Thrombin microcapsules, preparation and uses thereof
10869912 ยท 2020-12-22
Assignee
Inventors
Cpc classification
A61K9/1688
HUMAN NECESSITIES
A61K9/1694
HUMAN NECESSITIES
A61K9/1623
HUMAN NECESSITIES
International classification
A61K9/16
HUMAN NECESSITIES
Abstract
Provided are spray-dried thrombin powders comprising microcapsules, methods of preparation and uses thereof.
Claims
1. A method for preparing thrombin microcapsules, the method comprising the step of: a) spray drying an aqueous solution comprising: i) thrombin at a concentration ranging from 100 to 3000 IU/ml, ii) a carrier protein at a concentration ranging from 0.6 to 60 mg/ml and iii) a carbohydrate at a concentration ranging from 2 to 200 mg/ml, wherein said spray drying comprises producing droplets from the solution, and evaporating water from the droplets, wherein said evaporating is carried out by a drying gas and comprises heating said gas to a temperature ranging from about 100 to 170 C., thereby obtaining the thrombin microcapsules; and b) cooling the thrombin microcapsules by exposure to a cold gas flow.
2. The method according to claim 1, wherein said drying gas is set to a flow rate of 0.1 to about 1.0 m.sup.3/min.
3. The method according to claim 2, wherein said setting and said heating are carried out sequentially.
4. The method according to claim 2, wherein said drying gas flow rate is set from about 0.3 to about 0.6 m.sup.3/min.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) Some embodiments of the invention are described herein with reference to the accompanying figures. The description, together with the figures, makes apparent to a person having ordinary skill in the art how some embodiments of the invention may be practiced. The figures are for the purpose of illustrative discussion and no attempt is made to show structural details of an embodiment in more detail than is necessary for a fundamental understanding of the invention. For the sake of clarity, some objects depicted in the figures are not to scale.
(2) In the Figures:
(3)
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DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION
(8) The invention, in some embodiments thereof, relates to spray-dried thrombin powder comprising microcapsules, methods of preparation thereof and uses thereof.
(9) The principles, uses and implementations of the teachings herein may be better understood with reference to the accompanying description. Upon perusal of the description, one skilled in the art is able to implement the invention without undue effort or experimentation.
(10) Before explaining at least one embodiment in detail, it is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of the components and/or methods set forth in the following description. The invention is capable of other embodiments or of being practiced or carried out in various ways. The phraseology and terminology employed herein are for descriptive purpose and should not be regarded as limiting.
(11) As demonstrated in the Results section below (see Examples 1, 2 and
(12) As demonstrated in the Results section below, embodiments of reconstituted thrombin compositions disclosed herein that include both a carrier-protein (e.g., albumin) and a carbohydrate (e.g., a sugar alcohol such as mannitol) have exceptionally high thrombin activity-recovery (see Examples 2 and 4), surprisingly even at high thrombin concentrations (see Examples 6 and 6). Without wishing to be held to any one theory, it is currently believed that the combination of carrier protein and carbohydrate helps maintain thrombin structure and potency, inter alia, by providing heat protection.
(13) The thrombin activity recovery provided by embodiments of the teachings herein is superior to that provided by prior art. For example, US Patent Application Publication No. 2012/0315305 discloses spray-dried thrombin having a concentration of 25 to 1000 IU/g. The comparable Example 6 shows that a thrombin composition according to the teachings herein that includes a carrier protein provides a substantially higher thrombin activity recovery.
(14) It is believed that some of the advantages of some of the embodiments according to the teachings herein are a result of the spray-drying conditions used that lead to production of active thrombin powder at high mass and potency yields and at high concentrations. In some embodiments, a high level of thrombin activity recovery is achieved by at least one of: drying gas (e.g., dry air or nitrogen) at a temperature no higher than 190 C.; a ratio of thrombin solution flow rate: atomizing gas flow rate of at least 1:1000. i.e. when spray-drying 1 ml of thrombin solution per minute, atomizing gas flow rate is preferably at least 1 l/min. a ratio of thrombin solution flow rate: drying gas flow rate of at least 1:43,000. i.e. when drying 1 ml of thrombin per minute, the drying gas flow rate is preferably at least 43 l/min.
(15) Specifically, in Example 8a drying gas temperature of 190 C. yielded spray-dried thrombin powder with water content as low as 1.5%. In Example 9 is seen that to obtain spray-dried thrombin powder with a high thrombin activity recovery and low water content, it is preferable to set the inlet gas temperature to no more than about 170 C. In some embodiments, to reduce water content while maintaining thrombin activity recovery, drying gas flow rate is increased rather than drying gas temperature.
