METHOD FOR THE PRODUCTION OF FREEZE-DRIED PELLETS COMPRISING AN ANTI-COAGULATION FACTOR XIa (FXIa) ANTIBODY

Abstract

A method for the production of freeze-dried pellets comprising an anti-FXIa antibody comprises the steps of: a) freezing droplets of a solution comprising an anti-FXIa antibody to form pellets; b) freeze-drying the pellets; wherein in step a) the droplets are formed by means of droplet formation of the solution comprising an anti-FXIa antibody into a cooling tower which has a temperature-controllable inner wall surface and an interior temperature below the freezing temperature of the solution and wherein in step b) the pellets are freeze-dried in a rotating receptacle which is housed inside a vacuum chamber.

Claims

1. A method for the production of freeze-dried pellets comprising an anti-coagulation factor XIa (FXIa) antibody, the method comprising the steps of: a) freezing droplets of a solution comprising an anti-FXIa antibody to form pellets; and b) freeze-drying the pellets; wherein in step a) the droplets are formed by means of droplet formation of the solution comprising an anti-FXIa antibody into a cooling tower (100) which has a temperature-controllable inner wall surface (110) and an interior temperature below the freezing temperature of the solution; and in step b) the pellets are freeze-dried in a rotating receptacle (210) which is housed inside a vacuum chamber (200).

2. A method for reducing the reconstitution time of freeze-dried pellets comprising an anti-FXIa antibody as compared to anti-FXIa antibody comprising lyophilisates obtained by conventional freeze-drying, the method comprising the steps of: a) freezing droplets of a solution comprising an anti-FXIa antibody to form pellets; and b) freeze-drying the pellets; wherein in step a) the droplets are formed by means of droplet formation of the solution comprising an anti-FXIa antibody into a cooling tower (100) which has a temperature-controllable inner wall surface (110) and an interior temperature below the freezing temperature of the solution; and in step b) the pellets are freeze-dried in a rotating receptacle (210) which is housed inside a vacuum chamber (200).

3. The method according to claim 1, further comprising the steps c) and d) after step b): c) storing and homogenizing the freeze-dried pellets; and d) loading the freeze-dried pellets into containers.

4. The method according to claim 1, wherein in step a) the droplets are made by means of droplet formation by passing the solution through frequency-assisted nozzles.

5. The method according to claim 4, wherein the oscillating frequency is ≥200 Hz to ≤5000 Hz.

6. The method according to claim 1, wherein in step a) the inner surface (110) of the cooling tower (100) has a temperature of ≤−120° C.

7. The method according to claim 1, wherein the inner surface (110) of the cooling tower (100) is cooled by passing a coolant through one or more pipes (140) which are in thermal contact with the inner surface (110).

8. The method according to claim 1, wherein the pellet size median of the pellets obtained in step a) is about ≥200 μm to ≤1500 μm.

9. The method according to claim 1, wherein the solution comprising an anti-FXIa antibody in step a) has a content of dissolved solids of ≥5 weight-% to ≤30 weight %.

10. The method according to claim 1, wherein the solution comprising an anti-FXIa antibody in step a) has an antibody concentration of ≥5 mg/ml to ≤300 mg/ml.

11. The method according to claim 1, wherein the solution comprising an anti-FXIa antibody in step a) has the following composition with respect to 100 ml of the solution, the balance being water for injection: Anti-FXIa antibody ≥0.5 g to ≤30 g Trehalose ≥1 g to ≤25 g Histidine ≥50 mg to ≤1.5 g Glycine ≥50 mg to ≤1.5 g Arginine ≥50 mg to ≤5 g Polysorbate 80 ≥5 mg to ≤0.5 g

12. Freeze-dried pellets comprising an anti-FXIa antibody obtainable by the method according to claim 1.

13. The freeze-dried pellets comprising an anti-FXIa antibody according to claim 12, wherein the freeze-dried pellets comprising an anti-FXIa antibody exhibit a reduced reconstitution time as compared to anti-FXIa antibody comprising lyophilisates obtained by conventional freeze-drying.

Description

FIGURES

[0064] FIG. 1 schematically shows an apparatus for the method according to the invention.

[0065] FIG. 2 graphically depicts the temperature and pressure profile measured over time during conventional freeze-drying (Method 1) of the antibody solution.

