mixtures thereof.

The amino acids His and Glu, especially, surprisingly exhibited good stabilization during freeze-drying, even without any further additives.

On the other hand, when amino acids such as Gly, Met or Ala were used on their own, there was a perceptible decline in activity during the freeze-drying (Table I).

In some cases, the use of sugars or sugar alcohols on their own afforded good stabilization during the course of the freeze-drying (Table In particular, sugars or sugar alcohols such as sucrose, trehalose, lactose, maltose, sorbitol, mannitol, or the like, gave positive results. The decline in activity which takes place during freeze-drying when amino acids are added which were not sufficiently effective on their own (such as Met, Ala, inter alia) can be markedly reduced by combining the amino acids with sugars or sugar alcohols (Table I).

In a further embodiment, the present invention relates, therefore, to stable preparation forms of a transglutaminase which comprise the sugars or sugar alcohols sucrose, lactose, trehalose, maltose, sorbitol or mannitol, their derivatives, homologs or mixtures thereof.

Only negligible stabilization was achieved when buffering substances such as Tris or phosphate were employed on their own. However, the use of borates resulted in perceptible stabilization during freezedrying (Table I).

A slight protein precipitate which appeared on some occasions when the lyophilisate was dissolved was averted by using surface-active substances such as Tween 80 or Tween 20, polyethylene glycol (PEG) of molecular weights between 1000 and 35000 Da, cetyl alcohol, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), lanolin alcohol, sorbitan monooleate, inter alia, without the F XIII losing activity 20 during the freeze-drying (Tables II and III).

In a further preferred embodiment, the stable preparation forms consequently comprise, as a surfaceactive substance, Tween 80, Tween 20, PEG, cetyl alcohol, PVP, PVA, lanolin alcohol or sorbitan monooleate.

Stability during storage: A critical parameter of the formulated lyophilisate is its storage stability, which was determined at 4°C and at room temperature, and also, under accelerated conditions, at 37 0

C.

It was found that the combination of amino acids and sugars, sugar alcohols or sugar derivatives was advantageous for good storage stability (Table IV).

Investigation using different sugars showed that 6 while sugars such as sucrose, lactose, trehalose or maltose stabilize activity even during relatively long storageperiodsat elevated temperature, sugars such as glucose or fructose, as reducing sugars, are unable, to a sufficient extent, to prevent a slow decline in activity at 37 0 C (Table Combining the amino acids His or Glu, which give rise to good stability even on their own, with sugars also resulted in a stability which was still further elevated.

In a further preferred embodiment, the stable preparation forms consequently comprise, as stabilizer, sucrose, maltose, trehalose, lactose, sorbitol or mannitol, or their derivatives or homologs, or 15 mixtures thereof, in combination with the amino acid His, Glu, Ile and/or Ala.

*t As has already been mentioned, surface-active substances had no negative effects on storage stability when used in the appropriate concentration range 20 (Tables II and III).

Although F XIII does not possess any accessible SH groups, SH agents such as Cys, N-acetylcysteine, thioglycerol or glutathione surprisingly exhibit a positive effect on the storage stability of F XIII, especially at elevated temperatures. In this connection, chelating agents such as EDTA or citrate can be added to protect the SH functions.

In a further preferred embodiment, the novel, stable preparation form of a transglutaminase therefore comprises Cys, N-acetyl-Cys, thioglycerol, sodium sulfide or glutathione, or mixtures thereof, where appropriate in the presence of a chelating agent.

Very good results as regards maintaining the activity and the solubility of the lyophilisate were 7,achieved using ternary or quaternary mixtures .(amino acid(s), sugar and surface-active component), for example using His/Tween/sucrose mixtures, His/ PEG/sucrose mixtures or His/Ile/PEG/sucrose mixtures. In a further preferred embodiment, the novel, stable preparation form of a transglutaminase consequently comprises as additives, apart from (an) amino acid(s), a sugar or sugar alcohol and a surface-active substance, with the additive mixtures His/Tween 20/sucrose or His/Tween 80/sucrose, His/ PEG/sucrose or His/Ile/PEG/sucrose being particularly preferred in this context.