(16) It has surprisingly been found that in some embodiments increasing the drying gas flow rate leads to a decrease in the water content in the resulting dry thrombin composition despite the reduced retention time of the thrombin in the drying column.
(17) It is considered that increased drying gas flow rate would decrease the pressure over the drying particles, resulting in an increase in evaporation. Without wishing to be held to any one theory, it is noticed that when the powder reaches the bottom of the drying column, it is hot and particles would tend to adhere to the inner parts of the drying column and the tubes that deliver the powder flow from the drying column to the cyclone, resulting in reduced mass yield. This adhesion would be expected to occur when the powder is at a temperature greater than its glass transition temperature, which causes the surface of the particles to be rubbery or plastic in nature. Introducing cool gas into the bottom of the drying column would therefore be expected to reduce the temperature at which the particles flow, thus reducing surface temperature of the particles below their glass transition temperature, such that the particles would be present in a glassy or elastic state and preventing them from adhering to the inner parts of the system, such that mass yield is consequently increased.
(18) As shown in Example 7, embodiments with particularly low drying gas temperature (140 C.) and high drying gas flow rate (thrombin solution: drying gas flow rate 1:900000) provided a spray-dried thrombin powder that was dry, had a narrow particle size distribution, and had a high thrombin activity recovery.
(19) Embodiments of spray-dried thrombin compositions according to the teachings herein having a relatively high thrombin concentration provide a number of advantages. For example, when a fibrin patch comprising such compositions is used, there is a higher thrombin concentration in the formed fibrin clot formed, resulting in more fibrin fibers in the clot, leading to greater clot strength and adhesion to tissue (see Example 10), as well as faster clot formation (Time To Hemostasis (TTH) <3 minutes, see Example 11).
(20) Embodiments of spray-dried thrombin compositions according to the teachings herein can be stored at room temperature for extended periods of time without needing storage in a cool (i.e., 2-8 C.) or freezing (i.e. less than 18 C.).
(21) The flowability of spray dried thrombin compositions according to the teachings herein are shown in Example 12. As used herein, the term powder flowability refers to the ease with which a powder will flow under a specified set of conditions, as measured with a powder rheometer (FT4 powder rheometer, Freeman technology, Gloucestershire, UK), by moving a blade in upward lifting mode of displacement. Examples of such conditions include the pressure on the powder, the roughness of the particle surface, the humidity of the air around the powder and the equipment the powder is flowing through or from. The flowability is expressed as the energy required to displace the powder composition, divided by the displaced powder weight. The demonstrated low specific energy of the composition means that less energy is required to displace the composition. Practically, during a manufacturing process, a low specific energy composition is less likely to cause interlocking and friction which may clog machinery (e.g., an auger dozer) used to transfer the composition.
(22) Additionally, drying of embodiments of aqueous thrombin composition feed according to the teachings herein using embodiments of spray-drying processes according to the teachings herein for preparing spray-dried thrombin microcapsule compositions results in the production of highly concentrated thrombin compositions having dense particles. As shown in Table 17 (Example 12), the conditioned bulk density (i.e. the powder density wherein air gaps between the particles are not eliminated, for instance by tapping) of the spray-dried thrombin composition according to an embodiment of the teachings herein was 0.46 g/ml and the tapped density (the powder density after elimination of air gaps between the particles e.g. by tapping) was 0.58 g/ml, compared to conditioned bulk density of 0.37 g/ml and tapped density of 0.48 gr/ml for a comparable lyophilized composition.
EXAMPLES
Materials and Methods
(23) Materials
(24) Thrombin (NDC No. 63713-460, Manufactured by Omrix Biopharmaceuticals Ltd., Israel). In the experiments below, a thrombin component as in EVICEL Fibrin Sealant (Manufactured by Omrix Biopharmaceuticals Ltd., Israel) was used.
(25) Human Serum Albumin (Plasbumin 25, 25% sterile solution of albumin in aqueous diluent, NDC no. 13533-692-20) was acquired from Grifols USA, Research Triangle Park, N.C., USA.
(26) D-Mannitol (Cat. No. 443907), Sodium Acetate Trihydrate (Cat. No. 1370121000), Calcium Chloride Dihydrate (1371015000) were obtained from Merck Milipore (Billerica, Mass., USA).
(27) Until use, thrombin was stored in a standard laboratory freezer set to maintain a temperature of about 18 C., Human Serum Albumin and all solid materials were stored at room temperature (about 24 C.) not exceeding 30 C.
