WO2023115427A1

WO2023115427A1 - Spray dried thrombin

Info

Publication number: WO2023115427A1
Application number: PCT/CN2021/140664
Authority: WO
Inventors: Hai Li; Caixia Jiang; Yufu Li
Original assignee: Guangzhou Bioseal Biotech Co., Ltd.; Ethicon, Inc.
Priority date: 2021-12-23
Filing date: 2021-12-23
Publication date: 2023-06-29

Abstract

Disclosed is a powdered composition comprised of: (i) thrombin, (ii) total protein in an amount of less than 75%, by weight, (iii) albumin in an amount of more than 5% by weight and iv) a non-acidic amino acid, the composition being substantially free of carbohydrates. A Method for preparing thrombin powders is also disclosed herein.

Description

SPRAY DRIED THROMBIN

FIELD OF THE INVENTION

The present invention relates, inter alia, to a spray-dried thrombin powder, methods of preparation thereof and uses thereof.

BACKGROUND OF THE INVENTION

Thrombin is a proteolytic enzyme having multiple functions in blood coagulation. Thrombin is formed from prothrombin (coagulation Factor II) , a circulating zymogen precursor protein in the plasma. It is proteolytically cleaved to form thrombin in the coagulation cascade. Thrombin in turn acts as a serine protease that converts soluble fibrinogen into insoluble strands of fibrin, as well as catalyzing many other coagulation-related reactions.

Thrombin is widely used in clinical applications as a coagulation factor to staunch bleeding of wounds by conversion of fibrinogen to fibrin. It is a common component of surgical dressings, and has been used in combination with fibrinogen and other coagulation proteins in two-component hemostatic systems such as fibrin glues, adhesives, and sealants.

Typically, spray drying involves atomization of a liquid, such as a solution, suspension or emulsion, for example by spraying through a spray nozzle while contacting with an atomizing gas to form spray particles; by two-fluid nozzle atomization, wherein spray is created by combination of a liquid flow and a gas flow, in which the atomization energy is provided by the gas flow; or by centrifugal atomization, wherein the solution is delivered in a rotating disk, such that spray is created by the energy created by the rotation of the disc. Alternatively, the liquid flow may be sprayed using a pressure nozzle in which the liquid flow is forced through a small aperture, the change in pressure transforming the liquid flow into spray of small droplets.

EP3731885A1 discloses spray-dried thrombin materials obtained from feedstock solutions comprising less than 5%by weight albumin and excluding trehalose or other excipients as well as methods of manufacturing the thrombin materials and methods of treating bleeding wounds.

US9376674B2 relates to a thrombin composition which is stable in solution for therapeutic use as a component of fibrin adhesives or for other haemostatic uses, which may be subjected to double nanofiltration in order to retain viruses, and which may be preserved in the lyophilised or frozen condition.

US10869912B2 discloses spray-dried thrombin powders comprising microcapsules, methods of preparation and uses thereof.

US6113948A relates to soluble microparticles comprising fibrinogen or thrombin, in free-flowing form. These microparticles can be mixed to give a dry powder, to be used as a fibrin sealant that is activated only at a wound site.

WO2014135689A2 relates to sterile powder compositions suitable for medical use comprising thrombin and fibrinogen, and to methods for producing the same, wherein the thrombin powder is produced from a liquid feedstock, wherein the feedstock comprises a solution or a suspension of thrombin, preferably a solution, wherein the powder is produced by removal of liquid by a process selected from aseptic spray drying or aseptic fluid bed drying, and wherein the powder resulting from removal of liquid from the feedstock exhibits at least 80%of the thrombin potency or activity of the liquid feedstock, and wherein the fibrinogen powder is produced by removal of liquid from a feedstock, wherein the feedstock comprises a solution or a suspension of fibrinogen, preferably a solution, by aseptic spray drying or aseptic fluid bed drying, and wherein said composition is packaged as a sterile final pharmaceutical product for medical use

US9717821B2 relates to formulations comprising a dry powder fibrin sealant comprised of a mixture of fibrinogen and/or thrombin, for use in the treatment of wounds or injuries, in particular for use as a topical hemostatic composition or for surgical intervention.

US8846105B2 relates to a dry powder fibrin sealant which comprises a mixture of fibrinogen and thrombin for use in surgery, trauma and other wounds or injuries. It further relates to novel formulations comprising said dry powder fibrin sealant for use in the treatment of wounds or for surgical intervention or as a topical hemostat.

SUMMARY OF THE INVENTION

In previous studies, in order to protect thrombin from denature during spray drying process, carbohydrates were usually contained in the formulation of thrombin preparation as stabilizer. Carbohydrates contained in the medical drug could have a negative impact e.g., in controlling blood sugar level for diabetics. An object of the present invention is to provide a spray dried thrombin preparation with high potency and without carbohydrates contained therein.

The inventors have surprisingly found that implementing a spray drying of a thrombin solution, with the thrombin solution being substantially free of carbohydrates and comprising, inter alia, a non-acidic amino acid, avoids potential process-induced losses in thrombin potency, as well as retains low water content, high solid yield and desired powder particle size distribution.

Each aspect and embodiment of the present disclosure may be combined with any other aspect and embodiment unless specified otherwise.

According to an aspect of the present disclosure, there is provided a powdered composition comprising: (i) thrombin, (ii) total protein in an amount of less than 75%, by weight, (iii) albumin in an amount of more than 5%by weight, and iv) a non-acidic amino acid, wherein the composition is substantially free of carbohydrates.

In some embodiments, the composition is spray dried, that is, obtained by a spray-drying process.

In some embodiments, the non-acidic amino acid comprises glycine.

In some embodiments, the composition is substantially free of a neutral salt, e.g., NaCl.

In some embodiments, the weight ratio of the non-acidic amino acid to total protein ranges from 2:5 to 16:17, respectively.

In some embodiments, the weight ratio of albumin to total protein ranges from 1:17 to 3:5, respectively.

In some embodiments, the composition comprises total protein in an amount of at least 6%by weight.

In some embodiments, the albumin is in an amount of at least 5%, by weight.

In some embodiments, the non-acidic amino acid is in an amount ranging from 40%to 94%, by weight.

In some embodiments, the thrombin is present in an amount ranging from about 11800 to about 142000 Units per gram total protein.

According to an aspect of the present disclosure, there is provided a method for preparing thrombin powder ( "the preparation method aspect" ) , the method comprising the steps of: mixing thrombin, albumin, a non-acidic amino acid in an aqueous solution, wherein the solution is substantially free of carbohydrates; and spray drying the aqueous solution.

In some embodiments of the preparation method aspect, the non-acidic amino acid comprises glycine.

In some embodiments of the preparation method aspect, the solution comprises proteins at a concentration of 0.6 to 3.8 % (w/v) .

In some embodiments of the preparation method aspect, the solution comprises basic or non-acidic amino acid at a concentration ranging from 2 to 8% (w/v) .

In some embodiments of the preparation method aspect, the solution comprises non-acidic amino acid at a concentration ranging from 2 to 8% (w/v) .

In some embodiments of the preparation method aspect, the solution comprises albumin at a concentration ranging from 0.5 to 3% (w/v) .

In some embodiments of the preparation method aspect, the solution comprises thrombin at a concentration ranging from 450 to 850 IU/ml.

In some embodiments of the preparation method aspect, the solution is substantially free of a neutral salt.

In some embodiments of the preparation method aspect, the aqueous solution comprises:

from about 450 to about 850 IU/ml thrombin;

from about 0.5 to about 3%w/v albumin;

from about 2 to about 8%w/v non-acidic amino acid (e.g., glycine) ; and

from about 0.6 to about 3.8%w/v total protein.

