CN116194080A

CN116194080A - Hemostatic preparation and use thereof

Info

Publication number: CN116194080A
Application number: CN202180064844.0A
Authority: CN
Inventors: I·努尔; R·明茨; D·维斯诺维斯基; M·尤努索夫; M·莱西茨
Original assignee: Omrix Biopharmaceuticals Ltd
Current assignee: Omrix Biopharmaceuticals Ltd
Priority date: 2020-09-23
Filing date: 2021-09-13
Publication date: 2023-05-30
Also published as: WO2022064478A1; AU2021348528A1; AU2021348528A9; EP4216928A1

Abstract

Disclosed herein are formulations comprising concentrated factor V, vitamin K-dependent coagulation protein, and a stabilizer, wherein the concentration of the stabilizer is such that the factor V or the vitamin K-dependent coagulation protein is in zymogen form. The preparation method of the preparation is also disclosed.

Description

Hemostatic preparation and use thereof

Technical Field

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

Background

References deemed relevant to the background art of the presently disclosed subject matter are listed below:

-U.S. Pat. No. 3,560,475

-U.S. Pat. No. 9,814,765

-U.S. Pat. No. 7,371,722

U.S. patent application 2018/0036338

-U.S. Pat. No. 5,866,122

U.S. patent application 2018/0369346

U.S. Pat. No. RE38431

U.S. patent application 2014/0155498

U.S. patent application 2009/0011043

The admission of the above references herein should not be inferred as implying any patentability of the presently disclosed subject matter.

Background

Us patent 9,814,765 describes a one-component sealant formulation (e.g. in liquid form) comprising fibrinogen; vitamin K-dependent prothrombin comprising at least factor II (FII) and Factor X (FX), methods of its preparation and use are also described.

Us patent 7,371,722 relates to a pharmaceutically active substance comprising (a) prothrombin (factor II) obtained from plasma or by genetic engineering, (B) a factor V, VIII, IX, X obtained from plasma or by genetic engineering, which may be present at least partly in their activated state, and a factor XIa obtained from plasma or by genetic engineering, and (C) a prion-safe, coagulation-promoting phospholipid for use in the production of a medicament capable of producing thrombin or comprising thrombin and a composition comprising the same.

U.S. patent application 2018/0036338 relates to hemostatic compositions comprising polysaccharide-based polymers, such as amylopectin, amylose, dextrin, maltodextrin and icodextrin, and modified forms thereof, crosslinked with a crosslinking agent, which are useful as hemostatic compositions.

Us patent 5,866,122 describes a pharmaceutical preparation for the treatment of coagulation disorders comprising purified prothrombinase factor, in particular purified prothrombin and optionally purified factor Xa as active ingredient.

Us patent 3,560,475 describes a prothrombin complex prepared from a plasma fraction containing factors II, VII, IX and X by adsorption with tricalcium phosphate, elution with trisodium citrate and multiple precipitation with polyethylene glycol.

U.S. patent application 2018/0369346 provides a composition comprising: a pre-accelerator element (factor V) and at least one factor selected from the group consisting of: prothrombin (factor II), proconversion (factor VII) and Stuart-proar factor (factor X).

U.S. Pat. No. RE38431 relates to stable liquid formulations of factor VIII and factor IX, which can be administered by injection or infusion to provide a constant level of coagulation factor in the blood.

U.S. patent application 2014/0155498 describes diagnostic or therapeutic formulations for stabilizing liquid coagulation factors comprising at least one coagulation factor, at least one stabilizer and optionally additives.

U.S. patent application 2009/0011043 describes a tissue sealant comprising whole blood and an effective amount of an exogenous protein cross-linking agent for cross-linking the whole blood.

Disclosure of Invention

The present disclosure is based on the development of a formulation that comprises a coagulation factor in the zymogen form (i.e., inactive form) and is stable when stored in liquid form. In particular, and according to a first aspect thereof, the present disclosure provides a formulation comprising concentrated factor V, at least one vitamin K-dependent coagulation protein, and at least one stabilizer, wherein the concentration of the at least one stabilizer is such that the factor V and/or the at least one vitamin K-dependent coagulation protein is in zymogen form.

According to another aspect, the present disclosure also provides a formulation comprising concentrated factor V, at least one vitamin K-dependent clotting protein, and free calcium ion at a concentration of up to about 5mM, e.g., less than 5 mM.

According to another aspect, the present disclosure also provides 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 0.1mM to less than 5 mM.

According to another aspect, the present disclosure also provides a formulation comprising concentrated factor V and one or more co-purified vitamin K-dependent clotting proteins selected from 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 0.1mM to less than 5 mM. The formulation does not include exogenously added vitamin K-dependent coagulation proteins selected from the group consisting of factor II, factor VII, factor IX and factor X.

According to another aspect, the present disclosure also provides 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, such as in the range of 0.1mM to less than 5 mM. The formulation does not include exogenously added vitamin K-dependent coagulation proteins selected from the group consisting of 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 is not less than 0.5 factor II about 1 factor VII about 1 factor IX about 1 factor X about 1;

not less than 0.5 may be, for example, 0.5, 0.6, 0.7, 0.8, 0.9, or any value in the range of, for example, 0.7 to 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:

the corresponding ratio of factor V to at least one member from factor II, factor VII, factor IX, factor X is not less than 0.5 factor V with at least one member being about 1.

Basically, these factors are inactive.

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

According to another aspect, the present disclosure provides a method for treating, inhibiting, preventing or delaying bleeding or any disorder associated therewith in a subject in need thereof. The method of the invention comprises the step of administering to a subject an effective amount of the disclosed formulation.

According to another aspect, the present disclosure provides a formulation comprising concentrated factor V, at least one vitamin K-dependent coagulation protein, and at least one stabilizer for use in treating, inhibiting, preventing or delaying bleeding or any disorder associated therewith in a subject in need thereof, the at least one stabilizer being present in a concentration such that the factor V and/or the at least one vitamin K-dependent coagulation protein is in zymogen form.

According to another aspect, the invention also relates to a kit comprising a formulation as disclosed herein, and optionally instructions for using the formulation to treat, inhibit, prevent or delay bleeding or any disorder associated therewith.

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

Drawings

For a better understanding of the subject matter disclosed herein and to illustrate how the subject matter may be implemented 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 levels of procoagulants and inhibitors in the elution fractions at pH 8.0 for different AEX resins in batch mode, expressed in units/ml loading, with factors ranging from left to right as FII, FV, FVII, FIX, FX, protein C, protein S and antithrombin III (if present).

FIG. 2 is a bar graph showing the percent recovery of FII, FV, and FIX (bottom to top segments in each column) in a 20cm high AEX column, except for a 2.6cm diameter C61R2, where all columns are 1.0cm in diameter.

Fig. 3 shows the levels of procoagulant and inhibitor measured in the elution fractions, expressed as percent recovery (elution/loading) of the different runs, factors ranging from left to right as FII, FV, FVII, FIX, FX, protein S, protein C and antithrombin III (if present) using a 0.8cm wide x 10cm pre-packed Eshmuno Q column (Merck) in the design of experiment (DOE).

FIGS. 4A and 4B are bar graphs showing the amounts of FII (FIG. 4A) and FV (FIG. 4B) observed after 24 hours of incubation at different temperatures at pH 6.7, pH 7.2 and pH 7.7 (from left to right: -65 ℃, shown only at pH 6.7: room temperature; and 2 ℃ to 8 ℃).

FIG. 5 is a bar graph showing the amounts of FII and FV (IU/mL) in the loading fraction and in the fresh elution fraction after more than one freeze-thaw cycle at < -65 > C for 24 hours, and after incubation of the fresh fraction at different temperatures (see example 4B).

FIG. 6 is a bar graph showing the amount of FII and FV in the elution fractions after more than one freeze-thaw cycle at < -65℃for 24 hours using different stabilizers, control samples not including stabilizers.

