AU2021348528A1 - Hemostatic formulations and uses thereof - Google Patents

Abstract

Disclosed herein are formulations comprised of concentrated Factor V, vitamin Independent clotting protein and a stabilizer, wherein the stabilizer is at a concentration at which the Factor V or the vitamin K-dependent clotting protein are in the zymogen form. Also disclosed herein are processes for the preparation of the formulations.

Description

HEMOSTATIC FORMULATIONS AND USES THEREOF

TECHNOLOGICAL FIELD

The present disclosure inter alia relates to hemostatic formulations and uses thereof.

BACKGROUND ART

References considered to be relevant as background to the presently disclosed subject matter are listed below:

- US Patent No. 3,560,475

- US Patent No. 9,814,765

- US Patent No. 7,371,722

US Patent Application No. 2018/0036338

- US Patent No. 5,866,122

US Patent Application No.2018/0369346

- US Patent No. RE38431

US Patent Application No.2014/0155498

US Patent Application No.2009/0011043

Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.

BACKGROUND

US Patent No. 9,814,765 describes a single component sealant formulation (e.g. in a liquid form) that includes fibrinogen; vitamin K-dependent clotting zymogens comprising at least Factor II (FII) and Factor X (FX), methods for its preparation, and use.

US Patent No. 7,371 ,722 relates to a pharmaceutically active substance containing (A) prothrombin (coagulation factor II) obtained from plasma or by genetic engineering, (B) coagulation factors V, VIII, IX, X obtained from plasma or by genetic engineering that at least partially may be present in their activated state, and coagulation factor Xia obtained from plasma or by genetic engineering, and (C) prion-safe, coagulationpromoting phospholipids, the active substance is for producing a drug that is capable of generating thrombin or that contains thrombin and compositions comprising thereof.

US Patent Application No. 2018/0036338 relates to hemostatic compositions comprising polysaccharide-based polymers, such as amylopectins, amylose, dextrins, maltodextrins and icodextrin and modified forms thereof, cross-linked with a crosslinking agent that are useful as a hemostatic composition.

US Patent No. 5,866,122 describes a pharmaceutical preparation for treating blood coagulation disorders which comprises purified prothrombinase factors, in particular purified prothrombin and optionally purified factor Xa as active component.

US Patent No. 3,560,475 describes a prothrombin complex prepared from a plasma fraction containing Factors II, VII, IX and X by adsorption with tribasic calcium phosphate, elution with trisodium citrate, and multiple precipitations with polyethylene glycol.

US Patent Application No. 2018/0369346 provides a composition, comprising: proaccelerin (factor V) and at least one factor selected from the group consisting of: prothrombin (factor II), proconvertin (factor VII), and Stuart-Prower factor (factor X).

US Patent No. RE38431 relates to stable liquid formulations of Factor VIII and Factor IX that can be administered by injection or infusion to provide a constant level of the coagulation factor in the blood.

US Patent Application No. 2014/0155498 describes diagnostic or therapeutic formulation of stable liquid coagulation factors comprises at least one coagulation factor, at least one stabilizer, and optional additives.

US Patent Application No. 2009/0011043 describes tissue sealants that include whole blood and an effective amount of an exogenous protein cross-linker to cross-link the whole blood. GENERAL DESCRIPTION

The present disclosure is based on the development of a formulation that comprises coagulation factors in a zymogen form (i.e. inactive form) and is stable when stored in liquid form. Specifically, and in accordance with a first of its aspects, the present disclosure provides a formulation comprising concentrated Factor V, at least one vitamin K-dependent clotting protein and at least one stabilizer, wherein the at least one stabilizer is at a concentration at which the Factor V and/or the at least one vitamin K-dependent clotting protein are in a zymogen form.

The present disclosure also provides in accordance with a further aspect, a formulation comprising concentrated Factor V, at least one vitamin K-dependent clotting protein and free calcium ions at a concentration of at most about 5mM, for example less than 5mM.

The present disclosure also provides in accordance with a further aspect, a formulation comprising concentrated Factor V, Factor II, Factor VII, Factor IX and Factor X and free calcium ions at a concentration of less than about 5mM, for example in the range of O.lmM and less than 5mM.

The present disclosure also provides, in accordance with a further aspect, a formulation comprising concentrated Factor V and one or more co-purified vitamin K- dependent clotting protein selected from Factor II, Factor VII, Factor IX and Factor X and free calcium ions, the free calcium ions being at a concentration of less than about 5mM, for example in the range of O.lmM and less than 5mM. This formulation does not include exogenously added vitamin K-dependent clotting proteins selected from Factor II, Factor VII, Factor IX and Factor X.

The present disclosure also provides, in accordance with a further aspect, a formulation comprising concentrated Factor V, Factor II, Factor VII, Factor IX and Factor X and free calcium ions, the free calcium ions being at a concentration of less than about 5mM, for example in the range of O.lmM and less than 5mM. This formulation does not include exogenously added vitamin K-dependent clotting proteins selected from Factor II, Factor VII, Factor IX and Factor X. In one embodiment the ratio between Factor V, Factor II, Factor VII, Factor IX and Factor X is:

Factor V not less than 0.5: Factor II about 1: Factor VII about l:Factor IX about 1 and: Factor X about 1;

Not less than 0.5 may be e.g. 0.5, 0.6, 0.7, 0.8, 0.9 or e.g. in the range of 0.7-0.8 or 0.5 to 1 and including any value therebetween.

In one embodiment the ratio between Factor V, and at least one of Factor II, Factor VII, Factor IX and Factor X is:

Factor V to at least one member from Factor II, Factor VII , Factor IX, Factor X being in a respective ratio of Factor V being not less than 0.5 to the at least one member being about 1.

Essentially, the factors are non-activated.

The composition may further comprise antithrombin III, heparin, protein C, and/or Protein S.

In accordance with another aspect, the present disclosure provides a method for the treatment, inhibition, arresting or delaying bleeding or any disorder associated thereto in a subject in need thereof. The method of the invention comprises the step of administering to the subject an effective amount of a formulation as disclosed.

In accordance with yet another aspect, the present disclosure provides a formulation comprising concentrated Factor V, at least one vitamin K-dependent clotting protein and at least one stabilizer for use in the treatment, inhibition, arresting or delaying bleeding or any disorder associated thereto in a subject in need thereof, the at least one stabilizer is present at a concentration at which the Factor V and/or the at least one vitamin K-dependent clotting protein are in zymogen form.

The present disclosure also relates in accordance with some further aspect, to a kit comprising a formulation as disclosed herein, and optionally instructions for use of the formulation to treatment, inhibition, arresting or delaying bleeding or any disorder associated thereto.

Also, provided herein, in accordance with another aspect of the present disclosure, is a process for the preparation of a formulation comprising concentrated Factor V and at least one vitamin K-dependent clotting protein, the process comprising (i) subjecting blood, plasma or any product/fraction thereof to at least one separation step and (ii) recovering the Factor V and at least one vitamin K-dependent clotting protein obtained in step (i). In some embodiments, the at least one separation step comprises at least one chromatography step. In some other embodiments, the recovery comprises eluting a fraction bound to the at least one anion exchange chromatography with an elution solution into a collection solution.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. 1 is a bar graph showing the total level of procoagulants and inhibitors in the elution fraction in Units per ml of load at pH 8.0 by different AEX resins in batch mode, from left to right, the factors aligned FII, FV, FVII, FIX, FX, Protein C, Protein S and Antithrombin III (if present).

Fig. 2 is a bar graph showing the percentage recoveries of FII, FV and FIX (from bottom to upper fragment in each bar) in 20cm height AEX columns, all columns with a diameter of 1.0 cm, except for C61R2, with 2.6 cm.

Fig. 3 is a graph showing the levels of procoagulants and inhibitors determined in the elution fraction as percentage of recovery (Elution/Load) of the different runs in the Design of Experiment (DOE) using a prepacked Eshmuno Q column of 0.8cm width by 10cm (Merck), from left to right, the factors aligned FII, FV, FVII, FIX, FX, Protein S, Protein C and Antithrombin III (if present).

Figs. 4A and 4B are bar graphs showing the amount of FII (Fig. 4A) and FV (Fig. 4B) observed after 24 hours incubation at different temperatures (from left to right: -65 °C, shown only at pH 6.7; Room Temperature; and 2-8 °C) at pH 6.7, 7.2 and 7.7.

Fig. 5 is a bar graph showing the amount (lU/mL) of FII and FV in the load and in the elution fraction fresh, after 24h at <-65²C subjected to more than one freeze-thaw cycles and fresh fraction after 24h incubation at different temperatures (see Example 4B). Fig. 6 is a bar graph showing the amount of FII and FV in the elution fraction after 24h at <-65°C subjected to more than one freeze-thaw cycles using different stabilizers, control samples do not include stabilizers.

Figs. 7A and 7B are bar graphs showing the stability of FII (Fig. 7A) and FV (Fig. 7B) in the elution fraction of an AEX column (Eshmuno Q) after dilution *4 with PuW, 20mM Citrate or 50mM Glycine buffers at pH 7.0-7.3, containing whether 1 or 5 mM CaCh. Untreated and diluted samples in different solutions were tested for FII and FV at Time 0 and after 24h incubation (from left to right in each quartet) at <-65°C, 2-8°C and room temperature (RT). A-untreated pH 8.0, B- untreated pH 7.2, C- Elution diluted 1/4 in Purified water (PuW), D- Elution diluted 1/4 in PuW+ ImM Ca²⁺, E- Elution diluted 1/4 in Citrate buffer + ImM Ca²⁺, F- Elution diluted 1/4 in Citrate buffer + 5mM Ca²⁺, G- Elution diluted 1/4 in Glycine buffer + ImM Ca²⁺.

Fig. 8 is a graph showing the levels of FII and FV of the load and elution fraction of two different runs from an AEX column (Eshmuno Q), where A is the elution untreated, B is the elution diluted *4 with a buffer at pH 7.2 for a final concentration of ImM CaCh and llU/ml Heparin, and C is the elution diluted *4 with a buffer at pH 7.2 for a final concentration of ImM CaCh, llU/ml Heparin and 50 mM glycine.

Figs. 9A to 9C show the effect of calcium ion on recovery, Figs. 9A and 9B are graphs showing the levels of FV (Fig. 9 A) and FIX (Fig. 9B) in the elution fraction from an AEX column (Eshmuno Q) after 24h incubation at <-65°C, 2-8°C and RT (from left to right), untreated and diluted *4 with 50mM glycine solution at pH 7.2 and 0. l-5mM CaCh, TO denotes Time 0; Fig. 9C is a graph showing the recovery percent of FV in the elution fraction from an AEX column (Eshmuno Q) diluted *4 with 50mM glycine solution at pH 7.2 and 0.1-5mM CaCh after 24h incubation at 2-8°C..

Fig. 10 is a representation of the method used to lyophilize the elution fraction of an AEX (Eshmuno Q) concentrated 17 times from starting CDP material, showing the shelf temperature (solid line) and pressure (dashed line).