(28) Equipment
(29) Spray dryer. 4M8-TriX spray dryer was used (by ProCepT nv, Zelzate, Belgium).
(30) Methods
(31) Preparation of Thrombin Solution:
(32) For each experiment, a desired amount of frozen thrombin solution (Omrix Biopharmaceuticals, Israel) was allowed to warm to room temperature inside a sealed bottle. A thrombin solution was made by dissolving the warmed thrombin in triple-distilled water at the desired concentration (see tables, below).
(33) Spray Drying:
(34) The prepared thrombin solution was drawn into a syringe and placed inside the syringe pump of the spray dryer. The syringe pump was set to the desired flow rate with feed valve closed.
(35) While the syringe pump feed valve was closed, the spray dryer was activated and the desired atomizing gas flow rate, drying gas flow rate, drying gas temperature, cooling gas flow rate and cyclone gas flow rate were set.
(36) The cooling gas flow rate and temperature were selected such as not to disrupt laminar flow in the drying column, but to reduce the gas flow temperature to below the glass transition temperature of the composition in order to prevent powder from sticking to the glass parts.
(37) The spray dryer was allowed to run until a steady state was reached where the actually measured value of the parameters reached the set levels and remained steady. The feed valve was then opened, allowing the thrombin solution to flow through the feed inlet to the spray nozzle to be atomized by the atomizing gas flow to small droplets which then dried in the drying column. Spray dried thrombin powder was formed, which was collected in the powder outlet of the cyclone of the spray dryer.
(38) The spray dried thrombin powder recovered from the cyclone was weighed in a de-humidifier at a relative humidity of below 30% and divided into samples of between 100-200 mg. Each sample was individually sealed in a test tube with a plug and sealed with Parafilm (Bemis, Oshkosh, Wis., USA) until evaluation.
(39) Characterization of the Spray-Dried Thrombin Powder
(40) The spray-dried thrombin powder was characterized by determining the thrombin activity including thrombin activity recovery (compared to thrombin activity prior to spray drying), water content, particle size distribution, total and clotable protein, density (conditioned bulk and tapped), flowability and mass yield.
(41) Thrombin Activity Determination
(42) Thrombin activity of an aqueous thrombin solution was determined using a clotting time assay by measuring thrombin clotting activity in the solution according to the modified European Pharmacopeia Assay (0903/1997) procedure. Briefly, a calibration curve of thrombin concentration vs. clotting time was prepared by mixing a thrombin standard (Fibri-Prest 2 by Beijing Stago Diagnosis Trading Co. Ltd, Beijing, China) with a 0.1% fibrinogen solution (Omrix Biopharmaceuticals Ltd., Israel) at various different thrombin concentrations (4, 6, 8, and 10 IU/ml), and measuring the time taken for each sample to clot using a clotting measurement device (STart by Beijing Stago Diagnosis Trading Co. Ltd., Beijing, China).
(43) For each analysis, a sample of approximately 35 mg spray-dried thrombin was dissolved in 1 ml double distilled water (to give a thrombin solution having thrombin activity of 1000 IU/ml). This solution was diluted 1:200 to produce a solution having a potency of 5 IU/ml). 40 l of the diluted thrombin solution was mixed with 160 l of 0.1% fibrinogen solution in the clotting measurement device. The clotting time of the sample was determined and the thrombin activity was extrapolated with reference to the calibration curve.
(44) Water Content Determination
(45) Water content determination was carried out using the volumetric Karl Fischer Titration method (KFT), which is based on the US Pharmacopoeia assay (USP 27, <921>, P. 2398-2399). Prior to the titration, the water was extracted from the spray-dried thrombin powder by adding about 10 ml dried methanol to about 100 to about 200 mg of the spray dried thrombin powder.
(46) Particle Size Distribution Determination
(47) The size distribution of particles suspended in liquid is measured by using the principles of light scattering. Measurement were carried out by using the particle size analyzer LS 13 320 (Beckman Coulter Inc., Pasadena, Calif., USA). The pattern measured by the LS 13 320 is the sum of the patterns scattered by each constituent particle in the sample. The LS 13 320 consists of an optical bench and Micro Liquid Module (MLM) which includes a chemically resistant liquid cell and stir bar to keep the suspended particles homogenously mixed. The system is designed to work with small quantities of suspension fluid (12 ml). A powder sample of between 100 and 200 mg of a spray-dried thrombin was suspended in 10 ml hydrofluoroether (HFE-7000, manufactured by 3M Company, Two Harbors, Minn., USA). The sample was placed in a micro-liquid module (MLM) and the particle size distribution was determined.
