Hemostatic compositions and methods of making thereof

Abstract

The present invention is directed to hemostatic compositions comprising at least partially integrated agglomerated ORC fibers, fibrinogen, and thrombin and methods of forming a powdered hemostatic composition, comprising the steps of: forming a suspension of a mixture comprising particles of fibrinogen, thrombin, ORC fibers in a non-aqueous low boiling solvent, agitating and shearing said suspension in a high shear mixing reactor, adding water to allow particles to agglomerate, allowing the non-aqueous solvent to evaporate, drying and sieving the composition; and thus forming the powdered hemostatic composition.

Claims

1. A method of forming a powdered hemostatic composition, comprising the steps of: a) forming a suspension of a mixture comprising particles, the particles comprising fibrinogen, thrombin, oxidized regenerated cellulose (ORC) fibers, in a non-aqueous low boiling solvent comprising hydrofluoroether; b) agitating and shearing said suspension in a high shear mixing reactor; c) adding water in an amount of at least 9 g per 100 g of said particles, resulting in 0.1% to less than 25% of fibrinogen conversion into fibrin so as to form agglomerated particles comprising said fibrinogen, thrombin and ORC fibers, while preventing full clotting of the fibrinogen; d) allowing the non-aqueous solvent to evaporate; e) drying and sieving the composition; and thus forming the powdered hemostatic composition.

2. The method of claim 1, wherein said non-aqueous low boiling solvent comprises HFE7100.

3. The method of claim 1, wherein said suspension further comprises Lysine or Tris.

4. The method of claim 1, wherein said suspension further comprises calcium chloride.

5. The method of claim 1, wherein said water is added in an amount of 9 g per 100 g of said particles.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 is a schematic diagram of the manufacturing process.

(2) FIG. 2 is a photo showing test vials evaluating gelling of inventive and comparative compositions in water.

(3) FIG. 3 is a photo showing test vials evaluating clotting of blood in contact with inventive and comparative compositions.

(4) FIG. 4 is a photo showing test vials evaluating clotting of blood in contact with inventive and comparative compositions.

(5) FIG. 5 is a composite photo showing the results of solubilization testing of the comparative compositions.

(6) FIG. 6 is a composite photo showing the results of solubilization testing of the inventive compositions.

(7) FIG. 7 is a composite photo showing the results of solubilization testing of the comparative compositions.

(8) FIG. 8 is a composite photo showing the results of solubilization testing of the inventive compositions at varying particle size.

(9) FIG. 9 is showing SEM images of one of inventive compositions.

(10) FIG. 10 is a schematic diagram of the HSM manufacturing process.

(11) FIG. 11 is a schematic diagram of the HSM manufacturing reactor.

(12) FIG. 12 shows inventive hemostatic powder prepared by the HSM Method of Example 9.

(13) FIG. 13 shows a Chart of gel strength in kPa.

(14) FIG. 14 shows the results of testing in animal model of the hemostatic powders made by using different solvents/binders.

(15) FIG. 15 shows schematic block-diagram of the HSM manufacturing process for preparation of hemostatic powders using acetone and polymer RG502.

(16) FIG. 16 shows schematic block-diagram of the HSM manufacturing process for preparation of hemostatic powders using acetone and water.

(17) FIG. 17 shows schematic block-diagram of the HSM manufacturing process for preparation of hemostatic powders using 96% ethanol.

(18) FIG. 18 shows schematic block-diagram of the HSM manufacturing process for preparation of hemostatic powders using HFE/water.

(19) FIG. 19 shows density properties of hemostatic powders obtained with various solvents/binders.

(20) FIG. 20 shows gel strength properties of hemostatic powders obtained with various solvents/binders.

(21) FIG. 21 shows Lung leak model with 1.2 cm long incision.

(22) FIG. 22 shows the inventive hemostatic powder forming a gel (counted for 2 min) on the deflated lung.

(23) FIG. 23 shows the inventive hemostatic powder forming a gel that seals the lung leak at 30 cm H.sub.2O.

(24) FIG. 24 shows Lung leak model with 1 cm long incision.

(25) FIG. 25 shows the inventive hemostatic powder forming a gel that seals the lung leak at 30 cm H.sub.2O.

(26) FIG. 26 shows the inventive hemostatic powder forming a gel partially delaminated at 40 cm H.sub.2O pressure (arrow indicates the delamination area).

DETAILED DESCRIPTION

(27) The inventors have discovered hemostatic materials and process for making thereof, the hemostatic materials having surprising and highly beneficial properties for hemostasis.

(28) The hemostatic material according to the present invention is made from oxidized cellulose-based fiber materials, more preferably form oxidized regenerated cellulose powder, fibrinogen powder, and thrombin powder. The hemostatic material according to the present invention represents at least partially integrated ORC fibers, fibrinogen, and thrombin in a form of a powder.

(29) Referring to FIG. 1, a schematic block-diagram of the process of making the hemostatic material according to the present invention is shown and comprises the steps of: Preparing dry powders of fibrinogen, thrombin, and ORC Suspending a mixture of fibrinogen, thrombin, and ORC powders in a non-aqueous solvent capable of rapid evaporation under ambient conditions Spraying the suspension through a nozzle onto a substrate Allowing the non-aqueous solvent to evaporate and drying the resulting hemostatic material Removing/separating the hemostatic material from the substrate and sieving, thus forming at least partially integrated ORC fibers, fibrinogen, and thrombin in a form of a powder

(30) In one embodiment, Tris, or Tris(hydroxymethyl)aminomethane buffer in a powder form is added to the fibrinogen, thrombin, and ORC mixture for pH adjustment. Then the mixed composition was added to HFE to form a suspension. In one embodiment, the cooling/chilling effect of the material stream during spraying due to HFE evaporation allows some ambient moisture to be absorbed onto or into the resulting hemostatic material. Any excessive moisture thus absorbed is removed in the final drying step in vacuum oven drying.