FIGS. 7A and 7B are bar graphs showing the results of the use of PuW, 20mM citrate or 50mM glycine buffer (pH 7.0 to pH 7.3, containing 1mM or 5mM CaCl) ₂ ) Stability of FII (FIG. 7A) and FV (FIG. 7B) in the eluted fraction of the AEX column (Eshmuno Q) after 1/4 dilution. After incubation for 24 hours (from left to right in each quartet) at time 0 and at < 65 ℃, 2 ℃ to 8 ℃ and Room Temperature (RT), the different solutions were tested for absenceFII and FV of the treated and diluted samples. A-untreated pH 8.0, B-untreated pH 7.2, C-diluting the 1/4 eluted fraction with purified water (PuW), D-with PuW +1mM Ca ²⁺ Diluting 1/4 of the eluted fraction, E-with citrate buffer+1 mM Ca ²⁺ Diluting 1/4 of the eluted fraction, F-with citrate buffer+5 mM Ca ²⁺ Diluting 1/4 of the eluted fraction, G-with glycine buffer+1 mM Ca ²⁺ Diluting the eluted fraction by 1/4.

FIG. 8 shows FII and FV levels of loading and elution fractions from two different runs of an AEX column (Eshmuno Q), where A is the untreated elution fraction, B is the elution fraction diluted 1/4 with pH 7.2 buffer to a final concentration of 1mM CaCl ₂ And 1IU/ml heparin, C is an eluted fraction diluted 1/4 with buffer pH 7.2 to a final concentration of 1mM CaCl ₂ 1IU/ml heparin and 50mM glycine.

FIGS. 9A to 9C show the effect of calcium ions on recovery, FIGS. 9A and 9B show the levels of FV (FIG. 9A) and FIX (FIG. 9B) in the elution fractions of AEX column (Eshmuno Q) after 24 hours incubation at < 65 ℃, 2 ℃ to 8 ℃ and room temperature (left to right), untreated and with 50mM glycine solution pH 7.2 and 0.1mM to 5mM CaCl, respectively ₂ Dilution 1/4, T0 represents time 0; FIG. 9C shows the incubation with 50mM glycine solution pH 7.2 and 0.1mM to 5mM CaCl after 24 hours at 2℃to 8 ℃ ₂ Percent recovery of FV in the eluted fraction of the AEX column (Eshmuno Q) diluted 1/4.

Fig. 10 is a graphical representation of a method for lyophilizing an eluted fraction of AEX (Eshmuno Q) concentrated 17-fold from starting CDP material, showing shelf temperature (solid line) and pressure (dashed line).

Detailed Description

Blood coagulation is a tightly controlled cascade, comprising a series of reactions in which a coagulation factor in the form of a zymogen (an inactive enzyme precursor) is activated, ultimately leading to the formation of a blood clot. Thus, formulations comprising a coagulation factor, particularly in the form of a zymogen, may play an important role in initiating and mediating the blood clotting process. However, such preparations are neither readily available nor readily stable due to (inter alia) degradation and activation of the zymogen.

The present disclosure is based on the development of purified, highly stable and ready-to-use formulations comprising a coagulation factor in the form of an inactive zymogen. In particular, the inventors have surprisingly found that the presence of a stabilizing agent such as a divalent cation in a specific concentration range causes the coagulation factor, in particular factor V and vitamin K dependent coagulation protein, to remain in the zymogen form. The inventors have also found that most of these coagulation factors, some of which are known to be labile factors, can be maintained as zymogens in the formulation for at least 24 hours at about room temperature, specifically at a temperature of about 18 ℃ to about 26 ℃.

Thus, the inventors propose that the presence of a specific concentration of stabilizer does not promote activation of the coagulation cascade. After administration of the formulation, for example by intravenous administration, the coagulation cascade will be initiated, for example in the case of wounds.

Thus, according to some aspects, the present disclosure provides a formulation comprising concentrated factor V, at least one vitamin K-dependent coagulation protein, and at least one stabilizer, wherein the concentration of the at least one stabilizer is such that a majority of the factor V and/or the at least one vitamin K-dependent coagulation protein is present in the formulation in zymogen form.

Hereinafter, when referring to formulations, it is to be understood that the methods, kits and processes disclosed herein are also referred to. Thus, whenever a feature is provided in relation to a formulation, it is to be understood that the same feature in relation to the method, kit and process is defined, mutatis mutandis.

A formulation according to some embodiments of the present disclosure is understood to be a hemostatic, clot-forming formulation having a component that reacts upon contact with blood under certain conditions (e.g., the presence of a specific factor at the site of bleeding) to subsequently form an active component and thereby stop/arrest bleeding. In the context of the present disclosure, it will be appreciated that the formulation ingredients do not interact with each other and are essentially inactive, i.e., present in zymogen form, until they are brought into contact with blood under certain conditions, at which time clotting begins and blood clot formation.

The formulation may be in any form. According to some embodiments, the formulation is in liquid form. When referring to the liquid form, it is understood to be a form that is liquid at room temperature.

According to some embodiments, the formulation is solid, for example in powder form or lyophilized form. When in powder or lyophilized form, it is mixed with a suitable aqueous formulation such that, when used, the formulation is in liquid form.

In one embodiment, the solid composition is stable. By "stable" is meant herein that one or more of the 5 factors retain an activity equal to or greater than about 70% thereof, for example, upon storage for one month at room temperature.

For example, a solid composition reconstituted with an aqueous liquid is stable if one or more of the 5 factors retains about 70% or more of its activity when stored at room temperature for about 12 hours.

According to some embodiments, the formulation is in frozen form. When in frozen form, the formulation thaws at the time of use, being in its liquid form at room temperature.

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

As described herein, the formulation comprises a coagulation factor in the form of a zymogen. As used herein, a zymogen is understood to be an inactive precursor form of a factor (enzyme) that requires biochemical changes (such as hydrolysis reactions revealing active sites or changing conformation to reveal active sites) to become an active factor (enzyme). Since the coagulation factor is largely maintained/present in the formulation as a zymogen, the formulation is inactive, i.e., the activation of components in the formulation is minimal or non-existent.

The presence of the clotting factor in the zymogen form can be determined by testing the activity of the formulation under different conditions (e.g., different time points and different temperatures) using any method known in the art. Such methods include, for example, chromogenic kits such as HYPHEN ^TM Or clotting kits such as STAGO ^TM . The chromogenic assay uses the change in color when the substrate is cleaved by a factor acting as an enzyme, and the clotting assay uses the change in viscosity produced when fibrinogen is coagulated. For example, in a clotting assay, the activated partial thromboplastin clotting time (aPTT) can be used to test activity. aPTT is an example of a blood clotting test that measures how long it will take blood to form a clot. aPTT generally uses deficient plasma of a specific factor (i.e., without a specific factor) and uses normal plasma containing all factors close to 1IU/ml as a reference. For example, the lack of activation of the formulations described herein can be determined by using the unactivated partial thromboplastin time (natt) method, using phospholipids, and without using an activator (kaolin).

In some embodiments, the presence of a zymogen of factor V (i.e., lack of activity) and/or a zymogen of at least one vitamin K-dependent coagulation protein in the formulation is determined at a temperature of about 37 ℃.

In some embodiments, the majority of factor V and/or the at least one vitamin K-dependent thrombin is present in the formulation in zymogen form for at least 1 hour, sometimes at least 2 hours, sometimes at least 3 hours, sometimes at least 4 hours at a temperature of up to about 30 ℃.

In some embodiments, the majority of the factor V and/or the at least one vitamin K dependent thromboplastin is present in the formulation in zymogen form for at least 1 hour, sometimes at least 2 hours, sometimes at least 3 hours, sometimes at least 4 hours, at least one temperature of 0 ℃, 1 ℃, 2 ℃, 3 ℃, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃, 10 ℃, 11 ℃, 12 ℃, 13 ℃, 14 ℃, 15 ℃, 16 ℃, 17 ℃, 18 ℃, 19 ℃, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃ or 30 ℃.

In some embodiments, the majority of factor V and/or the at least one vitamin K-dependent thrombin is present in the formulation in zymogen form for at least 4 hours at a temperature of up to about 30 ℃, sometimes up to 26 ℃.

In some embodiments, the majority of factor V and/or the at least one vitamin K-dependent coagulation protein is present in the formulation in zymogen form for at least 4 hours at room temperature.