[0066] FIG. 3 graphically depicts the temperature and pressure profile measured over time during freezing and drying of the antibody solution according to the method described in WO 2006/008006 (Method 2).

[0067] FIG. 4 graphically depicts the temperature profile in the cooling tower measured over time during processing of the antibody solution according to the present invention (Method 3).

[0068] FIG. 5 graphically depicts the temperature and pressure profile measured over time during freezing and drying of the antibody solution according to the present invention (Method 3).

[0069] FIG. 6 shows Scanning Electron Microscopy (SEM) pictures of a pellet produced according to the present invention (Method 3).

[0070] FIG. 7 shows Scanning Electron Microscopy (SEM) pictures of a lyophilisate produced according to conventional freeze-drying (Method 1).

[0071] FIG. 8 shows Scanning Electron Microscopy (SEM) pictures of a lyophilisate produced according to the freeze-drying process disclosed in WO 2006/008006 (Method 2).

[0072] FIG. 1 schematically depicts an apparatus for conducting the method according to the invention. The apparatus comprises, as main components, the cooling tower 100 and the vacuum drying chamber 200.

[0073] The cooling tower comprises an inner wall 110 and an outer wall 120, thereby defining a space 130 between the inner wall 110 and the outer wall 120.

[0074] This space 130 houses a cooling means 140 in the form of piping. A coolant can enter and leave the cooling means 140 as indicated by the arrows of the drawing.

[0075] Coolant flowing through the cooling means 140 leads to a cooling of the inner wall 110 and thus to a cooling of the interior of the cooling tower 100. In the production of frozen pellets (cryopellets), liquid is sprayed into the cooling tower via nozzle 150. Liquid droplets are symbolized in accordance with reference numeral 160.

[0076] The liquid droplets eventually solidify (freeze) on their downward path, which is symbolized in accordance with reference numeral 170. Frozen pellets 170 travel down a chute 180 where a valve 190 permits entry into the vacuum drying chamber 200.

[0077] While not depicted here, it is of course also possible and even preferred that the chute 180 is temperature-controlled in such a way as to keep the pellets 170 in a frozen state while they are collecting before the closed valve 190.

[0078] Inside the vacuum drying chamber 200 a rotatable drum 210 is located to accommodate the frozen pellets to be dried. The rotation occurs around the horizontal axis in order to achieve an efficient energy transfer into the pellets. Heat can be introduced through the drum or via an encapsulated infrared heater. As an end result, freeze-dried pellets symbolized by the reference numeral 220 are obtained.

EXAMPLES

Example 1: Lyophilization by Conventional Freeze-Drying

[0079] This example describes conventional lyophilization (Method 1) of a liquid high-concentration composition comprising 076D-M007-H04-CDRL3-N110D. The composition comprised a histidine-glycine-arginine buffer system. Trehalose was added as stabilizer. 076D-M007-H04-CDRL3-N110D was formulated at approximately 150 mg/ml in:

[0080] 20 mM L-Histidine, 50 mM L-Arginine hydrochloride, 50 mM Glycine, 5% trehalose dihydrate, 0.10% polysorbate 80, pH 5.0 (composition 32).

[0081] To develop a suitable lyophilization process it was essential to determine the collapse temperature that decided at which temperature the primary drying could be conducted. The collapse temperature was measured using a lyo-microscope (Lyostat 2, Biopharma) by freezing the composition to −50° C. before drawing vacuum (0.1 mbar) and heating the sample with a ramp of 1° C./minute to 20.0° C. While heating up the composition pictures were taken and analyzed until a collapse of the tested system could be observed.

[0082] The collapse temperature of 076D-M007-H04-CDRL3-N110D was found to be −14.3° C. and is an essential parameter for selection of the following lyophilization cycle.

[0083] The liquid composition 32 comprising anti-FXIa antibody 076D-M007-H04-CDRL3-N110D was processed according to a conventional freeze-drying method (Method 1). The solution containing 150 mg/ml anti-FXIa antibody was filled into 10R type I glass vials and freeze-dried in a conventional vial freeze dryer. A total of 20 vials were filled with 2.25 ml solution per vial, semi-stoppered and loaded into a Virtis Genesis freeze dryer. The solution was frozen to −45° C., and primary drying was performed at +10° C., followed by a secondary drying step at 40° C. The complete freeze drying process required approx. 38 hours. The vials were stoppered within the freeze dryer and sealed directly after unloading.