(48) Total Protein and Clottable Protein
(49) A total protein test was used to determine the total amount of proteins in a fibrin sealant patch sample. Powder is extracted from a fibrin sealant patch and dissolved in a solubilizing solution which is capable of dissolving the proteins in the sample. The protein concentration is determined by the Lowry method against a calibration curve.
(50) Friability
(51) The friability of patches comprising fibrinogen and thrombin powders was intended to determine the degree of powder flaking or crumbling from a fibrin sealant patch after a force was applied to a sample of the patch. To measure the friability, a patch was cut to a specific size, and the weight of the sample was determined. The sample was subjected to a defined impact force by placing the sample within a vial and dropping the vial through a tube of fixed height onto a rubber stopper. The amount of powder loss was determined by weighing the patch sample and the percentage of weight reduction of the sample was calculated.
(52) Tissue Peel
(53) The adhesion strength of a fibrin sealant patch to calf corium tissue substrate was determined by using the 5000 Series Instron Tensiometer, Instron Corp. Norwood, Mass., USA). To measure the adhesion strength, a fibrin patch was applied to the surface of the corium and wetted with saline. The average force necessary to subsequently perform a 90 degree peel test of the fibrin patch from the calf corium tissue was determined by measuring the integral under the force vs distance curve.
(54) Tensile Strength
(55) The tensile strength of a dogbone-shaped fibrin patch sample having width on the sides of 25 mm, width in the middle of 15 mm and length 102 mm was determined. The sample was placed between Instron grips (5000 Series Instron Tensiometer, Instron Corp. Norwood, Mass., USA). The crosshead was moved at a pre-determined constant rate of extension of 305 mm/min. The test was concluded when a peak load drop of 30% was achieved, indicating a rupture occurrence.
(56) Density (Conditioned Bulk, Tapped)
(57) Conditioned bulk density, tapped density and specific energy were measured using a powder rheometer (FT4 powder rheometer, Freeman technology, Gloucestershire, UK). Tests were carried out in a 25 mm vessel with a 23.5 mm blade at 250 taps.
(58) The term density refers to the weight of solids per unit volume. The density can be measured in liquid, solid (e.g. powder) or gas form. For powder, the density can be conditioned bulk density or tapped density. The bulk density of a powder is the ratio of the mass of an untapped powder sample and its volume including the contribution of the interparticulate void volume.
(59) As used herein, the term bulk density refers to the density of a bulk powder sample of a known weight in a known volume size.
(60) As used herein, the term conditioned bulk density refers to the density of a bulk, powder sample, which has undergone a conditioning step, in which the powder is gently displaced using a rotating blade in order to loosen the powder to remove any pre compaction or excess air.
(61) As used herein, the term tapped density refers to the conditioned bulk density powder sample is tapped a constant number of times after which the density is measure.
(62) Flowability (Specific Energy)
(63) The FT4 powder rheometer (FT4 powder rheometer, Freeman technology, Gloucestershire, UK) was used to measure the resistance that the powder exerts on the rheometer blade (the resistance of the powder to flow), expressed as torque (radial resistance) and force (vertical resistance).
Specific energy(flowability)=[distance(torque+force)]/split mass
wherein distance refers to the distance travelled by the blade in an upward lifting mode of displacement.
(64) As used herein, the term split mass refers to the mass of powder that was displaced during the measurement.
(65) In the examples described below the test conditions were as follow: 5 helix and 100 mm/sec blade tip speed. A sample volume of 25 ml was used.
Example 1
Effect of Thrombin Solution Composition on Maintaining Thrombin Activity During Spray Drying
(66) Various thrombin solutions were prepared (including different salts and/or carbohydrates and/or proteins) in order to test the effect of the excipient(s) in solution on maintaining the thrombin activity during spray-drying.
(67) Eight different such thrombin solutions were prepared as shown in Table 1:
(68) TABLE-US-00001 TABLE 1 HSA (human Compo- Sodium Sodium Calcium D(-) serum sition Thrombin Acetate Chloride Chloride Mannitol albumin) # [IU/ml] [mM] [mM] [mM] [mg/ml] [mg/ml] 1 1000 7 67 3 2 1000 7 67 40 3 1000 20 200 40 4 800 7 67 3 20 5 1200 7 67 3 20 6 1000 7 67 3 6 7 1000 20 200 3 20 6 8 1000 20 20 40 20 6
(69) Compositions 1-8 were spray-dried as described above with the following parameters: 1. Thrombin sample flow rate: 400 ml/h; 2. Atomizing gas flow rate: 7 l/min; 3. Drying gas (dry air) flow rate: 600 l/min; 4. Drying gas (dry air) temperature: 140 C.; 5. Cooling gas (dry air) flow rate: 100 l/min; 6. Cyclone gas (dry air) flow rate: 150 l/min.