In some embodiments of the preparation method aspect,

from about 0.6 to about 3.8%w/v total protein.

In some embodiments of the preparation method aspect, there is provided spray-dried thrombin powder obtained by the method of any one of the embodiments.

In some embodiments of the preparation method aspect, the spray-dried thrombin powder is characterized in that it exhibits at least 80%of the thrombin potency or activity of the aqueous solution (e.g., the aqueous solution as tested upon lyophilization) . In some embodiments of the preparation method aspect, the spray-dried thrombin powder is characterized in that it exhibits about 80%to about 90%of the thrombin potency or activity of the aqueous solution (e.g., the aqueous solution as tested upon lyophilization) . In some embodiments of the preparation method aspect, the method is characterized in that it provides at least 80%, at least 90%solid yields, in some embodiments 80%to about 90%solid yields.

In another aspect, there is provided a matrix comprising the thrombin powder of any aspect and embodiments provided herein. In some embodiments, the matrix further comprises a fibrinogen powder.

In another aspect, there is provided an aqueous composition suitable for use in preparing thrombin powder, the aqueous composition comprising: from about 450 to about 850 IU/ml thrombin; from about 0.5 to about 3%w/v albumin; from about 2 to about 8%w/v non-acidic acid (e.g., glycine) ; and from about 0.6 to about 3.8%w/v total protein.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawing. With specific reference now to the drawing in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawing makes apparent to those skilled in the art how embodiments of the invention may be practiced.

Figures 1A-1D present main effects plots analysis by statistical tools (using Minitab software; mean values) based on Table 4 (Thrombin First Screening DOE Results) , against (upper panel from left to right) inlet air flow in the column, inlet air temperature, atomization air rate, and against (lower panel from left to right) cyclone gas rate, nozzle (mm) , and flow rate of solution input. Figure 1A presents the main effect plot for water content (%) ; Figure 1B presents the main effect plot for potency retain (%) ; Figure 1C presents the main effect plot for solid yields (%) ; Figure 1D presents the main effect plot for size distribution, D50.

Figures 2A-2D present graphs showing the effect of different parameters on water content of the spray-dried product. X-axes relate to different feed rates of the stock solution. Each graph line relates to a different atomizing air flow rate; dashed line in each graph represents the maximal desired water content (set for 3%) . Figure 2A relates to two drying columns with air cooling part ( "Mode I" ) and with glycine being contained in the formulation; Figure 2B relates to Mode I without glycine being contained in the formulation; Figure 2C relates to three drying columns without air cooling part ( "Mode II" ) with glycine being contained in the formulation. Figure 2D relates to Mode II without glycine being contained in the formulation.

Figure 3A-3D present graphs showing the effect of different parameters on the thrombin activity (potency) of the spray-dried product. X-axes relate to different feed rates of the stock solution. Each graph line relates to a different atomizing air flow rate. Figure 3A relates to Mode I (herein and below -Modes are as in Figures 2A-2D above) with glycine being contained in the formulation; Figure 3B relates to Mode I without glycine being contained in the formulation; Figure 3C relates to Mode II with glycine being contained in the formulation. Figure 3D relates to Mode II without glycine being contained in the formulation.

Figure 4A-4D present graphs showing the effect of different parameters on solid yields of the obtained spray-dried powder. X-axes relate to different feed rates of the stock solution. Each graph line relates to a different atomizing air flow rate. Figure 4A relates to Mode I with glycine being contained in the formulation; Figure 4B relates to Mode I without glycine being contained in the formulation; Figure 4C relates to Mode II with glycine being contained in the formulation. Figure 4D relates to Mode II without glycine being contained in the formulation.

Figure 5A-5D present graphs showing the effect of different parameters on the particle size distribution (D50, μm) of the obtained spray-dried powder. X-axes relate to different feed rates of the stock solution. Each graph line relates to a different atomizing air flow rate. Figure 5A relates to Mode I with glycine being contained in the formulation; Figure 5B relates to Mode I without glycine being contained in the formulation; Figure 5C relates to Mode II with glycine being contained in the formulation. Figure 5D relates to Mode II without glycine being contained in the formulation.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

In previous publications, in order to protect thrombin from denature during spray drying process, carbohydrates were usually contained in the formulation of thrombin preparation as stabilizer. Carbohydrates contained in the medical drug could have a negative impact e.g., in controlling blood sugar level for diabetics. An object of the present invention is to provide a spray dried thrombin preparation without carbohydrates contained in the formulation.

It was surprisingly found that implementing a spray drying of a thrombin solution, with the thrombin solution being substantially free of carbohydrates and comprising, inter alia, a non-acidic amino acid, avoids process-induced losses in thrombin potency.

The spray dried thrombin powder may be produced and optimized by controlling one or more of the following parameters: the composition of the thrombin solution used for the spray-drying process, spray parameters (e.g., solution flow rate, atomizing gas flow rate) , drying gas parameters (e.g., drying gas flow rate, drying gas temperature) , and powder collection parameters (e.g., cooling gas flow) .

According to an aspect of some embodiments of the present invention, there is provided a powdered composition comprising: (i) thrombin, (ii) total protein in an amount of less than 75%, by weight, (iii) a carrier protein, e.g., albumin, in an amount of more than 5%, by weight and iv) a basic, non-polar, or non-acidic amino acid, wherein the composition is substantially free of carbohydrates.

As used herein, the term a “powdered composition” typically refers to about 50%to about 100%, by weight of the composition being weight dry, solid, loose particles of small grain size.

By "about 50%to about 100%" it may be meant to include ranges such as: 50-55%, 50-60%, 50-65%, 50-70%, 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 50-99%, 55-60%, 55-65%, 55-70%, 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 55-99%, 60-65%, 60-70%, 60-75%, 60-80%, 60-85%, 60-90%, 60-95%, 60-99%, 65-70%, 65-75%, 65-80%, 65-85%, 65-90%, 65-95%, 65-99%, 70-75%, 70-80%, 70-85%, 70-90%, 70-95%, 70-99%, 75-80%, 75-85%, 75-90%, 75-95%, 75-99%, 80-85%, 80-90%, 80-95%, 80-99%, 85-90%, 85-95%, 85-99%, 90-95%, 90-99%, 95-99%, 90-100%, or 95-100%.

Typically, the powder comprises particles having diameters/length in the micrometer range and in the non-micrometer range, for example amorphous microcapsule debris (i.e. separate shell fragments, originating e.g., from a microcapsule that was burst or torn into separate shell fragments) . In some embodiments, a “powdered composition” according to the teachings herein relates essentially to the thrombin particles.

The term "solid" characterizes the state of the compound or composition at room temperature (e.g., 25 ℃) and at atmospheric pressure (760 mmHg) , i.e. a compound or a composition of high consistency which retains its form during storage. This term in the present application also relates to non-fluid particles, or dissolved substance. As opposed to "liquid" compounds and compositions, the solid does not flow under its own weight. "Powdered" and "particulate" may be used interchangeably herein.

In some embodiments, the thrombin particles have D50 of at least 1μm, at least 2μm, at least 3μm, at least 4μm, or at least 5μm. In some embodiments, a “powdered composition” according to the teachings herein have D90 of below 45μm, below 44μm, below 43μm, below 42μm, below 41μm, below 40μm, below 39μm, below 38μm, below 37μm, below 36μm, or below 35μm. In exemplary embodiments, the thrombin particle size distribution is: D50 of at least 5μm and D90 of up to 39μm.