(31) According to one aspect of the present invention, the ratio of fibrinogen/thrombin mixture to ORC powder in the inventive hemostatic material is from about 1:1 to about 10:1 by weight.

(32) According to one aspect of the present invention the inventive hemostatic material comprises particles having size of 250-850 microns.

(33) According to one aspect of the present invention the inventive hemostatic material comprises substantially uniformly distributed at least partially integrated ORC fibers, fibrinogen, and thrombin in a form of a powder

(34) According to one aspect of the present invention, the inventive hemostatic material has high uniformity, integration, fast gelling/clotting, and strong adhesion force.

(35) According to one aspect of the present invention, the collecting surface or substrate onto which the suspension is sprayed, comprises an inert non-woven felt or mesh or a steel plate.

(36) According to another embodiment of the present invention, the present invention is directed to a hemostatic material comprising aggregates comprising fibrinogen, thrombin, and oxidized regenerated cellulosic fibers. In some aspects, the hemostatic material further includes additives, such as calcium chloride, and a buffer, such as Tris and/or Lysine.

(37) In another aspect, the present invention is directed to a method of making the hemostatic materials described above by suspending a mixture of fibrinogen, thrombin, and ORC powders in a non-aqueous solvent in a High Shear Mixing (HSM) apparatus, and performing high shear mixing and regular mixing and/or agitation, while allowing the volatile non-aqueous solvent to evaporate through any suitable port(s) such as through air seals of mixing blades/impellers and/or through a vacuum tube. Optionally, a small amount of water or aqueous solution is introduced into the reactor during mixing to aid particles binding/aggregation/clumping. The non-aqueous low boiling solvent can be hydrofluoroether C.sub.4F.sub.9OCH.sub.3, such as but not limited to HFE7100.

(38) According to an embodiment of the present invention, a hemostatic aggregated powder comprises ORC fine fibers or powder, thrombin, fibrinogen, lysine (as a buffer/pH adjusting agent), calcium salt.

(39) According to an embodiment of the present invention, a process of making such powdered hemostatic product comprises the steps of mixing the individual components as powders with HFE, creating a suspension in HFE (volatile non-aqueous solvent) in High Shear mixing/shearing reactor, having a low speed mixing blade and high-speed shear blade. As mixing is performed, HFE can evaporate through air seals of mixing blades and/or vacuum tube. A small amount of water is introduced into the reactor to aid particles binding/aggregation/clumping.

(40) According to an embodiment of the present invention, a method of making hemostatic powder, comprising the steps of: Forming a suspension of ORC powder, thrombin, fibrinogen, lysine, calcium salt in HFE (volatile non-aqueous solvent), agitating the suspension with high speed shear blade (and optionally continuously mixing the components simultaneously with a low speed mixer); Adding a small amount of water to the reactor to aid particles binding/aggregation/clumping; allowing HFE to evaporate from the reactor completely; Thus forming the hemostatic powder.

Example 1. Preparation of Hemostatic Compositions

(41) The individual components of the hemostatic compositions of the present invention were prepared as described below.

(42) Fibrinogen. Any method of preparation of fibrinogen powder can be utilized, including lyophilization, freeze drying, etc. In the instant example, fibrinogen powder was prepared by spray drying method (Spray dryer manufacturer: ProCepT, Model: 4M8-TriX). Fibrinogen solution is a formulation commercially available from Bioseal Biotech CO. LTD, located in Guangzhou, China, and comprising fibrinogen, albumin, and other needed reagents in WFI. The fibrinogen solution was first atomized through a spray nozzle in a hot airflow, then dried instantly. The spray drying parameters were as shown in Table 1

(43) TABLE-US-00001 TABLE 1 Feed Rate 130 ml/h Drying Columns 3 (Mode II) Column Air Flow 0.6 m.sup.3/min Inlet Air Temperature 150° C. Nozzle Diameter 0.8 mm Atomizing Air Flow 12 L/min Cyclone Gas 0.15 m.sup.3/min

(44) Thrombin. Any method of preparation of thrombin powder can be utilized, including lyophilization, freeze drying, etc. In the instant example, thrombin powder was prepared by spray drying method with thrombin formulation solution. Thrombin solution was the formulation commercially available from Bioseal Biotech CO. LTD, located in Guangzhou, China, and comprising thrombin, albumin, and other needed reagents in WFI. The spray drying parameters were as shown in Table 2

(45) TABLE-US-00002 TABLE 2 Feed Rate 258 ± 20 ml/h Drying Columns 2 Column Air Flow 0.3 m.sup.3/min Cooling Air Flow 0.3 m.sup.3/min Inlet Air Temperature 160° C. Nozzle Diameter 0.4 mm Atomizing Air Flow 7 L/min Cyclone Gas 0.1 m.sup.3/min

(46) The thrombin and fibrinogen powders were then mixed together for preparation of the composite by the ratio of 89.7% of fibrinogen 7.8% of thrombin and 2.5% calcium chloride by weight, thus forming fibrin sealant powder.

(47) The source of fibrinogen and thrombin was Porcine blood plasma which was fractionated to obtain fibrinogen and thrombin. and supplied by Bioseal Biotech CO. LTD, Located in Guangzhou, China

(48) The ORC powder can be obtained by processing of the Surgicel original fabric. A reference is made to the U.S. Provisional Patent Application No. 62/251,773 by Yi-Lan Wang, filed 6 Nov. 2015 and titled “Compacted Hemostatic Cellulosic Aggregates”, which is incorporated by reference in its entirety for all purposes.