(70) After recovery from the cyclone, each spray-dried thrombin composition (dried thrombin with excipients) was reconstituted in water to the same concentration as the parent solution. The thrombin activity of each reconstituted solution was compared to that of the respective parent solution. The relative activities are presented in the graph of
(71) As shown in
Example 2
Effect of Thrombin and Human Serum Albumin (Carrier Protein) Concentration on Maintaining Thrombin Activity During Spray Drying of Thrombin Solutions
(72) Fifteen thrombin solutions having different concentrations of thrombin and albumin, serving as a carrier protein, were prepared as shown in Table 2:
(73) TABLE-US-00002 TABLE 2 Thrombin Human Serum Thrombin Activity Composition # [IU/ml] Albumin [mg/ml] Recovery (%) 9 100 0.06 55-77 10 100 0.6 91-100 11 100 1.8 79-100 12 100 2.25 52-98 13 100 6.0 100 14 1000 0.006 23-55 15 1000 0.06 31-69 16 1000 0.6 62-98 17 1000 1.8 92-94 18 1000 2.25 81-83 19 1000 6.0 86-100 20 3000 0.06 67-75 21 3000 0.6 94-96 22 3000 1.8 58-62 23 3000 2.25 77-81
(74) Compositions 9-23 were spray dried as described above. As discussed above, after spray drying, three 35 mg samples of each spray-dried thrombin composition were reconstituted in water to the same concentration as the parent solution. The thrombin activity of each reconstituted solution was measured twice and compared to that of the respective parent solution, giving a total of 6 measurements for each solution. The average thrombin activity recoveries are presented in Table 2.
(75) An additional parameter was calculated which shows the ratio of albumin weight to thrombin activity in the parent solution and its effect on thrombin activity recovery post spray drying, as can be seen in Table 2b (sorted from low to high according to Albumin/thrombin (g/IU) ratio). Thrombin activity recovery shown represents averagestandard deviation.
(76) TABLE-US-00003 TABLE 2b Albumin/ Thrombin Composition thrombin Activity # (g/IU) Recovery (%) 14 0.006 23-55 20 0.02 67-75 15 0.06 31-69 21 0.2 94-96 9 0.6 55-77 16 0.6 62-98 22 0.6 58-62 23 0.75 77-81 17 1.8 92-94 18 2.25 81-83 10 6 91-100 19 6 86-100 11 18 79-100 12 22.5 52-98 13 60 100
(77) The results indicate that the highest thrombin activity recoveries are achieved in compositions having relatively high albumin to thrombin levels (Table 2b) with only composition 21 as an outlier. However, having high ratio of albumin to thrombin does not guarantee high thrombin activity recovery post spray drying as can be seen by the large thrombin activity recovery variation obtained for compositions 11 and 12 and the relatively low thrombin activity recovery of compositions 22 and 23.
Example 3
Effect of Casein (Carrier Protein) Concentration on Maintaining Thrombin Activity During Spray Drying
(78) Four compositions of thrombin solution having different casein (carrier protein) concentrations were prepared, as shown in Table 3:
(79) TABLE-US-00004 TABLE 3 Composition Thrombin Casein Thrombin Activity # [IU/ml] [mg/ml] Recovery (%) 24 1000 0.06 44-54 25 1000 0.6 60-76 26 1000 1.8 92-98 27 1000 6.0 78-100
(80) Solutions 24-27 were spray dried as described above. As discussed above, after spray drying, three 35 mg samples of each spray-dried thrombin composition were reconstituted in water to the same concentration as the parent solution. The thrombin activity of each reconstituted solution was measured twice and compared to that of the respective parent solution, giving a total of 6 measurements for each solution. The thrombin activity recoveries obtained are presented in Table 3. The results achieved with casein are similar to the results achieved with albumin, meaning high thrombin activity recovery is obtained when ratio of carrier protein to thrombin is high however this does not guarantee high thrombin activity recovery as can be seen in high variance of the thrombin activity recovery in composition 27.