DETAILED DESCRIPTION

Blood clotting is a tightly controlled cascade, including a series of reactions in which among others, coagulation factors in zymogen form (inactive enzyme precursor) are activated, ultimately resulting in formation of a blood clot. Hence, formulations that comprise coagulation factors and specifically coagulation factors in zymogen form, may play an important role in initiating and mediating blood clotting processes. However, such formulations are not easily obtained nor maintained as stable formulations, due to, inter alia, degradation and activation of the zymogens.

The present disclosure is based on the development of purified, highly stabilized and ready to use formulations that comprise coagulation factors in an inactive, zymogen form. Specifically, the inventors have surprisingly found that the presence of a stabilizer, such as a divalent cation, in a specific concentration range, maintains the coagulation factors and specially factor V and vitamin K-dependent clotting proteins in zymogen form. The inventors further found that the majority of these coagulation factors, some of which are known to be labile factors, may be maintained as zymogens in the formulation for at least 24 hours at about room temperature, specifically at a temperature of between about 18°C to about 26°C.

Hence, it was suggested by the inventors that the presence of the stabilizer at specific concentrations, does not promote the activation of the clotting cascade. After the formulation is administrated, for example by intravenous administration, the clotting cascade would be initiated, for example in case of trauma.

Thus, the present disclosure provides in accordance with some aspects, a formulation comprising concentrated Factor V, at least one vitamin K-dependent clotting protein and at least one stabilizer, wherein the at least one stabilizer is at a concentration at which the majority of Factor V and/or the at least one vitamin K-dependent clotting protein is present in the formulation in zymogen form.

In the following text, when referring to the formulations it is to be understood as also referring to the methods, kits, and processes disclosed herein. Thus, whenever providing a feature with reference to the formulations, it is to be understood as defining the same feature with respect to the methods, kits, and processes, mutatis mutandis.

The formulation in accordance with some embodiments of the present disclosure is to be understood as a hemostatic, blood clot-forming formulation, having ingredients that upon contact with blood at certain conditions (e.g. presence of specific factors at the bleeding site) react to subsequently form active components and thereby stop/arrest bleeding. In the context of the present disclosure it is to be understood that formulation ingredients do not interact with each other and are essentially inactive, i.e. present in a zymogen form, until they are brought into contact with blood at certain conditions, at which time clotting initiates and a blood clot is formed.

The formulation can be in any form. In accordance with some embodiments, the formulation is in a liquid form. When referring to a liquid form it is to be understood as one being liquid at room temperature.

In accordance with some embodiments, the formulation is solid e.g. in powder form or in lyophilized form. When in powder or lyophilized form, it is mixed with a suitable aqueous formulation such that upon use, the formulation is in liquid form.

In one embodiment, the solid composition is stable. Herein "stable" means e.g. when one or more of the 5 factors keeps equal or more of about 70% of its activity when stored for one month at room temperature.

Reconstituted solid composition with an aqueous liquid is stable e.g. when one or more of the 5 factors keeps equal or more of about 70% of its activity when stored for about 12 hours at room temperature.

In accordance with some embodiments, the formulation is in a frozen form. When in frozen form, the formulation is thawed upon use to be, at room temperature, in its liquid form.

When in liquid form, the liquid carrier is typically a buffer to maintain the pH of the formulation at a pH value of between about 6.5 and about 8.5. In some embodiments, the pH is between about 6.5 and about 8.0, at times between about 6.5 and about 7.5, at times between about 7.0 and about 8.0, at times between about 7.0 and about 7.3, at times about 7.2.

As described herein, the formulation comprises coagulation factors in a zymogen form. A zymogen as used herein, is to be understood as the inactive precursor form of the factor (enzyme), that required a biochemical change (such as a hydrolysis reaction revealing the active site or changing the confirmation to reveal the active site) in order to become an active factor (enzyme). As the coagulation factors are maintained/present in its majority in the formulation as zymogens, the formulation is not active, i.e. activation of components in the formulation is minimal or absent.

The presence of the clotting factors in a zymogen form can be determined by testing the activity of the formulation by using any known method in the field at different conditions (e.g. different time points and different temperatures). Such methods include, for example, chromogenic kits such as HYPHEN™ or clotting kits such as STAGO™ . Chromogenic assays make use of a change in a color of a substrate when cleaved by the factor which is an enzyme and clotting assay uses the change in viscosity produced by the clotting of fibrinogen. For example, in clotting assays, the activity can be tested using activated partial thromboplastin clotting time (aPTT). The aPTT is an example of a blood coagulation test that measures how long it will take to the blood to form a clot. aPTT typically uses deficient plasma of a specific factors (i.e. does not contain a specific factor) and normal plasma that contain all factors close to 1 lU/ml as the reference. Lack of activation of the formulation described herein can be determined, for example, by using the non-activated partial thromboplastin time (NaPTT) method, using phospholipids and no activator (kaolin).

In some embodiments, the presence of zymogens (i.e. lack of activity) of Factor V and/or zymogens of at least one vitamin K-dependent clotting protein in the formulation is determined at a temperature of about 37°C.

In some embodiments, the majority of Factor V and/or the at least one vitamin Independent clotting protein is present in a zymogen form in the formulation for at least 1 hour, at times at least 2 hours, at times at least 3 hours, at times at least 4 hours, at a temperature of at most about 30°C.

In some embodiments, the majority of Factor V and/or the at least one vitamin Independent clotting protein is present in a zymogen form in the formulation for at least 1 hour, at times at least 2 hours, at times at least 3 hours, at times at least 4 hours, at a temperature of at least one of 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C,

11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C,

25°C, 26°C, 27°C, 28°C, 29°C or 30°C.

In some embodiments, the majority of Factor V and/or the at least one vitamin independent clotting protein is present in a zymogen form in the formulation at a temperature of at most about 30°C, at times at most 26°C, for at least 4 hours.

In some embodiments, the majority of Factor V and/or the at least one vitamin independent clotting protein is present in a zymogen form in the formulation at room temperature for at least 4 hours. When referring to a factor being in a zymogen form it should be understood as if the specific factor is at least partially in an inactive form, such that the amount/fraction of the active form (if present) is not sufficient to initiate the activation of the formulation to thereby form a blood clot. Typically, but not exclusively, when referring to a factor in the zymogen form, includes a factor having 70% and above in inactive form. As described herein, such activation would be initiated upon contact with injured or exposed tissue where the negatively charged phospholipids and collagen is abundant. Such a condition would activate the tissue factor(s) and initiate a full coagulation cascade. For example, upon administration into the blood circulation this treatment can stop exsanguination which is the loss of blood to a degree sufficient to cause death.

For example, the formulation comprises at least 20% of Factor V (i.e. as a zymogen) and 80% in an activated form (i.e. as Factor Va), at times at least 50% as Factor V and 50% as Factor Va, at times at least 60% as Factor V and 40% as Factor Va, at times at least 70% as Factor V and 30% as Factor Va, at times at least 80% as Factor V and 20% as Factor Va, at times at least 90% as Factor V and 10% as Factor Va, at times at least 98%, or at least 99% as Factor V is a zymogen.

In some embodiments, at least 50%, at times at least 60%, at times at least 70%, at times at least 80%, at times at least 90%, at times at least 98%, or at least 99% of Factor V is a zymogen in the formulation when measured at room temperature, at times at a temperature below 23°C, for at least 4 hours, at times for at least 8 hours, at times for at least 12 hours, at times for at least 18 hours or for at least 24 hours.

In some embodiments, between about 50% and about 100%, at times between about 80% and about 100%, at times between about 98% and about 100%, at times between about 80% and about 90% of Factor V is in the zymogen form in the formulation when measured at room temperature for at least 4 hours, at times for at least 8 hours, at times for at least 12 hours, at times for at least 18 hours, or for at least 24 hours.

In some embodiments, at least 50% of the factor V is in zymogen form in the formulation when measured at room temperature for at least 24 hours.

When referring to room temperature it should be noted as referring to a temperature of at most 30°C, at times at most 26°C, at times at most 25°C, at times at most 24°C, at times between 20°C-26°C, at times between 23°C-26°C, at times between 17°C-26°C, at times between 20°C-23.5°C. As described here, the formulation comprises Factor V ("FV") also known as proaccelerin which is a protein of approximately 330kDa produced in the liver. Factor V circulates in plasma as a single-chain glycoprotein in a zymogen form having essentially no activity in the blood clotting cascade and needs to be first cleaved and activated to express procoagulant activity. The activated Factor V is denoted in the art as Factor Va.

In addition to Factor V, the formulation comprises at least one vitamin K-dependent coagulation protein. Vitamin K is known to participant in synthesis of several proteins that mediate both coagulation and anticoagulation. Vitamin K-dependent coagulation protein as used herein refers to a protein, typically synthesized in the liver that is being involved in the coagulation process.

In some embodiments, the vitamin K-dependent coagulation protein is a procoagulation protein.

In some embodiments, the vitamin K-dependent coagulation protein is an anticoagulation protein.

In some embodiments, the at least one vitamin K-dependent coagulation protein is pro-coagulation protein comprising at least one of Factor II, Factor VII, Factor IX or Factor X.

Factor II (FII) also denoted as prothrombin (EC 3.4.21.5) refers to a protein of approximately 72 kDa, that on activation, is proteolytically cleaved to form thrombin, and in turn acts as a serine protease that converts fibrinogen to fibrin. The activated form of Factor II is denoted Factor Ila.

Factor VII (FVII) also denoted as proconvertin (EC 3.4.21.21) is activated to FVIIa by different proteases. The activated form of Factor VII is denoted Factor Vila.

Factor IX (FIX) also denoted a Christmas factor (EC 3.4.21.22) is a blood clotting factor, a zymogen of serine protease that upon activation, is converted into the active serine protease and, in the presence of Ca²⁺ and membrane phospholipids, it forms the activated factor IX (IXa). Hence, the activated form of Factor IX is denoted Factor IXa.

Factor X also denoted as Stuart-Prower factor, (EC 3.4.21.6) is a serine endopeptidase and requires vitamin K for its synthesis in the liver. Factor X is activated, by hydrolysis, into factor Xa. In some embodiments, the pro-coagulation protein comprises Factor II, Factor VII, Factor IX and Factor X.

In some embodiments, the formulation comprises zymogens of Factor II, Factor VII, Factor IX and Factor X.

In some embodiments, the formulation comprises zymogens of Factor V, Factor II, Factor VII, Factor IX and Factor X.

The amounts of the various zymogens in the formulation may vary. As detailed herein, the formulation comprises concentrated zymogens. As used herein the term “concentrated” denotes that the amount of at least one of the zymogens in the formulation is at least 1 unit/ml of zymogen, at times at least 2 units/ml of zymogen. Typically, but not exclusively, concentrated refers to the concentration of one or more of Factor V, Factor II, Factor VII, Factor IX and Factor X being equal to or more than about 5 times its corresponding concentration in plasma.