In some embodiments, the thrombin particles have D50 of about 1μm, about 2μm, about 3μm, about 4μm, about 5μm, about 6μm, about 7μm, about 8μm, about 9μm, about 10μm, about 11μm, about 12μm, about 13μm, about 14μm, about 15μm, about 16μm, about 17μm, about 18μm, about 19μm, about 20μm, about 21μm, about 22μm, about 23μm, about 24μm, or about 25μm. In some embodiments, a “powdered composition” according to the teachings herein have D90 of about 35μm, about 34μm, about 33μm, about 32μm, about 31μm, about 30μm, about 29μm, about 28μm, about 27μm, about 26μm, or about 25μm. In exemplary embodiments, the thrombin particle size distribution is: D50 of at least 5μm and D90 of up to 39μm.

Size distribution D50 is also known as the median diameter or the medium value of the units in the powder/aggregates size distribution, it is the value of the units' diameter at 50%in the cumulative distribution. For example, if D50 is X pm, then 50%of the units in the sample are larger than X μm, and 50%are smaller than X μm. Size distribution is the number of units that fall into each of the various size ranges given as a percentage of the total number of all units' sizes in the sample of interest. Accordingly, D90 value refers to 90%of the units having a size that is smaller than the D90 value. All ranges disclosed herein include the upper and lower limit, where applicable.

In some embodiments, at least part of the powder is in the form of aggregate (s) or granulate (s) . The term "aggregate" describes a particle formed from assembled components. Aggregates may optionally be made by a step of humidifying the powder composition; compacting, e.g., by roller and/or slugging the powder to form aggregates; dehumidifying; milling; sieving the aggregates; and optionally dosing the resulting aggregates into a storage container or into a delivery device. The term "granulate" or "granulate material" may particularly denote a conglomeration of particles typically below 900 micron, below 800 micron, below 700 micron, or even below 500 micron.

The term “at least part of the powder is in an aggregated form” refers to a part of the population of the particles in the powder e.g., at least between 10%and 100%forming an aggregate.

In some embodiments, the composition is spray-dried, that is, obtainable, or, in some embodiments, obtained by, a spray draying process.

The term "spray drying" is used herein in a broad sense, which include, without being limited thereto, processes for transforming a solid dissolved or suspended in a liquid form, e.g., a stock solution, into a powdery. Typically, spray drying is a drying method used to create powder from a solution, suspension or emulsion that is atomized through a spray nozzle in a hot airflow and dried instantly. Spray drying process for producing thrombin powders, which maintain their activity may be controlled by several process parameters, among them are, without limitation: column air flow (also referred to as "air cooling part" ) , temperatures, nozzle size, atomizing air flow rate, and material flow rate. Non-limiting exemplary parameters used in order to establish a robust process to produce a blend of thrombin and optionally additional proteins (e.g., in a powder form) which maintains its activity are provided in the Examples section below.

The term "liquid" relates to a substance that can flow, has not fixed shape, and is not a solid or gas. The term "solution" relates to dispersed or dissolved substance (s) and the medium in which it is dispersed or dissolved, or to a single homogeneous liquid phase that is a mixture in which the components are uniformly distributed throughout the mixture.

As used herein, "thrombin" denotes an activated enzyme which results from the proteolytic cleavage of prothrombin (factor II) . Thrombin may be produced by a variety of methods of production known in the art, and includes, but is not limited to, recombinant thrombin and plasma derived thrombin.

Human thrombin is a 295 amino acid protein composed of two polypeptide chains joined by a disulfide bond. Both human and non-human (e.g., bovine) thrombin may be used within the scope of the present disclosure.

The origin for thrombin used in the present disclosure may be from one or several sources including but not limited to: plasma (e.g., porcine plasma) , recombinant bacteria and/or cells (see e.g., Vu et al., 2016, J. Viet. Env. 8 (1) : 21-25) , whole blood (pooled from several donations or not) and/or blood fraction (that may be pooled from several donations, e.g., plasma) .

In some embodiments, the powdered composition also comprises albumin. The composition may comprise at least 5%albumin by weight, or 5.5%to 6%albumin or 5%to 30%or 5%to 25%or 5%to 10%, by weight.

In some embodiments of the present disclosure, the composition, e.g., such that comprises the spray-dried thrombin is substantially free of any carbohydrates. For example, the composition does not contain trehalose.

The term "substantially free of any carbohydrates" means that the composition contains no significant amount any carbohydrates, e.g., less than 1%, less than 0.5%, by weight, or, in some embodiments, is completely free (i.e. absent) of any carbohydrates.

"Carbohydrate" refers to a sugar alcohol, such as mannitol. In some the carbohydrate is a saccharide, such as a saccharide selected from the group consisting of a monosaccharide, a disaccharide (optionally selected from the group consisting of sucrose, trehalose and a combination thereof) , an oligosaccharide, a polysaccharide and a combination thereof. Thus, in some embodiments, the composition also does not contain mannitol or any other stabilizing sugar. In some embodiments, the disclosed spray dried composition maintains stability and performance in the absence of trehalose and mannitol. The compositions may be substantially free of trehalose and/or substantially free of mannitol.

By "basic or neutral amino acid" , or "non-acidic amino acid" , it is meant the naturally occurring basic to neutral amino acids, such as, without limitation, arginine, glycine, lysine, and histidine, as well as any basic amino acid having a carboxyl group and an amino group in the molecule, which is water-soluble and provides an aqueous solution with a pH of about 7 or greater. Accordingly, basic amino acids include, but are not limited to, arginine, lysine, salts thereof or combinations thereof. In certain embodiments, the amino acid is glycine.

As shown in the Examples section that follows, the results demonstrated that it is difficult to control water content below 3% (by weight) without glycine contained in the thrombin formulation (see Figures 2A-2D) .

In some embodiments, the weight ratio of the non-acidic amino acid to total protein ranges from 2:5 to 16: 17, respectively. In some embodiments, the weight ratio of the non-acidic amino acid, e.g., glycine, to total protein ranges from 2:5 to 16:17, respectively. In some embodiments, the weight ratio of the non-acidic amino acid, e.g., glycine, to total protein ranges from 2:5 to 16:17, respectively. In some embodiments, the weight ratio of the non-acidic amino acid, e.g., glycine, to total protein ranges from 2:6 to 16:20, respectively. In some embodiments, the weight ratio of the non-acidic amino acid, e.g., glycine, to total protein ranges from 2:5 to 16:17, respectively. In some embodiments, the weight ratio of the non-acidic amino acid, e.g., glycine, to total protein ranges from 2:7 to 14:17, respectively. In some embodiments, the weight ratio of the non-acidic amino acid, e.g., glycine, to total protein ranges from 2:5 to 16:17, respectively.

In some embodiments of the powdered composition, the non-acidic amino acid, e.g., glycine, is present in an amount ranging from 30 to 98%, by weight. In some embodiments, the non-acidic amino acid, e.g., glycine, is present in an amount ranging from 40 to 94%, by weight. In some embodiments, the non-acidic amino acid, e.g., glycine, is present in an amount ranging from 35 to 96%, by weight. In some embodiments, the non-acidic amino acid, e.g., glycine, is present in an amount ranging from 40 to 94%, by weight. In some embodiments, the non-acidic amino acid, e.g., glycine, is present in an amount of 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 94, or 96%, by weight, including any value and range therebetween. In exemplary embodiments, the non-acidic amino acid, e.g., glycine, is present in an amount of about 73%, by weight.

In some embodiments, the carrier protein is selected from albumin, casein, keratin and a combination thereof.

In some embodiments, the carrier protein comprises albumin, optionally human serum albumin. In some embodiments, the carrier protein is albumin, optionally human serum albumin.

In some embodiments, the carrier protein e.g., albumin, is in an amount of at least 5%by weight. In some embodiments, the carrier protein e.g., albumin, is in an amount of more than 5%by weight. In some embodiments, the carrier protein e.g., albumin, is in an amount of 10 to 25%, by weight. In some embodiments, the carrier protein e.g., albumin, is in an amount of 10 to 18%, by weight. In some embodiments, the carrier protein e.g., albumin, is in an amount of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, by weight, including any value and range therebetween. In exemplary embodiments, the albumin, is present in an amount of about 18%, by weight.