When referring to a factor in the zymogen form, it is understood as if the particular factor is at least partially in an inactive form, such that the amount/fraction of active form (if present) is insufficient to initiate activation of the formulation to form a blood clot. Typically, but not exclusively, when referring to factors in the zymogen form, factors having inactive forms of 70% and above are included. As described herein, such activation can be initiated upon contact with injured or exposed tissue enriched in negatively charged phospholipids and collagen. Such conditions activate tissue factors and initiate the complete coagulation cascade. For example, such treatment may stop massive hemorrhage, which is a blood loss sufficient to cause death, when administered into the blood circulation.

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

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

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

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

When referring to room temperature, it is noted that temperatures of up to 30 ℃, sometimes up to 26 ℃, sometimes up to 25 ℃, sometimes up to 24 ℃, sometimes between 20 ℃ and 26 ℃, sometimes between 23 ℃ and 26 ℃, sometimes between 17 ℃ and 26 ℃, sometimes between 20 ℃ and 23.5 ℃.

As described herein, the formulation comprises factor V ("FV"), also known as pre-accelerator, is a protein of about 330kDa produced in the liver. Factor V circulates in plasma as a single chain glycoprotein in the form of a zymogen, is essentially inactive in the blood clotting cascade, and requires first cleavage and activation to express procoagulant activity. Activated factor V is known 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 be involved in the synthesis of several proteins that mediate coagulation and anticoagulation. As used herein, vitamin K-dependent coagulation proteins refer to proteins that are normally synthesized in the liver and involved in the coagulation process.

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

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

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

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

Factor VII (FVII), also known as proconversion (EC 3.4.21.21), is activated to FVIIa by a different protease. The activated form of factor VII is referred to as factor VIIa.

Factor IX (FIX), also known as Christmas factor (EC 3.4.21.22), is a blood clotting factor, is a zymogen of a serine protease that is converted to an active serine protease upon activation and is expressed in Ca ²⁺ And the presence of membrane phospholipidsUnder this, activated factor IX (IXa) is formed. Thus, the activated form of factor IX is referred to as factor IXa.

Factor X, also known as Stuart protein factor (EC 3.4.21.6), is a serine endopeptidase and requires vitamin K for its synthesis in the liver. Factor X is activated to factor Xa by hydrolysis.

In some embodiments, the procoagulant 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 amount of each zymogen in the formulation may vary. As detailed herein, the formulation comprises a concentrated zymogen. As used herein, the term "concentrate" means that the amount of at least one zymogen in the formulation is at least 1 unit/ml zymogen, sometimes at least 2 units/ml zymogen. Typically, but not exclusively, concentration refers to a concentration of one or more of factor V, factor II, factor VII, factor IX and factor X equal to or greater than about 5 times its corresponding concentration in plasma.

In some examples, the formulation comprises at least 1IU/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 1IU/ml of factor IX.

Herein, "U" means the unit of blood coagulation factor, i.e., the unit of physiological measurement of blood coagulation factor in 1ml of normal human plasma.

"IU" refers to the international unit of coagulation factor, which is a physiological measurement in a given sample compared to an appropriate intentional standard, e.g., as determined by a coagulation assay performed with respect to an internal reference standard for potency concentration measurement that has been calibrated against, e.g., world Health Organization (WHO) second international standard 01/580 for thrombin.

In some embodiments, factor V is present in the formulation at a concentration of at least 20%, at least 30% of the concentration of the at least one vitamin K-dependent clotting protein.

In some other embodiments, the concentration of factor V present in the formulation 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, the concentration of factor V present in the formulation is at least 20% or at least 30% of the concentration of factor II.

In some embodiments, the concentration of factor V present in the formulation 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 of at least 20% or at least 30% of the concentration of factor IX.

In some embodiments, the concentration of factor V present in the formulation 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, at least one stabilizer is present in the formulation at a concentration that stabilizes (i.e., minimally degrades or does not degrade) and/or is inactive (i.e., in the form of a zymogen) the formulation components. In other words, the presence of the stabilizer may prevent or reduce degradation of the coagulation factor.

As understood, coagulation factors have a short half-life in plasma, for example 5h (FVII) or 100h (FII) or 12h to 36h (FV). However, it has surprisingly been found that the factor in the formulation is stable in the presence of a stabilizer, in particular at the concentrations described herein. The stability of the formulation (activity% of factor V) is determined during storage, in particular when the formulation is in liquid form, using coagulation and chromogenic as described herein after storage at a temperature below room temperature for at least 24 hours. In some examples, stability is measured at a temperature between about 2 ℃ and 8 ℃ and/or at about room temperature after storage for at least 24 hours. Typically, but not exclusively, the term stable refers to one or more of the 5 factors remaining equal to or greater than about 70% of their activity.

Furthermore, when referring to a stable formulation according to the present disclosure, it is also understood as a formulation whose clotting time is not or minimally influenced by its storage conditions at the time of use. In other words, in use, the formulation will set in substantially the same period of time, whether the formulation is stored at room temperature or at a lower temperature (e.g., between 1 ℃ and room temperature or between 2 ℃ and 8 ℃).

In some embodiments, the stabilizing agent is present at a concentration of less than 5 mM. In some other embodiments, the stabilizing agent is present at a concentration of about 0.1mM to about 5 mM. In some embodiments, the stabilizing agent is present at a concentration of about 0.1mM to about 3 mM. In some embodiments, the stabilizing agent is present at a concentration of about 0.1mM to about 2mM, sometimes about 0.1mM to about 1mM, sometimes about 0.5mM to about 3mM, sometimes about 0.5mM to 2 mM. In some embodiments, the stabilizing agent is present at a concentration of about 1 mM.

In some embodiments, the stabilizing agent comprises at least one divalent cation. Non-limiting examples of divalent cations include magnesium, calcium.

In some embodiments, the at least one stabilizer is calcium ions. In some embodiments, the calcium ion is free calcium ion. As understood, the term free calcium ion refers to ionized and non-sequestered calcium ions. It should be noted that the presence of the chelating agent may reduce the concentration of free calcium ions. The term free also applies to other divalent cations encompassed by the present disclosure that are non-chelating cations and thus free cations. The fine balance between chelating agent concentration and calcium and the ratio of chelating agent to calcium should allow free calcium concentrations in the range of above 0.1mM up to below 5 mM.

As shown in fig. 9C, in the absence of a stabilizer, in particular in the absence of free calcium ions, only 17% of factor V remains in stable form, i.e. undegraded, whereas by increasing the amount of stabilizer, in particular increasing the amount of free calcium ions, an increase is observed in the presence of factor V. 2mM stabilizer was added to maintain 80% of factor V. Without being bound by theory, the inventors propose that a particular concentration of stabilizer, i.e., free calcium ion, successfully maintains the stability and lack of activity of the component.

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

In some embodiments, the formulation comprises free calcium ions at a concentration of between about 0.1mM and about 5mM, sometimes between about 0.1mM and less than 5mM, sometimes between about 0.1mM and about 3mM, sometimes between about 0.1mM and about 2mM, sometimes between about 0.1mM and about 1mM, sometimes between about 0.5mM and about 3mM, sometimes between about 0.5mM and 2 mM.

In some embodiments, the formulation comprises free calcium ions at a concentration of about 0.1mM, sometimes about 0.3mM, sometimes about 0.5mM, sometimes about 0.8mM, sometimes about 1mM, sometimes about 1.3mM, sometimes about 1.5mM, sometimes about 1.8mM, sometimes about 2mM, sometimes about 2.5mM, sometimes about 3mM, sometimes about 3.5mM, sometimes about 4mM, or about 4.5 mM.

The formulation may also comprise anticoagulant proteins. In some embodiments, the anticoagulant protein is in the zymogen form, i.e., inactive anticoagulant protein.

In some embodiments, the anticoagulant protein is a vitamin K dependent anticoagulant protein.

In some embodiments, the anticoagulant protein is a non-vitamin K dependent anticoagulant protein.

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

In some embodiments, the anticoagulant protein is at least one of protein C or protein S.

In some embodiments, the anticoagulant protein is antithrombin III (AT III).

In some embodiments, the anticoagulant protein is AT least one of protein C, protein S, or AT III.