In some examples, the formulation comprises at least 1 lU/ml of at least one of Factor V, Factor II, Factor VII, Factor IX and Factor X. In some examples, the formulation comprises at least 1 lU/ml of Factor IX.

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, Factor V is present in the formulation at a concentration that is at least 20%, at least 30% of the concentration of the at least one vitamin K-dependent coagulation protein.

In some other embodiments, Factor V is present in the formulation at a concentration that is at least 20%, at least 30% of the concentration of at least one of Factor II, Factor VII, Factor IX or Factor X. In some embodiments, Factor V is present in the formulation at a concentration that is at least 20%, or at least 30% of the concentration of Factor II.

In some embodiments, Factor V is present in the formulation at a concentration that is at least 20%, or at least 30% of the concentration of Factor VII.

In some embodiments, Factor V is present in the formulation at a concentration that is at least 20%, or at least 30% of the concentration of Factor IX.

In some embodiments, Factor V is present in the formulation at a concentration that is at least 20%, or at least 30% of the concentration of Factor X.

In some embodiments, the formulation comprises at least one stabilizer. As detailed herein, the at least one stabilizer is present in the formulation at a concentration such that the formulation components are stable (i.e. minimally or not degraded) and/or inactive (i.e. in a zymogen form). In other words, the presence of the stabilizer prevents or reduces the degradation of the coagulation factors.

As appreciated, coagulation factors have a short half-life in plasma, for example 5h (FVII) or lOOh (FII), or 12-36h (FV). However, it was surprisingly found that in the presence of a stabilizer and specifically a stabilizer at concentrations as described herein, the factors in the formulation are stable. The stability of the formulation is determined as % activity of Factor V using clotting and chromogenic as described herein during the storage, particularly when the formulation is in liquid form, after storage at a temperature of below room temperature for at least 24 hours. In some examples, the stability is measured at a temperature of between about 2°C and 8°C and/or at about room temperature after at least 24 hours storage. Typically, but not exclusively, the term stable refers to one or more of the 5 factors keeping equal or more of about 70% of its activity.

In addition, and in accordance with the present disclosure, when referring to a stable formulation it is also to be understood as one that, upon use, has clotting time that is not affected or is minimally affected by its storage conditions. In other words, upon use, the formulation will clot at essentially the same time period irrespective of whether it was stored at room temperature or at lower temperatures, e.g. between 1°C to room temperature, or 2°C-8°C.

In some embodiments, the stabilizer is present at a concentration below 5mM. In some other embodiments, the stabilizer is present at a concentration of between about 0. ImM and about 5mM. In some embodiments, the stabilizer is present at a concentration of between about O.lmM and about 3mM. In some embodiments, the stabilizer is present at a concentration of between about O.lmM and about 2mM, at times between about 0. ImM and about ImM, at times between about 0.5mM and about 3mM, at times between about 0.5mM and 2mM. In some embodiments, the stabilizer is present at a concentration of about ImM.

In some embodiments, the stabilizer comprises at least one divalent cation. Nonlimiting examples of divalent cation include magnesium, calcium.

In some embodiments, the at least one stabilizer is calcium ions. In some embodiments, the calcium ions are free calcium ions. As appreciated, the term free calcium ions refer to ionized and non-chelated calcium ions. It should be noted that the presence of a chelating agent may reduce the concentration of free calcium ions. The term free also applies to other divalent cations that are encompassed by the present disclosures as being non-chelated cations and hence free cations. The fine balance between the chelator concentration and the calcium and the ratio of chelator to calcium should be one that allows for a free calcium at concentrations in the range of above O.lmM and up to below 5mM.

As shown in Fig. 9C, in the absence of a stabilizer and specifically in the absence of free calcium ions, only 17% of Factor V is maintained as a stable form, i.e. not degraded, while by increasing the amount of the stabilizer, and specifically increasing the amount of free calcium ions, an increase is observed in the presence of Factor V. With the addition of 2mM stabilizer 80% of the Factor V is maintained. Without being bound by theory, it was suggested by the inventors that the specific concentration of the stabilizer, i.e. free calcium ions, successfully maintains both the stability and the lack of activity of the components.

In some embodiments, the formulation comprises free calcium ions at a concentration of at most about 5mM.

In some embodiments, the formulation comprises free calcium ions at a concentration of between about O.lmM and about 5mM, at times between about O.lmM and less than 5mM, at times between about O.lmM and about 3mM, at times between about O.lmM and about 2mM, at times between about O.lmM and about ImM, at times between about 0.5mM and about 3mM, at times between about 0.5mM and 2mM. In some embodiments, the formulation comprises free calcium ions at a concentration of about O.lmM, at times of about 0.3mM, at times of about 0.5mM, at times of about 0.8mM, at times of about ImM, at times of about 1.3mM, at times of about 1.5mM, at times of about 1.8mM, at times of about 2mM, at times of about 2.5mM, at times of about 3mM, at times of about 3.5mM, about 4mM, or about 4.5mM.

The formulation may also comprise an anti-coagulant protein. In some embodiments, the anticoagulant protein is in a zymogen form, i.e. an inactive anticoagulant protein.

In some embodiments, the anti-coagulant protein is a vitamin K-dependent anticoagulation protein.

In some embodiments, the anti-coagulant protein is a nonvitamin K-dependent anticoagulation protein.

In some embodiments, the anti-coagulant protein is at least one of protein C, protein S or protein Z.

In some embodiments, the anti-coagulant protein is at least one of protein C or protein S.

In some embodiments, the anti-coagulant protein is anti-thrombin III (AT III).

In some embodiments, the anti-coagulant protein is at least one of protein C, protein S or AT III.

The formulation may comprise additional components.

In some embodiments the formulation comprises at least one excipient. In some embodiments, the at least one excipient is at least one sugar.

In some embodiments, the at least one excipient is at least one of mannitol, maltose, sucrose, glycerol, citrate or glycine. In some examples, the at least one excipient is glycine. It was suggested that glycine may be added to the formulation as it is suitable as a non-chelating (unlike e.g. citrate which has chelating activity) buffer hence, keeping calcium ions in a free non-chelated form, i.e. free calcium ions.

The coagulation factors as described herein including for example, Factor V, at least one vitamin K-dependent coagulation protein and the anti-coagulant protein, can be isolated from blood or plasma of human beings or mammals or can be recombinant factor(s).

In some examples, the coagulation factors as described herein are derived from blood, plasma or any product/fraction thereof. Blood, plasma or any product/fraction thereof as used herein refers to at least one of whole blood, fresh frozen plasma (FFP), platelet rich plasma (PRP), cryo-depleted plasma (CDP), cryo-precipitated plasma, recovered plasma, or source plasma obtained from plasma apheresis.

"Fresh frozen plasma" as used herein refers to the fluid portion of one unit of human blood that has been centrifuged, separated, and frozen solid. FFP contains all components (factors/proteins) of the coagulation.

"Platelet rich plasma" is a concentrate of platelet-rich plasma protein derived from whole blood and centrifuged to remove red blood cells.

“Cryo-depleted plasma” as used herein relates to a component made from FFP, that remains once cryoprecipitate has been removed from FFP. CDP includes most of the clotting factors.

"Cryoprecipitate" as used herein, relates to precipitated proteins of plasma obtained from a single unit of fresh plasma and rapid thawing. Cryoprecipitate is rich in Factor VIII, factor XIII, von Willebrand factor and fibrinogen.

"Recovered plasma" as used herein, relates a fraction collected through whole blood donation and being separated from its cellular components.

“Apheresis” as used herein refers to blood of a person that passes through an apparatus that separates out one particular constituent and returns the remainder to the circulation.

In some embodiments, the coagulation factors are derived from CDP.

In some examples, Factor V is derived from CDP.

In some examples, the at least one vitamin K-dependent coagulation protein is derived from CDP. In some examples, Factor II, Factor VII, Factor IX and Factor X are derived from CDP.

In some examples, the at least one anti-coagulant protein is derived from CDP. In some examples, at least one of protein C, protein S or AT III is derived from CDP. The formulation described herein is suitable for systemic administration. "Systemic administration” as used herein mean that the administration of a formulation directly intravenously into the central blood system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes. The phrases "parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, intravenous injection or intraarterial.

In some embodiments, the formulation is suitable for intravenous injection. In some embodiments, the formulation is suitable for intravenous infusion.

As described herein, the formulation may be used for the treatment of a variety of disorders associated with bleeding such as various coagulation abnormalities as well as spontaneous bleeding episodes, trauma and in surgical procedures.

Thus, the present disclosure provides in accordance with some other aspects, a method for the treatment, inhibition, arresting or delaying bleeding or any disorder associated thereto.

The methods of the invention comprise a step of administering to a subject in need thereof an effective amount of the formulation described herein. In some specific embodiments, the methods of the invention employ administering to a bleeding subject a therapeutically effective amount of a formulation of the invention.

In some embodiments, the methods comprise administering to a subject in need thereof an effective amount of the formulation comprising Factor V, at least one vitamin K-dependent clotting protein and at least one stabilizer, wherein the at least one stabilizer is present in the formulation at a concentration at which the Factor V and the at least one vitamin K-dependent clotting protein are zymogenes.

In accordance with some embodiments, the methods of the invention comprise administering to a subject in need thereof an effective amount of the formulation comprising Factor V, at least one vitamin K-dependent clotting protein and at least one stabilizer at a concertation of below about 5mM, at times between about 0. ImM and about 5mM, at times about 2mM.

In accordance with some embodiments, the methods of the invention comprise administering to a subject in need thereof an effective amount of the formulation comprising Factor V, at least one vitamin K-dependent clotting protein and free calcium ions at a concertation of below about 5mM, at times between about O.lmM and about 5mM, at times about 2mM.

In accordance with some embodiments, the methods of the invention comprise administering to a subject in need thereof an effective amount of the formulation comprising Factor V, at least one of Factor II, Factor VII, Factor IX and Factor X and free calcium ions at a concertation of below about 5mM, at times between about O.lmM and about 5mM, at times about 2mM.

In accordance with some embodiments, the methods of the invention comprise administering to a subject in need thereof an effective amount of the formulation comprising Factor V, at least one vitamin K-dependent clotting protein, a stabilizer and at least one anti-coagulation protein as described herein.

In accordance with some embodiments, the methods of the invention comprise administering to a subject in need thereof an effective amount of the formulation comprising Factor V, at least one of vitamin K-dependent clotting protein, a stabilizer and at least one anti-coagulation protein as described herein. The anti-coagulation protein in some examples is at least one of protein C, protein S or AT III.

In some embodiments, the methods of the invention comprise administration of the formulation of the invention that is derived from at least one of whole blood, fresh frozen plasma (FFP), platelet rich plasma (PRP), cryo-depleted plasma (CDP), cryo-precipitated plasma, recovered plasma, or source plasma obtained from plasma apheresis.

In some embodiments the methods of the invention comprise administration of the formulation that is derived from CDP.