Example 4
Effect of Carbohydrate Type and Concentration on Maintaining Thrombin Activity during Spray Drying
(81) Twenty-five thrombin solutions having different Thrombin concentrations, carbohydrate types and carbohydrate concentrations, with or without a carrier protein were prepared, as shown in Tables 4 to 8:
(82) TABLE-US-00005 TABLE 4 Composition Thrombin Mannitol Thrombin Activity # [IU/ml] [mg/ml] Recovery (%) 28 800 2.0 43 29 1200 2.0 54 30 2000 2.0 49 31 3300 2.0 42
(83) TABLE-US-00006 TABLE 5 Composition Thrombin Trehalose Albumin Thrombin Activity # (IU/ml) [mg/ml] [mg/ml] Recovery (%) 32 200 20 34-48 33 400 20 40-44 34 454 20 54-64 35 600 20 43-47 36 800 20 38-44 37 1000 20 57-63 38 454 20 0.6 76-86
(84) TABLE-US-00007 TABLE 6 Composition Thrombin Sucrose Albumin Thrombin Activity # (IU/ml) [mg/ml] [mg/ml] Recovery (%) 39 1000 0.2 45 40 1000 2.0 71-75 41 1000 20 66-80 42 1000 0.2 0.6 54-64 43 1000 2.0 0.6 93 44 1000 20 0.6 81-93
(85) TABLE-US-00008 TABLE 7 Composition Thrombin Starch Thrombin Activity # (IU/ml) [mg/ml] Recovery (%) 45 1000 0.06 44-48 46 1000 0.6 36-46 47 1000 1.8 51-85 48 1000 6.0 57-81
(86) TABLE-US-00009 TABLE 8 Composition Thrombin Maltodextrin Thrombin Activity # (IU/ml) [mg/ml] Recovery (%) 49 1000 0.06 37-49 50 1000 0.6 48-56 51 1000 1.8 71-77 52 1000 6.0 59-89
(87) Compositions 28-52 were spray dried. Samples were recovered and reconstituted in water as described above, and the thrombin activity of each reconstituted solution was compared to that of the respective parent solution. The relative activities are presented in the respective Tables 4-8.
(88) The results show:
(89) Table 4: addition of a sugar alcohol (mannitol) alone to the thrombin solution was not sufficient to maintain thrombin activity during spray drying.
(90) Tables 5 and 6: addition of a disaccharide (trehalose in Table 5, sucrose in Table 6) alone to the thrombin solution was not sufficient to maintain thrombin activity during spray drying. However, when the disaccharide was added together with a carrier protein (albumin) there was substantial increase in thrombin activity recovery.
(91) Tables 7 and 8: addition of a polysaccharide (starch in Table 7, maltodextrin in Table 8) alone to the thrombin solution was not sufficient to maintain thrombin activity during spray drying.
Example 5
Effect of Thrombin Concentration with Fixed Molar Ratio of Thrombin to Human Serum Albumin (Carrier Protein) on Maintaining Thrombin Activity During Spray Drying
(92) Three thrombin compositions having different thrombin and albumin concentrations were prepared, as shown in Table 9, wherein the molar ratio of thrombin to albumin was 1:6.4 in all three solutions:
(93) TABLE-US-00010 TABLE 9 Composition Thrombin Albumin Thrombin Activity # (IU/ml) (wt %) Recovery (%) 53 100 0.06 55-77 54 1000 0.6 62-98 55 3000 1.8 58-62
(94) Samples of compositions 53-55 were spray dried. Samples were recovered and reconstituted in water as described above, and the thrombin activity of each reconstituted solution was compared to that of the respective parent solution. The thrombin activity recoveries are presented in Table 9. It is seen that the thrombin activities of the three reconstituted solutions was roughly the same, either low thrombin activity recovery (compositions 53 and 55) or high variance of thrombin activity recovery (composition 54).
Example 6
Effect of Trehalose (Disaccharide) on Maintaining Thrombin Activity During Spray Drying
(95) US Patent Application Publication No. 2012/0315305 discloses spray drying of a thrombin composition comprising trehalose and reports 97% thrombin activity recovery.
(96) Solutions 56 and 57, similar to those described in US 2012/0315305 (except that the source of the thrombin was different and the spray dryer was of a different type), were prepared, as shown in Table 10. Additionally, a novel thrombin solution 58 including trehalose and a protein (human serum albumin) was prepared.
