METHOD TO PRODUCE A MEDICINAL PRODUCT COMPRISING A BIOLOGICALLY ACTIVE PROTEIN AND THE RESULTING PRODUCT

Abstract

The present invention pertains to a method for producing a medicinal product comprising a biologically active protein comprising the steps of providing an aqueous composition comprising a solvent, the biologically active protein and between 20% w/w and 60% w/w of a non-polymeric sugar, freezing the composition, thereby forming at least one frozen body comprising the solvent in frozen form, putting the frozen body in a drying apparatus while being carried by a support, the support comprising one or more restraining elements that define one or more boundaries of the support, wherein at most 30% of the surface of the body is contiguous with the one or more restraining elements, reducing the pressure in the drying apparatus below atmospheric pressure, providing heat to the body in order to sublimate the frozen solvent of the body and obtain a dried body. The invention also pertains to a product obtainable by this method.

Claims

1. A method for producing a medicinal product comprising a biologically active protein comprising the steps of: providing an aqueous composition comprising a solvent, the biologically active protein and between 20% w/w and 60% w/w of a non-polymeric sugar, freezing the composition, thereby forming at least one frozen body comprising the solvent in frozen form, putting the frozen body in a drying apparatus while being carried by a support, the support comprising one or more restraining elements that define one or more boundaries of the support, wherein at most 30% of the surface of the body is contiguous with the one or more restraining elements, reducing the pressure in the drying apparatus below atmospheric pressure, providing heat to the body in order to sublimate the frozen solvent of the body and obtain a dried body.

2. A method according to claim 1, characterized in that the frozen solvent is sublimated in less than 48 hours.

3. A method according to claim 2, characterized in that the frozen solvent is sublimated in less than 36 hours.

4. A method according to claim 3, characterized in that the frozen solvent is sublimated in 16 to 24 hours.

5. The method of claim 1 wherein at most 20% of the surface of the body is contiguous with the one or more restraining elements of the support.

6. A method according to claim 5, characterized in that at most 10% of the surface of the body is contiguous with the one or more restraining elements of the support.

7. The method of claim 1 wherein the frozen body is a spheroid.

8. A method according to claim 7, characterized in that the spheroid has a volume between 50 ?l and 1000 ?l.

9. The method of claim 1, wherein the restraining element of the support is a floor, the body being provided lying on this floor, and the support is open to allow radiation to freely pass to the said floor, characterized in that at least a part of the heat is provided by emitting heat radiation from a radiation source present in the drying apparatus above the support, to reach the frozen body.

10. The method of claim 1, characterized in that at least a part of the heat is provided by conduction of heat via the restraining element that is contiguous with the frozen body.

11. The method of claim 1, wherein the restraining element of the support is a floor, the body being provided lying on this floor, and the support is open to allow radiation to freely pass to the said floor, characterized in that the heat is provided by emitting heat radiation from a radiation source present in the drying apparatus above the support to reach the body in combination with conduction of heat via the restraining element that is contiguous with the frozen body.

12. The method of claim 1 wherein the amount of the sugar in the aqueous composition is chosen from the group that consists of the ranges 20-55% w/w, 20-50% w/w, 20-45% w/w, 25-45% w/w, 25-40% w/w, 25-35% w/w and 27-30% w/w.

13. The method of claim 12 wherein the sugar comprises monomeric and/or dimeric molecules.

14. A method according to claim 13, characterised in that the sugar comprises glucose, galactose, maltose, sucrose, trehalose, fructose, lactose, saccharose, mannitol, sorbitol and/or xylitol.

15. The method of claim 1 wherein the restraining element of the support is a floor, characterized in that multiple frozen bodies are positioned in the form of a monolayer on the said floor while being dried.

16. The method of claim 1 wherein a residual moisture content in the dried body is less than 2% w/w.

17. A medicinal product in the form of a substantial homogenous freeze-dried body having a volume between 50 and 1000 ?l comprising a biologically active protein, the protein being dispersed in a solid matrix of a non-polymeric sugar, characterized in that the body comprises between about 21 and 72% w/v of the said sugar.