It should be appreciated that the formulation of the invention comprises zymogens and hence the formulation is not active prior to its administration. The formulation is activated after being administered into the subject by the methods described herein. Specifically, the formulation of the invention is to be administered, as detailed herein, to a subject suffering from bleeding or any disorder associated thereto, such that upon administration as detailed herein, the zymogens may be eventually activated by upstream clotting factors in the normal flow of the clotting cascade. As used herein the term bleeding or bleeding disorder refers to at least one defect in hemostasis resulting in an increased susceptibility to bleeding (also known as hemorrhagic diathesis). The bleeding in accordance with some embodiment is associated with hemostatic disorders.

In some embodiments, the method of the invention may be applicable for reversing exsanguination. Exsanguination refers to loss of blood to a degree sufficient to cause death.

In some embodiments, the method of the invention may be applicable for the treatment, prevention, arresting, prophylaxis, amelioration, inhibition of bleeding, hemostatic disorders and any bleeding or disorder associated therewith in a subject in need thereof, the method comprising the step of administering to said subject a therapeutically effective amount of the formulation of the invention.

Bleeding disorders can be caused by various physiological processes. For example, bleeding disorders may be caused by platelet disorders (primary hemostasis defect), coagulation defects (secondary hemostasis defect), or a combination thereof. Platelet disorders are typically associated with mucocutaneous bleeding (e.g., epistaxis, petechiae, gastrointestinal bleeding) whereas coagulation defects are typically associated with bleeding into potential spaces (e.g., hemarthrosis, muscular bleeding).

In some embodiments, the methods of the invention may be applicable for subjects suffering from a bleeding disorder that may be an inherited or acquired bleeding disorder.

In some embodiments, the methods of the invention may be applicable for subjects suffering from acquired bleeding disorders.

Acquired bleeding disorders are caused by an external stimulus. In some embodiments, the acquired bleeding disorder may be at least one of injury-induced bleeding, surgery -induced bleeding or trauma-induced bleeding. In some embodiments, the method of the invention may be applicable for treating, inhibiting, arresting or delaying bleeding associated with surgical procedures.

In a further embodiment the method of the invention is suitable for treating trauma- induced bleeding. In some embodiments, the methods of the invention may be applicable for subjects suffering from a trauma, for example from trauma induced coagulopathy. In some embodiments, the methods of the invention may be applicable for subjects suffering from a bleeding disorder that may be an inherent bleeding disorder. "Inherent bleeding disorder" as used herein relates to an inherent deficiency in at least one coagulation factor.

In some embodiments, the methods of the invention may be applicable for subjects suffering from at least one of Factor I deficiency, Factor II deficiency, Factor V deficiency, Factor VII deficiency, Factor VIII deficiency (also known as hemophilia A), Factor IX deficiency (also known as hemophilia B), Factor X deficiency, Factor XI deficiency, Factor XII deficiency or Von Willebrand disease (VWD).

In yet another embodiment, the invention may be particularly applicable for treating, inhibiting, arresting or delaying bleeding associated with factor V deficiency.

In some specific embodiments, the method of the invention may be applicable for treating, inhibiting, arresting or delaying bleeding associated with factor XI deficiency.

In other embodiments, the method of the invention may be applicable for treating, inhibiting, arresting or delaying bleeding associated with factor VII deficiency.

In yet further embodiments, the method of the invention may be applicable for treating, inhibiting, arresting or delaying bleeding associated with factor X deficiency.

In certain embodiments, the methods of the invention may be applicable for treating, inhibiting, arresting or delaying bleeding associated with factor II deficiency.

As used herein, “disease”, “disorder”, “condition” and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all of such terms. It is understood that the interchangeably used terms "associated" and "related", when referring to pathologies herein, mean diseases, disorders, conditions, or any pathologies which at least one of: share causalities, co-exist at a higher than coincidental frequency, or where at least one disease, disorder, condition or pathology causes a second disease, disorder, condition or pathology.

As noted above, the invention provides methods for treating disorders specified above. The term “treatment” as used herein refers to the administering of a therapeutic amount of the formulation of the present invention which is effective to improve or avoid one or more undesired symptoms associated with a disease or condition as described herein. As indicated above, the method of the invention involves the administration of a therapeutically effective amount of the combinations of the invention. The term “effective amount” or “therapeutically effective” for purposes disclosed herein indicates that the amount of formulation is effective to treat, inhibit or delay one or more symptoms of a disease as described herein.

As used herein, the term “subject” refers to a living organism that is treated with the formulation as described herein, including, but not limited to, any mammal, such as a human.

It should be appreciated that the methods of the invention are not limited to any route of administration. Specifically, the formulations may be administered systemically. In some embodiments, the method comprises systemic administration of the formulations of the invention. In some other embodiments, method comprises injection or infusion of the formulations.

The present disclosure provides in accordance with some other aspects, a formulation for use in the treatment, inhibition, arresting or delaying bleeding or any disorder associated thereto. Thus, in accordance with some embodiments, the formulations of the invention may be relevant for the treatment of bleeding in a subject in need thereof, wherein the formulations comprise Factor V, at least one vitamin K-dependent clotting protein and at least one stabilizer, wherein the at least one stabilizer is present in the formulation at a concentration at which the Factor V and the at least one vitamin K-dependent clotting proteins are zymogenes. In some embodiments, the formulation of the invention is applicable for systemic use.

Further, the present disclosure provides a kit (package). The kit of the invention may comprise a formulation comprising concentrated Factor V, at least one vitamin K- dependent clotting protein and at least one stabilizer at a concertation of below about 5mM, at times between about O.lmM and about 5mM, at times about 2mM.

According to some embodiments, the kit of the invention is for use in a subject suffering from bleeding or any disorder associated thereto as described herein above. It should be further appreciated that the kit of the invention may be also used for preventing bleeding in subjects having an increased risk for bleeding. In some embodiments, the kit further comprises instructions for use of the formulation to treat, inhibit or delay bleeding or any disorder associated thereto.

In some embodiments, the instructions comprise administrating the formulation in liquid form.

In some embodiments, the kit of the invention may be particularly suitable for systemic use. Hence, the instructions comprise administrating the formulation by systemic administration.

The kit may further include container means for containing the formulation. In some embodiments, the kit comprises an applicator configured to allow administration of the formulation, preferably by systemic administration such as infusion or injection. In some embodiments, the kit comprises the formulation within an applicator. In some embodiments, the applicator is a syringe.

A further aspect of the invention relates to a process for the preparation of the formulation. Specifically, the process of the invention comprises the step of subjecting blood or plasma as described herein to at least one separation step followed by recovering the formulation of the invention.

Hence, the present disclosure provides a process for the preparation of a formulation comprising concentrated Factor V and at least one vitamin K-dependent clotting protein in zymogen form.

The process may comprise the step of subjecting at least one of whole blood, fresh frozen plasma (FFP), platelet rich plasma (PRP), cryo-depleted plasma (CDP), cryoprecipitated plasma, recovered plasma, or source plasma obtained from plasma apheresis to at least one separation step.

The at least one separation step as used herein refers to any method known in the art for separating substances from blood, plasma or any product/fraction thereof as described herein.

For example, the process may comprise use of a surface of a solid phase capable of binding at least a portion of the plasma/blood components and hence separating plasma/blood components from each other. The at least one separation step may comprise a chromatography method, a centrifugation method or any combination thereof.

For example, the at least one separation step may comprise a chromatography method by column or batch, a centrifugation method, salting out or saturation with heparin, PEG or ammonium sulfate precipitation or any combination thereof.

In some embodiments, the separation step comprises a chromatographic step.

In some embodiments, the chromatographic step comprises at least one of a column chromatography, an ion-exchange chromatography, a gel-permeation chromatography, an affinity chromatography, a size-exclusion chromatography, or a hydrophobic interaction chromatography.

Regardless of the specific of chromatography method that is used, the separation may be done by a column setup, a batch setup or any combination thereof such as a hybrid setup. As appreciated, in a column setup, a solid medium is packed onto a column, and the mixture such as, plasma, is allowed to move through the column to allow settling, a wash buffer run through the column and the elution buffer subsequently applied to the column and collected. A batch set up, includes the following: adding the initial mixture, for example a plasma sample to the solid phase in a vessel, mixing, separating the solid phase, removing the liquid phase, washing, re-centrifuging, adding the elution buffer, recentrifuging and removing the elute.

In some embodiments, the chromatographic step comprises an ion exchange chromatography. This chromatography method is based on electrostatic interactions between charged protein groups, and solid support material (matrix). Ion exchange chromatography includes anion-exchange chromatography and cation-exchange chromatography, with the former using positively charged resins while adsorbing negatively charged components and the latter vice versa.

In some embodiments, the chromatographic step comprises at least one anion exchange chromatography step.

Hence, in accordance with some examples, the process comprises subjecting at least one of whole blood, FFP, PRP, CDP, cryo-precipitated plasma, recovered plasma, or source plasma obtained from plasma apheresis to at least one chromatography step and specifically to at least one anion exchange chromatography step and collecting an eluent into a solution comprising a stabilizer.

Additionally or alternatively, the process comprises subjecting at least one of whole blood, FFP, PRP, CDP, cryo-precipitated plasma, recovered plasma, or source plasma obtained from plasma apheresis to at least one chromatography step and specifically to at least one anion exchange chromatography step and transferring an eluent into a solution comprising a stabilizer.

In some embodiments, the stabilizer is present in the collection solution at a concentration such that the coagulation factors recovered by the process of the invention are at a zymogen form. In some embodiments, the collection solution (denoted herein at times also as the formulation of the invention) comprises at least one stabilizer at a concentration of at most about 5mM.

In some embodiments, at which the collection solution does not comprise a stabilizer, the eluent is immediately transferred to a storage solution comprising at least one stabilizer at a concentration of at most about 5mM. The storage solution in accordance with such embodiments is referred to herein as the formulation of the invention.

It was surprisingly found by the inventors that subjecting a plasma fraction such as CDP, to at least one chromatographic separation step, such as an anion exchange chromatography, and collecting the eluent into a formulation comprising specific concentration of a stabilizer as described herein successfully recovers the components in the eluent in a zymogen form.

In some embodiments, the process comprises subjecting CDP to at least one anion exchange chromatography step and collecting the eluent into a formulation comprising a divalent cation such as calcium ions.

In some examples, the process comprises collecting the eluent into a formulation comprising free calcium ions at a concentration of below about 5mM. In some examples, the process comprises collecting the eluent into a formulation comprising free calcium ions at a concentration of between about 0. ImM and below about 5mM, at times between about O.lmM and about 3mM, at times between about O.lmM and about 2mM, at times between about O.lmM and about ImM, at times between about 0.5mM and about 3mM, at times between about 0.5mM and 2mM. In some embodiments, the process comprises collecting the eluent into a formulation comprising calcium ions at a concentration of about 0. ImM, at times of about 0.3mM, at times of about 0.5mM, at times of about 0.8mM, at times of about ImM, at times of about 1.3mM, at times of about 1.5mM, at times of about 1.8mM, at times of about 2mM, at times of about 2.5mM, at times of about 3mM, at times of about 3.5mM or about 4mM.