(97) TABLE-US-00011 TABLE 10 Composition Thrombin Trehalose CaCl.sub.2 Albumin Thrombin Activity # (IU/ml) (mg/ml) (mM) (wt %) Recovery (%) 56 454 307 40 54-64 57 200 200 34-48 58 200 200 0.6 76-86
(98) Compositions 56-58 were spray dried in similar spray drying conditions according to the publication. Samples were recovered and reconstituted in water, and the thrombin activity of each reconstituted solution was compared to that of the respective parent solution. The thrombin activity recoveries are presented in Table 10.
(99) The results indicate that contrary to that reported in US 2012/0315305, solutions 56 and 57 showed a thrombin activity recovery of less than 65%. However, the addition of a carrier protein (human serum albumin) in composition 58 in accordance with some embodiments of the teachings herein led to a substantial increase in thrombin activity recovery.
Example 7
Spray Drying Process and Product Characterization of a Preferred Thrombin Composition
(100) Three liters of a composition 59, based on composition 8 of example 1, consisting of thrombin, albumin, mannitol, acetate, calcium and sodium chloride in water as shown in Table 11 were prepared.
(101) TABLE-US-00012 TABLE 11 Ingredient Concentration Presumed Role in Composition Thrombin 1019 IU/ml API (Active Drug Product) Human Serum Albumin 5.6 mg/ml Carrier and Stabilizer protein Mannitol 19.5 mg/ml Cryoprotectant, stabilizes protein 3D structure Acetate 18.8 mM Buffer Calcium 42 mM Clot stability Sodium Chloride 200 mM Used to dilute Thrombin concentration in dry form of product
(102) The parameters of the spray dryer were set to the following:
(103) Thrombin sample flow rate: 400 ml/h; Atomizing gas flow rate: 7 l/min; Drying gas flow rate: 600 l/min; Drying gas temperature: 140 C.; Cooling gas flow rate: 100 l/min; Cyclone gas flow rate: 130 l/min; Two-fluid nozzle with tip diameter: 0.4 mm.
(104) 3 liters of Composition 59 was spray dried as described above. During the spray drying, the real values of the various spray drying parameters were recorded every 1 minute. 63.25 g spray dried composition was collected in a 200 ml glass bottle designated Bottle #1, and when Bottle #1 was full, 48.41 g spray dried composition was collected in a 200 ml glass bottle designated Bottle #2. After being filled with the dried composition, the bottles were immediately capped and sealed with Parafilm until analysis was performed.
(105) The average measured spray drying parameters as well as the spray dried thrombin composition attributes were as shown in Table 12:
(106) TABLE-US-00013 TABLE 12 Parameter Bottle #1 Bottle #2 Atomizing gas flow rate 6.99 0.2 7.01 0.17 (l/min) Drying gas flow rate (l/min) 600 10 600 10 Drying gas temperature ( C.) 139.97 0.55 139.94 0.46 Gas temperature at the drying 82.48 1.8 81.62 1.71 column outlet ( C.) Gas temperature at the inlet 76.32 1.63 75.93 1.51 of the cyclone ( C.) Pressure in the drying column 13.9 0.47 13.82 0.48 (mBar) Pressure drop over the 46.72 1.06 47.25 0.8 cyclone (mBar) Spray dried thrombin - Water 2.36 0.08 content (%) Spray dried thrombin - D50 - 7.70 0.39 Particle size distribution (m) D90 - 15.92 0.64 Spray dried thrombin activity 24.1 0.7 (IU/mg) Spray dried thrombin - 98.8 Thrombin activity recovery (%) Spray dried thrombin - mass 91.2 yield (%)
(107) The spray dried thrombin powder obtained was dry with narrow particle size distribution, and most importantly, maintained the thrombin activity of the parent solution.
Example 8
Effect of Drying Gas Temperature on Spray Dried Thrombin Attributes
(108) The preferred thrombin composition (composition 8) was spray dried in several spray drying runs, when only the drying gas temperature was changed from run to run: Thrombin composition flow rate: 7 ml/min Atomizing gas flow rate: 7 l/min Drying gas flow rate: 300 l/min Drying gas temperature: 100, 110, 120, 130, 140, 150, 160, 170, 180 and 190 C. Cooling gas flow rate: 150 l/min Cyclone gas flow rate: 300 l/min
(109) Dry samples of the spray dried thrombin composition with different drying gas temperatures were collected from the cyclone and the thrombin activity recovery of a respective reconstituted solution was measured as described above. The results of the thrombin activity are shown in
(110) As shown in
Example 9
Effect of Drying Gas Flow Rate on Spray Dried Thrombin Attributes
(111) The preferred thrombin composition (composition 8) was spray dried in several spray drying runs, when only the drying gas flow rate was changed from run to run: Thrombin composition flow rate: 7 ml/min Atomizing gas flow rate: 7 l/min Drying gas flow rate: 300, 350, 400, 450, 500 and 550 l/min Drying gas temperature: 155 C. Cyclone gas flow rate: Flow rate which supplement to spray dryer total flow rate of 600 l/min (i.e., 300, 250, 200, 150, 100 and 50 l/min).
