Method for the production of freeze-dried pellets comprising factor VIII

Abstract

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

2. The method of claim 1, further comprising c) storing and homogenizing the freeze-dried pellets comprising factor VIII; d) assaying the freeze-dried pellets comprising factor VIII while they are being stored and homogenized; and e) loading the freeze-dried pellets comprising factor VIII into containers.

3. The method of claim 1, wherein freezing droplets of a solution comprising factor VIII to form pellets comprises passing the solution comprising factor VIII through frequency-assisted nozzles.

4. The method of claim 3, wherein an oscillating frequency of the frequency-assisted nozzles is 1000 Hz and 2000 Hz.

5. The method of claim 1, wherein the inner wall surface of the cooling tower comprises a temperature of 120 C.

6. The method of claim 1, wherein the inner wall surface of the cooling tower is cooled by passing a coolant through one or more pipes, wherein the one or more pipes are in thermal contact with the inner surface.

7. The method of claim 2, wherein factor VIII of the freeze-dried pellets comprising factor VIII comprises a target dosage, wherein assaying the freeze-dried pellets comprising factor VIII while they are being stored and homogenized comprises determining an active content of factor VIII in the freeze-dried pellets comprising factor VIII, and wherein loading the freeze-dried pellets comprising factor VIII into containers comprises loading an amount of freeze-dried pellets comprising factor VIII into the containers such that a dosage of factor VIII in the amount of freeze-dried pellets comprising factor VIII equals or exceeds the target dosage by 25%.

8. The method of claim 1, wherein the pellets of step a) comprise a maximum of the particle size distribution d50 of 200 m to 1500 m.

9. The method of claim 1, wherein the solution comprising factor VIII in step a) comprises a content of dissolved solids of 8 weight-% and 12 weight-%.

10. The method of claim 1, wherein 1 gram of the solution comprising factor VIII in step a) comprises the following composition, the balance being water for injection: TABLE-US-00005 Factor VIII 99 IU to 101 IU Sucrose 68 mg to 72 mg Histidine 2 mg to 4 mg Glycine 23 mg to 26 mg NaCl 1 mg to 3 mg CaCl.sub.2 0.2 mg to 0.4 mg Polysorbate 80 0.07 mg to 0.1 mg.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention will be further described with reference to the following figures and examples without wishing to be limited by them.

(2) FIG. 1 schematically shows an apparatus for the method according to some embodiments;

(3) FIG. 2 shows the temperature profile over time in a cooling tower according to some embodiments;

(4) FIG. 3 shows a temperature and pressure profile during a freeze-drying step according to some embodiments;

(5) FIG. 4 shows another temperature and pressure profile during a freeze-drying step according to some embodiments;

(6) FIG. 5 shows the potency development of samples after different times of storage at room temperature (RT) or at 2-8 C.

(7) FIG. 6 shows the product aggregate/fragment distribution of samples after different times of storage at room temperature (RT) or at 2-8 C. according to some embodiments;

(8) FIG. 7 shows a Scanning Electron Microscopy (SEM) picture of a pellet produced according to some embodiments in 200-fold magnification;

(9) FIG. 8 shows a Scanning Electron Microscopy (SEM) picture of a pellet produced according to some embodiments in 2000-fold magnification;

(10) FIG. 9 shows a Scanning Electron Microscopy (SEM) picture of a pellet produced according to the method described in WO 2006/008006 A1 in 200-fold magnification;

(11) FIG. 10 shows a Scanning Electron Microscopy (SEM) picture of a pellet produced according to the method described in WO 2006/008006 A1 in 2000-fold magnification;

(12) FIG. 11 shows a Scanning Electron Microscopy (SEM) picture of a lyophilisate produced according to standard lyophilisation in 200-fold magnification; and

(13) FIG. 12 shows a Scanning Electron Microscopy (SEM) picture of a lyophilisate produced according to standard lyophilisation in 2000-fold magnification.

DETAILED DESCRIPTION OF THE INVENTION

(14) FIG. 1 schematically depicts an apparatus for conducting the method according to some embodiments. The apparatus comprises, as main components, the cooling tower 100 and the vacuum drying chamber 200. 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.

(15) 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.

(16) 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.

(17) 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.

(18) 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.

(19) 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

(20) GeneralDetermination of Potency the rFVIII Drug Product

(21) Potency has been measured by the use of a Chromogenic assay using the Coatest FVIII kit. The chromogenic assay method consists of two consecutive steps where the intensity of color is proportional to the Factor VIII activity. In the first step, Factor X is activated to Factor Xa by Factor IXa with its cofactor, Factor VIIIa, in the presence of optimal amounts of calcium ions and phospholipids, with 5 minutes incubation at 37 C. Excess amounts of Factor X are present such that the rate of activation of Factor X is solely dependent on the amount of Factor VIII. In the second step, Factor Xa hydrolyzes the chromogenic substrate to yield a chromophore and the color intensity is read photometrically at 405 nm. The validity of the assay is confirmed using a linear regression statistical method against a standard of established potency and the potency of an unknown sample is calculated. Potency is reported in International Units per mL (IU/mL). In case potency is hereinafter referred to as a value of [%], such value is consistently to be understood as a percentage of a target potency in UI/ml (normalized values). Obviously bigger values of %-potency are preferred.