In some embodiments, the elution solution comprises glycine. In some other embodiments, the elution solution comprises sodium chloride. In some other embodiments, the elution solution has a pH of between about 6.0 and about 10.0.

In some other embodiments, the collection solution has a pH of between about 6.0 and about 10.0, at times between about 6.5 and about 9, at times between about 6.8 and about 8.5, at times between about 6.8 and about 8.0, at times between about 7.0 and about 8.0.

The process of the invention may comprise additional steps. In some embodiments, the process comprises a step of washing a fraction bound to the ion exchange with a washing solution prior to eluting the bound fraction with an elution solution into the collection solution.

The present disclosure also provides a formulation obtainable by the process of the present disclosure.

The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof. It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word “comprise” , and variations such as “comprises” and “comprising” , will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

NON-LIMITING EXAMPLES

Test methods for determination coagulation factors

Determination of Procoagulants Concentration in Tested Samples using Clotting Assay (Modified Activated Partial Thromboplastin Time).

These assays determined specific procoagulants concentration of both the extrinsic and the intrinsic pathways by measuring clotting time of tested sample and using specific relevant deficient plasma. The clotting time is measured using the STart®4 (Stago) clotting machine according to the manufacturer instructions.

Determination of common pathway procoagulant concentrations (F II and FV)

The determination of procoagulants concentration (FII and FV) was performed according to the manufacturer’s instructions (Stago Start-4 SOP ref 26987). The principle of the test consists of the use of calcium thromboplastin (tissue factor) to measure the clotting time of the tested sample and to compare with that of the normal standard (Uni-calibrator Cat. #00625). Specific factors concentration is determined using the relevant specific deficient plasma (i.e. deficient factor II and deficient factor V). The deficient plasma supplies all the factors in excess except for the factor being tested which is supplied by the tested sample. The concentration of the tested factor (%) is interpolated from the calibration curve which was prepared by diluting the Unicalibrator in Owren-Koller buffer (Cat. #00360) to achieve the following dilutions: 1:10, 1:20, 1:40 and 1:80. Samples were diluted according to the estimated concentration, in order to fit the calibration curve range. The results are expressed by the STart®4 as a percentage of the activity of normal plasma. The assay calibration curve and samples were performed in duplicates. The results of the duplicate preparations of each sample needed to be within RSD of 10%. The test is carried out at 37°C.

Determination of intrinsic pathway procoagulant concentrations (FIX)

The determination of the intrinsic pathway procoagulant concentration (FIX) was performed according to the manufacturer’s instructions (Stago Start-4 SOP ref 26987) using C.K. PREST® kit (Cat. #00847). The test involves the recalcification of plasma in the presence of a standardized amount of cephalin (Platelet substitute) and a specific activator. This procedure minimizes test variables by standardizing the contact activation and by optimizing the concentration of the platelet like phospholipids. The clotting time of the tested sample is measured and compared with that of the normal standard (Unicalibrator Cat. #00625). Specific factor’s concentration is determined using the relevant specific deficient plasma (i.e. deficient IX). The rest of the assay was performed as described above for the common pathway factors.

Determination of procoagulant inhibitor concentration (Protein S)

The determination of the procoagulant inhibitor concentration (Protein S) was performed according to the manufacturer’s instructions (Stago Start-4 SOP ref 26987) using Staclot® Protein S kit (Cat. #00746). The principle of the assay is based upon the cofactor activity of Protein S which enhances the anticoagulant action of activated Protein C. This enhancement is reflected by the prolongation of the clotting time of a system enriched with factor Va which is a physiological substrate for activated protein C. The assay was performed as described above for the common pathway factors. Determination of Procoagulants Concentration in Tested Samples using Chromogenic Assay

These assays determined specific procoagulants concentration of FVII, FX, Protein C and Antithrombin III by using chromogenic method according to the EU Pharmacopeia. The OD is measured using an ELISA reader.

Determination of FVII concentration

The determination of Factor VII was performed according to the manufacturer’s instructions (Hyphen Biomed) using BIOPHEN FVII kit (Cat. # 221304). In the presence of tissue factor (rabbit thromboplastin) and calcium, factor FVII forms an enzymatic complex which activates the factor X, present at a constant concentration and in excess, to factor Xa. The amount of factor Xa formed depends on the concentration of Factor VII to be assayed. This factor Xa formed cleaves the specific substrate Xa (SXa-11) and releases para- nitroaniline (pNA). The amount of pNA generated is directly proportional to the Factor Xa activity. The amount of Factor VII in the assayed sample is directly proportional to the Factor Xa activity generated, measured by the amount of pNA released, determined by color development at 405 nm.

Determination of FX

The determination of Factor X was performed according to the manufacturer’s instructions (Hyphen Biomed) using BIOPHEN FX kit (Cat. # 221705). Using the BIOPHEN Factor X assay, Factor X is measured following a specific activation with RVV, an enzyme extracted from snake venom (Russell’s viper venom). Activated Factor X (FXa) then specifically cleaves the specific substrate SXa-11, releasing paranitroaniline (pNA), which color is measured at 405nm. There is a direct relationship between color development and Factor X activity in the tested plasma.

Determination of Protein C

The determination of Protein C was performed according to the manufacturer’s instructions (Hyphen Biomed) using BIOPHEN™ Protein C (LRT) (Cat. # 221211- RUO). Using this assay, Protein C is measured following a specific activation with Protaca, an enzyme extracted from snake venom (Agkistrodom C Contortrix). Activated protein C (APC) then specifically cleaves the specific substrate SaPC-21, releasing para- nitroaniline (pNA), which color is measured at 405nm. There is a direct relationship between color development and Protein C activity in the tested plasma.

Determination of Antithrombin (AT)

The determination of Antithrombin was performed according to the manufacturer’s instructions (Hyphen Biomed) using BIOPHEN™ Antithrombin (Cat. #221102). BIOPHEN™ Antithrombin assay is a kinetic method based on the inhibition of FXa, which is at a constant concentration and in excess, by AT in presence of heparin. The remaining FXa is then measured by its amydolitic activity on a FXa specific chromogenic substrate, which releases pNA. The amount of pNA generated is inversely proportional to the AT concentration present in the tested plasma. The assay is insensitive to heparin.

Determination of Heparin (UFH)

The determination of Heparin was performed according to the manufacturer’ s instructions (Hyphen Biomed) using BIOPHEN™ ANTI-Xa (2 Stages Heparin Assay) (Cat. # 221005). This kit is a chromogenic anti-Xa method, developed for measuring Unfractionated Heparins (UFH) in plasma or in purified solutions, for their Anti-Xa activity. The BIOPHEN™ ANTI-Xa (2 Stages Heparin Assay) assay is a method based on the inhibition of a constant amount of Factor Xa (FXa), by the tested heparin in presence of exogenous antithrombin (stage 1), and hydrolysis of a Factor Xa specific chromogenic substrate (CS 11(65)), by the Factor Xa in excess (stage 2). pNA is then released from the substrate. The amount of pNA released (measured at 405 nm) is then a relation of the residual Factor Xa activity. There is an inverse relationship between the concentration of heparin and color development.

Other tests performed

In addition to coagulation factors, other parameters of the formulation in-process samples were also tested as detailed below.

Total protein

For the total protein assay, determination of protein concentration by Biuret Method (SOP# TM-98211) was used. In this assay, copper ions of the alkaline Biuret reagent form a purple-colored complex with serum proteins, whose maximum absorption is at 540 nm. The color intensity is proportional to the protein concentration. Batch screening study

The following experiments was aimed to determine the ability of various anion exchange resins to bind and elute FIT, FV, FVII, FIX, FX, Protein C, Protein S and ATIII with high efficiency.

Example 1 - Characterization of anion exchange resins

The resins, two strong and two weak anion exchange resins as detailed in Table 1, were tested at different pH values: 7.0, 7.5, 8.0 and 8.5. Cryo-Depleted Plasma (CDP) produced at Omrix (Plasma Fractionation Institute, Tel-Hashomer, Israel) was used as the starting material for the screening process. Protein Concentration: >47 mg/ml protein. CDP batch # X22Y730 was thawed, filtered with 0.8/0.2 pm PES filter and aliquoted. The aliquots were pH adjusted to 4 different pH values: 7.0, 7.5, 8.0 or 8.5. Then, the aliquots were frozen again and thawed before each assay. Equilibration buffer, pH adjusted to 7.0, 7.5, 8.0 and 8.5. Elution 1 Buffer: 20mM Sodium citrate, 0.25M NaCl, pH 7.0. Elution 2 Buffer: 20mM Sodium citrate, IM NaCl, pH 7.0.

Table 1 Resins tested

Pre-treatment for Fractogel TMAE (M) and GigaCap DEAE-650M resins:

The resins were washed with 3CV's (column volume) of Purified water (PuW) and 3CV's of IM NaCl. These steps were carried out by rolling 5min at room temperature (RT) and then left standing until full decantation was obtained.

Incubation and elution:

150pl (which is consider as 1CV) of each of the resins were used for the experiment. Each process was executed in duplicates and the samples generated in each step were combined before testing. During the Load, Wash and Elution steps shown in Table 2A, the resins were incubated with the sample/buffers at each of 4 different pH values (7.0, 7.5, 8.0 and pH 8.5). Each tested resin was subjected to 1 min rolling, then centrifuged for 1 minute at 500g, and then the supernatant was collected, except of the equilibration step which was discarded.

Table 2A Process for the resins batch screening

* Solutions adjusted to pH 7.0, 7.5, 8.0 and pH 8.5

Sampling:

Samples were taken from the supernatant after the centrifugation step, from Unbound (~ 1.8ml each), Wash (~2ml) and Elutions 1 and 2 (~0.6ml each). The duplicates were combined into one sample, and then aliquoted into 4 aliquots and stored at <-65°C for testing.

Pre-treatment for Cap to DEAE and UNOsphere Q resins:

Table 2B provides details on the process. Cryo Depleted Plasma (CDP) produced at Omrix (Plasma Fractionation Institute, Tel-Hashomer, Israel) was used as the starting material for the screening process. Protein Concentration: >47 mg/ml protein. CDP batch # X22Y730 was thawed, supplemented with llU/ml Heparin, filtered with 0.8/0.2 pm PES filter and aliquoted. The aliquots were pH adjusted to two different pH values: 7.0 and 8.0. Then, the aliquots were frozen again and thawed before each assay. Equilibration buffer: 10 mM citrate, pH adjusted to 7.0 and 8.0. Elution Buffer: lOmM Sodium citrate, IM NaCl, pH 8.0.

0.5ml (which is consider as 1CV) of each of the resins were used for the experiment. Each process was executed in duplicates and the samples generated in each step were combined before testing. The screening was carried out in two different pH values (7.0 and 8.0). For steps 1-3 (see Table 2B), the incubation was carried out for 1 minute and then the sample was left standing until complete decantation of the resin was obtained. During the Load, Wash and Elution (steps 4-6) the sample/buffers were incubated for 1 min while rolling and then centrifuged for 1 minute at 500g and the supernatant was collected.