With regard to stablity during storage, mixtures composed of (an) amino acid(s) and/or sugar or sugar 15 alcohol, a surface-active substance and a reducing agent, e.g. mixtures-composed of amino acid(s)/Cys or N-acetyl-Cys/PEG/sucrose, amino acid(s)/thioglycerol/ PEG/sucrose and sugar/reducing agent/PEG likewise proved to be suitable formulation systems, 20 especially at higher temperatures as well (see Tables III and VI). In a further preferred embodiment, the novel preparation form therefore comprises, as additive, a mixture composed of (an) 0: amino acid(s) and/ or sugar or sugar alcohol, a surface-active substance and a reducing agent, with the mixtures amino acid(s)/Cys/PEG/sucrose, amino acid(s)/N-acetyl-Cys/PEG/sucrose, amino acid(s)/thioglycerol/PEG/ sucrose and sugar/reducing agent/PEG, where appropriate in the presence of a chelating agent, being particularly preferred.

In the described formulations, the concentration of F XIII which is employed can be varied in a wide range and is preferably in the range of 0.003-50 mg/ ml.

The concentrations of the amino acids which are employed are preferably in a range of 0.01-10% (w 8 v particularly, however, in a range of 0.1-3% (w The sugar concentrations are preferably 0.1-20% (w particularly preferably, however, between 0.2 and 10% /v Surface-active components may be employed in a preferred concentration range of 0.00001-5% (w /v particularly preferably between 0.0002% and The concentrations of the reducing agents are preferably between 0.001% and 2% /v particularly, however, between 0.005% and The residual moisture is also of importance for the stability of the lyophilized protein during storage.

Using the given additives, the temperatures can be raised to 50-60°C for several hours, insofar as this 15 is necessary for reducing the residual moisture, during a final phase of the freeze-drying, without any loss of activity.

For the purpose of freeze-drying transglutaminases, or F XIII, and also for their subsequent stability 20 during storage, the pH of the solutions should preferably be in the range of 6-9, particularly preferably between 7 and 8.The described arino acids or phosphate buffers, borate buffers or Tris buffers having a pH in the range of 6 to 9, which are used, where appropriate, in combination with a chelating agent, are preferred for effecting the buffering.

The present invention also encompasses the use of the above-described stabilization additives for preparing stable liquid preparations which comprise protein(s), since the results obtained from the experiments carried out on transglutaminases, by way of example, are applicable to other preparations.

The present invention furthermore encompasses a process for stabilizing proteins, preferably transglutaminases, in which the purified proteins or the purified protein, which is preferably a transglutaminase, are/is mixed, in accordance with the customary procedure, as a solution or precipitate, with a solution which contains one or more of the novel additives and is adjusted to a pH range which is advantageous for stability, after which the whole is freeze-dried.

Due to the outstanding properties of the stabilized transglutaminases which are prepared in accordance with the novel process, these transglutaminases are very well suited for formulating a pharmaceutical.

The present invention also includes the use of stable preparation forms which comprise factor XIII, or biologically active fragments, derivatives or muteins thereof, for preparing a pharmaceutical for treating, for example, diseases which are characterized by F XIII deficiency.

Where appropriate, the novel pharmaceuticals can be formulated, in accordance with known methods, with suitable, pharmaceutically tolerated, well-known excipients. They can be administered in an appropriate dose which can be determined by the doctor in attendance. Administration can be effected by a variety of routes, for example intravenously, intraperitoneally, subcutaneously, intramuscularly, intradermally or topically.

The examples illustrate the invention.

10 Example 1 rF XIII, which was prepared by expression in yeast cells and purified to a purity of >98% by means of suitable methods, and also placental F XIII, likewise in purified form, were treated, as a solution or as a precipitate, with solutions of the stabilizers in order to attain the given activities. The F XIII solutions were filtered, used to fill small glass bottles and dried in accordance with a suitable freeze-drying program. The lyophilisates were then reconstituted once again to the original volume using distilled water. The F XIII activities were determined before and after the freeze-drying. In addition, the reconstituted solution was assessed with regard to ,turbidity.

15 The activity of the F XIII was determined using the commercially available Berichrom F XIII R test kit.

The results presented in Tables I, II and III demonstrate unambiguously that it is necessary to add the novel stabilizing constituents when freeze-drying F XIII and 20 that the enzymic activity and the solubility can be preserved by adding amino acids or sugars or amino acids and sugars even without the additional use of HSA. The solubility, in particular, can in some cases be still further improved by adding surface-active agents.