The formulation may contain 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 embodiments, at least one excipient is glycine. It is proposed that glycine can be added to the formulation because it is suitable as a non-chelating (as opposed to e.g. citrate having chelating activity) buffer, thus keeping the calcium ions in free non-chelating form, i.e. free calcium ions.

The clotting factors described herein, including, for example, factor V, at least one vitamin K dependent clotting protein, and an anticoagulant protein, may be isolated from human or mammalian blood or plasma, or may be recombinant factors.

In some examples, the clotting factors described herein are derived from blood, plasma, or any product/fraction thereof. As used herein, blood, plasma, or any product/fraction thereof refers to at least one of whole blood, fresh Frozen Plasma (FFP), platelet Rich Plasma (PRP), cryoprecipitated plasma (CDP), cryoprecipitated plasma, recovered plasma, or source plasma obtained from plasma apheresis.

As used herein, "freshly frozen plasma" refers to the fluid portion of a unit of human blood that has been centrifuged, separated, and frozen for solids. FFP contains all components (factors/proteins) of coagulation.

"platelet-rich plasma" is a concentrate of platelet-rich plasma proteins derived from whole blood and centrifuged to remove erythrocytes.

As used herein, "cryoprecipitated plasma" refers to a component made from FFP that remains after removal of cryoprecipitation from FFP. CDP includes a majority of coagulation factors.

As used herein, "cryoprecipitate" refers to precipitated plasma proteins obtained from a single unit of fresh plasma and thawed quickly. Cryoprecipitate is rich in factor VIII, factor XIII, von Willebrand factor and fibrinogen.

As used herein, "recovering plasma" refers to the fraction collected by whole blood donation and separated from its cellular components.

As used herein, "apheresis" refers to a device in which human blood is subjected to separation of a particular component and the remainder returned to the circulation.

In some embodiments, the clotting factor is derived from CDP.

In some examples, the factor V is derived from CDP.

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

In some examples, at least one anticoagulant protein is derived from CDP. In some examples, AT least one of protein C, protein S, or AT III is derived from CDP.

The formulations described herein are suitable for systemic administration. As used herein, "systemic administration" refers to the administration of a formulation directly into the central blood system intravenously, such that it enters the patient's system and is therefore subject to metabolism and other similar processes. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, typically by injection, and includes intravenous injection or intra-arterial administration.

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

As described herein, the formulations are useful for treating a variety of disorders associated with bleeding, such as various coagulation abnormalities, as well as spontaneous bleeding episodes, wounds, and surgical procedures.

Thus, according to some other aspects, the present disclosure provides methods for treating, inhibiting, preventing, or delaying bleeding or any disorder associated therewith.

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

In some embodiments, the method comprises administering to a subject in need thereof an effective amount of a formulation comprising factor V, at least one vitamin K-dependent coagulation protein, and at least one stabilizer, wherein the concentration of the at least one stabilizer in the formulation is such that the factor V and the at least one vitamin K-dependent coagulation protein are zymogens.

According to some embodiments, the methods of the invention comprise administering to a subject in need thereof an effective amount of a formulation comprising factor V, at least one vitamin K-dependent clotting protein, and at least one stabilizer at a concentration of less than about 5mM, sometimes between about 0.1mM and about 5mM, sometimes about 2mM.

According to some embodiments, the methods of the invention comprise administering to a subject in need thereof an effective amount of a formulation comprising factor V, at least one vitamin K-dependent coagulation protein, and free calcium ions at a concentration of less than about 5mM, sometimes between about 0.1mM and about 5mM, sometimes about 2mM.

According to some embodiments, the method of the invention comprises administering an effective amount of a factor V; the formulation of free calcium ions and at least one of factor II, factor VII, factor IX and factor X is administered to a subject in need thereof at a concentration of free calcium ions of less than about 5mM, sometimes between about 0.1mM and about 5mM, sometimes about 2mM.

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

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

In some embodiments, the methods of the invention comprise administering a formulation of the invention derived from at least one of whole blood, fresh Frozen Plasma (FFP), platelet Rich Plasma (PRP), cryoprecipitated plasma (CDP), cryoprecipitated plasma, recovered plasma, or source plasma obtained from plasma apheresis.

In some embodiments, the methods of the invention comprise administering a formulation derived from CDP.

It will be appreciated that the formulation of the invention comprises a zymogen and thus the formulation is inactive prior to its administration. The formulation is activated after administration to a subject by the methods described herein. In particular, as detailed herein, the formulations of the invention will be administered to a subject suffering from bleeding or any disorder associated therewith, such that upon administration as detailed herein, the zymogen may eventually be activated by an upstream coagulation factor in the normal flow of the coagulation cascade.

As used herein, the term bleeding or bleeding disorder refers to at least one hemostatic defect (also referred to as hemorrhagic diathesis) that results in an increased susceptibility to bleeding. According to some embodiments, the bleeding is associated with a hemostatic disorder.

In some embodiments, the methods of the invention may be useful for reversing massive hemorrhages. Major bleeding refers to blood loss to a degree sufficient to cause death.

In some embodiments, the methods of the invention may be useful for treating, preventing, arresting, preventing, ameliorating, inhibiting bleeding, hemostatic disorders, and any bleeding and disorders associated therewith in a subject in need thereof, the method comprising the step of administering to the subject a therapeutically effective amount of a formulation of the invention.

Bleeding disorders can be caused by a variety of physiological processes. For example, bleeding disorders may be caused by platelet disorders (primary hemostatic defects), coagulation defects (secondary hemostatic defects), or combinations thereof. Platelet disorders are often associated with skin mucosal hemorrhages (e.g., nose bleeding, petechiae, gastrointestinal hemorrhages), while coagulation defects are often associated with potential interstitial hemorrhages (e.g., joint bleeding, muscle bleeding).

In some embodiments, the methods of the invention may be applicable to subjects with bleeding disorders, which may be genetic or acquired bleeding disorders.

In some embodiments, the methods of the invention are applicable to subjects with acquired bleeding disorders.

Acquired bleeding disorders are caused by external stimuli. 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 methods of the invention may be useful for treating, inhibiting, preventing, or delaying bleeding associated with surgery.

In another embodiment, the methods of the invention are suitable for treating trauma induced hemorrhage. In some embodiments, the methods of the invention may be applicable to subjects suffering from a wound, such as a wound-induced coagulopathy.

In some embodiments, the methods of the invention may be applicable to subjects with bleeding disorders, which may be congenital bleeding disorders. As used herein, a "congenital bleeding disorder" relates to a congenital deficiency of at least one coagulation factor.

In some embodiments, the methods of the invention may be applicable to subjects having at least one of a factor I deficiency, a factor II deficiency, a factor V deficiency, a factor VII deficiency, a factor VIII deficiency (also known as hemophilia a), a factor IX deficiency (also known as hemophilia B), a factor X deficiency, a factor XI deficiency, a factor XII deficiency, or Von Willebrand Disease (VWD).

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

In some specific embodiments, the methods of the invention may be useful for treating, inhibiting, preventing, or delaying bleeding associated with factor XI deficiency.

In other embodiments, the methods of the invention may be useful for treating, inhibiting, preventing, or delaying bleeding associated with factor VII deficiency.

In another embodiment, the methods of the invention may be useful for treating, inhibiting, preventing, or delaying bleeding associated with factor X deficiency.

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

As used herein, "disease," "disorder," "condition," and the like, are used interchangeably and have the meaning given to each and all of such terms as they relate to the health of a subject. It is to be understood that the terms "associated" and "related," as they relate to pathologies herein, refer to a disease, disorder, condition, or any pathology having at least one of the following: sharing causal relationships coexist at a frequency higher than coincidences, or wherein at least one disease, disorder, condition or pathology causes a second disease, disorder, condition or pathology.

As described above, the present invention provides methods of treating the disorders specified above. As used herein, the term "treatment" refers to the administration of a therapeutic amount of a formulation of the present invention that is effective to ameliorate or avoid one or more undesirable symptoms associated with a disease or condition described herein.

As indicated above, the methods of the invention comprise administering a therapeutically effective amount of a combination of the invention. The term "effective amount" or "therapeutically effective" for purposes disclosed herein means an amount of the formulation effective to treat, inhibit or delay one or more symptoms of the diseases described herein.