Table 2B Process for the resins batch screening

* Solutions adjusted to pH 7.0 and 8.0 Samples were collected from the supernatants after centrifugation, from Unbound (~5ml), Wash and Elution (~2ml each). The duplicates were combined into one sample, divided into 4 aliquots and stored at <-65°C for testing.

Results

A summary of recoveries of all factors and inhibitors is presented in Table 3. According to the results obtained, regardless of the pH used in the process all FIT, FVII, FIX and FX were found mostly in the elution fractions, while FV seemed to have eluted in the unbound (UB) fraction, with small amounts found in the elution fractions.

Table 3 Recovery percentage* of coagulation factors and inhibitors, using different resins at pH 7.0-8.5

* Recoveries are calculated as the activities combined of Elution 1 and 2 over the activity in the load, in percentage (%). ATIII = Anti-Thrombin III. N/D = Not determined. Fig. 1 shows the levels of procoagulants and inhibitors in the elution fraction for each of the different resins studied in Units of activity per ml of load at pH 8.0. The levels of FIT, FVII, FIX and FX are overall satisfactory however, FV is present in very low levels (<0.10IU/ml), from which an important part did not bind to the resins (present in the UB fraction) and another part may have been degraded during the purification process.

Based on the summary of results in Fig. 1, it is suggested that all the four resins D- Fractogel TMAE, F-Gigacap DEAE, I-Capto DEAE and M-UNOsphereQ, from which Fractogel TMAE and UNOsphereQ are strong anion exchange resins and Gigacap DEAE and Capto DEAE are weak ones had good results at pH 8.0. This suggests that the strength of the resin is not a key parameter in obtaining the various members from the plasma.

Example 2 - AEX resins for column chromatography performance analysis

In order to further analyze the performance of the resins, Fractogel TMAE (M), Gigacap DEAE, Capto DEAE and Unosphere Q, were packed into 1.0cm width x 20cm height (15-16ml) columns. In addition, a strong anion exchange resin, Eshmuno Q (Merck) was also tested. Eshmuno Q was packed into a 20cm height X 2.6cm diameter (~ 100ml) column. In these runs, the maximum pressure during the run, and the purification performance for FII, FV and FIX were studied. Table 4 shows the packing parameters of the columns used in the study.

Table 4 AEX column packing (bed height 20cm) technical parameters

The method varied slightly between runs, however all runs included the following steps/conditions:

1) The runs were carried out at room temperature ("RT").

2) The CDP (loading material) was spiked with llU/ml Heparin, filtered through a PES 0.2pm filter and then adjusted to pH 8.0 (pH 7.0 for C67 and C68). 3) The loading material and all fractions were kept/collected on ice.

4) Column preparation with 2-5 CV of PuW.

5) Equilibration with equilibration buffer (20mM Sodium citrate, 5mM CaCh, except for C55R1 (see Table 5), where no buffer was used for equilibration) until effluent has reached the buffer pH (7.0 for all runs except for C67R1 at pH 8.0). In some cases, the pH was not reached until CDP started to load.

6) Loading (see linear velocity in Table 5).

7) 3-4CV flush with equilibration buffer.

8) Elution with IM NaCl + 20mM Sodium citrate.

9) Regeneration method (150cm/h): IM NaCl 3CV, 0.5M HC1 2CV, PuW 2CV, 0.5M NaOH 2CV, NaOH incubation 60min, IM NaCl 3CV, PuW 2CV, 20-25% Ethanol 2CV.

10) All steps were performed automatically using AKTA Avant system monitored at 280 nm.

The hardware used was Tricorn 10x200mm for Fractogel TMAE (M), Gigacap DEAE, Capto DEAE and Unosphere Q and Hi Scale 26 for Eshmuno Q.

Results

Table 5 20cm AEX pressure performance

* Max pressure > 1.0 bar

Fig. 2 shows the activity of FIT, FV and FIX in the elution fraction of 5 different packed resins.

High recoveries levels of FII were demonstrated in the elution of all runs, ranging from 73% for 30CV in Capto DEAE to 93% for 30CV in Unosphere Q. On another hand FV recoveries varies between runs, with the lowest level in the elution of Capto DEAE (23%), and the highest levels in Eshmuno Q 100ml (97%). It is worth mentioning that ImM Calcium was added to the elution fraction for the latter ones.

Apart from that, Fractogel TMAE has shown very high levels with 57% (15CV) and 88% (17CV) of FV. In all other runs, FV ranged between 30-35%. Finally, FIX was found in high levels in at least one of the runs for each resin, except for Capto DEAE, where only 14% were detected. The highest value was observed using Eshmuno Q.

Based on these results, it is possible to conclude that overall resins Fractogel TMAE (M), Gigacap DEAE, Unosphere Q and Eshmuno Q are all suitable to purify a set of coagulation factors.

Example 3 - Design of experiments (DOE) for Eshmuno Q Chromatography

The aim of this experiment was to analyze of the effect of the pH, flowrate and temperature on the performance of one of the tested resins. The Eshmuno Q column was used as an exemplary resin in the purification of the full set of proteins for a formulation from CDP. The experiments were conducted with Eshmuno Q, using a prepacked column with dimensions 8x100mm (5ml CV). Three variables were selected for this aim: chromatography temperature, pH of equilibration buffer and load, and the linear velocity of the loading step. Eight runs were carried out combining extreme values:

1) pH of the equilibration buffer and load: 7.0 and 8.5.

2) Temperature of the column during loading: whether on ice or at 23°C.

3) Linear velocity: 120 and 480cm/h.

An additional run (#9) at pH 7.5, 240cm/h and 23°C was executed with the purpose to confirm that no reuse issues are found in the column subjected to the study, and to have an additional value at intermediate condition values. The plan is shown in Table 6. Except the variables studied, all other conditions were kept constant, except the use of another CDP batch during Run 6 of the DOE. The levels of the coagulation factors were compared and the effect of each of the variables was determined separately and in combination. In addition, protein recovery was analyzed. Table 6 Design of experiments (DOE) plan

Preparation of Test Material

During the day of the experiment, two aliquots of 50ml containing HU/ml Heparin were defrosted at 23°C, filtered through a 0.2pm polyether sulfone filter (Pall), the pH was adjusted according to the design of experiments (pH 7.0, 7.5 or 8.5) and kept on ice until and during the loading to the column.

Chromatography method

The starting material loaded into the Eshmuno Q. For the equilibration of the column, 2CV of purified water and 10CV of equilibration buffer (20 mM Sodium citrate, 5 mM CaCh, pH = 7.0, 7.5, 8.0 or 8.5) were passed through a 5ml column (8x100mm), then 15CV CDP at pH = 7.0, 7.5, 8.0 or 8.5 were loaded into the column at 120 cm/h, 240 cm/h or 480 cm/h. The column was washed with 4CV equilibration buffer and then 3CV were eluted with a solution containing IM NaCl and 20mM Citrate at pH 7.0. The 3CV elution (~15ml) was collected into 45ml of a solution containing CaCh, Glycine and Heparin at pH 7.2, targeting for a final concentration of ImM CaCh, 50mM Glycine and llU/ml Heparin.

Results

General information of the runs is described in Table 7. All steps were performed automatically using AKTA Avant system monitored at 280nm. The protein recovery observed was satisfactory in all runs, ranging from 89-97% (Table 7) Low velocity (120cm/h) presented the lowest values (89-92%), while Run #2 presented the highest value of 97%. The elution fraction presented variable absolute values of protein from 11.5 to 39.1mg, where the lowest values were observed at pH 7.0, meaning that despite the higher pro-coagulant recoveries observed, less contaminants were bound at pH 7.0.

Table 7 Fractions weight and total protein* recovery of runs

*Total protein determined as described above.

Coagulants recoveries and analysis

FV recoveries were particularly high when working at pH 7.0 (67-101%) compared to results at pH 8.5 (41-65%). While FII presented the best result at pH 7.0 (Run #2, 94%), acceptable values were found across the DOE.

The rest of the procoagulants and inhibitors presented overall consistent high values, except for Anti-thrombin III (<LOD in all runs), which eluted mostly in the UB fraction in all the conditions studied. Finally, Run #9 at intermediate values of pH and velocity (pH 7.5 and 240cm/h) showed excellent levels of FV and protein C, and an acceptable value of 68% recovery for FII. Fig. 3 shows procoagulants and inhibitors determined in the elution fraction as percentage of recovery (Elution/Load) of the different runs in the DOE for Eshmuno Q. The effect of different conditions on the binding of formulation to Eshmuno Q was studied in the context of the DOE. Run #2 showed the best combined recovery corresponding to the lowest pH, high temperature and highest velocity. Example 4: Stability study

Coagulation factors in the plasma are known to have a half-life between 5h (FVII) to lOOh (FII), however, in the elution fraction of the AEX chromatographic column, the stability is reduced significantly, especially that of FV, which presents a challenge for the purification process. Further, Calcium, Mannitol, Maltose, Sucrose, Glycerol, and Glycine were tested when incubating at different temperatures for 24h, and after freezethaw cycles at <-65°C. In addition, the optimal pH was analyzed in this study.

Example 4A - pH optimization

The following experiment was aimed to determine the effect of the pH in a range of 6.7-7.7 in order to determine the optimal pH for the stability of FII and FV. The elution fraction of a chromatographic run in an Eshmuno Q 2ml column was used for the experiment, where 25ml of CDP (batch #X33Y 140) were loaded into the column and the elution fraction was stored at <-65°C. The elution sample was then thawed and adjusted to three different pH values A) 6.7, B) 7.2 and C) 7.7. Each sample was aliquoted and incubated overnight (24h) in two different temperatures: Al, Bl, Cl at RT, and A2, B2, C2 at 2-8°C. In addition, an untreated control (no pH adjustment) was stored at <-65°C during the 24h.

Results

FII and FV levels are shown in Fig. 4A and 4B, respectively. As shown in Fig. 4A, FII in the control was 0.77 lU/ml. At RT, the sample at pH 6.7 showed a similar value to that of the control, while increasing the pH seemed to improve the stability, reaching 0.97 and 1.15 lU/ml respectively for pH 7.2 and 7.7. On the other hand, when incubating at 2-8°C no clear trend was found throughout the pH range, with values of 0.90, 0.77 and 0.85 lU/ml respectively at pH 6.7, 7.2 and 7.7. As shown in Fig. 4B, FV concentration of the control was 0.35IU/ml, suggesting that freeze and thaw cycle has a major effect on the stability of FV. Here, low pH seems to give the best protection from degradation at RT, although the value is half of the control (0.14IU/ml). Higher pH values showed no protection at RT. During incubation at 2-8°C FV degraded completely throughout the pH range. The experiment suggested that a stabilizing agent for FV is needed in order to keep this factor present until the end of the production process. Example 4B - Effect of temperatures on stability

The aim of this study is to determine how the stability of an elution sample obtained from an AEX column is affected by the storage temperature.