Example 2 Lyophilisates of rF XIII and placental F XIII, which had been prepared as indicated in Example 1, were stored at 4°C or at 37 0 C and reconstituted with Aqua Injectab. or with sodium chloride solution after various times in order to determine the enzyme activity which remained.

The results presented in Tables II to VI demonstrate that adequate long-term stabilization can be achieved using the already described mixtures comprising an amino acid or amino acids and/or sugars or sugar derivatives. The 11' addition of a reducing component leads in some cases to a further increase in stability, particularly under accelerated conditions.

S

S

*o* 12- Table I: Freeze-dryincg FXIII U

U

Activity before lyophilis. Activity of the lyophilisate (in of the starting value) Turbidity after dissolving the lyophilisate a. a. a 100 2 mM Tris* HCI, pH 7.4 100 8 mM Na borate pH 8.0 100 68 H- 1% L-His, phys. NaCI, pH 7.4 100 39 1% L-His, pH 7.6 100 100 1 L-Arg, pH 7.6 100 96 1 L-Gly, pH 7.6 100 41+- 1 L-Ala, pH 7.6 100 67 10 1% L-Glu, pH 7.6 100 88 1% L-Met, pH 7.6 100 10 1% L-His, 0.1% L-Ile, pH 7.6 100 99 1 sucrose 100 92 H- 2.5% sucrose 100 91 H- 5% sucrose 100 83 H- 2.5% lactose 100 100 H- 2.5% sorbitol 100 92 Htrehalose 100 99 Hmaltose 100 100 H- 1% L-His, 2.5% sucr., pH 7.6 1100 96 H 1% L-Arg, 2.5% sucr., pH 7.6 100 90 1+ 13 Continuation: Table 1 'U

U

Activity before lyophilis. Activity of the lyophilisate (in of the starting value) Turbidity after dissolving the lyophilisate S.

S

S.

S

S

*S.S

*S

S S

S

1% L Ala 2.5 su H 7 6 10 1% L-Ala, 2.5% sucr., pH 7.6 100 100+ 1% L-Lu, 2.5% sucr., pH 7.6 100 87

H

1% L-Lys, 2.5% sucr., pH 7.6 100 92

H-

1% L-Thr, 2.5% 100 94 sucr., pH 7.6 1% L-His, 0.1% L-lle, 2.5% 100 94

H-

sucr., pH 7.6 1% L-His, 0.1% L-lle, 2.5% 100 gluc., pH 7.6 1% L-His, 0.1% L-lHe, 2.5% 100 99 Hlact., pH 7.6 1% L-His, 0.1% L-lle, 2.5% 100 92 fruct.,_pH_7.6 1% L-His, 0.1% L-lle, 2.5% 100 91(sorbitol, pH 7.6 L-His, 0.1% L-lle, 2.5% 100 [H5 sucr., pH 7.6 1% L-His, 0.1% L-lle, 1% 100 93 H sucr., pH 7.6 1% L-His, 0.1% L-Cys, 2.5% 100 107sucr., pH 7.6 Activity test: Berichrom FXIII Turbidity: no turbidity (-)minimal turbidity +/-very low turbidity +slight, but very distinct, turbidity ++marked turbidity +ver-y marked turbidity Table II: Ef fect of sur ac-a iL:a~ a s n*l ;rtablty of an FXIII ivophiligate *Activity before ITurbidity after I Activitv IJ/mIl at t= .y MiMii~iNMM Ium d slvn 0 0.5 1 2 3 6 12 24 mon.

1% L-His, 0.1% L-11e 2.5% 153 150 144 139 139 143 136 sucr., pH 7.2 1% L-His, 0.1% L-I1e, 2.5%/6 154 141 151 132 132 139 136 sucr., pH 7.6 1% L-His, 0.1% L-Ile, 2.5% 152 148 143 141 141 138 137 sucr., pH 8.0 11- 1% L-His, 0.1% L-I1e, 2.5% 129 124 122 113 115 111 115 122 sucr.

1% L-His, 2.5% sucr. 131 H- 12 5 126 114 117 108 118 120 1% L-His,0.01% PEG 4000, 128 119 124 114 ill 111 114 119 sucr.

1% L-His, 0.001% PEG 130 123 125 112 114- 112 114 119 4000, 2.5% sucr.