(49) Briefly, the ORC powder was obtained by processing of the Surgicel original fabric in the following process:

(50) 1) split and cut the fabric into about 2″×8″ pieces,

(51) 2) mill the fabrics into the powder particle size (D50 less than 94 microns) using known milling methods. One of the methods used in the preparations is the ball mill method—place ˜100 grams of fabric into a 500-ml zirconia jar, then place 12 to 13 pieces of 20 mm zirconia balls (agates) into the same jar, cover and fix the jar in a Retsch planetary ball mill (Model PM100), mill the fabric with 450 rpm for 20 minutes, transfer the milled powders onto a 8″ diameter and 300-micron opening sieve, separate the agates and the powders by slightly shaking, and collect the powders.

(52) The inventive hemostatic compositions were prepared as follows by using co-spraying methods. 1 part of ORC fiber was combined with 1 part, or 2 parts, or 5 parts, or 10 parts of fibrin sealant powders, by weight. Thus, as an example, 10 g of ORC powder was combined with 10 g, 20 g, 50 g, or 100 g of fibrin sealant mixed powders, to produce 20 g, 30 g, 50 g, or 100 g of mixture.

(53) A small amount of Tris was added to adjust pH to 7.0 for each respected ORC: Fibrin sealant ratio. pH was adjusted by placing the powder on a wetting surface and measuring the resulting pH and evaluating the amount of Tris needed for obtaining a neutral pH of 7. The sample was then discarded. A corresponding proportional amount of dry powder of Tris was then added to the powder mixture, prior to co-spraying.

(54) ORC is not per se neutralized as Tris is added in dry form. ORC is being neutralized when the whole powder formulation is wetted during the application and the Tris is dissolved. A low boiling non-aqueous solvent was utilized for making a suspension of FS and ORC powders. Hydrofluoroether C4F9OCH3 was used, obtained as HFE 7100, for instance supplied by 3M as Novec 7100 Engineered Fluid, having boiling point of 61° C. HFE7100 solvent was added to the powders compositions and filtered through 150 μm sieve. The evenly distributed suspension was created by constantly agitating at 90 rpm/min in the reservoir at 20±5° C. temperature. The suspended components were sprayed through a 0.7 mm diameter nozzle. The type of the nozzle used was B1/8VAU-316SS+SUV67-316SS manufactured by Spraying Systems Co. The flow through the nozzle was at a flow rate of 130 ml/min, onto a non-woven fabric or stainless steel substrate at 20±5° C. temperature.

(55) Upon spraying the suspension, most of the HFE 7100 solvent was evaporating. The HFE 7100 solvent residues and the absorbed ambient moisture in the powder, if any, could evaporate in a vacuum drying oven for 24 h±5 h at 20±5° C.

(56) The resulting hemostatic composition was scooped off or peeled off or otherwise separated from the substrate, and sequentially passed through sieves having 850 μm, 355 μm, 250 μm openings.

(57) Table 3 shows parameters used for co-spraying compositions

(58) TABLE-US-00003 TABLE 3 PARAMETERS FOR CO-SPRAYING THE COMPOSITION -fan pressure 0.3 bar atomizing pressure 3 bar flow rate 130 ml/min liquid pressure 16 kpa Nozzle Diameter 0.7 mm agitation speed 90 rpm/min

(59) As comparative examples, pure fibrin sealant mixture composition with no ORC and no Tris was also prepared by the same spray method HFE7100.

Example 2. Comparison of Mechanically Mixed Vs. Spray Mixed Hemostatic Compositions Gelling

(60) Rapid gelling and formation of strong gels is important for hemostatic materials.

(61) The inventive hemostatic composition was prepared as described above by co-spray method.

(62) The comparative mechanically mixed composition was prepared by manually shaking dry powders, i.e. FS and ORC powders obtained as described above, by manually shaking in a container into an evenly mixed composition, with no co-spraying performed. The compositions included all the same components, including Tris, with the difference being methods of mixing.

(63) Fibrin sealant powder to ORC ratios tested were: Fibrin sealant (FS): ORC ratios—1:1; 2:1; 5:1; 10:1 (by weight). Thus for 1:1 FS/ORC ratio, 1 part of fibrin sealant powder (comprising fibrinogen, thrombin, calcium chloride) is combined with 1 part of ORC powder (by weight).

(64) Mechanically mixed composition and the inventive spray mixed composition were then added to 20 ml of water in a 50-ml vial in the amount of 200 mg on the top of the water surface. After 2 min for gelling, the vial was turned upside down and observations made if a gelled layer of composition was formed in which case the water was observed sealed by the gelled layer and was held on the bottom of the vial by the formed gel layer.

(65) Referring now to FIG. 2, showing an image of the test vials turned upside down at the end of the test, with test vial 1 showing mechanically mixed composition 1:1 FS/ORC ratio test vial 2 showing mechanically mixed composition 2:1 FS/ORC ratio test vial 3 showing mechanically mixed composition 5:1 FS/ORC ratio test vial 4 showing mechanically mixed composition 10:1 FS/ORC ratio test vial 5 showing mechanically mixed composition with no ORC powder (fibrin sealant powder only) test vial 6 showing inventive hemostatic composition 1:1 FS/ORC ratio

(66) Analysis of the results shown in FIG. 2 indicates that in test vials 1-5 gelling was insufficient to hold the fluid and fluid is visible in the lower part of the vial. In test vial 6, fluid is visible in the upper part of the vial, i.e. water is being held by the gelled layer and prevented from moving to the lower part of the vial under gravitational force. Thus, for mechanically mixed compositions in all ratios and for fibrin sealant composition without ORC gelling was insufficient, while the inventive hemostatic composition prepared in 1:1 ratio exhibited surprisingly strong gelling.