Fig. 5 shows results of this study. The levels of FII and FV in the fresh condition and after 24h at three different temperatures: <-65°C, 2-8°C and Room Temperature (RT) were analyzed, including the effect of 2 cycles of freeze-thaw at <-65°C.

A chromatographic run (C65R2) was carried out using Fractogel TMAE (M) Medcap prepacked 1 ml column at pH 8.0. CDP (batch #X33Y140). Loading material was spiked with HU/ml Heparin, filtered through a PES 0.2pm Supor filter and pH was adjusted to 8.0. 32.5 ml were loaded into the column and the bound material was eluted with a IM NaCl + 20mM Sodium citrate buffer. The 3.5CV of elution collected were diluted 1/5 with a buffer at pH 7.2 containing a concentration of 20mM Sodium Citrate, ImM CaCh. In this eluted sample, the concentration factor (volume of Load / volume of Elution) was 1.7. This elution was tested for FII and FV fresh and after 24h at different temperatures:

A) Fresh (tested in the same day after collection)

B) 24h at <-65°C (1^st cycle of Freeze-Thaw)

C) Another 24h at <-65°C (2^nd cycle of Freeze-Thaw)

D) 24h at 2-8°C

E) 24h at RT

Results

The load presented 1.06IU/ml FII, from which 0.56IU/ml were recovered in the fresh elution. When incubating the sample 24h at 2-8°C and RT, 0.64 and 0.69IU/ml FII were obtained, slightly higher than the fresh sample. The 24h freeze-thaw cycles presented 0.64 and 0.55IU/ml consecutively, showing a trend of degradation equivalent to 0. llU/ml decrease per cycle. On another hand, 0.76IU/ml FV were obtained in the load, from which only O.33IU/ml were recovered, representing less than half of the activity. Samples incubated 24h at 2-8°C and RT presented an almost complete degradation with 0.12 and O.lOIU/ml FV respectively. It can be suggested that due the presence of a chelating agent such as Sodium citrate, that addition of ImM CaCh to the elution was not sufficient to protect FV. The 24h freeze-thaw cycles presented 0.21 and O.l lIU/ml consecutively, showing a similar trend of degradation as FII, with O.lIU/ml per cycle decrease (Fig. 5).

Example 4C - The effect of freeze-thaw cycles on the FII and FV levels using sugars at 10%

The aim of this study was to study the effect of different sugars on the stability of FII and FV in an elution sample obtained from an AEX column, after two Freeze-thaw cycles at <-65°C. The elution fraction was tested for FII and FV fresh and after cycles of 24h Freeze-Thaw with and without 10% sugars.

A) Fresh

B) 1 cycle control (no sugar)

C) 2 cycles of Freeze-Thaw at <-65°C control (no sugar)

D) 2 cycles at <-65°C, containing 10% Mannitol

E) 2 cycles at <-65°C, containing 10% Maltose

F) 2 cycles at <-65°C, containing 10% Sucrose

G) 2 cycles at <-65°C, containing 10% Glycerol

Results

When analyzing the levels of FII, fresh and after two consecutive 24h freeze-thaw cycles at <-65°C, no clear difference is evidenced, where the fresh sample presented 0.56IU/ml, while 0.51 and 0.58IU/ml were observed after 1 and 2 cycles respectively, where the load presents 1.06IU/ml. When the sample was treated with Mannitol, the activity was reduced to 0.46IU/ml after 2 cycles of Freeze-Thaw, while the other sugars ranged between 0.58 to 0.60IU/ml.

On another hand, FV is affected significantly by the first Freeze-Thaw cycle, decreasing from O.33IU/ml in the fresh sample to 0.16IU/ml. A second cycle presented 0.14IU/ml, slightly lower than the previous. This degradation was observed in the same measure when using 10% of any of the sugars studied, with values ranging from 0.12IU/ml (mannitol) to 18 (glycerol) (Fig. 6). Example 5 - Dilution ' with a buffer at pH 7.2 containing Calcium

The aim of this experiment is to study the effect of diluting the elution fraction obtained from an AEX column with PuW, Citrate and Glycine buffers, containing small amounts of CaCh inside the range of pH 7.0-7.3 as potential stabilizer for FV. Samples untreated and diluted in different solutions were tested for FII and FV at T-0 and after 24h incubation at < -65°C, 2-8°C and RT.

A chromatographic run (C63R2) was carried out using Eshmuno Q prepacked 5ml column, at pH 8.0. CDP loading material was spiked with HU/ml Heparin, filtered through a PES 0.2pm Supor filter and pH was adjusted to 7.9. 75 ml of CDP (batch #X33Y 140) were loaded into the column and 86ml of UB were collected (which includes 2CV of the flush). The bound material was eluted with a IM NaCl + 20mM Sodium citrate buffer, and then the volume was divided into 7 samples:

A) Untreated (pH 8.0)

B) Untreated adjusted to pH 7.2

C) PuW

D) PuW + ImM CaCh

E) 20mM Citrate + 1 mM CaCh

F) 20mM Citrate+ 5 mM CaCh

G) 50mM Glycine + ImM CaCh

All samples were incubated at 3 different temperatures for 24h: <-65°C, 2-8°C and RT, while an additional sample was tested in the same day (T-0).

Results

The starting material presented 1.14 lU/ml FII and 0.80 lU/ml FV.

Untreated samples of FII should contain the same composition of the buffer in which it was eluted, which is 20mM Citrate + 5 mM CaCh. When the pH was left at pH 8.0 (A), the samples presented very similar values when incubated at 24h at different temperatures (values between 0.90-0.97IU/ml) compared to T-0 (0.96IU/ml). Adjusting the pH to 7.2 (B) did not show much improvement, with a lower value at T-0 (O.83IU/ml), and values of 0.92IU/ml, 0.82IU/ml and l.OHU/ml after 24h at <-65°C, at 2-8°C and RT respectively. When analyzing the treated samples, FII seems to be stable after 24h across all conditions and incubation temperatures, without evidencing any benefit by diluting *4 with PuW (C and D), Citrate (E and F) or Glycine (G). All values after 24h incubation ranged between 0.80 to 0.95 lU/ml, representing recoveries between 0.70 to 0.83% (Fig. 7A).

Untreated samples of FV at pH 8.0 presented O.38IU/ml at T-0, and a slightly lower value after 24h at <-65°C (freeze-thaw involved) at O.33IU/ml, while a great degradation is observed after 24h at 2-8°C and RT. Similarly, untreated samples adjusted to pH 7.2 presented 0.44 and 0.40 lU/ml for T-0 and 24h at <-65°C respectively, which is slightly better than pH 8.0. Complete degradation was observed only for 2-8°C, while 0.3IU/ml were observed at RT. Treated samples showed a different pattern: an increase in the stability seems to occur for all dilutions at T-0 when compared to the untreated (A and B), with associated values of 0.70 (C), 0.78 (D), 0.53 (E), 0.73 (F) and 0.85IU/ml (G), representing >88% recoveries to that of the load (0.80IU/ml), being G the best result (50mM Glycine, ImM Calcium).

Considering these values were obtained just after collection, it seems that a rapid degradation occurs at the moment of collection, unless the elution is immediately diluted by one of these solutions. When analyzing the stability after 24h at <-65°C , the effect of freeze-thaw seems to reduce the activity in exactly 0.2U/ml in all cases when compared to T-0: 0.70 to 0.50IU/ml (C), 0.78 to 0.60IU/ml (D), 0.53 to 0.32IU/ml (E), 0.73 to 0.52IU/ml (F) and 0.85 to 0.65IU/ml (G), the latter one being again the higher result, representing 81% recovery to the load. Diluting the sample with 20 mM Citrate + ImM Calcium (E) seemed to lead to almost complete degradation at both, 2-8°C and RT (0.08 and 0.10 lU/ml respectively).

On the other extreme, C and G proved to be the most stabilizing conditions, presenting very similar results: 0.42 and 0.43 lU/ml at 2-8°C, and 0.47 and 0.50 lU/ml at RT. This suggests that it is the reduction of Citrate concentration when diluting the sample and not the use of a specific buffer as Glycine, that stabilizes FV, probably due to the fact that Citrate chelates the free calcium in the solution, which is important for the stabilization. Other possibility is that the high concentration of salts in the elution buffer (IM NaCl) interferes FV activity. Dilution of NaCl to 250nM may reduce this interference. In addition, adding ImM Calcium (on the final concentration) seems to increase the stability, evidenced by the significantly lower levels obtained in C, where no calcium was added to the PuW (0.23 and 0.18IU/ml at 2-8°C and RT respectively). It was observed that even when diluting with a Citrate solution, adding a higher concentration of Calcium (5mM in the final solution) seems to compensate the negative effect of Citrate, probably because it out-weighs the equilibrium Citrate-Calcium towards the latter one. Finally, overall it seems that incubating the samples at RT is slightly beneficial than 2- 8°C for reasons not clear; a possible partial activation of some pro-coagulants or the degradation of some inhibitor may explain the phenomenon (Fig. 7B).

Example 6 - Dilution ( with a buffer at pH 7.2 containing Calcium

The aim of this experiment is the verification of results obtained in the stability of FII and FV when diluting the elution fraction obtained from an AEX column by *4 with a buffer containing a final concentration of ImM CaCh at pH 7.2. Two chromatographic runs (C63R3 and C63R4) were carried out using Eshmuno Q prepacked 5ml column, at pH 8.0. CDP loading material was spiked with llU/ml Heparin, filtered through a PES 0.2pm Supor filter and pH was adjusted to 8.0. 75 ml of CDP (batch #X33Y140) were loaded into the column and 80-87ml of UB were collected (including 2CV of the flush). The bound material was eluted with 3CV of IM NaCl + 20mM Sodium citrate and tested immediately for FII and FV as follows:

A) Untreated

B) Diluted 1/4 with a buffer at pH 7.2. Final concentration ImM CaCh and llU/ml Heparin.

C) Diluted 1/4 with a buffer at pH 7.2. Final concentration ImM CaCh, llU/ml Heparin and 50mM glycine.

Results

Based on the Fig. 8, in both experiments, FII presented similar values for the elution fraction whether untreated or diluted with a buffer containing calcium, all of them ranging between 0.78 and O.88IU/ml with no observable trend, where the load presented 1.14 and 1.16 lU/ml respectively for each experiment. On the other hand, it is evident that collecting the load in the dilution buffer has a positive effect on the stabilization of FV. Experiment C63R3 started with a load at 0.80IU/ml and only 0.26IU/ml were observed in the untreated elution fraction, while the *4 dilution with calcium presented 0.46 (B) and 0.52IU/ml (C). In addition, experiment C63R4 showed a similar trend, with a starting material at 0.68IU/ml, an elution at 0.26IU/ml when untreated (A), while 0.46IU/ml were obtained when diluting *4 in calcium buffer.