(22) GeneralSize Exclusion Chromatography (SEC) to Determine Distribution of Product Fragments/Aggregates

(23) The principle components of rFVIII preparations are separated into regions based on their hydrodynamic volume, or molecular size on a TSK gel G4000SWXL column with dimensions 7.8 mm ID30 cm, 8 mm particle size;450 Angstrom pore size.

(24) They are then quantitated based on their fluorescence emission at 340 nm after excitation at 276 nm. Quantitative results are expressed as relative % peak area for these regions. The procedure reports results for specific regions of the chromatogram and is used to measure rFVIII aggregates and integrity of chains.

(25) Three regions (Region 1, Region 2, and Region 3) are determined, while Region 2 (in % of total sample) is desired to be maximum, as therein all rFVIII molecules not being aggregated nor fragmented are summarized.

(26) GeneralSpecific Surface Area According to BET

(27) Determination of the specific surface via BET was performed in a multi-point measurement (nitrogen adsorption at 77 Kelvin) and for each sample, two independent amount of material were filled into BET containers and analyzed separately. The containers were tightly closed with stoppers, transferred to the sample preparation station, evacuated and pre-treated for 16 h at 30 C. in vacuum (<0.2, bar) to remove volatile components. Subsequently the samples were vented with nitrogen, weighed and measured according to DIN ISO 9277 using nitrogen.

(28) GeneralScanning Electron Microscopy (SEM) Measurements

(29) 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.

Example 1

(30) Cryopellets of a solution of Kogenate PF were manufactured. Kogenate PF is a plasma protein-free recombinant human factor VIII. The formulation for 1 g of the solution is given below:

(31) TABLE-US-00001 Solids Target: 10% Actual: 10.3% Kogenate PF 100 IU 100 IU Sucrose 70.87 mg 71.79 mg Histidine 3.32 mg 3.59 mg Glycine 23.6 mg 25.54 mg Sodium chloride 1.88 mg 2.03 mg Calcium chloride 0.28 mg 0.30 mg Polysorbate 80 0.08 mg 0.09 mg Water for injection ad 1 g ad 1 g

(32) The bulk solution was sprayed into a wall-cooled cooling tower in accordance with the method of some embodiments. The spraying nozzle had one aperture with a diameter of 400 m. This corresponds to target droplet size of 800 m. The oscillation frequency was 1375 Hz, the deflection pressure 0.2 bar and the pump was operated at 22 rpm. After a total duration of 35 minutes 879.3 g of frozen pellets were collected (96% yield).

(33) The interior temperatures of the cooling tower were monitored and their development over time is depicted in FIG. 2. Curve 1000 represents the sensor reading from an upper part of the cooling tower, curve 1010 the sensor reading from a central part of the cooling tower and curve 1020 the sensor reading from a lower part of the cooling tower. At 14:45 o'clock, when the temperatures had reached 126.0 C. (upper sensor), 129.7 C. (central sensor) and 133.1 C. (lower sensor), the spraying operation was initiated. This is represented by mark 1030 in FIG. 2. At 15:20 o'clock the spraying operation was halted (mark 1040) with recorded temperatures of 130.7 C. (upper sensor), 133.5 C. (central sensor) and 135.2 C. (lower sensor). The frozen pellets were collected in a cooled container having a temperature between 55 C. and 53 C.

Example 2

(34) This example concerns the freeze-drying of a sample of cryopellets obtained in example 1. A LyoMotion freeze-dryer from Meridion was employed in this step. This machine comprises a rotating drum in which the cryopellets were agitated and subjected to drying.

(35) A total of 21.3 g freeze-dried pellets (72.6% yield) having a residual moisture of 0.95% were isolated.

(36) The temperature and pressure profiles of the freeze-drying step are shown in FIG. 3. Curve 1050 represents the product temperature, curve 1060 the condenser temperature of the freeze-drying machine and curve 1070 the internal pressure inside the vacuum chamber of the freeze-drying machine.

Example 3

(37) This example concerns the freeze-drying of another sample of cryopellets obtained in example 1. A LyoMotion freeze-dryer from Meridion was employed in this step. This machine comprises a rotating drum in which the cryopellets were agitated and subjected to drying.

(38) A total of 21.4 g freeze-dried pellets (73.7% yield) having a residual moisture of 0.70% were isolated.