Example 3. In Vitro Testing of Blood Clotting

(67) In vitro clotting of blood by several inventive and comparative compositions was tested as follows.

(68) 20 ml of citrated whole blood (porcine) was added to a 50-ml vial. 200 mg of hemostatic compositions being tested were added on the top of the blood surface. After 2 min for clotting, the vial was turned upside down and observations of blood clotting were made. In case of complete clotting, the clotted blood stays in the upper part of the vial turned upside down. In case of incomplete clotting, blood remains fluid and drains towards the lower part of the vial due to the gravitational force.

(69) Comparative excipients added to fibrin sealant powder instead of ORC were Trehalose, PEG4000, Mannitol, and Alfa-cellulose (α-cellulose). All excipients were purchased from Aladdin industrial corporation. Fibrin sealant powder mixtures with Trehalose, PEG 4000, Mannitol, or Alfa-cellulose were prepared by spray method as described above in 10:1 ratio, i.e. with 10 parts of fibrin sealant (FS) powder combined with 1 part of the respective excipient. The total amount of inventive and comparative compositions added to 20 ml of blood was 200 mg.

(70) Referring now to FIG. 3, showing an image of the test vials turned upside down at the end of the test, with test vial 8 showing the inventive hemostatic composition having 2:1 FS/ORC ratio test vial 9 showing the inventive hemostatic composition having 5:1 FS/ORC ratio test vial 10 showing the inventive hemostatic composition having 1:1 FS/ORC ratio test vial 11 showing comparative composition comprising fibrin sealant powder only with no ORC made by co-spray test vial 12 showing comparative composition comprising fibrin sealant powder with addition of α-cellulose in 10:1 FS/α-cellulose ratio by weight test vial 13 showing comparative composition comprising fibrin sealant powder with addition of trehalose in 10:1 FS/trehalose ratio by weight test vial 14 showing comparative composition comprising fibrin sealant powder with addition of PEG4000 in 10:1 FS/PEG4000 ratio by weight test vial 15 showing comparative composition comprising fibrin sealant powder with addition of mannitol in 10:1 FS/mannitol ratio by weight

(71) Analysis of the results presented in FIG. 3 indicates that in test vials 8-10 containing the inventive hemostatic composition blood has clotted, with blood clot visible in the upper part of the vial turned upside down with clotted blood prevented from moving to the lower part of the vial under gravitational force. Thus, the inventive hemostatic composition prepared in 2:1; 5:1; 1:1 FS/ORC ratio exhibited surprisingly strong clotting of blood. Also, comparative sample containing α-cellulose in vial 12 shows clotting of blood. Comparative examples in vial 11 (fibrin sealant powder only with no ORC); vial 13 (fibrin sealant powder with addition of trehalose); vial 14 (fibrin sealant powder with addition of PEG4000); vial 15 (fibrin sealant powder with addition of mannitol) show no clotting or insufficient clotting, whereby clotting was insufficient to hold the fluid and fluid is visible in the lower part of the vial, i.e. blood remains fluid and drains towards the lower part of the vial due to the gravitational force. The inventive hemostatic compositions exhibited surprisingly strong blood clotting.

(72) Using the same testing methods, additional in vitro blood clotting testing was performed for inventive hemostatic composition and comparative mechanically mixed compositions prepared by manually shaking dry powders in a container as well as for fibrin sealant powder only with no ORC

(73) Referring now to FIG. 4, showing an image of the test vials turned upside down at the end of the test, with test vial 1 showing mechanically mixed composition 1:1 FS/ORC ratio test vial 2 showing mechanically mixed composition 2:1 FS/ORC ratio test vial 3 showing mechanically mixed composition 5:1 FS/ORC ratio test vial 4 showing mechanically mixed composition 10:1 FS/ORC ratio test vial 5 showing comparative composition (200 mg) comprising compacted ORC powder aggregates prepared as described in the U.S. Provisional Patent Application No. 62/251,773 by Yi-Lan Wang, filed 6 Nov. 2015 and titled “Compacted Hemostatic Cellulosic Aggregates” test vial 6 showing comparative composition comprising fibrin sealant powder only with no ORC prepared by co-spray method test vial 7 showing the inventive hemostatic composition having 1:1 FS/ORC ratio test vial 8 showing the inventive hemostatic composition having 2:1 FS/ORC ratio test vial 9 showing the inventive hemostatic composition having 5:1 FS/ORC ratio test vial 10 showing the inventive hemostatic composition having 10:1 FS/ORC ratio

(74) Analysis of the results presented in FIG. 4 indicates that comparative examples in test vials 1-6, containing mechanically mixed compositions in all ratios; ORC powder only; and fibrin sealant powder without ORC, show no clotting or insufficient clotting, whereby clotting was insufficient to hold the fluid and fluid is visible in the lower part of the vial, i.e. blood remains fluid and drains towards the lower part of the vial due to the gravitational force. On the contrary, and like the results presented in FIG. 3, in test vials 7-10, containing the inventive hemostatic composition, the blood has clotted, with blood clot visible in the upper part of the vial turned upside down with clotted blood prevented from moving to the lower part of the vial under gravitational force. Thus, the inventive hemostatic compositions prepared in 1:1-10:1 FS/ORC ratios exhibited surprisingly strong clotting of blood.