Example 7 - Calcium concentration range

The aim of this experiment was to determine the range in which calcium serves as a stabilizer of coagulation factors V and IX in the elution fraction without provoking activation. A suitable amount of CDP (batch #W35Y440) containing HU/ml Heparin was filtered through a PES 0.2pm Supor filter and pH was adjusted to 7.2. 20ml were loaded into a 1ml Eshmuno Q column, where 21ml of UB were collected, and the bound proteins were eluted with 3ml (3CV) of a buffer containing IM NaCl and 50mM Glycine at pH 7.2. The elution sample was divided into 6 aliquots, from which one sample was kept untreated (A) and 5 of them were diluted 1/4 with 50mM glycine solution at pH 7.2 containing different concentrations of CaCh to obtain a final concentration of: B) 0. ImM, C) 0.5 mM, D) 1.0 mM, E) 2.0 mM and F) 5.0mM. FV and FIX was determined in all samples just after treatment (To), and after 24h incubation at <-65°C (freeze-thaw), 2-8°C and at room temperature (RT).

Results

As observed in Fig. 9A, the activity of FV was dramatically reduced in untreated samples after 24h incubation at 2-8°C and RT, passing from 0.82IU/ml in the load to 0.14 and 0.15IU/ml respectively. While 24h freeze-thaw affected the activity of FV as well but in less extent (0.55IU/ml). Samples containing between 0.1-2.0 mM calcium presented low variability when tested at To with a small FV reduction (0.68-0.73IU/ml), while incubation at <-65°C did not compromise the activity.

As shown in Fig. 9B FIX activity was affected as well in untreated samples after 24h incubation, passing from 0.96IU/ml in the load to 0.83, 0.72 and 0.74IU/ml respectively at <-65°C, 2-8°C and RT. The presence of calcium between 0.1-2.0mM stabilized the activity of FIX in all conditions, while, as mentioned above, the addition of 5mM provoked the activation of the cascade, evidenced by an immediate clotting after addition of 25mM calcium (FV and FIX deficient clotting assay). As shown in Fig. 9C, incubations at 2-8°C and RT presented improved FV stability with the increase in calcium concentration, with a maximum at 2mM at 2-8°C (0.66IU/ml), while the addition of 5mM provoked the activation of the cascade.

Based on the results described herein, it was suggested that FII may be stable in a wide range of conditions. However, FV is highly unstable during its purification from plasma using AEX, experiencing immediate degradation from the moment it elutes from the column and probably even during the run itself (higher flowrates show higher recoveries for FV). It was suggested that one effective way to stabilize coagulation factors during the production of a formulation is the use of a buffer at pH 7.2 containing ImM calcium.

In addition, it is important to keep low levels of citrate when present in any of the purification steps to avoid chelation of the free calcium in the solution, which is critical for the stabilization of FV. The buffer may also comprise Glycine as a good option for a parenteral drug production process, and heparin to keep pro-coagulants inactive. When working with ion exchange columns in the binding mode, it is suggested to collect the elution fraction directly into dilution buffer previously adjusted to pH 7.2. In this way, FII and FV may be stable for at least 24h at <23°C. It is preferable to avoid freeze-thawing in any of the conditions. Finally, a range between 0.1-2.0mM CaCh in the absence of chelating agents like citrate is suitable for stabilization, obtaining optimum results at 2.0mM. Exceeding this value might provoke the activation of the coagulation cascade.

Example 8 - Lyophilization of the formulation

In order to improve the stabilization of the final product for long term storage, a lyophilization process was executed. For this purpose, 320ml of CDP (batch #W33Y 140) at pH 7.0 were loaded into a 16ml Eshmuno Q column at 200 cm/h. The bound material was eluted with 56 ml of a buffer containing IM NaCl and 20mM Glycine at pH 7.2, concentrating the purified proteins 6.3 times (load/elution ratio). The elution was divided into 3 aliquots: A) no calcium was added, B) added O.lmM CaCh and C) added 2mM CaCh. The samples were centrifuged in a lOKDa PES Ultra Spin column (Thermo) for 5 hours between 6000-7000g at 4 °C, concentrating the protein content 11 times. The samples were diluted between 1/3.8 to 1/4.3 with 20mM glycine at pH 7.2, while calcium concentration was compensated to keep the original values for A, B and C, achieving an osmolarity of 282 for A, 233 for B and 295 mOsm for C. 1ml samples of the three samples were freeze-dried in 5ml vials using a Christ Freeze-dryer model Epsilon 2-8D using the method depicted in Table 8 and Fig. 10.

Table 8 Lyophilization method

Fig. 10 shows lyophilization method. By the moment the samples were inserted in the vials prior to freeze-drying, the product was approximately 17 times more concentrated in coagulation factors than in the starting material, considering no degradation. The samples were reconstituted after 60 days with 1ml PuW.

Results

The cakes resulting from the freeze-dried samples presented a normal visual appearance, meaning no collapse was observed. The lyophilized samples took no more than 10 minutes to reconstitute with 1ml PuW. It is suggested that, at least in the conditions mentioned above, a calcium stabilized formulation obtained from an AEX column can be concentrated and lyophilized for its posterior reconstitution and use.

Claims (47)

- 48 - CLAIMS

1. A formulation comprising concentrated Factor V, at least one vitamin Independent clotting protein and at least one stabilizer, wherein said at least one stabilizer is at a concentration at which said Factor V and/or said at least one vitamin K-dependent clotting protein are in zymogen form.

2. The formulation of claim 1, wherein said Factor V and said at least one vitamin K-dependent clotting protein are in zymogen form in the formulation at a temperature of at most about 30°C for at least 4 hours.

3. The formulation of claim 1 or 2, wherein at least 50% of said factor V is in zymogen form at room temperature for at least 24 hours.

4. The formulation of any one of claims 1 to 3, wherein said Factor V is at a concentration that is at least 20% of at least one vitamin K-dependent clotting protein.

5. The formulation of any one of claims 1 to 4, wherein said at least one vitamin K- dependent clotting protein is at least one of Factor II, Factor VII, Factor IX or Factor X.

6. The sealant formulation of claims 1 to 5, comprising zymogens of Factor II, Factor VII, Factor IX and Factor X.

7. The formulation of any one of claims 1 to 6, wherein the stabilizer comprises at least one divalent cation.

8. The formulation of claim 7, wherein said divalent cation is at a concentration of at most about 5mM.

9. The formulation of any one of claims 1 to 8, comprising an anticoagulant protein.

10. The formulation of claim 9, wherein said anticoagulant protein is at least one of protein C, protein S or Anti-thrombin III (AT III).

11. The formulation of any one of claims 1 to 10, having a pH of between about 6.5 and about 8.0.

12. The formulation of any one of claims 1 to 11, further comprising at least one excipient.

13. The formulation of claim 12, wherein said excipient is at least one of mannitol, maltose, sucrose, glycerol or glycine. - 49 -

14. The formulation of any one of claims 1 to 13, derived from cryo-depleted plasma (CDP).

15. The formulation of any one of claims 1 to 14, being in powder, liquid or frozen form.

16. The formulation of any one of Claims 1 to 15, suitable for systemic administration.

17. A formulation comprising concentrated Factor V, at least one vitamin Independent clotting protein and free calcium ion at a concentration of below about 5mM.

18. The formulation according to claim 17, comprising zymogens of Factor II, Factor VII, Factor IX and Factor X.

19. A method for the treatment, inhibition, arresting or delaying bleeding or any disorder associated thereto in a subject in need thereof, the method comprising the step of administering to said subject an effective amount of a formulation, said formulation comprises concentrated Factor V, at least one vitamin K-dependent clotting protein and at least one stabilizer.

20. The method of claim 19, wherein said formulation is as defined in any one of claims 1 to 18.

21. The method of claim 19 or 20, wherein said bleeding disorder is an inherited or acquired bleeding disorder.

22. The method of any one of claims 19 to 21 , wherein said bleeding disorder is Factor V deficiency.

23. The method of any one of claims 19 to 21, wherein said bleeding disorder is an injury-induced bleeding, surgery-induced bleeding or trauma-induced bleeding.

24. The method of any one of claims 19 to 23, comprising administration said formulation in liquid form.

25. The method of any one of claims 19 to 24, wherein said administration is by systemic administration.

26. A formulation comprising concentrated Factor V, at least one vitamin Independent clotting protein and at least one stabilizer for use in the treatment, inhibition, - 50 - arresting or delaying bleeding or any disorder associated thereto in a subject in need thereof.

27. The formulation for use of claim 26, wherein said formulation is as defined in any one of claims 1 to 18.

28. The formulation for use of claim 26 or 27, wherein said bleeding disorder is an inherited or acquired bleeding disorder.

29. The formulation for use of any one of 26 to 28, wherein said bleeding disorder is Factor V deficiency.

30. The formulation for use of any one of claims 26 to 28, wherein said bleeding disorder is an injury-induced bleeding, surgery-induced bleeding or trauma-induced bleeding.

31. A kit comprising: (a) a formulation comprising concentrated Factor V, at least one vitamin K-dependent clotting protein and free calcium ion at a concentration of at most about 5mM and (b) instructions for use of the formulation in the treatment, inhibition, arresting or delaying bleeding or any disorder associated thereto in a subject in need thereof.

32. The kit of claim 31 , wherein said instructions comprise providing said formulation in liquid form

33. The kit of claim 31 or 32, wherein said instructions comprises applying the formulation by systemic administration.

34. The kit of any one of claims 31 to 33, comprising an applicator configured to allow said systemic administration.

35. The kit of any one of claims 31 to 34, comprising said formulation within an applicator.

36. The kit of any one of claim 35, wherein said applicator is a syringe.

37. The kit of any one of claims 31 to 36, wherein said bleeding disorder is an inherited or acquired bleeding disorder.

38. The kit of any one of claims 31 to 37, wherein said bleeding disorder is Factor V deficiency. - 51 -

39. The kit of any one of claims 31 to 37, wherein said bleeding disorder is an injury- induced bleeding, surgery-induced bleeding or trauma-induced bleeding.

40. A process for the preparation of a formulation comprising concentrated Factor V and at least one vitamin K-dependent clotting protein, the process comprising (i) subjecting blood, plasma or any product/fraction thereof to at least one separation step and (ii) recovering said Factor V and at least one vitamin K-dependent clotting protein obtained in step (i).

41. The process of claim 40, wherein said blood, plasma or any product/fraction is cryo-depleted plasma (CDP).

42. The process of claim 40 or 41, wherein said at least one separation step comprises at least one chromatography step.

43. The process of claim 42, wherein said at least one chromatography step comprises at least one anion exchange chromatography step.

44. The process of any one of claims 40 to 43, wherein said recovering comprises eluting a fraction bound to the at least one anion exchange chromatography with an elution solution into a collection solution.

45. The process of claim 44, said collection solution comprises a divalent cation at a concentration of at most about 5.0mM.

46. The process of any one of claims 40 to 45, comprising washing the bound fraction with a washing solution prior to eluting the bound fraction with the elution solution.

47. A formulation obtainable by the process of any one of claims 40 to 46.