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

It should be understood that the methods of the present invention are not limited to any route of administration. In particular, the formulation may be administered systemically. In some embodiments, the method comprises systemically administering the formulation of the invention. In some other embodiments, the method comprises injecting or infusing the formulation.

According to some other aspects, the present disclosure provides a formulation for use in treating, inhibiting, preventing or delaying bleeding or any disorder associated therewith. Thus, according to some embodiments, the formulation of the invention may be associated with treating bleeding in a subject in need thereof, wherein the formulation comprises factor V, at least one vitamin K-dependent coagulation protein, and at least one stabilizer, wherein the concentration of the at least one stabilizer in the formulation is such that the factor V and the at least one vitamin K-dependent coagulation protein are zymogens. In some embodiments, the formulations of the present invention are suitable for systemic use.

Furthermore, the present disclosure provides kits (packages). 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 concentration of less than about 5mM, sometimes between about 0.1mM and about 5mM, sometimes about 2mM.

According to some embodiments, the kit of the invention is used in a subject having bleeding or any disorder associated therewith as described above. It will also be appreciated that the kits of the invention may also be used to prevent bleeding in subjects having an increased risk of bleeding.

In some embodiments, the kit further comprises instructions for using the formulation to treat, inhibit or delay bleeding or any disorder associated therewith.

In some embodiments, the instructions include administration of the formulation in liquid form.

In some embodiments, the kits of the invention may be particularly suitable for systemic use. Thus, the instructions include administration of the formulation by systemic administration.

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

Another aspect of the invention relates to a method of preparing the formulation. In particular, the methods of the invention comprise the step of subjecting the blood or plasma described herein to at least one separation step followed by recovery of the formulation of the invention.

Thus, the present disclosure provides a method of preparing a formulation comprising concentrated factor V in zymogen form and at least one vitamin K-dependent coagulation protein.

The method may include the step of subjecting at least one of whole blood, fresh Frozen Plasma (FFP), platelet Rich Plasma (PRP), cryoprecipitated plasma (CDP), cryoprecipitated plasma, recovered plasma, or source plasma obtained from plasma apheresis to at least one separation step.

As used herein, at least one separation step 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 method may include using a solid phase surface capable of binding at least a portion of the plasma/blood components and thereby separating the plasma/blood components from each other.

The at least one separation step may comprise chromatography, centrifugation, or any combination thereof.

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

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

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

Regardless of which chromatographic method is used, separation can be performed by a column set-up, a batch set-up, or any combination thereof, such as a mixing set-up. As will be appreciated, in a column arrangement, a solid medium is packed onto the column and the mixture, such as plasma, is allowed to move through the column to allow sedimentation, wash buffer flows through the column and then elution buffer is applied to the column and collected. The batch setup included the following: adding an initial mixture, such as a plasma sample, to the solid phase in the container, mixing, separating the solid phase, removing the liquid phase, washing, re-centrifuging, adding an elution buffer, re-centrifuging and removing the elution buffer.

In one embodiment, the chromatography step comprises ion exchange chromatography. The chromatographic method is based on electrostatic interactions between charged protein groups and solid support materials (matrices). Ion exchange chromatography includes anion exchange chromatography using positively charged resins while adsorbing negatively charged components and cation exchange chromatography, and vice versa.

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

Thus, according to some examples, the method comprises subjecting at least one of whole blood, FFP, PRP, CDP, cryoprecipitated plasma, recovered plasma, or source plasma obtained from plasma apheresis to at least one chromatography step, specifically to at least one anion exchange chromatography step, and collecting the eluate into a solution comprising a stabilizing agent.

Additionally or alternatively, the method comprises subjecting at least one of whole blood, FFP, PRP, CDP, cryoprecipitated plasma, recovered plasma or source plasma obtained from plasma apheresis to at least one chromatography step, in particular to at least one anion exchange chromatography step, and transferring the eluate into a solution comprising a stabilizing agent.

In some embodiments, the stabilizing agent is present in the collection solution at a concentration such that the coagulation factor recovered by the methods of the invention is in the zymogen form. In some embodiments, the collection solution (also sometimes referred to herein as a formulation of the invention) comprises at least one stabilizer at a concentration of up to about 5 mM.

In some embodiments where the collection solution does not contain a stabilizer, the eluate is immediately transferred to a storage solution containing at least one stabilizer at a concentration of up to about 5 mM. According to such embodiments, the storage solution is referred to herein as the formulation of the present invention.

The inventors have surprisingly found that components of the eluate in the form of zymogens can be successfully recovered by subjecting a plasma fraction such as CDP to at least one chromatographic separation step such as anion exchange chromatography and collecting the eluate into a formulation comprising a specific concentration of a stabilizing agent as described herein.

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

In some examples, the method includes collecting the eluate into a formulation comprising free calcium ions at a concentration of less than about 5 mM. In some examples, the method includes collecting the eluate into a formulation including free calcium ions at a concentration between about 0.1mM and less than about 5mM, sometimes between about 0.1mM and about 3mM, sometimes between about 0.1mM and about 2mM, sometimes between about 0.1mM and about 1mM, sometimes between about 0.5mM and about 3mM, sometimes between about 0.5mM and 2 mM.

In some embodiments, the method comprises collecting the eluate into a formulation comprising calcium ions at a concentration of about 0.1mM, sometimes about 0.3mM, sometimes about 0.5mM, sometimes about 0.8mM, sometimes about 1mM, sometimes about 1.3mM, sometimes about 1.5mM, sometimes about 1.8mM, sometimes about 2mM, sometimes about 2.5mM, sometimes about 3mM, sometimes about 3.5mM, or about 4mM.

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

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

The method of the present invention may comprise additional steps. In some embodiments, the method includes the step of washing the bound fraction with a wash solution before eluting the bound fraction with the elution solution into the collection solution.

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

As used herein, the term "about" indicates a value that may deviate by at most 1%, more specifically 5%, more specifically 10%, more specifically 15% and in some cases at most 20% from the mentioned value, the range of deviations includes integer values and, if applicable, non-integer values, thereby constituting a continuous range. As disclosed and described, it is to be understood that this invention is not limited to the particular examples, method steps, and compositions disclosed herein, as such method steps and compositions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It should 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 the techniques employed by the inventors in practicing aspects of the present invention. It should be understood that while these techniques are examples of preferred embodiments for practicing the invention, those skilled in the art will recognize from this disclosure that many modifications may be made without departing from the spirit and intended scope of the invention.

Non-limiting examples

Test method for determining coagulation factors

The clotting assay (modified activated partial thromboplastin time) was used to determine the procoagulant concentration in the samples tested.

These assays determine specific procoagulant concentrations for both exogenous and endogenous pathways by measuring clotting time of a test sample and using specific associated deficient plasma. Using

(Stago) coagulometer the clotting time was measured according to the manufacturer's instructions.

Determination of common pathway procoagulant concentrations (FII and FV)

The determination of procoagulant concentrations (FII and FV) was performed according to the manufacturer's instructions (Stago Start-4 SOP ref 26987). The principle of the test consists in measuring the clotting time of the tested sample using calcium thromboplastin (tissue factor) and comparing it with the normal standard (Uni-calibrator catalog number 00625)) Is compared to the set time of (c). Specific factor concentrations were determined using the relevant specific deficient plasma (i.e., deficient in factor II and deficient in factor V). The lack of plasma provides an excess of all factors except the tested factor provided by the sample being tested. The concentration (%) of the tested factor was interpolated from a calibration curve drawn by diluting unicalifier in Owren-Koller buffer (catalog No. 00360) to obtain the following dilutions: 1:10, 1:20, 1:40 and 1:80. The samples were diluted according to the estimated concentration in order to fit the calibration curve range. For results

Expressed as a percentage of normal plasma activity. The calibration curve and sample were assayed and repeated. The results of the duplicate preparations for each sample need to be in the 10% RSD range. The test was performed at 37 ℃.