Example 4. Composition Solubilisation

(75) Rapid solubilisation or solubility of a powdered hemostatic composition when in contact with bodily fluids can help to establish rapid hemostasis and indicates rapid interaction with fluids. The visual test of solubilisation was performed as follows: 1 gram of tested hemostatic powdered composition was evenly applied to an area of a wetting substrate which comprising a non-woven fabric positioned on top of a sponge material which was placed in a tray with pure water. After the tested powdered hemostatic composition was applied to the surface of the wetting substrate, visual observation of the composition solubility was performed and results recorded at zero time (immediately after applying the composition, at 1 min and at 2 min after applying the tested composition.

(76) Referring now to FIG. 5, a composite image is shown representing the results of testing of the comparative mechanically mixed composition prepared by manually shaking dry powders in a container. The images taken at 0, 1, and 2 min for FS/ORC ratios—1:1; 2:1; 5:1; 10:1 as well as for FS powder with no ORC. The results indicate poor solubilisation even at 2 min time point for the comparative examples.

(77) Referring now to FIG. 6, a composite image is shown representing the results of testing of the inventive hemostatic composition prepared by the spray method. The images taken at 0, 1, and 2 min for FS/ORC ratios—1:1; 2:1; 5:1; 10:1 as well as for FS powder with no ORC. The results indicate good solubilisation even at 1 min time point and very good solubilisation at 2 min time point, with rapid full solubilisation observed for 1:1 and 2:1 ratios already at 1 min and good solubilisation observed for all ratios at 2 min. Pure FS is showing poor solubilisation even at 2 min time point for the comparative example.

(78) Referring now to FIG. 7, a composite image is shown representing the results of testing of the comparative compositions comprising FS with excipients added to fibrin sealant powder instead of ORC as well as for FS powder with no ORC. The excipients were Trehalose, PEG4000, Mannitol. Fibrin sealant powder mixtures with Trehalose, PEG 4000, Mannitol, were prepared by spray method as described above in 10:1 FS/excipient ratio. The images taken at 0, 1, and 2 min are shown. The results indicate poor solubilisation even at 2 min time point for the comparative examples.

(79) The solubility of inventive hemostatic composition prepared by the spray method was affected by the concentration of the ORC component. Even at low concentrations of ORC, the solubility of the composition has improved.

Example 5. Particle Size Effects

(80) The effects of the particle size on the performance of the inventive hemostatic compositions were evaluated. Particle size was controlled by sequentially passing the composition through sieves with apertures of 850 μm, 355 μm and 250 μm. Inventive composition powders were separated into size groups of predominantly above 850 μm, predominantly 355-850 μm, predominantly 250-355 μm and predominantly below 250 μm. Inventive hemostatic compositions made with 5:1 FS/ORC ratio were tested for solubility.

(81) Referring now to FIG. 8, a composite image is shown representing the results of testing of the inventive hemostatic composition prepared by the spray method. The images taken at 0, 1, and 2 min for different powder size ranges. The results indicate particularly excellent solubilisation at 2 min point for predominantly 355-850 μm and good solubilisation for predominantly 250-355 μm compositions, with less effective solubilisation for compositions predominantly above 850 μm, and predominantly below 250 μm. Thus, the range of predominantly 250-850 μm is showing good solubilisation and is the preferred range for particle size, with particles predominantly in the 355-850 μm range particularly preferred. The resulting powder is an agglomerate of fibrinogen, thrombin, and ORC, and has many particles with size larger than the starting materials particle size.

Example 6. Effects of Tris Addition

(82) A peel test of the inventive compositions with added Tris and without added Tris was performed. Tris additions were titrated to achieve pH=7. Tris powder was ground and passed through a 150 μm sieve. The powder of below 150 μm was collected and added the pre-determined amount into the dry mixture to adjust the pH of the composition, prior to co-spray.

(83) The peel test was performed as follows. 0.5 g of the Inventive composition was applied to the corium tissue, covered by a composite bi-layer matrix which was pressed into the powder for 3 minutes, the sample-from-tissue separation force was measured by an Instron tensile testing machine and recorded as force per unit width (N/m). The composite bi-layer matrix comprised a layer of synthetic absorbable poly (glycolide-co-lactide) (PGL, 90/10 mol/mol) nonwoven fabric needlepunched into a knitted carboxylic-oxidized regenerated cellulose fabric (ORC), as described in U.S. Pat. No. 7,666,803 by D. Shetty et al., titled “Reinforced absorbable multilayered fabric for use in medical devices”, which is incorporated by reference herein.

(84) Referring now to Table NN, adhesion forces of inventive formulations are shown as a function of ORC addition. While the adhesion is lower at higher ORC content even 1:1 FS powder: ORC fiber formulation has appreciable peel force.

(85) Referring now to Table 4, adhesion Forces of inventive formulations with and without Tris are shown for different FS/ORC ratios along with corresponding pH values. Tris was added in weight percentages listed to adjust pH to 7.0. While all compositions have exhibited high peel forces, presence of Tris clearly resulted in higher peel forces for the same FS/ORC ratios, with some showing 2-4 times higher peel force.