1% L-His, 0.0001% PEG 130 118 120 107 114 107 107 116 4000, 2.5% sucr.

1% L-His. 0.001% Tween20, 128 123 124 112 114 113 119 119 sucr.

1% L-His, 0.0001% Tween 130 -1-2 2 124 112 11 05 12 120 2.5% sucr.

I- -01 -1 1% L-His, 10mM citrate, 156 H- 149 146 138 143 136 147 sucr.

1% L-His, 0.01% PEG 4000, 155 -147 138 137 140 134 135 sucr.

1% L-His, 0.1% L-Cys, 2.5% 156 -156 156 146 148 153 151 sucr.I Storage of the samples at +4 0

C

Turbidity: no turbidity pH 7.6, unless otherwise Activity test: Berichrom minimal turbidity indicated FXIII (U/mi) slight, but very distinct, turbidity Table III: Storage of FXIII lvophilisate Storage of the samples at room temperature Activity before storage (U/mi) Activity (U/mi) at t Turbidity Iafter t

I

mon.

(UI mI) 3 mon.

mI) 6 mon.

9 mon.

12 mon.

I mon.

S S

S.

S

*y

S

5..5

*SSS

S *5 5 1% His/0.001% PEG j85 F99 89 84 I86 69 4000/2.5% sucr./pH 7.6 1% His/0.1% 92 110 97 103 110 91 cysteine/0.001 PEG

IK

4000/2.5% sucr./pH 7.6 1% His/0.01% 87 108 96 198 104 85 cysteine/0.0O1

PEGI

4000/2.5% sucr./pH 7.6 1% His/0.005% PEG ~99 '109 103 99 14 79 4000/2.5% sucr./pH 7.6 9 1His/0.01% PVP 194 105 96 96 88 '78 15/2.5% sucr./pH 7.6 EC__ ___f9 His/0.1% ie/ 0.001- 11 106 92 PEG 4000/ 2.5% sucr./ pH 7.6 .3 84 90 69 Activity test: Berichrom

FXIII

Turbidity: no turbidity minimal turbidity 16 Table IV: Storage of rhuFXIII lyophilisate at 4°C 1% L-His pH 125 150 112 95 111 7.6 82 100 108 100 0@SO 0e S. 0

S

0000..

0

S

0SOS es

S

00 00 0 5

S

0000 0 0550 005* 0 000@ 0* 00 S 0 0000 *000 .0g.

0S 00 0 0 000000 0 1% L-Gly pH 125 61 26 48 34 30 29 25 16 7.6 1% L-Glu pH 125 132 127 116 110 77 114 83 93 7.6 1% L-His, 125 144 112 106 133 97 111 109 94 2.5% sucr., pH 7.6 1% L-Arg, 125 134 113 106 118 88 113 60 89 2.5% sucr., pH 7.6 1% L-Ala, 125 150 124 98 114 71 127 102 118 2.5% sucr., pH 7.6 1% L-Glu, 125 130 108 110 122 84 140 104 2.5% sucr., pH 7.6 1% sucrose 160 153 130 139 136 137 141 129 146 2.5% sucrose 160 148 127 135 128 135 130 126 141 5% sucrose 160 130 109 117 113 109 114 107 121 2.5% lactose 160 163 136 146 137 137 137 131 158 2.5% sorbitol 160 146 122 132 125 122 123 124 148 2.5% trehal- 160 160 136 142 137 141 136 127 138 ose maltose 160 155 134 139 132 118 139 123 150 1% L-His, 125 148 136 126 93 100 116 105 102 0.1% L-lie, pH 7.6 L-His, 125 142 123 113 119 93 116 106 100 0.1% L-lie, sucr., pH 7.6 1% L-His, 125 139 112 109 127 94 87 108 103 0.1% L-lie, 1% sucr., pH 7.6 1% L-His, 125 161 135 120 113 91 112 112 101 0.1% L-Cys, sucr., pH 7.6 Activity test: Berichrom FXIII 17 Table V: Storage of FXIII lyophilisate at 37 0

C

IActivity before I~ (U/m I) I Activity (UlmI) of the lyophilisate at t yophil.