18. A body according to claim 17, characterized in that the body is a spheroid.

19. A product in the form of a substantial homogenous freeze-dried body having a volume between 50 and 1000 ?l, comprising a biologically active protein, the body being obtainable via the method of claim 8.

20. The method of claim 14, wherein a residual moisture content in the dried body is less than 2% w/w.

Description

[0034] The invention will be explained in more detail using the following examples and figures.

[0035] FIG. 1 schematically shows a first type of support, viz. a glass vial, filled with frozen spheroids or a liquid composition, frozen after being filled in the support.

[0036] FIG. 2 schematically shows a drying set-up in a lyophilising apparatus.

[0037] FIG. 3 schematically shows an alternative drying set-up in a lyophilising apparatus.

[0038] FIG. 4 schematically shows a further alternative drying set-up in a lyophilising apparatus.

[0039] Example 1 is a first example of freeze-drying high sugar compositions.

[0040] Example 2 is a second example of freeze-drying high sugar compositions.

[0041] Example 3 is a third example of freeze-drying high sugar compositions.

[0042] Example 4 is a fourth example of freeze-drying high sugar compositions.

[0043] Example 5 is a fifth example of freeze-drying high sugar compositions.

[0044] Example 6 provides stability data for medicinal products according to the invention.

[0045] FIG. 1

[0046] FIG. 1 schematically shows a first type of support, viz. a glass vial, filled with frozen spheroids or a liquid composition, frozen after being filled in the support. The figure depicts a first glass vial (1) which is filled with about 15 frozen spheres (5) of about 110 ?l each. The figure also depicts a second glass vial (1) filled with 1.5 ml of liquid composition that has been frozen to from one frozen cake (6).

[0047] FIG. 2

[0048] FIG. 2 schematically shows a drying set-up in a lyophilising apparatus (apparatus as a whole not shown). The figure depicts a shelf (10) which carries vials 1 and 1. The vials 1 are filled with frozen spheres 5 and the vials 1 are filled with a cake of frozen liquid (6). The shelf can be heated via built-in electrical heaters (not shown).

[0049] FIG. 3

[0050] FIG. 3 schematically shows an alternative drying set-up in a lyophilising apparatus. In this set up the vials are put on a heightened support (20) which is virtually thermally insulated from the heated shelf (10). This way, the vials will receive virtually no heat via conduction of heat generated in the shelf (10). Above the vials there is a second heated shelf (10), which shelf is provided with a black coating (11). This black coating provides that the shelf will act as a good radiating source, emitting heat radiation 12 (mainly infra-red light) towards the vials.

[0051] FIG. 4

[0052] FIG. 4 schematically shows a further alternative drying set-up in a lyophilising apparatus. This set-up uses two shelves, one heated shelf 10 which acts as a first heat source and one heated shelf 10 which acts as a second heat source (via black coating 11 that provides a radiating surface for the bottom of shelf 10, corresponding to the set-up as depicted in FIG. 3). Standing on the first shelf 10 are open containers 15 and 15. Container 15 is filled with a monolayer of frozen spheres (5), whereas the second container 15 is filled with a frozen liquid (6).

[0053] Examples 1 to 5 are proof-of-principle examples showing that using the method according to the invention, a substantially homogenous dried body can be obtained within a reasonable fast drying cycle, despite the fact that over 20% w/w of a non-polymeric sugar is present in the initial aqueous composition. For these examples a simple solution of 30% sucrose (w/v) in water was used (which approximates 27% w/w). Part of this aqueous composition was used to make frozen spheres having a volume of approximately 100 ?l. For this a process as described in WO 2010/125084 was used, in particular the process as mentioned on line 33, page 20, to line 2, page 21 (in conjunction with FIGS. 1, 2, 3 and 4 of the same international application). The frozen spheres are used in the examples as described below.

[0054] In example 1, a first set of three 10 ml glass lyophilising vials (1) having a diameter of approximately 20 mm, are filled with about 15 spheres (5), equating about 1.5 ml of the initial aqueous composition. In the vials, this results in about 1? layer of spheres. In this set-up, less then 10% of the surface of the frozen spheres (about 1-3%) is contiguous with the one or more walls (restraining elements) of the supporting vial. A second set of three of corresponding vials (1) is filled with 1.5 ml of the liquid composition as described here above, whereafter the liquid is frozen. This way, about 60% of the surface of the frozen body is contiguous with the one or more walls of the supporting vial. The resulting filled vials are schematically shown in FIG. 1.