In some embodiments, the weight ratio of the carrier protein e.g., albumin, to the total protein ranges from 1:20 to 3:4, respectively. In some embodiments, the weight ratio of the carrier protein e.g., albumin, to the total protein ranges from 1:18 to 3:4, respectively. In some embodiments, the weight ratio of the carrier protein e.g., albumin, to the total protein ranges from 1:17 to 3:4, respectively. In some embodiments, the weight ratio of the carrier protein e.g., albumin, to the total protein ranges from 1:20 to 3:5, respectively. In some embodiments, the weight ratio of the carrier protein e.g., albumin, to the total protein ranges from 1:18 to 3:5, respectively. In some embodiments, the weight ratio of the carrier protein e.g., albumin, to the total protein ranges from 1:17 to 3:5, respectively.

In some embodiments, the composition, e.g., spray-dried composition, comprises protein (including e.g., thrombin and albumin) having a total weight of at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, or at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%.

In some embodiments, the composition is substantially free of neutral salt. In some embodiments, the salt being substantially free of is sodium chloride.

The term "substantially free of neutral salt" means that the composition contains no significant amount of salt, e.g., sodium chloride, e.g., less than 1%, less than 0.5%, by weight, or, in some embodiments, is completely free of salt, e.g., sodium chloride.

The term “neutral salt” refers to a salt that is not substantially acidic or basic, i.e., has little or no effect on the pH of the formulation when dissolved. It was found that no significant loss of activity of thrombin was detected, even without using a neutral salt.

In some embodiments, the disclosed composition is dry.

It was surprisingly found that use of the processes described herein e.g., spray-drying method using aqueous compositions described herein result in production of thrombin powders which have high levels of thrombin activity (see below, e.g., Table 7) , in some embodiments, having a thrombin content of from 10000 to about 150000 Units per gram total protein and are dry. By "dry" it means, in some embodiments, having less than 5%w/w water content, or, in some embodiments, less than about 3%w/w water content.

Herein, "activity" “thrombin activity” , or “thrombin biological activity” are meant to include thrombin mediated conversion of heterologous substrates, including proteins e.g., fibrinogen into fibrin, as well as the conversion of Factor VIII to Factor Villa, XI to XIa, XIII to Xllla, and Factor V to Va. A “heterologous substrate” is a substrate, preferably a protein substrate, other than thrombin. In some embodiments, the thrombin activity refers to conversion of fibrinogen into fibrin. Methods for measuring thrombin activity are known in the art, and determination of thrombin activity by clotting time is described in the Examples section hereinbelow.

In some embodiments, the thrombin content ranges from 10 to about 150 IU/mg solid. In some embodiments, the thrombin content ranges from 10 to 150 IU/mg solid. In some embodiments, the thrombin content ranges from 11 to about 145 IU/mg solid. In some embodiments, the thrombin content ranges from 11.8 to about 142 IU/mg solid. In some embodiments, the thrombin content ranges from 30 to about 60 IU/mg solid.

Herein "U" denotes a coagulation factor unit, that is a unit of a physiological measurement of a coagulation factor in 1 ml of human normal plasma. "IU" refers to International Unit of a coagulation factor which is a physiological measurement in a given sample as it compared to an adequate intentional standard, e.g., as determined by the clotting assay against an internal reference standard for potency concentration measurement that has been calibrated against, for example, the World Health Organization (WHO) Second International Standard for Thrombin, 01/580.

In some embodiments, there is provided a spray-dried powdered composition comprising: (i) thrombin, in a content ranging from 10 to about 150 IU/mg solid; (ii) total protein in an amount of less than 75%, by weight; (iii) albumin in an amount of more than 5%to about 30%, by weight; and (iv) a glycine in an amount of about 70%wherein the composition is substantially free of carbohydrates.

In some embodiments, spray-drying droplets are formed by spraying an aqueous thrombin solution (also referred to as "stock solution" ) through an atomizing spray nozzle tip into a drying chamber, and then evaporating the water from the droplets to form the desired powder by a hot drying gas flow. As used herein, the term “aqueous thrombin composition” refers to a solution which comprises thrombin and solutes.

Any suitable droplet size may be used to implement the teachings herein. As known in the art, droplets may be formed for spray drying by use of an atomizing gas that flows through the atomizing nozzle e.g., having a diameter of about 0.4-0.8 mm, coaxially and in the same direction as the aqueous thrombin composition. Any suitable inert gas may be used as an atomizing gas including air, nitrogen and argon. Preferably, the atomizing gas is dry, containing as little water as possible, in some embodiments the atomizing gas has up to about 30%relative humidity. As known in the art, droplets formed for spray drying may be sprayed into a drying gas inside the drying chamber and are carried away. Any suitable gas may be used as a drying gas at a temperature in the range of from about 100 to about 190℃, e.g., 160℃, including air, nitrogen and argon. In some embodiments, the drying gas is dry, containing as little water as possible, such as having up to about 30%relative humidity.

As used herein, the term “drying gas” refers to a gas, which upon contact with the droplets, dries the droplets into a powder.

In certain embodiments, the droplets are dried by passing through three drying columns.

As used herein, the term “drying column” or “drying chamber” refers to a column in which the drying of the droplets takes place, implemented by a flow of drying gas at high temperature (such as from about 100 to about 190 ℃) .

In certain embodiments, the droplets are dried by passing through e.g., two drying columns (sequentially) , followed by an air-cooling part.

As used herein, the term “air-cooling part” refers to a part of the spray-drying device in which a gas which enters the spray dryer and merges with the drying gas flow in contact with the powder flow in order to reduce the temperature of the drying gas flow and of the powder.

In some embodiments, the rate of the inlet air flow is from about 0.1 to about 1.0 m ³/min, optionally from about 0.3 to about 0.6 m ³/min. In some embodiments, the rate of the cooling air flow is from about 0.1 to about 1.0 m ³/min, optionally from about 0.2 to about 0.3 m ³/min.

In certain embodiments, the rate flow of the atomizing air ranges from 5 to 12 L/min. In certain embodiments, the rate flow of atomizing air ranges from 6 to 12 L/min. In certain embodiments, the rate flow of atomizing air ranges from 7 to 12 L/min. In certain embodiments, the rate flow of atomizing air ranges from about 5 to about 7 L/min.

In some embodiments, the feed rate ranges from about 200 to about 600 ml/hour. In some embodiments, the feed rate ranges from 200 to 480 ml/hour. In some embodiments, the feed rate ranges from 230 to about 280 ml/hour. In some embodiments, the feed rate ranges from about 200 to less than 360 ml/hour.

According to an aspect of some embodiments of the invention, there is provided method for preparing thrombin powder, the method comprising the steps of: mixing thrombin, albumin, a non-acidic amino acid in an aqueous solution, wherein the solution is substantially free of carbohydrates; and spray drying the aqueous solution.

According to a further aspect of some embodiments described herein, there is provided an aqueous composition suitable for use in preparing thrombin powder, the aqueous composition comprising: thrombin, albumin, a non-acidic amino acid in an aqueous solution, wherein the solution is substantially free of carbohydrates.

In some embodiments of any aspect, the aqueous solution comprises from about 1 to about 8 mg/ml (about 450 to 850 IU/ml) thrombin.

As used herein, the term “mixing” refers to, but is not limited to, combining components in any physical form, e.g., powder, blend, solution, suspension, dispersion, or the like.