Determination of intrinsic pathway procoagulant concentration (FIX)

Determination of the concentration of endogenous pathway procoagulant (FIX)

The kit (catalog number 00847) was carried out according to the manufacturer's instructions (Stago Start-4 SOP ref 26987). The test involves recalcification of plasma in the presence of standard amounts of cephalin (platelet substitute) and a specific activator. This procedure minimizes the test variables by normalizing contact activation and optimizing the concentration of platelet-like phospholipids. The clotting time of the tested sample was measured and compared to the clotting time of a normal standard (unicalifier catalog number 00625). The concentration of the specific factor was determined using the relevant specific deficient plasma (i.e. deficient in IX). The remainder of the assay was performed as described above for the common pathway factor.

Determination of procoagulant inhibitor concentration (protein S)

Determination of procoagulant inhibitor concentration (protein S) use

Root of protein S kit (catalog number 00746)According to the manufacturer's instructions (Stago Start-4 SOP ref 26987). The principle of this assay is based on the cofactor activity of protein S, which enhances the anticoagulant effect of activated protein C. This enhancement is manifested by an extended clotting time in the factor Va-rich system, factor Va being a physiological substrate for activating protein C. Assays were performed as described above for common pathway factors.

Determination of procoagulant concentration in a sample tested using chromogenic assay

These assays determine the specific procoagulant concentrations of FVII, FX, protein C and antithrombin III by using a chromogenic method according to the EU pharmacopoeia. OD was measured using ELISA reader.

Determination of FVII concentration

Factor VII was determined using the biphen FVII kit (catalog No. 221304) according to the manufacturer's instructions (Hyphen Biomed). In the presence of tissue factor (rabbit thromboplastin) and calcium, factor FVII forms an enzyme complex that activates factor X to factor Xa, both present at a constant concentration and in excess. The amount of factor Xa formed depends on the concentration of factor VII to be determined. The formed factor Xa cleaves the specific substrate Xa (SXa-11) and releases p-nitroaniline (pNA). The amount of pNA produced is directly proportional to factor Xa activity. The amount of factor VII in the sample determined is proportional to the activity of factor Xa produced, as measured by the amount of pNA released, as determined by color development at 405 nm.

Determination of FX

The determination of factor X was performed using the BIOPHEN FX kit (catalog number 221705) according to the manufacturer's instructions (Hyphen Biomed). Factor X is measured after specific activation with RVV, an enzyme extracted from snake venom (Russell viper venom), using a BIOPHEN factor X assay. The activated factor X (FXa) then specifically cleaves the Jie Teding substrate SXa-11, releasing p-nitroaniline (pNA), the color of which is measured at 405 nm. There is a direct relationship between color development and factor X activity in the plasma tested.

Determination of protein C

Protein CDetermining usage of BIOPHEN ^TM Protein C (LRT) (catalog number 221211-RUO) was performed according to the manufacturer's instructions (Hyphen Biomed). Using this assay, in use

Measuring protein C, < > -after specific activation>

Is an enzyme extracted from snake venom (Agkistrodom C Contortrix). The Activated Protein C (APC) then specifically cleaves the Jie Teding substrate SaPC-21 releasing p-nitroaniline (pNA), the color of which is measured at 405 nm. There is a direct relationship between color development and protein C activity in the plasma tested.

Determination of Antithrombin (AT)

Determination of antithrombin Using BIOPHEN ^TM Antithrombin (catalog No. 221102) was performed according to manufacturer's instructions (Hyphen Biomed). BIOPHEN ^TM Antithrombin assays are based on a kinetic approach to inhibit constant concentration and excess FXa by AT in the presence of heparin. The remaining FXa is then measured by its amylase activity on FXa specific chromogenic substrate, which releases pNA. The amount of pNA produced is inversely proportional to the concentration of AT present in the plasma tested. The assay is insensitive to heparin.

Determination of heparin (UFH)

Heparin determination Using BIOPHEN ^TM ANTI-Xa (stage 2 heparin assay) (catalog No. 221005) was performed according to manufacturer's instructions (Hyphen Biomed). The kit is a chromogenic anti-Xa method developed for measuring the anti-Xa activity of unfractionated heparin (UFH) in plasma or purified solutions. BIOPHEN ^TM ANTI-Xa (2-stage heparin assay) assays are based on the inhibition of a constant amount of factor Xa (FXa) by the tested heparin in the presence of exogenous antithrombin (stage 1), and the hydrolysis of a factor Xa specific chromogenic substrate (CS 11 (65)) by excess factor Xa (stage 2). The pNA is then released from the substrate. The amount of pNA released (measured at 405 nm) is then related to the residual factor Xa activity. The concentration of heparin is inversely related to the color development.

Other tests performed

In addition to the clotting factors, other parameters of the samples during formulation were tested as detailed below.

Total protein

For the total protein assay, protein concentration was determined using the biuret method (SOP# TM-98211). In this assay, the copper ions of the basic biuret reagent form a purple complex with serum proteins, the maximum absorption of which is at 540 nm. The color intensity is proportional to the protein concentration.

Batch screening study

The following experiments were aimed at determining the ability of various anion exchange resins to bind and elute FII, FV, FVII, FIX, FX, protein C, protein S and ATIII with high efficiency.

EXAMPLE 1 characterization of anion exchange resin

At different pH values: resins were tested at 7.0, 7.5, 8.0 and 8.5 as two strong anion exchange resins and two weak anion exchange resins detailed in table 1. Cryoprecipitated plasma (CDP) produced by Omrix (Plasma Fractionation Institute, tel-hashimer, israel) was used as starting material for the screening process. Protein concentration: 47mg/ml protein. CDP batch #X22Y730 was thawed, filtered through a 0.8/0.2 μm PES filter and aliquoted. The pH of the aliquots was adjusted to 4 different pH values: 7.0, 7.5, 8.0 or 8.5. The aliquots were then re-frozen and thawed prior to 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, 1M NaCl, pH 7.0.

Table 1 resins tested

Pretreatment of Fractogel TMAE (M) and GigaCap DEAE-650M resins：

The resin was washed with 3CV (column volume) of purified water (PuW) and 3CV of 1M NaCl. These steps were performed by rolling at Room Temperature (RT) for 5 minutes and then standing until complete decantation was obtained.

Incubation and elution：

150 μl (considered 1 CV) of each resin was used for the experiment. Each process was repeated twice and the samples generated in each step were pooled prior to testing. During the loading, washing and elution steps shown in table 2A, the resin was incubated with sample/buffer at each of 4 different pH values (7.0, 7.5, 8.0 and pH 8.5). Each tested resin was rolled for 1 minute and then centrifuged at 500g for 1 minute, after which the supernatant was collected except for the equilibration step, where it was discarded.

Table 2A resin batch screening method

Step (a)	Step name	Column volume	Reagent/buffer name
				1	Washing of resin storage solutions	2x6.7	PuW
2	Balancing	2x10	10mM sodium citrate buffer
				3	Load(s)	6	CDP, lot #x22y730 ×
4	Washing	6.7	10mM sodium citrate buffer
				5	Elution 1	2	0.25M NaCl+20mM sodium citrate pH 7.0
6	Elution 2	2	1M NaCl+20mM sodium citrate pH 7.0

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

Sample of：

Samples were taken from the supernatant after the centrifugation step, unbound (1.8 ml each), wash (2 ml), and eluates 1 and 2 (0.6 ml each). Duplicate samples were combined into one sample, which was then divided into 4 aliquots and stored at < -65 ℃ for testing.

Pretreatment of Capto DEAE and UNOsphere Q resins：

Details of this approach are provided in table 2B. Cryoprecipitated plasma (CDP) produced by Omrix (Plasma Fractionation Institute, tel-hashimer, israel) was used as starting material for the screening process. Protein concentration: 47mg/ml protein. CDP batch #X22Y730 was thawed, supplemented with 1IU/ml heparin, filtered through a 0.8/0.2 μm PES filter and aliquoted. The pH of the aliquots was adjusted to two different pH values: 7.0 and 8.0. The aliquots were then re-frozen and thawed prior to each assay. Equilibration buffer: 10mM citrate, pH was adjusted to 7.0 and 8.0. Elution buffer: 10mM sodium citrate, 1M NaCl, pH 8.0.

0.5ml (considered 1 CV) of each resin was taken for the experiment. Each process was repeated twice and the samples generated in each step were pooled prior to testing. Screening was performed at two different pH values (7.0 and 8.0). For steps 1 to 3 (see table 2B), the incubation was performed for 1 minute, then the sample was left to stand until complete decantation of the resin was obtained. During loading, washing and elution (steps 4 to 6), the sample/buffer was incubated for 1 min while rolling, and then centrifuged at 500g for 1 min, and the supernatant was collected.