[0055] These vials are put on a shelf in a standard lyophilising apparatus, the prime parts of which are schematically shown in FIG. 2, which has beforehand been brought to a temperature of about ?45? C. This lyophiliser is subjected to the following freeze-drying cycle (Table 1).

TABLE-US-00001 TABLE 1 Phase Freezing Temp [? C.] ?45 ?45 Time [m] 0 1 Vacuum [mbar] NA NA Initial Temp [? C.] ?45 Sublimation Time [m] 0 Vacuum [mbar] 0.04 Sublimation Temp [? C.] ?45 ?45 35 35 35 Time [m] 0 10 123 960 240 Vacuum [mbar] 0.04 0.04 0.04 0.04 0.34

[0056] As can be seen in Table 1, after loading the shelf with the filled containers the shelf (10) is firstly kept at a temperature of ?45? C. for 1 minute (the Freezing phase). Herewith the frozen bodies are brought to a temperature of ?45? C. The pressure is kept atmospheric. Then, the pressure is lowered to 0.04 mbar while the temperature of the shelf is kept at minus 45? C. (Initial sublimation). Under these conditions, the frozen liquid already sublimates and heat is supplied to the pellets via conduction through the shelf. However, the speed of sublimation under these conditions is relatively low. To increase the speed of sublimation, the shelf is brought to a temperature of 35? C. (Sublimation), and kept at that temperature for about 22 hours (total of 1333 minutes, as indicated under Sublimation). The pressure is kept at the low value of 0.04 mbar. After that, the sublimation process is completed and up to about 98% of the frozen liquid has left the frozen bodies, thereby transforming into dried bodies. Then, dried nitrogen gas with a temperature of about 20? C. is led into the lyophiliser until the pressure is about atmospheric. This takes about 2 minutes. Then the door can be opened to take out the vials.

[0057] Within the drying time of about 22 hours, the spheres were in general dry and in good shape, with some insignificant incomplete drying at some spots. In the second set of vials however, the frozen body had turned into a mess of foam and syrupy liquid.

[0058] In example 2 the same lyophilising apparatus as used in example 1 is used, albeit that the bottom of the shelf above the shelf carrying the vials is provided with a black PTFE (polytetrafluoroethylene) plate. By intimate contact between this black plate and the shelf, this plate is warmed to virtually the same temperature as the shelf itself and hence will act as a radiation heat source. Still, only a small portion of the radiation will reach the frozen bodies directly since the glass wall of the vial will absorb and reflect most radiation. A comparable set-up is described in WO 2010/125084 in conjunction with FIG. 5 of that reference. By having a heat flow to the vials not only by conduction via the vials standing on a shelf, but also by radiation from the shelf above, the drying performance can be improved. Still, only for the spheres this leads to an improved drying result, namely that the spheres can be dried in about 20 hours. The liquid filled vials however still lead to a very poor inhomogenous drying result.

[0059] In example 3 the lyophilising set-up as described in conjunction with example 2 is used, albeit that a heat flow via conduction is virtually ruled out by putting the vials on a heightened support, keeping them virtually insulated from the shelf by which they are carried. This set-up is shown in FIG. 3. By choosing this set-up, the required heat flow towards the frozen bodies is virtually completely obtained via radiation from the shelf above. The drying cycle, which now includes the actual freezing of the material from a liquid into a solid frozen body, is shown beneath in Table 2.

TABLE-US-00002 TABLE 2 Phase Freez- Temp [? C.] ?45 ?45 ?20 ?20 ?45 ?45 ing Time [m] 0 10 15 60 15 20 Vacuum NA NA NA NA NA NA [mbar] Initial Temp [? C.] ?45 Subli- Time [m] 0 mation Vacuum 0.04 [mbar] Subli- Temp [? C.] ?45 ?45 35 35 mation Time [m] 0 10 123 960 part 1 Vacuum 0.04 0.04 0.04 0.04 [mbar] Subli- Temp [? C.] 35 mation Time [m] 240 part 2 Vacuum 0.34 [mbar]

[0060] This gave the same drying result as described in conjunction with example 1, in about the same drying time.