(112) Samples of dry spray dried thrombin were collected from the cyclone and the thrombin activity recovery of a respective reconstituted solution was measured as described above. The results of the thrombin activity are shown in
(113) As seen in
Example 10
Production of Fibrin Patches with Lyo-Milled BAC2 and Spray Dried Thrombin
(114) The thrombin composition 8 described in Example 1 was spray dried under the conditions described in Example 1. The resulting spray dried thrombin powder recovered from the cyclone was used together with lyophilized-milled BAC2 to produce fibrin patches as described in WO2007117237.
(115) The characteristics of the fibrin patch were analyzed, as shown in Table 13:
(116) TABLE-US-00014 TABLE 13 Parameter Spray dried thrombin Water content (%) 2.4 Particle size distribution (m) D50 = 8; D90 = 16 Thrombin activity [IU/mg solids] 24.1
(117) The characteristics of the fibrin sealant patch were analyzed as shown in Table 14:
(118) TABLE-US-00015 TABLE 14 Fibrin sealant patch made with spray dried thrombin Parameter composition water content [%] 2.1 thrombin activity [IU/cm.sup.2] 36 2.7 total protein [mg/cm.sup.2] 12 Clottable protein [mg/cm.sup.2] 8.4 Friability [weight reduction %] 8.1 Tissue Peel Test [N/m] 155 Tensile Strength [N/cm] 21.4
(119) It was seen that the fibrin sealant patch made with the spray dried thrombin powder had low water content (2.1%) and high thrombin activity (362.7 IU/cm.sup.2).
(120) In the tissue peel test, the adhesion strength of a wetted fibrin sealant patch to a corium tissue substrate was determined by determining the force required to peel the patch from the tissue. The patch made with the spray dried thrombin composition exhibited high adhesion.
Example 11
In Vivo Efficacy of Fibrin Sealant Patch
(121) The time to hemostasis (TTH) of the fibrin sealant patches described in example 10 was tested.
(122) Two pigs of similar size and weight were anesthetized for the entire surgical procedure and the spleens surgically exposed. Five 3 mm deep 15 mm long wounds were made in each one of the two spleens using an appropriate blade and fibrin patches were applied to cover each one of the wounds. The results after 3 minutes are seen in Table 15 and after 10 minutes are seen in Table 16, where a check indicates complete hemostasis.
(123) TABLE-US-00016 TABLE 15 Animal #1-Different sites Animal #2-Different sites Site #1 Site #2 Site #3 Site #4 Site #5 Site #1 Site #2 Site #3 Site #4 Site #5 % Pass 100
(124) TABLE-US-00017 TABLE 16 Animal #1-Different sites Animal #2-Different sites Site #1 Site #2 Site #3 Site #4 Site #5 Site #1 Site #2 Site #3 Site #4 Site #5 % Pass 100
(125) As can be seen in tables 15 and 16, patches achieved 100% hemostasis within 3 minutes, and no re-bleeding was observed after 10 minutes.
Example 12
Characterization of Spray Dried Thrombin Composition
(126) The thrombin composition 8 of Example 1 was either spray dried under conditions described in Example 1 or lyophilized. The two resulting dried thrombin powders were analyzed for bulk density, tapped density and specific energy (Particle Technology Labs, US) as shown in Table 17.
(127) TABLE-US-00018 TABLE 17 Conditioned Bulk Tapped Density Flowability Density (gr/ml) (gr/ml) (Specific Energy mJ/gr) Spray dried 0.46 0.58 9.52 composition Lyophilized 0.37 0.48 20.3 composition
(128) The results presented in Table 17 indicate that the spray-dried thrombin powder is denser than the lyophilized thrombin powder. The results in Table 17 also indicate that the spray-dried thrombin powder has a lower specific energy for flowability than the lyophilized thrombin powder, meaning the spray dried thrombin flows more easily. Practically, the spray dried thrombin powder is less likely to cause mechanical interlocking and friction in machinery, for example, reducing the risk of clogging of transfer devices, feeding mechanism and tubing.