In some embodiments of any aspect, the aqueous composition comprises from about 5 to 30 mg/ml (0.5 to 3 % (w/v) ) carrier protein e.g., albumin. In some embodiments of the method disclosed herein, the carrier protein is selected from the group consisting of albumin, casein, and keratin. In some embodiments of the method disclosed herein, the carrier protein comprises albumin. In some embodiments of the method disclosed herein, the carrier protein is albumin.

In some embodiments of any aspect, the aqueous composition comprises from about 6 to 12 mg/ml carrier protein e.g., albumin. In exemplary embodiments, the aqueous composition comprises about 10 mg/ml albumin.

In some embodiments of any aspect, the aqueous composition comprises is substantially free of carbohydrate. As noted above, "carbohydrate" refers to a sugar alcohol, such as mannitol. In some the carbohydrate is a saccharide, such as a saccharide selected from the group consisting of a monosaccharide, a disaccharide (optionally selected from the group consisting of sucrose, trehalose and a combination thereof) , an oligosaccharide, a polysaccharide and a combination thereof. Thus, in some embodiments, the solution also does not contain mannitol or any other stabilizing sugar. In some embodiments, the solution maintains stability and performance in the absence of trehalose and mannitol. The compositions may be substantially free of trehalose and/or substantially free of mannitol.

Embodiments of "non-acidic amino acid" are described hereinabove and are incorporated into the method aspects.

In some embodiments of any aspect, the aqueous composition comprises non-acidic amino acid (e.g., glycine at a concentration of from about 20 to about 80 mg/ml (2 to 8 %(w/v) ) . In some embodiments, the aqueous composition comprises non-acidic amino acid e.g., glycine at a concentration of from about 30 to about 50 mg/ml. In exemplary embodiments, the aqueous composition comprises glycine at a concentration of about 40 mg/ml. In some embodiments, the aqueous composition comprises non-acidic amino acid e.g., glycine at a concentration of about 20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, or about 80 mg/ml, including any value and range therebetween.

In some embodiments of any aspect, the aqueous composition comprises from about 6 to 40 mg/ml (0.6 to 4 % (w/v) ) total proteins. In some embodiments, the aqueous composition comprises from about 8 to 38 mg/ml total proteins. In exemplary embodiments, the aqueous composition comprises about 2.5 mg/ml proteins other than albumin and thrombin.

In some embodiments of any aspect, the pH range of the aqueous composition (e.g., solution) is from about 5 to about 8, such as from 6.9 to 7.1. In some embodiments, the aqueous composition is substantially free of neutral salt. In some embodiments, the salt being substantially free of is sodium chloride. Embodiments of neutral salt are described hereinabove and are incorporated herein.

In some embodiments, the spray dried thrombin powder e.g., obtainable by the aqueous solution, comprises thrombin at about 5-25% (e.g., about 9%) albumin at about 5-30% (e.g., about 18%) , glycine at 50 to 80% (e.g., about 73%) , and is devoid of carbohydrates (%are by weight) .

The term "aqueous solution" refers to a solution comprising water as the solvent.

In some embodiments, the spray-dried thrombin powder is characterized in that it exhibits at least about 70%of the thrombin potency or activity of the corresponding aqueous solution. In some embodiments, the spray-dried thrombin powder is characterized in that it exhibits about 80%to about 98%of the thrombin potency or activity of the corresponding aqueous solution. In some embodiments, the spray-dried thrombin powder is characterized in that it exhibits about 90%to about 99%of the thrombin potency or activity of the corresponding aqueous solution, such that from about 10 to about 150 IU/mg solid.

In some embodiments, the content or potency of thrombin in the spray-dried thrombin powder ranges from about 10 to about 150 IU/mg solid. In some embodiments, the content or potency of thrombin in the spray-dried thrombin powder ranges from about 10 to 150 IU/mg solid. In some embodiments, the content or potency of thrombin in the spray-dried thrombin powder ranges from about 11 to about 145 IU/mg solid. In some embodiments, the content or potency of thrombin in the spray-dried thrombin powder ranges from about 11.8 to about 142 IU/mg solid. In some embodiments, the content or potency of thrombin in the spray-dried thrombin powder ranges from about 30 to about 60 IU/mg solid.

In an aspect of some embodiments, there is provided a kit comprising a container comprising the spray-dried thrombin powder according to any of the embodiments disclosed herein, as a first component. In some embodiments, the kit further comprises a second component selected from the group consisting of fibrinogen, gelatin, collagen, oxidized regenerated cellulose and a combination thereof.

The spray-dried thrombin powder as disclosed herein may be used for any therapeutic purpose. The term “any therapeutic purpose” refers to any curative or preventive treatment in a subject. Exemplary therapeutic purposes include, but are not limited to, hemostasis, sealing a bore hole formed in a tissue or organ e.g., a bone; anastomosis at blood vessels; joining tissue parts e.g., soft tissue parts; treating or preventing dura defects e.g., tears and leaks following dural injections, fissures or cracks; treating or preventing bleeding; treating or preventing air leaks such as following pulmonary lung resection; treating or preventing defects following intestinal perforation; treating or preventing defects following anastomosis procedure carried out in any tissue e.g., uterine, esophagus, stomach, pancreas, pancreatic duct, gall bladder, bile duct, intestinal (including the small intestine and the large intestine) , and rectum; treating or preventing post-operation leaks in any tissue e.g., uterine, esophagus, stomach, pancreas, pancreatic duct, gall bladder, bile duct, intestinal (including the small intestine and the large intestine) , and rectum; preventing or diminishing the occurrence of post-operative leaks at the staple or suture line e.g., by applying the powder according to the invention, either alone or combined with a matrix e.g., a patch, onto at least a part of a defect such as a staple/suture line; for strongly affixing prosthesis e.g., during a hernia operation; for staple/suture line reinforcement; to prevent or diminish alveolar air leakage; treating or preventing renal defects; treating or preventing fistulas; treating or preventing heart defects e.g., penetrating heart wounds; reinforcing of a vascular graft prosthesis; and treating or preventing cerebrospinal fluid leakage. In some embodiments, the spray-dried thrombin powder, or matrix according to any of the embodiments discloses herein is for use in providing assistance in hemostasis, sealing leaks and/or joining structures. In some embodiments, there is provided a method of providing hemostasis, sealing leaks and/or joining structures in a subject in need thereof, the method comprising use of the spray-dried thrombin powder or the matrix according to any of the embodiments disclosed herein.

As used herein, the term "bleeding" refers to extravasation of blood from any component of the circulatory system. A "bleeding" thus encompasses unwanted, uncontrolled and often excessive bleeding in connection with surgery, trauma, or other forms of tissue damage, as well as unwanted bleedings in patients having bleeding disorders. The term "blood" , or any grammatical inflection thereof, also includes blood fractions, such as plasma.

"Wound" as used herein refers to any damage to any tissue of a patient which results in the loss of blood from the circulatory system and/or any other fluid from the patient's body. The damage may have been caused by any agent or source, including traumatic injury, infection or surgical intervention. A wound may be in a soft tissue, such as an organ, or in hard tissue, such as bone. The tissue may be an internal tissue, such as an organ or blood vessel, or an external tissue, such as the skin. The loss of blood may be internal, such as from a ruptured organ, or external, such as from a laceration.

As used herein, and unless stated otherwise, the terms “weight ratio” , “by weight” , “w/w” , “weight percent” , or “wt. %” , which are used herein interchangeably describe the concentration of a particular substance out of the total weight of the corresponding mixture, solution, suspension, formulation or composition. As used herein, and unless stated otherwise, the terms “volume ratio” , “by volume” , “v/v” , “volume percent” , or “v %” , which are used herein interchangeably describe the concentration of a particular substance out of the total volume of the corresponding mixture, dispersant, solution, suspension, formulation or composition.

In some embodiments, the composition is stable.