[0084] The details of the lyophilization cycle according to a conventional freeze-drying method (Method 1) for composition 32 are summarized in Table 1.

TABLE-US-00001 TABLE 1 Lyophilization cycle of composition 32 (Method 1) Time Temp Pressure [hh:mm] [° C.] [mbar] Loading 00:01 20.0 1000 Freezing 00:30 −5.0 1000 Freezing 01:00 −5.0 1000 Freezing 00:40 −45.0 1000 Freezing 03:30 −45.0 1000 Evacuation 00:01 −45.0 0.100 Primary drying 01:00 10 0.1 Primary drying 19:00 10 0.1 Secondary drying 01:00 40 0.04 Secondary drying 10:00 40 0.04 Time Loading 00:01 Summary Freezing 05:41 Primary drying 20:00 Secondary drying 11:00 Total 36:42

[0085] The pressure and temperature profile measured over time during the thus conducted conventional freeze-drying process is graphically depicted in FIG. 2.

[0086] The conventional lyophilization method described above resulted in a yellowish cake or powder which can subsequently be reconstituted.

[0087] For reconstitution of the lyophilisate 2 ml sterile water for injection as reconstitution medium was injected into each of the vials. The vials were then gently agitated for about 10 to 20 seconds. Reconstitution of this lyophilisate obtained by conventional freeze-drying resulted in a reconstitution time of 137 min.

[0088] After reconstitution a clear, yellowish solution without any visible particles was observed. No aggregation or hints of aggregation were detected.

Example 2: Lyophilization by Two Different Spray-Freeze-Drying Methods

[0089] As the reconstitution time of the lyophilisate obtained by a conventional freeze-drying method as described in Example 1 (Method 1) was, with more than 2 hours, unacceptably long, two different other freeze-drying methods were applied and compared to the conventional freeze-drying as described above.

[0090] Firstly, the liquid composition 32 comprising anti-FXIa antibody 076D-M007-H04-CDRL3-N110D was processed according to the method described in WO 2006/008006 (Method 2). 138 ml solution containing 150 mg/ml anti-FXIa antibody were sprayed through a 400 μm nozzle and atomized at a frequency of 470 Hz with a rate of about 19.5 g/min and a pressure overlay of 220 mbar. The droplets were frozen in an isolated vessel filled with liquid nitrogen that was positioned approx. 25 cm below the nozzle and stirred throughout the process. After completion of spraying the frozen pellets were removed by pouring the liquid nitrogen through a pre-cooled sieve and placed in a steel rack lined with plastic foil onto the pre-cooled shelves of a Virtis Advantage Pro freeze dryer and lyophilized. Primary drying was conducted at 0° C. shelf temperature over a duration of 33 hours, followed by secondary drying for 5 hours at 30° C. After completion of drying, the dry pellets were instantly transferred into glass bottles which were firmly closed. Subsequently, 520 mg of pellets were weighed into 10R type I glass vials under a dry nitrogen atmosphere. The pressure and temperature profile measured over time during freezing and drying of the antibody solution according to the method described in WO 2006/008006 is graphically depicted in FIG. 3.

[0091] Secondly, the liquid composition 32 comprising anti-FXIa antibody 076D-M007-H04-CDRL3-N110D was processed according to the spray-freeze-drying based method for reducing the reconstitution time of freeze-dried pellets according to the present invention (Method 3) which comprises the steps of:

a) freezing droplets of a solution comprising an anti-FXIa antibody to form pellets;
b) freeze-drying the pellets;
wherein in step a) the droplets are formed by means of droplet formation of the solution comprising an anti-FXIa antibody into a cooling tower which has a temperature-controllable inner wall surface and an interior temperature below the freezing temperature of the solution and in step b) the pellets are freeze-dried in a rotating receptacle which is housed inside a vacuum chamber.