(86) TABLE-US-00004 TABLE 4 Adhesion Forces of inventive formulations with and without Tris With no Tris added With Tris added Composition Peel Force Peel Force Tris % by FS/ORC ratio N/m pH N/m pH weight 1:1 26.7 2 72.6 7 20 2:1 53.8 2 127.5 7 14.3 5:1 41.2 5 235.9 7 7.7 10:1  221.7 5.5 >340 7 4.4 (Above the upper limit of measurement)

(87) The analysis of data indicates surprising improvements in adhesive force or peel force for inventive composition having neutral pH achieved by Tris addition. While the force is somewhat lower

Example 7. Characterization of Particles

(88) Referring now to FIG. 9, showing magnified SEM images of 5:1 FS/ORC inventive composition, it is apparent that the components of the composition are at least partially integrated, i.e. attached to each other or coated onto one another, and are not in a simple mechanical mixture.

(89) Examination of the inventive composition in powder form shows the components well mixed and the biologics were closely attached on the ORC fibers.

Example 8. Hemostasis Testing

(90) An in vivo test of hemostatic efficacy in liver abrasion model using the inventive hemostatic compositions was performed as follows. A liver abrasion model was created by creating an oozing area of 3 cm×3 cm on the surface of the porcine liver. 0.5 g of the inventive hemostatic composition having FS/ORC ratio of 5:1 was applied to cover the oozing area without any tamponade applied. Hemostasis was achieved in under 2 min.

(91) An in vivo test of hemostatic efficacy in liver resection model using the inventive hemostatic compositions was performed as follows. A liver resection model was created by using the Pringle maneuver which is a surgical maneuver used in some abdominal operations whereby a large atraumatic hemostat is applied as a clamp. The Pringle maneuver was applied to control bleeding first, then a cut 5 cm long and 5 cm wide of the liver tissue was created along the liver edge to expose bile duct. Immediately after, the inventive hemostatic composition powder was applied to cover the transection plane, spraying saline simultaneously until bleeding was stopped. The Pringle clamp was then released to examine the results. It was observed that hemostasis was achieved and bile leak was prevented after the Pringle clamp was released. The hemostasis was achieved in 2 min.

Example 9. Method of Manufacturing in High Shear Reactor

(92) According to an embodiment of the present invention, a process of making powdered hemostatic product comprises the steps of mixing the individual components as powders with HFE, creating a suspension in HFE (volatile non-aqueous solvent) in High Shear mixing/shearing reactor, having a low speed mixing blade and high-speed shear blade. As mixing is performed, HFE can evaporate through air seals of mixing blades and/or vacuum tube. A small amount of water is introduced into the reactor to aid particles binding/aggregation/clumping. The amount of water is sufficient for a small portion of the biologics to react and form particles, however it is not sufficient for all biologics i.e. thrombin and fibrinogen to fully react so that all fibrinogen is converted into fibrin. The portion of fibrinogen converted into fibrin is from about 0.1% to about 50%, more preferably 1% to 25%, even more preferably 2% to 10%. There is always an amount of clottable fibrinogen contained in the hemostatic powders prepared according to the present invention, available for reaction when powder is used on or in a wound.

(93) According to an embodiment of the present invention, a method of making hemostatic powder, comprising the steps of: Forming a suspension of ORC powder, thrombin, fibrinogen, lysine, calcium salt in HFE (volatile non-aqueous solvent), agitating the suspension with high speed shear blade (and optionally continuously mixing the components simultaneously with a low speed mixer); Adding a small amount of water to the reactor to aid particles binding/aggregation/clumping; allowing HFE to evaporate from the reactor completely; Thus forming the hemostatic powder.

(94) Referring to FIG. 10, a schematic block-diagram of the process of making the hemostatic material according to the present invention is shown and comprises the steps of: Preparing dry powders mixture of fibrinogen, thrombin, ORC, optionally calcium chloride, and optionally buffer compound such as Tris and/or lysine; Suspending dry powders mixture in a non-aqueous solvent capable of rapid evaporation under ambient conditions in a High Shear Mixer reactor Agitating the suspension with high speed shear blade and optionally continuously mixing the components simultaneously with a low speed mixer blade Introducing a small amount of water into the reactor to aid particles binding/aggregation/clumping Allowing the non-aqueous solvent to evaporate from the reactor drying the resulting hemostatic material Removing the resulting hemostatic material from the reactor and optionally sieving, thus forming at least partially integrated ORC fibers, fibrinogen, and thrombin in a form of an aggregated powder

(95) In one embodiment, Lysine or Tris in a powder form is added to the fibrinogen, thrombin, and ORC mixture for pH adjustment.

(96) According to one aspect of the present invention the inventive hemostatic material comprises substantially uniformly distributed at least partially integrated ORC fibers, fibrinogen, and thrombin in a form of a powder

(97) According to one aspect of the present invention, the inventive hemostatic material has high uniformity, integration, fast gelling/clotting, and strong adhesion force.

(98) Referring now to FIG. 11, a schematic rendering of a High Shear mixing (HSM) reactor is shown, with container or bowl 5, Impeller 1 for the raw materials mixing and suspending (low speed mixer); High speed shearing blade or chopper knife 2; Spray nozzle 3 for the water (binder) additions; Vent 4 for the removal of HFE volatile fluid; Pathways 6 for ingress of optional air or gas for purging HFE volatile fluid through mixing and/or shearing blades seals. Alternatively, a dedicated air intake can be provided (no shown). Alternatively, a vacuum can be connected to vent 4.

(99) HSM reactors are available commercially. The reactor used in the present examples was HSM model Mini-CG, available from Chuangzhi Electrical and Mechanical Co., Ltd. (China).

Example 10. Comparative Testing of Hemostatic Powders Made by Spray Methods of Example 1 and HSM Methods of Example 9

(100) The inventors compared the HSM method (Example 9) particles vs. particles made by spraying through a nozzle of HFE suspension (Example 1). The test results showed the hemostatic powders made by the HSM method had comparable physiochemical properties and functional performance like tensile strength and adhesion strength, and had comparable hemostatic efficacy in animal study to the powders made by the spaying method of Example 1.