Table 2B resin batch screening method

Step (a)	Step name	Column volume	Reagent/buffer name
				1	Washing of resin storage solutions	2×20	PuW
2	Preconditioning of	20	1M NaCl
				3	Balancing	3×20	10mM sodium citrate
4	Load(s)	9	CDP,1IU/ml heparin
				5	Washing	4	10mM sodium citrate
6	Elution	4	1M NaCl+10mM sodium citrate pH 8.0

* Solutions adjusted to pH 7.0 and 8.0

Samples were collected from the supernatant after centrifugation, unbound (5 ml), washed and eluted (2 ml each). Duplicate samples were combined into one sample, divided into 4 aliquots and stored at < -65 ℃ for testing.

Results

Recovery of all factors and inhibitors is summarized in table 3. According to the results obtained, all FII, FVII, FIX and FX were mainly present in the eluted fraction, whereas FV appeared to have eluted in the Unbound (UB) fraction, and a small amount was present in the eluted fraction, regardless of the pH used in the process.

TABLE 3 percent recovery of coagulation factors and inhibitors using different resins at pH 7.0 to 8.5*

* Recovery was calculated as the combined activity of

elution

1 and 2 divided by the activity in the sample, in percent (%). ATIII = antithrombin III. N/D = undetermined.

Fig. 1 shows the levels of procoagulants and inhibitors in the eluted fractions of each of the different resins studied, expressed in units of activity loaded per milliliter at pH 8.0. FII, FVII, FIX and FX levels are generally satisfactory, however FV is present at very low levels <0.10 IU/ml) where a significant portion is not bound to the resin (present in the UB fraction) and another portion may have been degraded during purification.

Based on a summary of the results in FIG. 1, it was shown that all four resins D-Fractogel TMAE, F-Gigacap DEAE, I-Capto DEAE and M-UNOspheeQ were good at pH 8.0, with Fractogel TMAE and UNOspheeQ being strong anion exchange resins and Gigacap DEAE and Capto DEAE being weak anion exchange resins. This suggests that the strength of the resin is not a critical parameter in obtaining the various components from plasma.

EXAMPLE 2 AEX resin for column chromatography Performance analysis

To further analyze the properties of the resin, fractogel TMAE (M), gigacap DEAE, capto DEAE and Unosphere Q were packed into columns 1.0cm wide by 20cm high (15 ml to 16 ml). In addition, the strong anion exchange resin Eshmuno Q (Merck) was also tested. Eshmuno Q was packed into a 20cm high X2.6cm diameter (-100 ml) column. In these runs, the maximum pressure during the run and the purification performance of FII, FV and FIX were studied. Table 4 shows the packing parameters of the columns used for the study.

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

The method varies slightly from run to run, however all runs include the following steps/conditions:

1) The run was performed at room temperature ("RT").

2) 1IU/ml heparin was added to CDP (loading material), filtered through PES 0.2 μm 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 was performed with 2CV to 5CV PuW.

5) With equilibration buffer (20 mM sodium citrate, 5mM CaCl ₂ Except for C55R1 (see table 5), where no equilibration was performed with buffer) until the effluent reached buffer pH (all runs were 7.0 except for C67R1, which was pH 8.0). In some cases, this pH is not reached until CDP begins to load.

6) Loading (see line speed in table 5).

7) A3 CV to 4CV rinse was performed with equilibration buffer.

8) Elution was performed with 1M NaCl+20mM sodium citrate.

9) Regeneration method (150 cm/h): 1M NaCl 3CV, 0.5M HCl 2CV, puW CV, 0.5M NaOH 2CV, naOH incubation for 60 minutes, 1M NaCl 3CV, puW CV, 20% to 25% ethanol 2CV.

10 All steps were performed automatically using AKTA Avant system under monitoring at 280 nm.

The hardware used by Fractogel TMAE (M), gigacap DEAE, capto DEAE and Unosphere Q, tricore 10x200mm,Eshmuno Q, was Hi Scale 26.

Results

TABLE 5 20cm AEX pressure Properties

* Maximum pressure >1.0 bar

FIG. 2 shows the activities of FII, FV and FIX in the elution fractions of 5 different packing resins.

FII was shown to have high recovery levels in all runs, ranging from 73% in Capto DEAE to 93% in Unosphere Q, 30 CV.

On the other hand, FV recovery varies from run to run, being the lowest level (23%) in Capto DEAE elution and the highest level (97%) in Eshmuno Q100 ml. It is worth mentioning that for the latter, 1mM calcium was added to the eluted fraction.

In addition, fractogel TMAE showed very high levels, with FV of 57% (15 CV) and 88% (17 CV). In all other runs FV is in the range between 30% and 35%. Finally, high levels of FIX, except Capto DEAE, were found in at least one run of each resin, with only 14% detected. The highest value was observed with Eshmuno Q.

Based on these results, it can be concluded that: all resins Fractogel TMAE (M), gigacap DEAE, unosphere Q and Eshmuno Q are suitable for purification of a set of clotting factors.

Example 3 for Eshmuno Experimental design of Q chromatograph (DOE)

The purpose of this experiment was to analyze the effect of pH, flow rate and temperature on the performance of one of the resins tested. The Eshmuno Q column was used as an exemplary resin to purify the complete set of proteins for formulation from CDP. Experiments were performed with Eshmuno Q using a pre-packed column with dimensions of 8X 100mm (5 ml CV). Three variables were chosen for this purpose: chromatographic temperature, equilibration buffer and pH of loading, and linear speed of loading step. Eight runs were performed in combination with extrema:

1) Equilibration buffer and pH of loading: 7.0 and 8.5.

2) Column temperature during loading: on ice or at 23 ℃.

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

Additional runs were performed at pH 7.5, 240cm/h and 23℃ (# 9) in order to confirm that no reuse problems were found in the column under investigation and that there were additional values at intermediate condition values. The data are shown in table 6. All other conditions were kept constant except for the variables studied, except that another CDP batch was used during run 6 of the DOE. The levels of coagulation factors are compared and the effect of each of the variables is determined individually and in combination. In addition, protein recovery was analyzed.

TABLE 6 design of experiments (DOE) plan

Preparation of test materials

On the day of the experiment, 50ml aliquots containing 1IU/ml heparin were thawed at 23 ℃, filtered through a 0.2 μm polyethersulfone filter (Pall), pH adjusted according to the experimental design (pH 7.0, 7.5 or 8.5) and kept on ice before and during loading onto the column.

Chromatographic method

The starting material was loaded into Eshmuno Q. For column equilibration, 2CV of purified water and 10CV of equilibration buffer (20 mM sodium citrate, 5mM CaCl) ₂ Ph=7.0, 7.5, 8.0 or 8.5) was passed through a 5ml column (8×100 mm) and then 15CV CDP at ph=7.0, 7.5, 8.0 or 8.5 was loaded into the column at 120cm/h, 240cm/h or 480cm/h. The column was washed with 4CV equilibration buffer and then eluted with a pH7.0 solution containing 1M NaCl and 20mM citrate for 3CV. 3CV eluate (. About.15 ml) was collected to 45ml containing CaCl ₂ In pH 7.2 solutions of glycine and heparin, the target final concentration was 1mM CaCl ₂ 50mM glycine and 1IU/ml heparin.

Results

General information of the operation is as described in table 7. All steps were performed automatically using the AKTA Avant system under monitoring at 280 nm. The observed protein recovery was satisfactory in all runs, ranging from 89% to 97% (table 7). Low speed (120 cm/h) exhibited the lowest value (89% to 92%) while run #2 exhibited the highest value of 97%. The eluted fraction exhibited a variable absolute protein value of from 11.5mg to 39.1mg, with the lowest value observed at pH 7.0, which means that less contaminant was bound at pH 7.0 despite higher procoagulant recovery was observed.