(39) The temperature and pressure profiles of the freeze-drying step are shown in FIG. 4. Curve 1080 represents the product temperature, curve 1090 the condenser temperature of the freeze-drying machine and curve 1100 the internal pressure inside the vacuum chamber of the freeze-drying machine.

(40) Results

(41) Potency assays and size exclusion chromatography analyses of the products obtained are given in the table below for samples taken from the process directly after manufacturing.

(42) TABLE-US-00002 Size exclusion chromatography Potency (average of 2 samples) [% of [relative area-%] target Region 1 Sample potency] (HMW) Region 2 Region 3 Ex. 2 86.2% 0.6 75.0 17.2 Ex. 2 89.9% 0.5 74.6 17.5 Ex. 3 87.9% 0.5 73.8 18.3 Ex. 3 88.4% 0.5 74.5 17.6

(43) Two samples from each example were analyzed with respect to the potency of the factor VIII therein. Target potencies were 250.0 mg/vial. A loss of potency during the processing of the bulk solution and freeze-drying is to be expected. The determined actual potencies between 86.2% and 89.9% were considerably lower in variation than those observed in conventional freeze-drying in a vial. Here potencies ranging from 80.9% to 91.2% can be observed depending on the position of the individual vial in the drying chamber.

(44) For reference, the following table gives analytical data for the precursors of examples 2 and 3 (IPC values).

(45) TABLE-US-00003 Size exclusion chromatography Potency (average of 2 samples) Target [% of [relative area-%] potency target Region 1 Sample [IU/ml] potency] (HMW) Region 2 Region 3 Kogenate 1220.0 101.2% 0.4 74.5 17.6 PF solution after thawing Kogenate 100.0 96.8% 0.6 73.6 17.5 PF bulk solution after dilution Ex. 1 100.0 111.7% 0.6 73.0 18.0 (cryopellets)

(46) Furthermore the above properties of the samples were evaluated for up to 6 months of storage at room temperature (RT) and at 2-8 C.

(47) It can be seen from FIG's 5 and 6 (samples were prepared from different starting material of lower activity) that no significant changes in potency or fragment/aggregate composition of the samples has occurred, which underlines the robustness of the process according to some embodiments with regard to the properties of the thereby produced pellets.

Example 4

Direct Comparison to the Prior Art

(48) Solutions of the identical batch of Factor VIII drug substance with the same formulation composition as provided in Example 1 were prepared and processed via different drying methods. 3000 ml solution were frozen in a cooling tower according to Example 1, and the frozen pellets were lyophilized in two different rotary freeze dryers (LyoMotion) with a batch size of 319 g and 2614 g.

(49) A separate part of the solution was processed according to the method described in WO 2006/008006 A1. A total of 1200 ml solution were sprayed in portions of 200 ml through a 400 m nozzle and atomized at a frequency of 900 Hz with a rate of about 16.5 g/min. 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 each portion, the frozen pellets were removed by pouring the liquid nitrogen through a pre-cooled sieve and storing them at low temperature. Once all portions were collected, they were placed in 2 racks lined with plastic foil onto the pre-cooled shelved of a Virtis Advantage Pro freeze dryer and lyophilized. Primary drying was conducted at 10 C. shelf temperature over a duration of 60 hours, followed by secondary drying for 8 hours at 25 C. After completion of drying, the dry pellets were instantly transferred into glass bottles and firmly closed. Subsequently, 250 mg of pellets were weighed into 10R type I glass vials under a nitrogen atmosphere.

(50) A third separate part of the solution was filled into 10R type I glass vials and freeze dried in a conventional vial freeze dryer. A total of 488 vials were filled with 2.5 ml solution per vial (1241 g solution in total), semi-stoppered and loaded into a HOF freeze dryer. The solution was frozen to 45 C., and primary drying was performed at 20 C., followed by a secondary drying step at 25 C. The complete freeze drying process required approx. 65 hours. The vials were stoppered within the freeze dryer and sealed directly after unloading.

(51) All three samplesthose from the first processing according to Example 1, those processed as described in WO 2006/008006 A1 and those from a conventional vial freeze drying processwhere thereafter made subject to specific surface area according to BET and Scanning Electron Microscopy (SEM) measurements.

(52) It can be seen that the pellets produced pursuant to some embodiments display a higher specific surface areawhich improves reconstitution of the freeze-dried solid in a liquid for administrationand more homogeneous morphology, which improves handling properties in later process steps for those pellets.

(53) The respective specific surface area according to BET are summarized as follows:

(54) TABLE-US-00004 Specific surface area according Pellet produced . . . to BET [m.sup.2/g] according to embodiments disclosed 5.2 herein according to WO 2006/008006 A1 0.8 standard lyophilisation 0.4

(55) It's apparent that the specific surface area of pellets that are produced by some embodiments disclosed herein is significantly bigger than that of pellets produced according to similar prior art processes (such as WO 2006/008006 A1) and particularly compared to standard lyophilisation.