(101) Comparison of powders of Example 1 formulated with Tris buffer to powders formulated in Example 9 (HSM method) using Lysine as buffering compound showed no substantial difference in hemostatic powder performance.

(102) Advantageously, HSM method of preparing hemostatic powder granulations has advantages of providing HSM equipment that is a substantially sealed system so that powder can be granulated in a sealed environment, and no material is lost during the process. The bioburden could be minimized as compared to spraying method of Example 1.

(103) Referring to FIG. 12, a hemostatic powder prepared by the HSM Method of Example 9 is shown.

Example 11. Preparation of Hemostatic Compositions Using HSM Method: Comparison of Various Solvents/Binders

(104) The inventors used the HSM method (Example 9) to manufacture hemostatic powders using several different solvents/binders. Referring to FIG. 13, a Chart of gel strength in kPa is shown, with the gel formed by the hemostatic powders of the present invention, whereby such powders were made by using one of four different solvents and/or binders. The gel strength is shown for several different testing points. First segment of the chart shows gel strength for acetone as a volatile suspension solvent and polymer RG502 (D, L-lactide-co-glycolide) as a binder. The second segment shows gel strength for ethanol/water combination as volatile solvent and binder. The third segment shows gel strength for acetone/water combination as volatile solvent and binder. The fourth segment shows gel strength for HFE/water combination as volatile solvent and binder. As can be seen from Chart of FIG. 13, HFE/water combination shows the highest gel strength.

(105) Referring now to FIG. 14, the results of testing in animal models of the hemostatic powders made by using different solvents/binders are presented (Hemostasis testing on dermatome liver surface model). As can be seen from the FIG. 14, combinations of acetone/RG502; ethanol/water; acetone/water all showing bleeding after 3 min after application. To the contrary, the inventive HFE/water combination shows cessation of bleeding after only 1 minute.

Example 12. Preparation of Hemostatic Compositions Using HSM Method and Polymer RG502/Acetone

(106) 200 g of raw material powder including Fibrinogen powder/Thrombin powder/ORC powder/Calcium chloride powder and Tris was prepared according to the formulation shown in Table 5. The binder/suspension solution was prepared from 74.7 g acetone and 8.3 g polymer RG502(D, L-lactide-co-glycolide), with polymer fully dissolved and mixed thoroughly.

(107) TABLE-US-00005 TABLE 5 Formulation for Example 12 Raw material Fibrinogen (g) 153.54 Thrombin (g) 15.08 Calcium chloride (g) 5.24 ORC (g) 17.40 Tris (g) 8.70 Binder Polymer RG502 (g) 8.30 D,L-lactide-co-glycolide Acetone (g) 74.70

(108) The schematic block-diagram of the manufacturing process is shown in FIG. 16. All dry material was transferred into the bowl of HSM, premixed for 5 minutes using the mixing blade impeller speed at 75 rpm, high shear blade chopper speed at 1000 rpm. The binder/suspension solution (RG502/acetone) was then sprayed onto the dry materials by a Peristaltic pump and a spray nozzle, with the feed flowrate 60 g/min. The solution of polymer/acetone starts binding the raw material particles to form powder.

(109) The Impeller speed was then increased to 150 rpm and the chopper speed increased to 3000 rpm, continuing the post granulation process for 5 mins.

(110) After granulation, a sieve (the pore size is 1.7 mm) was used to sieve the materials and collect the resulting powder under the sieve. The powder was transferred to vacuum drying box and dried for 1 hour. Two sieves (pore size 106 and 425 μm) were then used to sieve the product. The final powder fraction was collected between the two sieves.

Example 13. Preparation of Hemostatic Compositions Using HSM Method and Water/Acetone

(111) 200 g of raw material powder including Fibrinogen powder/Thrombin powder/ORC powder/Calcium chloride powder and Tris was prepared according to the formulation shown in Table 6. The binder/suspension solution was prepared from 68 g acetone and 17 g of purified water, mixed thoroughly.

(112) TABLE-US-00006 TABLE 6 Formulation for Example 13 Raw material Fibrinogen (g) 153.54 Thrombin (g) 15.08 Calcium chloride (g) 5.24 ORC (g) 17.40 Tris (g) 8.70 Binder Water (g) 17.00 Acetone (g) 68.00

(113) The schematic block-diagram of the manufacturing process is shown in FIG. 18. All dry material was transferred into the bowl of HSM, premixed for 5 minutes using the mixing blade impeller speed at 75 rpm, high shear blade chopper speed at 1000 rpm. The binder/suspension solution (water/acetone) was then sprayed onto the dry materials by a Peristaltic pump and a spray nozzle, with the feed flowrate 60 g/min. The solution starts binding the raw material particles to form powder.

(114) The Impeller speed was then increased to 120 rpm and the chopper speed increased to 3000 rpm, continuing the post granulation process for 3 mins.

(115) After granulation, a sieve (the pore size is 1.7 mm) was used to sieve the materials and collect the resulting powder under the sieve. The powder was transferred to vacuum drying box and dried for 1 hour under vacuum. Two sieves (pore size 106 and 425 μm) were then used to sieve the product. The final powder fraction was collected between the two sieves

Example 14. Preparation of Hemostatic Compositions Using HSM Method and 96% Ethanol

(116) 150 g of raw material powder including Fibrinogen powder/Thrombin powder/ORC powder/Calcium chloride powder and Tris was prepared according to the formulation shown in Table 7. The binder/suspension solution was prepared from 60 g of 96% ethanol.