[0061] In the next example, example 4, it is tried to obtain a reasonably good drying result of the body as present in the second set of vials as described in example 1. These vials were put on a shelf of the same lyophilising apparatus, thus only using a heat flow via conduction through the shelf. A drying cycle according to Table 3 was used, which led to a long drying time of over 76 hours.

[0062] Although a markedly improved drying result was obtained when compared with the result of example 1, the resulting dried body still had substantial melted regions, deep cracks and some foaming. This means that even longer drying times would be needed to achieve a drying result which is comparable with that obtainable using the method according to the invention.

TABLE-US-00003 TABLE 3 Phase Freezing Temp [? C.] ?45 ?45 Time [m] 0 30 Vacuum [mbar] NA NA Initial Temp [? C.] ?45 Sublimation Time [m] 0 Vacuum [mbar] 0.04 Sublimation 1 Temp [? C.] ?25 ?25 20 20 35 35 Time [m] 15 2880 600 360 240 240 Vacuum[mbar] 0.04 0.04 0.04 0.04 0.04 0.20 Sublimation 2 Temp [? C.] 35 Time [m] 200 Vacuum[mbar] 0.27

[0063] In example 5, another type of support is used for drying the frozen bodies (see FIG. 4). This support is an open container (15, 15) having a width of about 20 cm, a length of about 30 cm and a height of about 4 cm. The container is made of a heat conducting plastic material, in this case carbon black filled polyethyleneterephtalate. The container can be put in a heat conducting contact with a shelf upon which they rest. The difference with such a container when comparing it with a standard freeze-drying vial is that such a container is in essence open, and thus allows radiation to freely pass to the floor of said container to reach the frozen material. In the arrangement shown in FIG. 4, one container 15 is filled with a monolayer of frozen spheres 5 and the other container 15 is filled with a frozen layer 6 of the sucrose containing composition (with a thickness of about 5? mm). The drying set-up is the set up as described in conjunction with example 2. By heating the shelves, the frozen bodies may receive heat via the heated bottom and side walls of the containers and by irradiation from the heated shelf above. A drying cycle as indicated in Table 4 is used from drying the frozen material.

[0064] The result of this drying cycle is that the individual spheres are nicely dried, being of good shape and have virtually no incomplete drying spots. The layer of frozen sucrose composition however is inhomogeneously dried, with substantial incomplete drying and foaming.

TABLE-US-00004 TABLE 4 Phase Freez- Temp [? C.] ?45 ?45 ?20 ?20 ?45 ?45 ing Time [m] 0 10 15 60 15 20 Vacuum NA NA NA NA NA NA [mbar] Initial Temp [? C.] ?45 Subli- Time [m] 0 mation Vacuum 0.04 [mbar] Subli- Temp [? C.] ?45 ?45 35 35 mation Time [m] 0 10 123 960 1 Vacuum 0.04 0.04 0.04 0.04 [mbar] Subli- Temp [? C.] 35 mation Time [m] 240 2 Vacuum 0.34 [mbar]

[0065] Example 6 provides some embodiments of medicinal products comprising a biologically active protein that are obtained using a method according to the invention, in particular a method as described here above in conjunction with example 2. The first product (denoted as CDV) is vaccine in the form of lyospheres that can serve to formulate a vaccine for injection by re-suspension of one or more spheres in water-for-injection. Each sphere, having a volume of approximately 100 ?l, contains live attenuated canine distemper virus at a titre of about 7 (log10 of the TCID50). This product is obtained in two forms, both at pH 7.2 using a 10 mM KPO.sub.4 buffer, a first form according to the prior art having 3.7% (w/w) of sucrose as a stabilising agent (and in addition, 0.8% w/v gelatin and 1.0% w/v NZ amine as bulking agents), and a second form having 21.4% (w/w) of non-polymeric sugar (15.3% (w/w) sucrose and 6.1% (w/w) trehalose) as a stabilising agent (next to 5.2% w/v arginine, 0.8% w/v gelatin and 1.0% w/v NZ amine as bulking agents).These spheres were subjected to a test wherein the spheres were stored at 45? C. for up to 4 weeks. The resulting titre was determined after 1, 2 and 4 weeks of storage. The results are indicated in table 5.