The terms "stable" and "stability" when referring to the disclosed paste, mean that an active component within, e.g., thrombin at a certain temperature and after certain time duration remains at least 70 %active, e.g., capable of forming a fibrin clot.

The powder may be an intermediate suitable for combination with another powder or material, suitably to produce a sterile final composition or material. Compositions described herein, as well as the contents of the abovementioned kits, may, if desired, be presented in a pack or dispenser device, which may contain one or more unit dosage forms containing the composition and additional ingredients (e.g., fibrinogen) and/or reagents (e.g., dispersant) for preparing the composition. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser device may also be accompanied by a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions for human or veterinary administration. Such notice, for example, may include labeling approved by the U.S. Food and Drug Administration (FDA) for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a pharmaceutically acceptable carrier may also be prepared, placed in an appropriate container, and labeled for use for an indicated application and/or for treatment of an indicated condition, as further detailed herein. The term "preparation" refers to a physiologically suitable for therapeutic use.

It will be appreciated that compositions of embodiments of the present disclosure may be attached to or included in medical devices, such as for promoting wound healing.

In another aspect of the present disclosure, the disclosed composition in any aspect or embodiment thereof is for use in a method for preparing a fibrin sealant in/on an injured tissue of a subject, e.g., by applying the disclosed composition in any aspect and/or embodiment thereof on a surface of the tissue.

In another aspect of the present disclosure, there is provided a method for preparing a fibrin sealant in/on an injured tissue of a subject, e.g., by applying the disclosed composition in any aspect and/or embodiment thereof for a hemostatic treatment as a hemostat on a surface of the tissue.

The term "hemostatic" refers to an ability to prevent, reduce, or stop blood loss e.g., from wounds, such as surgical or traumatic wounds, e.g., by promoting blood clot formation.

"Hemostasis" (or "haemostasis" ) refers to the first stage of wound healing. It is a process which causes bleeding to stop. By “assistance in hemostasis” it is meant to help reduce or stop bleeding. By "applied to a bleeding tissue" it is meant to refer to e.g., a topical application of the composition at the bleeding site, e.g., at a surgical site to control bleeding. Control of bleeding is needed in various situations including treatment of wounds.

As used herein, the terms "controlling" , "preventing" , or "reducing" , which may be used herein interchangeably in the context of the bleeding, including any grammatical inflection thereof, indicate that the rate of the blood extravagated is essentially nullified or is reduced e.g., by 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, or even by 100 %, of the initial rate of bleeding, compared to situation lacking the contact of the disclosed composition in/on the bleeding site. Methods for determining a level of appearance of bleeding are known in the art.

Any aspect and embodiment of the hereinthroughout disclosed composition may be incorporated to the aspect and embodiments of the kit, including embodiments of the composition, the dispersant, and/or the powder.

Optionally, fibrinogen and the disclosed thrombin powder are each packaged individually (e.g., in dry form or in solution) in the kit. Thus, each of the two ingredients is packaged in separate packaging material, in addition to the packaging material of the whole kit.

Alternatively, the fibrinogen, and thrombin are packaged together (e.g., as a dry powder) in the kit in the same packaging material, and optionally the dispersant is provided in an additional container. In some embodiments, the kit further comprises instructions on how to combine the ingredients of the kit and/or how to combine the ingredients of the kit with an additional ingredient (e.g., a dispersant) , in order to produce the desired composition.

Optionally, the kit further comprises a dispersant. Dispersant may be in a pure form or in a solution with another liquid (e.g., water, saline or aqueous buffer) . Optionally, the dispersant is packaged individually, apart from the powder. Alternatively, the dispersant is packaged in combination with the powder, for example, as a ready-for-use composition described herein. In such embodiments, the kit may further contain a measuring means, e.g., a measuring cylinder, to measure the volume of the dispersant or a component thereof.

Additionally or alternatively, the hemostatic kit may comprise an applicator, such as syringe, containing the blend, mixture or powder and another applicator containing the dispersant. For example, dual-syringe mixing devices may produce a substantially homogenous paste mixture by combining initially separate liquid and powders and then passing the blended contents back and forth between two connected syringes via interconnected outlet (s) . Therefore, a low expression force for dispensing the paste from a syringe may be preferred for ease of mixing and ultimately for deployment of the resulting paste. The desired expression force may be less than 1.51 lbf.

In some embodiments, at least one of the containers in the kit is a pre-filled syringe. In some embodiments, a syringe is provided in addition to the container (s) of the kit. In some embodiments, the container is in a specific type, such as a vial or an applicator such as syringe.

In some embodiments, at least one of the containers in the kit is a pre-filled syringe. In some embodiments, a syringe is provided in addition to the container (s) of the kit. The term "container" may refer to any generic structure such as a vessel or a vial, that may contain the powder or dispersant.

The kit may be applied using an applicator device which may be used for administering several and sequential injections of the composition. In one embodiment, the applicator device enables multiple injections of a fixed-dose of the mixed components on a 2-D surface of a tissue while moving the device. In one embodiment, the applicator has a syringe with an injection needle, which is optionally automatically retracted from the patient's skin after the injection is completed without the need for the administrator to lift the device upward from the injection surface. In one embodiment, the kit may be used for the administration of a sealant.

The kit e.g., hemostatic kit of the invention may be a kit for use in reducing, preventing or stopping blood flow, e.g., in open wounds, and it may be used for reducing, preventing or stopping blood flow during a procedure, such as during, before, or after a surgical procedure such as, for example, laparoscopic surgery, neurosurgery, abdominal surgery, cardiovascular surgery, thoracic surgery, head and neck surgery, pelvic surgery and skin and subcutaneous tissue procedures. The kit may be used for reducing or preventing blood flow from the skin, or in internal organs.

In one embodiment, this kit may be stored at room temperature, such as at a temperature in the range of 8 to 40 ℃, or at lower temperatures.

In some embodiments of any aspect of the kit or the composition disclosed herein, the powder or blend may comprise an additive e.g., calcium salt and/or one or more excipients, e.g., selected from, without being limited thereto, one or more amino acids, saccharides, and/or saccharide derivatives.

The term "additive" is meant to be understood as any substance that can be added to a composition, and may also include an active additive such as calcium salt as described below.

The term "excipient" as used herein denotes a non-active or non-therapeutic agent added to a pharmaceutical composition e.g., to provide a desired consistency or stabilizing effect.

Calcium is an important element in the clotting cascade, and may be needed for activation of factor XIII into factor XIIIa, which cross-links and stabilizes fibrin to generate an insoluble clot.

Accordingly, in some embodiments of any aspect of the disclosed kit and/or composition, the blend, mixture or powder further comprises an additive such as, without limitation, calcium. Calcium used with the invention may be in the form of a salt, e.g., calcium chloride salt. Alternatively, additional salts may be used, such as calcium acetate and/or calcium citrate. In the kit, the calcium salt may be provided in the composition comprising the powder. Alternatively, the excipient (s) , and/or the calcium salt, may be provided in the kit in a separate container, or the excipient (s) , and/or the calcium salt, may be provided in the kit in the same container comprising the blend/mixture/powder component.

In some embodiments of any aspect of the kit and/or compositions, the powder further comprises one or more excipients selected from, without being limited thereto, sucrose (e.g., for the fibrinogen component) , histidine, arginine, sodium chloride, sodium citrate, tween 80, and glycine.

In some embodiments of any aspect of the kit and/or compositions, the disclosed composition may be used in conjunction with a backing, pad, bandage, gauze, sponge, scaffold, or matrix to provide mechanical strength to cover the wound surface. In this case, the instant matrix is supported on a pad for ease of application or tamponade.