1 mon 1 3 mon 1 6 mon I on 6 mn I12 mon 1% L-His, 0.1% L- 444 394 405 311 300 212 Ile, 2.5% sucr., pH 7.6 1% L-His. 0.1% L- 440 373 358 152 34 3 Ile, 2.5% glucose pH 7.6 1% L-His, 0.1% L- 424 419 385 322 293 285 Ile, 2.5% lactose pH 7.6 1% L-His, 0.1% L- 423 387 385 37 11 3 Ilie, 2.5% fructose, pH 7.6 1% L-His, 0.1% L- 420 380 298 120 64 21 Ile, 2.5% sorbitol pH 7.6 1% L-His, 0.1% L- 425 366 393 316 293 293 Ile, 2.5% maltose pH 7.6 1% L-His, 0.1% L- 413 408 418 350 354 393 Ile, 0.1% L-Cys, sucrose, pH 7.6 1% L-Glu, 0. 1% L- 424 395 350 281 301 314 Ilie, 2.5% sucr., pH 7.6 1% L-Lys, 0.1% L- 423 350 403 328 318 333 Ilie, 2.5% sucr., pH 7.6 Activity test: Berichrom FXIII -18 Table VI: Storacre of FXIII lyophilisate at various temperatures Activity Activity (U/mI) after various storage times at +41C in months lyophil. (U/mI) 0 0.5 1 mon 3 mon 7 mon 12 24 E mon mon mon mn 1% His/ 0.001% 112P 118 119 117 116 115 122 120 P sucr./0.2% pH 7.6 1% His/ 0.001% 112 113 123 120 125 118 120 125 sucr./ 0.2% N-acetylcysteine/ pH 7.6 1% His/f0.001% 119 109 125 122 129 116 129 131 sucr./ 0.2% thioglycerol/ pH 7.61 1% His/ 0.001% 119 109 117 110 118 109 113 116 sucr./ pH 7.6 11 11- Storag~e at room temperature 0 0.5 1 3 7 12 24 mo mon mon man] mon mon n 1% His/0.001% 112 118 119 115 116 114 120 112 sucr./0.2% cysteine/ pH 7.6 1% His/0.001% 112 113 112 114 120 122 122 120 sucr./O.2% Nacetylcysteine/pH 7.6 1% His/0.001% 119 109 .123 121 120 116 118 116 sucr./0.2% thioglycerol/pH 7.6 1% His/0.001%/ 119 109 117 108 109 105 100 98 sucr./pH 7.6 19 Continuation of Table VI: Storagre of FXIII 1yophilisa.te at various temperatures Storage at +37'C 0 0.5 1 3 7 12 24 man mon m on m on Mon m on 1% His/0.001% 112 118 121 114 119 119 104 89 sucr./0.2% cysteinel pH 7.6 1% His/.001% PEG/ 112 113 119 111 112 102 107 89 sucr./O.2% N -a c e ty lc y s te in e /p H8 9 1% His/0.001% 119 109 119 120 113 95 98 thiogiycerol/pH 7.6 1% His/0.001% 119 109 107 108 100 85 78 57 PEG/2.5% sucr./pH 7.6 1 1 1 a a Activity test: Berichrom FXIII "Comprises/comprising" when used in this specificati-on is taken integers, 25 presence integers, to specify the presence of stated f eatures, steps or components but does not preclude the or addition of one or more other f eatures, steps, components or groups thereof.

Claims (22)

1. A stable, pharmaceutical preparation of a transglutaminase which, after lyophilisation is readily soluble without any turbidity which preparation comprises the transglutaminase (ii) at least one D- or L- amino acid, their salts, derivatives and homologues, or dimers or oligomers thereof or mixtures thereof with the exception of glycine, and (iii)at least one sugar or sugar alcohol, in a buffered medium.

2. The stable preparation of claim 1, additionally containing a surface-active agent.

3. The stable preparation of claims 1 or 2, additionally containing a reducing agent.

4. The stable preparation of any one of claims 1 to 3, wherein the transglutaminase is factor XIII (FXIII) or a biologically active fragment, a derivative or a mutein thereof. The stable preparation as claimed in claim 4, wherein the FXIII is FXIII from plasma, placenta, thrombocytes or macrophages/monocytes or is rFXIII, or is biologically active fragments, derivatives or muteins thereof.