[0092] For this purpose, 250 ml solution containing 150 mg/ml anti-FXIa antibody was freeze-dried by spraying the solution into a wall-cooled cooling tower. The spraying nozzle had one aperture with a diameter of 400 μm. This corresponds to a droplet size of about 800 μm. The oscillation frequency was 1445 Hz, the deflection pressure 0.4 bar and the pump was operated at 14 rpm. After completion of drying, the dry pellets were instantly transferred into glass bottles which were firmly closed. Subsequently, 520 mg of pellets were weighed into 10R type I glass vials under a dry nitrogen atmosphere. The temperature profile in the cooling tower measured over time is graphically depicted in FIG. 4. The temperature and pressure profile measured over time during freezing and drying of the antibody solution is graphically depicted in FIG. 5.

[0093] The freeze-drying method according to the present invention (Method 3) yielded uniform pellets exhibiting a narrow size and weight distribution and a high surface area. The residual humidity in the pellets obtained by this method was 0.268%. The lyophilisates obtained by conventional freeze-drying (Method 1) comprised 0.15% residual moisture.

[0094] Size exclusion chromatography analyses of the pellets obtained by the three different freeze-drying processes are given in the Table 2.

TABLE-US-00002 TABLE 2 Size exclusion chromatography analyses of the pellets obtained by the three different freeze-drying processes SEC Sum high Sum low molecular molecular Dimer weight (HMW) weight (LMW) Monomer Sample [% area] aggregates aggregates [% Area] Method 3 1.66 1.82 1.20 96.96 (as described herein) Method 1 1.35 1.41 1.13 97.45 (Conventional Lyophilization) Method 2 1.57 1.77 1.15 97.07 (W02006/ 008006)

[0095] Overall, comparable analytical data were obtained by size exclusion chromatography for the three freeze-drying methods.

[0096] To determine the quantity of intact antibody relative to the overall proteinaceous components present in the sample, IgG purity was analyzed by Capillary SDS-Gel Electrophoresis (CGE). Test and reference samples were separated by CGE using a bare fused-silica capillary in the presence of sodium dodecyl sulfate (SDS). The test was performed under non-reducing conditions. The separated samples were monitored by absorbance at 220 nm. The intention of the assay was to integrate the peak area of the main peak and analyze the byproducts after reduction.

[0097] The results of capillary gel electrophoresis (CGE) and ELISA analyses are given in the Table 3.

TABLE-US-00003 TABLE 3 Capillary gel electrophoresis (CGE) and ELISA analyses of the pellets obtained by the three different freeze-drying processes CGE IgG HHL HH HL [% corr. [% corr. [% corr. [% corr. Sample Area] Area] Area] Area] ELISA Method 3 95.82 2.51 0.38 0.18 112 (111.95) Method 1 95.80 2.60 0.36 0.17 101 (100.73) Method 2 95.83 2.55 0.38 0.17 87 (86.87)

[0098] Reconstitution times of the pellets obtained by the three different freeze-drying methods were compared as follows. 2 ml sterile water for injection as reconstitution medium was injected into each of the vials. After taking photographs the vials were gently agitated for about 10 to 20 seconds. Reconstitution of the pellets over time was visually observed and documented photographically.

[0099] The reconstitution times of the pellets obtained by the three different freeze-drying methods are given below:

TABLE-US-00004 Freeze-Drying method Reconstitution Time Ab Concentration Method 1 137 min  150 mg/ml Method 2 16 min 150 mg/ml Method 3 11 min 150 mg/ml

[0100] The reconstitution of the freeze-dried anti-FXIa antibody comprising pellets obtained with the method according to the present invention (Method 3) was significantly faster than the reconstitution of equivalent anti-FXIa antibody comprising lyophilisates obtained by conventional freeze-drying (Method 1), but also faster compared to freeze-dried pellets obtained according to WO 2006/008006 (Method 2).

[0101] The pellets obtained by the three different freeze-drying methods were thereafter subjected to Scanning Electron Microscopy (SEM) measurements. Therefore, preparation of samples was performed in a glove bag under nitrogen atmosphere, each sample was prepared individually. The sample was placed on a holder and sputtered with gold. Subsequently the scanning electron microscopy measurement was performed. SEM pictures are shown in FIGS. 6 to 8.

[0102] It can be seen that the pellets produced pursuant to the method according to the invention display a particularly homogeneous morphology, which may improve handling properties in later process steps.