In some embodiments of any aspect of the kit and/or compositions, the composition is sterile. Especially when handling blood products, the sterility issue is crucial, and specifically the issue of viral inactivation. In general, viral inactivation may be carried out by any number of methods, including solvent detergent, heat inactivation, irradiation, and nanofiltration. Typically, the standard for viral inactivation requires using two different methods. Additionally, FDA standard for sterility requires filtration.

The term “sterile” as used herein means having a low bioburden, effectively being germ-free, e.g., essentially or even absolutely being free from microorganisms, e.g., bacteria and viruses. Sterilization is the process of reducing the bioburden to an effectively germ-free level.

The terms "comprises" , "comprising" , "includes" , "including" , "having" , and their conjugates mean "including but not limited to" . The term “consisting of” means “including and limited to” . The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed compositions or methods or structures.

The word “exemplary” is used herein to mean “serving as an example, instance or illustration” . Any embodiment described as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments and/or to exclude the incorporation of features from other embodiments.

The word “optionally” is used herein to mean “is provided in some embodiments and not provided in other embodiments” . Any particular embodiment of the invention may include a plurality of “optional” features unless such features conflict.

Embodiments of the present disclosure which may be combined with any other embodiments described hereinthroughout unless specified otherwise.

As used herein, the singular form "a" , "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

In those instances where a convention analogous to "at least one of A, B, and C, etc. " is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a composition having at least one of A, B, and C" would include but not be limited to compositions that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc. ) . It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B. "

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

The term “about” , or "approximately" , as used herein means that values that are 10%above or below the indicated value are also intended to be included. Unless otherwise indicated, all numbers such as those expressing, for example, ratios, weight, mole/mole, amounts, temperatures, etc., are to be understood as being modified in all instances by the term "about" . Accordingly, unless indicated to the contrary, the numerical parameters set forth in this description and attached claims are approximations that may vary by up to plus or minus 10%depending upon the desired properties sought to be obtained by the present invention.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

Materials and Methods

The source of thrombin was porcine blood plasma which was fractionated.

Spray drying is a drying method used to create powder from a solution, suspension or emulsion that is atomized through a spray nozzle in a hot airflow and dried instantly. Spray drying process is controlled by several process parameters, among them are: column air flow and temperatures, nozzle size and atomizing air flow rate, material flow rate, etc.

● (Spray dryer: 4M8-TriX spray dryer was used (by ProCepT NV, Zelzate, Belgium)

● Balance: JJ1000, G&G

● Auto-Coagulation Analyzer: STA-Compact, Stago

● Formulated thrombin solution without carbohydrates: Batches manufactured by Guangzhou Bioseal Biotech Co., Ltd. Each ml of the formulated thrombin solution produced approximately 55 mg of solid powder, in which including 73%glycine (40 mg/ml in the tested solutions, typically in the range of 20-80mg/ml) ; 18%albumin (10 mg/ml in the tested solutions, typically 5-30mg/ml) and approximately 9%thrombin (2mg/ml in the tested solutions, typically in the range of 1-8mg/ml with potency of 11842 IU/g to 141667 IU/g) and other proteins (1 to 8 mg/ml in the tested solution e.g., typically about 2.5 mg/ml) (storage condition: -20℃～-30℃ until use) .

● Sucrose: Lot#102420200301, Manufactured by Hunan Erkang pharmacy Co, Ltd.

● Mannitol: Lot#K1412030, Manufactured by Aladdin Industrial Corporation.

EXAMPLE 1: EFFECT OF CARBOHYDRATES ON THROMBIN POTENCY

In exemplary procedures, three batches of formulated thrombin solution (without carbohydrates contain in the formulation) were taken out from -20℃～-30℃ refrigerator and incubated at 37℃ of water bath for around 2 hours. Each batch contained approximately 500 ml of thrombin solution.

After the thrombin solution was thawed completely, it was aliquoted (approximately 150 ml) into polypropylene bottles for 3 portions. To the first portion of thrombin solution sucrose was added to 30 g/L of final concentration. To the second portion mannitol was added to 30 g/L of final concentration. To the third portion no carbohydrate was added. Each portion of thrombin activity was then determined by auto-coagulation analyzer.

Each portion of thrombin solution was spray-dried at the same parameters, which can be seen below in Table 1.

Table 1: Spray-Dried Parameters

The spray dried powder was collected and tested for the thrombin potency. The retention of thrombin potency after spray drying can be seen below in Table 2. Thrombin activity was measured by Stago compact, an automatic blood coagulation analyzer.

Table 2: Retention of Thrombin Potency w/wo Carbohydrate

The result demonstrate that after spray drying, there is no significant different for retention of thrombin potency with or without carbohydrate contained in the formulation of thrombin solution.

EXAMPLE 2: METHODS AND SELECTED PARAMETER RANGES

In order to establish a robust process to produce thrombin powder which maintains its activity, low water content, high solid yield and desired powder particle size and distributions, the main process parameter ranges (according to Table 3) were further investigated and optimized in a set of experiments.

Table 3: Process Parameter Ranges

In exemplary procedures, thrombin powders were produced using (each one separately) a spray drying method (Spray dryer: 4M8-TriX spray dryer was used (by ProCepT NV, Zelzate, Belgium) ) as follows.

Prepared thrombin solutions were drawn into a syringe and placed inside the peristaltic pump of the spray dryer at various amounts. The syringe pump was set to the desired flow rate with feed valve closed. 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 (air) flow rate were set.

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.

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 solutions to flow through the feed inlet to the spray nozzle to be atomized by the atomizing gas flow to small droplets which were then dried in the drying column. Spray dried powder was formed, and was then collected in the powder outlet of the cyclone of the spray dryer.

The spray dried 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

(Bemis, Oshkosh, Wisconsin, USA) until evaluation.

Determination of Thrombin Activity by Clotting Time: Thrombin activity is measured by Stago compact, an automatic blood coagulation analyzer. Desired specifications for Fibrinogen Activity: between 80-100%.

Water Content Determination by Carl Fischer’s Titration: Karl Fischer Titration (KFT) is an analytical method for quantification of water content in dry powder sample. The basis of the method is the reaction between iodine and sulfur dioxide in the presence of water. Karl Fischer modified this reaction to be used for the determination of water in non-aqueous system containing an excess of sulfur dioxide. Methanol is used as a solvent, and a base (pyridine) as the buffering agent. Briefly, 100-200 mg spray dried thrombin sample was suspended in 10 ml dry methanol which was further tested for water content. Required specifications for water content: <3%.

Particle Size Distribution -Light Scattering Method: The size distribution of particles is measured using the principle of light scattering. The pattern measured is the sum of the patterns scattered by each constituent particle in the sample. The particle size distribution of spray dried thrombin powder was determined. Recommended specifications for thrombin particle size distribution is: D50 ≥ 5μm, D90≤39μm which is suitable for hemostasis effect and usability.

EXAMPLE 3: DESIGN OF EXPERIMENTS (DOE) FOR THROMBIN SPRAY DRYING

A fractural factorial screening DOE (Resolution III) with six factors and two levels were designed for a first round of experiments, were conducted (Table 2) . After a first round of thrombin spray drying experiment, the results are shown as below in Table 4.

Table 4: Thrombin First Screening DOE Results

*Solid yield is the total powder weight after spray dry divided the total solid weight in the formulation.

**Potency retention is the thrombin potency of the spray dried powder per unit weight divided by the potency of the non-spray-dried (lyophilized) powder per unit weight.

Figures 1A-D present main effects plots analysis by statistical tools.

According to analysis result, atomizing air flow is a critical factor that can affect the solid yield, water content, potency retain, and particle size of the powder as shown in the sloping lines in Figure 1A-D. Also, as the graph slopes show, the inlet air flow is also one of the main factors. The overall solid yields were low in most of the runs. It may be caused by the small quantity produced.