(117) TABLE-US-00007 TABLE 7 Formulation for Example 14 Raw material Fibrinogen (g) 115.16 Thrombin (g) 11.31 Calcium chloride (g) 3.93 ORC (g) 13.05 Tris (g) 6.53 Binder 96% Ethanol (g) 60.00

(118) The schematic block-diagram of the manufacturing process is shown in FIG. 20. All dry material was transferred into the bowl of HSM, premixed for 3 minutes using the mixing blade impeller speed at 75 rpm, high shear blade chopper speed at 1000 rpm. The binder/suspension solution (96% ethanol) was then sprayed onto the dry materials by a Peristaltic pump and a spray nozzle, with the feed flowrate 4 g/min. The solution starts binding the raw material particles to form powder.

(119) The Impeller speed was then increased to 120 rpm and the chopper speed increased to 1500 rpm, continuing the post granulation process for 3 mins.

(120) After granulation, a sieve (the pore size 710 μm) was used to sieve the materials and collect the resulting powder under the sieve. The powder was transferred to tray oven for drying and dried at 45° C. for 0.5 hr.

(121) Two sieves (pore size 100 and 315 μm) were then used to sieve the product. The final powder fraction was collected between the two sieves

Example 15. Preparation of Hemostatic Compositions Using HSM Method and HFE/Water

(122) 100 g of raw material powder including Fibrinogen powder/Thrombin powder/ORC powder/Calcium chloride powder and Tris was prepared according to the formulation shown in Table 8. 500 g of HFE7100 was utilized.

(123) TABLE-US-00008 TABLE 8 Formulation for Example 15 Raw material Fibrinogen (g) 76.77 Thrombin (g) 7.54 Calcium chloride (g) 2.62 ORC (g) 8.70 Tris (g) 4.35 Suspending agent HFE 7100 (g) 500.00 Binder Water (g) 9.00

(124) The schematic block-diagram of the manufacturing process is shown in FIG. 22. All dry material was transferred into the bowl of HSM and 500 g of HFE7100 added. The composition was premixed for 3 minutes to form a suspension, using the mixing blade impeller speed at 200 rpm. 9 g of water was then sprayed into the suspension by a Peristaltic pump and a spray nozzle, with the feed flowrate of 4.5 g/min. The solution starts binding the raw material particles to form powder.

(125) The Impeller speed was then adjusted to 100-300 rpm and the chopper speed set at 150-1000 rpm, continuing the granulation process for 10 mins. Sealing pressure of impeller and chopper was adjusted to 0.02 MPa to blow off HFE and dry the composition.

(126) After granulation, a sieve (the pore size 710 μm) was used to sieve the materials and collect the resulting powder under the sieve. The powder was transferred vacuum box for drying and dried for 12-24 hours at vacuum from 0-10 Pa.

(127) Two sieves (pore size 106 and 355 μm) were then used to sieve the product. The final powder fraction was collected between the two sieves.

Example 16. Comparison of Hemostatic Powder Formulations Obtained in Examples 12, 13, 14, 15

(128) Hemostatic powders obtained as described in Examples 12, 13, 14, 15 were compared for their performance. Water content was tested by Karl Fischer method; Thrombin potency was tested based on clotting time; Clottable protein was tested by quantifying fibrinogen; Particle size was tested by Laser particle size analyzer; Density was measured as Bulk density; Gel strength was measured as Tensile strength test. The results of the testing are shown in Table 9 and in FIGS. 19, 20.

(129) TABLE-US-00009 TABLE 9 Properties of hemostatic powders obtained with various solvents/binders Particle Water Thrombin Clottable size Gel content potency Protein Density D50 strength (%) (IU/mg) (mg/g) (g/ml) (μm) (Kpa) Example 2.32 1.78 290.11 0.33 181.97 61.13 12 Example 2.44 2.19 324.55 0.38 201.57 55.35 13 Example 3.87 2.03 284.08 0.34 71.98 55.00 14 Example 2.80 1.80 326.76 0.29 57.66 121.15 15

(130) Advantageously, the volatile properties of HFE potentially increase the porosity of final hemostatic powder product, which will lead to a decrease in the density of the final product, and speeds up the dissolution or re-dispersion time. The density of the powders of Example 15 is lowest, while the strength of formed gel is highest vs. other methods. The hemostatic performance was also better as was shown in FIG. 14.

Example 17. Testing of the Inventive Hemostatic Powder in Lung Sealing Model

(131) Two studies were conducted to test the hemostatic powders of the present invention for sealing function on lung leak model. Both studies showed at 30 cm H2O pressure, the present powder formed gel that was tightly adhering to the lung tissue and sealed the leak very well. When the pressure was 40 cm H2O, the gel started to delaminate, but still could seal the leak.

(132) FIG. 21 shows Lung leak model with 1.2 cm long incision;

(133) FIG. 22 shows the inventive hemostatic powder forming a gel (counted for 2 min) on the deflated lung;

(134) FIG. 23 shows the inventive hemostatic powder forming a gel that seals the lung leak at 30 cm H2O;

(135) FIG. 24 shows Lung leak model with 1 cm long incision;

(136) FIG. 25 shows the inventive hemostatic powder forming a gel that seals the lung leak at 30 cm H2O;

(137) FIG. 26 shows the inventive hemostatic powder forming a gel partially delaminated at 40 cm H2O pressure (arrow indicates the delamination area).

(138) Advantageously, the inventive hemostatic powder is shown to be able to seal lung leaks.