6. The stable preparation as claimed in any one of claims 1 to 5, wherein the amino acids are His, Glu, Met, Thr, Lys, Ala, IIe, or Cys, or their salts, derivatives or homologs, or dimers or oligomers thereof, or mixtures thereof. 21

7. The stable preparation as claimed in any one of claims 1 to 6, wherein the sugar or sugar alcohol is sucrose, maltose, trehalose, lactose, sorbitol or mannitol, or their derivatives, homologs or mixtures.

8. The stable preparation as claimed in claim 7, wherein the sugar alcohols are present in combination with the amino acid His, Glu, IIe and/or Ala.

9. The stable preparation as claimed in any one of claims 2 to 8, wherein the surface-active agent is Tween 80, Tween PEG, cetyl alcohol, PVP, PVA, lanolin alcohol or sorbitan monooleate. The stable preparation as claimed in any one of claims 3 to 9, wherein the reducing agent is Cys, N-acetyl-Cys, thioglycerol, sodium sulfide or glutathione or mixtures thereof, where appropriate in the presence of a chelating agent.

11. The stable preparation as claimed in any one of claims 1 to 10 which comprises, as additives in addition to (an) amino acid(s), a sugar or sugar alcohol and a surface-active substance.

12. The stable preparation as claimed in claim 11 which comprises, as additives, His/Tween 80/sucrose, His/Tween His/PEG/sucrose or His/IIe/PEG/sucrose.

13. The stable preparation as claimed in any one of claims 1 to 12, which comprises, as additives, (an) amino acid(s) and/or a sugar or sugar alcohol, a surface-active substance and, where appropriate, a reducing agent.

14. The stable preparation as claimed in claim 13 which comprises, as additives, (an) amino acid(s)/Cys/PEG/sucrose, (an) amino acid(s)/N-acetyl-Cys/PEG/sucrose, (an) amino Sacid(s)/thioglycerol/PEG/sucrose or sugar/reducing 22 agent/PEG, where appropriate in the presence of a chelating agent. The stable preparation as claimed in any one of claims 1 to 14, wherein the concentration of the transglutaminase is in the range from 0.003 to 50 mg/ml.

16. The stable preparation as claimed in any one of claims 1 to 15, wherein the concentration of the amino acids, their salts, derivatives or homologs is in the range from 0.01 to preferably from 0.1 to 3%

17. The stable preparation as claimed in any one of claims 1 to 16, wherein the concentration of the sugar or sugar alcohol is between 0.1 and 20% preferably between 0.2 and 10%

18. The stable preparation as claimed in any one of claims 1 to 17, wherein the concentration of the surface-active agent is between 0.00001 and 5% preferably between 0.0002 and 0.1% (w/v)

19. The stable preparation as claimed in any one of claims 1 to 18, wherein the concentration of the reducing agent is between 0.001 and 2% preferably between 0.005 and 0.5% (w/v) The stable preparation as claimed in any one of claims 1 to 19, wherein the pH is in a range from 6 to 9, preferably between 7 and 8.

21. The stable preparation as claimed in any one of claims 1 to 20 which comprises a borate buffer having a pH in the range from 6 to 9 and, where appropriate, a chelating agent.

22. The stable preparation as claimed in any one of claims 1 to 21 which comprises a Tris buffer having a pH in the R range from 6 to 9 and, where appropriate, a chelating agent. 23

23. The use of one or more stabilizing additives as claimed in any one of claims 1 to 22 for preparing a stable liquid transglutaminase preparation.

24. A process for preparing a stable transglutaminase preparation, which comprises mixing the purified protein(s), as a solution or precipitate, with a solution which contains one or more additives as claimed in any one of claims 1 to 22, and freeze-drying. The use of the transglutaminase which has been stabilized in accordance with the process of claim 24 for preparing a pharmaceutical.

26. The use- of the stable preparation form of a transglutaminase as claimed in any one of claims 1 to 22 for preparing a pharmaceutical for treating diseases which are characterised by FXIII deficiency.

27. The use of the stable preparation form of a transglutaminase as claimed in any one of claims 1 to 22 for preparing a pharmaceutical for treating diseases which can be positively influenced by the topical or parenteral administration of this transglutaminase. S DATED this 4 th day of June 2001 AVENTIS BEHRING GmbH WATERMARK PATENT TRADEMARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA CJH:JPF:VRH P8357AU01.DOC