EXAMPLE 4: OPTIMIZATION EXPERIMENTS

In second round of experiments, the nozzle tip diameter was fixed at 0.4 mm, inlet air temperature was fixed at 160℃. The atomizing air flow ranged from 6 to 12L/min, the feed rate ranged from 200 to 480 ml/hour. There are two working modes. The first working mode is spray drying with 2 drying columns plus tangential air-cooling part. The second one is spray drying with 3 drying columns without tangential air-cooling part. The formulation with or without glycine were also a test factor in this round of experiments. Water content, thrombin activity, solid yield and particle size were measured for evaluation the best parameters (see Table 5) .

Table 5: Parameters in Second Round of Experiments

In each gram of spray dried powder, there was around 73%of glycine (if present in the formulation) , 18%of albumin and 9%of thrombin and other proteins

Water Content: for each run of spray drying, water content was measured and then summarized. Figures2A-D show the effect of different parameters on water content of the spray-dried product. Figure 2A relates to two drying columns with air cooling part ( "Mode I" ) and with glycine being contained in the formulation; Figure 2B relates to Mode I without glycine being contained in the formulation; Figure 2C relates to three drying columns without air cooling part ( "Mode II" ) with glycine being contained in the formulation. Figure 2D relates to Mode II without glycine being contained in the formulation.

The results demonstrated that it is difficult to control water content below 3%without glycine contained in the thrombin formulation. The feed rate and the atomizing air flow rate did not affect water content significantly.

Thrombin Potency: for each run of spray drying, thrombin potency of powder was measured and summarized as shown in Figures 3A-D. Figure 3A relates to Mode I with glycine being contained in the formulation; Figure 3B relates to Mode I without glycine being contained in the formulation; Figure 3C relates to Mode II with glycine being contained in the formulation. Figure 3D relates to Mode II without glycine being contained in the formulation.

The results demonstrated that increase of the feed rate from 200 to 360 ml/hr caused decrease of the thrombin potency of the powder by 10～20%.

Solid Yields: for each run of spray drying, thrombin potency of powder was measured and summarized as shown in Figures 4A-D. Figure 4A relates to Mode I with glycine being contained in the formulation; Figure 4B relates to Mode I without glycine being contained in the formulation; Figure 4C relates to Mode II with glycine being contained in the formulation. Figure 4D relates to Mode II without glycine being contained in the formulation.

The results demonstrated that over 90%of solid yield could be achieved when the spray drying thrombin formulation contained glycine, working with two drying columns plus tangential air-cooling part. Generally, the higher atomizing air flow, the lower yield was achieved. When spray drying formulated thrombin contained glycine, the powder accumulated on the wall could be blew off with higher cyclone gas flow (0.2～0.3m ³/min) after all solution is feed. It appears that the cooling air part is preferred since it can provide more fierce air flow, and the solid yield could be up to 90%.

Particle Size: for each run of spray drying, particle size of spray dried powder is measured and summarized in Figures 5A-D. Figure 5A relates to Mode I with glycine being contained in the formulation; Figure 5B relates to Mode I without glycine being contained in the formulation; Figure 5C relates to Mode II with glycine being contained in the formulation. Figure 5D relates to Mode II without glycine being contained in the formulation. The results show that the higher atomizing air flows in spray drying process, the smaller particle size the powders. The particle size distribution (D50) of spray dried powder is 5～10 μm when the atomizing flow is 6 L/min. There is no significant change in particle size when feed rate is change from 200ml/h to 480ml/h.

Confirmation Runs: After two sets of experiments, parameters are identified as the optimized condition for spray drying of thrombin formulation, which can be seen below in Table 6.

Table 6: Optimized Conditions for Spray Drying

*The atomizing air flow was adjusted to 7 L/min since when working at 6 L/min of atomizing air flow, part of small liquid can be observed splashing on upper drying column. The atomizing air flow is therefore increased to 7 L/min to achieve a stronger atomizing effect.

Formulated thrombin (from a specific batch) was spray dried for four times at this set of parameters to confirm the process can prepare qualified powder steady. The results can be seen below in Table 7.

Table 7: Dried Powder Parameters

The results demonstrate that spray drying at these parameters can prepare dried powder with water content below 3%, the thrombin potency retention is about 96%, the solid yield is around 84%and the particle size D50 is between 7 μm and 10 μm.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

A powdered composition comprising: (i) thrombin, (ii) total protein in an amount of less than 75%, by weight, (iii) albumin in an amount of more than 5%by weight and iv) a non-acidic amino acid, wherein the composition is substantially free of carbohydrates.
The composition of claim 1, being spray dried.
The composition of claim 1 or 2, wherein the non-acidic amino acid comprises glycine.
The composition of any one of claims 1 to 3, being substantially free of a neutral salt, e.g., sodium chloride.
The composition of any one of claims 1 to 4, wherein the weight ratio of the non-acidic amino acid to total protein ranges from 2: 5 to 16: 17, respectively.
The composition of any one of claims 1 to 5, wherein the weight ratio of albumin to total protein ranges from 1: 17 to 3: 5, respectively.
The composition of any one of claims 1 to 6, comprising total protein in an amount of at least 6%by weight.
The composition of any one of claims 1 to 7, wherein the albumin is in an amount of at least 15%by weight.
The composition of any one of claims 1 to 8, wherein the non-acidic amino acid is in an amount ranging from 40%to 94%by weight.
The composition of any one of claims 1 to 9, wherein the thrombin is present in an amount ranging from about 11800 to about 142000 Units per gram total protein.
A method for preparing thrombin powder, the method comprising the steps of: mixing thrombin, albumin, a non-acidic amino acid in an aqueous solution, wherein the solution is substantially free of carbohydrates; and spray drying the aqueous solution.
The method of claim 11, wherein the non-acidic amino acid comprises glycine.
The method of claim 11 or 12, wherein the solution comprises proteins at a concentration of 0.6 to 3.8 % (w/v) .
The method of any one of claims 11 to 13, wherein the solution comprises non-acidic amino acid at a concentration ranging from 2 to 8% (w/v) .
The method of any one of claims 11 to 14, wherein the solution comprises albumin at a concentration ranging from 0.5 to 3% (w/v) .
The method of any one of claims 11 to 15, wherein the solution comprises thrombin at a concentration ranging from 450 to 850 IU/ml.
The method of claim any one of claims 11 to 16, wherein the solution is substantially free of a neutral salt.
The method of any one of claims 11 to 17, wherein the aqueous solution comprises:

from about 450 to about 850 IU/ml thrombin;

from about 0.5 to about 3%w/v albumin;

from about 2 to about 8%w/v non-acidic amino acid (e.g., glycine) ; and

from about 0.6 to about 3.8%w/v total protein.
The method of any one of claims 11 to 18, being substantially free of a neutral salt, e.g., sodium chloride.
A spray-dried thrombin powder obtainable by the method of any one of claim 11 to 19.
The spray-dried thrombin powder of claim 19, characterized in that it exhibits at least 80%of the thrombin potency or activity of the aqueous solution.
A matrix comprising the thrombin powder of any one of claims 1 to 10 and/or the spray-dried thrombin powder of claim 20 or 21.
The matrix of claim 22, further comprising a fibrinogen powder.
An aqueous composition suitable for use in preparing thrombin powder, the aqueous composition comprising:

from about 450 to about 850 IU/ml thrombin;

from about 0.5 to about 3%w/v albumin;

from about 2 to about 8%w/v non-acidic acid (e.g., glycine) ; and

from about 0.6 to about 3.8%w/v total protein;

and being substantially free of carbohydrates.
The aqueous composition of claim 24, being substantially free of a neutral salt, e.g., sodium chloride.