TABLE-US-00005 TABLE 5 Titres of CDV spheres after storage at 45? C. storage time CDV Sphere 3.7% (w/w) CDV Sphere 21.4% (w/w) in weeks sugar log 10 TCID50 sugar log 10 TCID50 0 7.5 7.2 1 4.6 4.7 2 4.3 5.1 3 (estimate, 3.4 5.0 interpolation) 4 2.6 5.0

[0066] The second product (denoted as CPI) is also a vaccine in the form of lyospheres that can serve to formulate a vaccine for injection by re-suspension of one or more spheres in water-for-injection. Each sphere, having a volume of approximately 100 ?l, contains live attenuated canine parainfluenza virus at a titre of about 7 (log10 of the TCID50). This product is obtained in two forms, a first form according to the prior art having 3.7% (w/w) of sucrose as a stabilizing agent and a second form having 21.4% (w/w) of non-polymeric sugar as a stabilizing agent (both as indicated here-above). These spheres were subjected to a test wherein the spheres were stored at 45? C. for up to 4 weeks. The resulting titre was determined after 1, 2 and 4 weeks of storage. The results are indicated in table 6.

TABLE-US-00006 TABLE 6 Titres of CPI spheres after storage at 45? C. storage time CPI Sphere 3.7% (w/w) CPI Sphere 21.4% (w/w) in weeks sugar log 10 TCID50 sugar log 10 TCID50 0 6.7 6.9 1 4.6 5.0 2 4.2 5.3 3 (estimate, 3.3 5.1 interpolation) 4 2.5 4.9

[0067] The third product (denoted as CAV2) is also a vaccine in the form of lyospheres that can serve to formulate a vaccine for injection by re-suspension of one or more spheres in water-for-injection. Each sphere, having a volume of approximately 100 ?l, contains live attenuated canine adeno virus type 2 at a titre of about 5 (log10 of the TCID50). This product is obtained in two forms, a first form according to the prior art having 3.7% (w/w) of sucrose as a stabilizing agent, and a second form having 22.8% (w/w) of sucrose as a stabilizing agent. These spheres were subjected to a test wherein the spheres were stored at 45? C. for up to 4 weeks. The resulting titre was determined after 1, 2, 3 and 4 weeks of storage. The results are indicated in table 7.

TABLE-US-00007 TABLE 7 Titres of CAV2 spheres after storage at 45? C. storage time CAV2 Sphere 3.7% (w/w) CAV2 Sphere 22.8% (w/w) in weeks sugar log 10 TCID50 sugar log 10 TCID50 0 5.0 4.7 1 2.6 4.3 2 2.8 3.9 3 (estimate 2.7 4.1 interpolation) 4 2.5 4.2

[0068] The above experiments with the CPi, CAV2 and CDV antigens were repeated with a different non-polymeric sugar mixture above 20% w/w, to see whether comparable results could be obtained. In this experiment the stabilizer contained 21.6% (w/w) of non-polymeric sugar (6.1% (w/w) sucrose and 15.5% (w/w) trehalose) in a 10 mM KPO4 buffer at pH 7.2. The bulking agent was the same as in the other experiment (5.2% w/v arginine, 0.8% w/v gelatin and 1.0% w/v NZ amine). The spheres were again subjected to a test wherein the spheres were stored at 45? C. for up to 4 weeks. The resulting titre was determined after 1, 2 and 4 weeks of storage. The results are shown in table 8. An equivalent result as the previous formulation could be obtained with this stabilizer.

TABLE-US-00008 TABLE 8 CPi Sphere CAV2 Sphere CDV Sphere 21.6% (w/w) 21.6% (w/w) 21.6% (w/w) storage time sugar log sugar log sugar log in weeks 10 TCID50 10 TCID50 10 TCID50 0 6.50 4.42 7.08 1 5.50 4.33 5.83 2 5.67 4.33 5.42 4 4.67 4.33 5.33