Table 7 fraction weight and total protein recovery for each run

Run #, run #	UB weight (g)	Weight of eluent (g)	UB protein recovery
				1	85.5	15.3	91％
2	84.7	15.3	97％
				3	82.8	15.6	89％
4	83.7	15.8	89％
				5	82.9	15.7	89％
6	87.0	15.5	92％
				7	86.6	15.6	93％
8	85.2	15.7	91％
				9	87.1	15.6	94％

* Total protein determined as described above.

Coagulant recovery and analysis

FV recovery was particularly high (67% to 101%) when working at pH 7.0 compared to the results at pH 8.5 (41% to 65%). Although FII exhibited the best results at pH 7.0 (

run #

2, 94%), acceptable values were found throughout the DOE.

The remaining procoagulants and inhibitors exhibited high values that were generally consistent except for antithrombin III (in all runs < LOD), which eluted predominantly in the UB fraction under all study conditions. Finally, run #9 showed excellent FV and protein C levels at intermediate pH and speed values (pH 7.5 and 240 cm/h), and a FII recovery of 68%, which was an acceptable value. Fig. 3 shows the percent procoagulant and inhibitor recovery (elution/loading) determined in elution fractions for different runs in DOE for Eshmuno Q. The effect of different conditions on the binding of the formulation to Eshmuno Q was studied in the DOE context. Run #2 shows the best combined recovery corresponding to the lowest pH, high temperature and highest velocity.

Example 4: stability study

The coagulation factors in plasma are known to have half-lives between 5 hours (FVII) and 100 hours (FII), however, stability is significantly reduced in the eluted fraction of the AEX column, especially FV, which presents challenges to the purification process. In addition, calcium, mannitol, maltose, sucrose, glycerol and glycine were tested when incubated at different temperatures for 24 hours, and after freeze-thaw cycles at < 65 ℃. Furthermore, the optimal pH was analyzed in this study.

Example 4A-pH optimization

The following experiments were aimed at determining the effect of pH in the range of 6.7 to 7.7 in order to determine the optimal pH for FII and FV stabilization. The experiment used an eluted fraction of a chromatographic run in Eshmuno Q2 ml column, wherein 25ml of CDP (batch # X33Y 140) was loaded into the column and the eluted fraction was stored at < 65 ℃. The eluted samples were 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 (24 hours) at two different temperatures: a1, B1, C1 at room temperature, and A2, B2, C2 at 2 ℃ to 8 ℃. In addition, untreated controls (without pH adjustment) were stored at less than or equal to-65℃for 24 hours.

Results

FII and FV levels are shown in fig. 4A and 4B, respectively. As shown in FIG. 4A, the FII in the control was 0.77IU/ml. Samples at pH 6.7 showed similar values to the control at room temperature, while increasing pH appeared to improve stability, reaching 0.97IU/ml and 1.15IU/ml at pH 7.2 and 7.7, respectively. On the other hand, no significant trend was observed throughout the pH range when incubated at 2℃to 8℃with values of 0.90IU/ml, 0.77IU/ml and 0.85IU/ml at pH 6.7, 7.2 and 7.7, respectively. As shown in FIG. 4B, the control FV concentration was 0.35IU/ml, indicating that the freeze and thaw cycles have a major effect on FV stability. Here, the low pH seems to be able to optimally prevent degradation at room temperature, although this value is half that of the control (0.14 IU/ml). Higher pH values showed that degradation at room temperature was not prevented. During incubation at 2 ℃ to 8 ℃, FV was completely degraded over the entire pH range. This experiment shows that a stabilizer for FV is required in order to keep the factor present until the end of the production process.

EXAMPLE 4B Effect of temperature on stability

The purpose of this study was to determine how the stability of the eluted sample obtained from the AEX column was affected by the storage temperature.

The results of this study are shown in fig. 5. FII and FV levels were analyzed in the fresh state and after 24 hours at three different temperatures: the effects of 2 freeze-thaw cycles at-65 ℃, 2 ℃ to 8 ℃ and Room Temperature (RT), inclusive.

Chromatography (C65R 2) was performed using a 1ml column pre-packed with Fractogel TMAE (M) Medcap at pH 8.0. CDP (lot #x33Y 140). 1IU/ml heparin was added to the sample material, filtered through PES 0.2 μm Supor filter, and the pH was adjusted to 8.0. 32.5ml was loaded onto the column and bound material eluted with 1M NaCl+20mM sodium citrate buffer. The collected 3.5CV eluate was eluted with a solution containing sodium citrate at a concentration of 20mM and CaCl at a concentration of 1mM ₂ 1/5 dilution was performed in pH 7.2 buffer. In this eluted sample, the concentration factor (loading volume/elution volume) was 1.7. This eluate of fresh FII and FV after 24 hours at different temperatures was tested:

a) Fresh (same day after collection test)

B) 24 hours at minus 65℃ (1 st freeze-thaw cycle)

C) At-65℃for a further 24 hours (2 nd freeze-thaw cycle)

D) At 2 ℃ to 8 ℃ for 24 hours

E) 24 hours at room temperature

Results

The loading exhibited FII of 1.06IU/ml, from which 0.56IU/ml was recovered in fresh eluent. When the samples were incubated at 2℃to 8℃and room temperature for 24 hours, FII of 0.64IU/ml and 0.69IU/ml was obtained, slightly higher than for fresh samples. The 24 hour freeze-thaw cycle exhibited 0.64IU/ml and 0.55IU/ml in succession, showing a degradation trend equivalent to a decrease of 0.1IU/ml per cycle. On the other hand, FV of 0.76IU/ml was obtained in the sample, from which only 0.33IU/ml was recovered, representing less than half of the activity. Samples incubated for 24 hours at 2℃to 8℃and room temperature exhibited almost complete degradation of FV, 0.12IU/ml and 0.10IU/ml, respectively. It can be shown that due to the presence of a chelating agent such as sodium citrate, 1mM CaCl is added to the eluate ₂ Is insufficient to protect FV. The 24 hour freeze-thaw cycle exhibited 0.21IU/ml and 0.11IU/ml in succession, showing a similar degradation trend as FII, decreasing by 0.1IU/ml per cycle (FIG. 5).

Example 4C-Effect of freeze-thaw cycle with 10% sugar on FII and FV levels

The purpose of this study was to investigate the effect of different sugars after two freeze-thaw cycles at < -65 ℃ on the stability of FII and FV in eluted samples obtained from AEX columns. The fresh eluted fractions were tested for FII and FV with and without the eluted fractions after a 24 hour freeze-thaw cycle of 10% sugar.

A) Fresh and fresh

B) 1 cycle control (sugar-free)

C) 2 freeze-thaw cycle controls at-65℃or less (sugar-free)

D) 2 cycles at-65 ℃ and 10% mannitol

E) 2 cycles at-65℃or less, containing 10% maltose

F) 2 cycles at-65 ℃ and 10% sucrose

F) 2 cycles at-65 ℃ and containing 10% glycerol

Results

There was no significant difference when analyzing the FII levels of the fresh eluted fraction and the eluted fraction after two consecutive 24h freeze-thaw cycles at < 65℃where the fresh sample exhibited 0.56IU/ml and 0.51IU/ml and 0.58IU/ml were observed after 1 and 2 cycles, respectively, where the loading exhibited 1.06IU/ml. When the sample was treated with mannitol, the activity dropped to 0.46IU/ml after 2 freeze-thaw cycles, while the other sugars were in the range of 0.58IU/ml to 0.60 IU/ml.

FV, on the other hand, was significantly affected by the first freeze-thaw cycle, decreasing from 0.33IU/ml to 0.16IU/ml in the fresh sample. The second cycle exhibited 0.14IU/ml, slightly lower than the previous cycle. This degradation was observed in the same measurement when 10% of any sugar studied was used, with values ranging from 0.12IU/ml (mannitol) to 18IU/ml (glycerol) (fig. 6).

EXAMPLE 5 pH with calcium 7.2 dilution with buffer 1/4

The purpose of this experiment was to investigate the effect of diluting the eluted fractions obtained from the AEX column with PuW, citrate and glycine buffer containing a small amount of CaCl ₂ The pH is in the range of 7.0 to 7.3, and is used as a potential stabilizer for FV. After incubation at T-0 and at < 65 ℃, 2 ℃ to 8 ℃ and room temperature for 24 hours, the untreated samples and the samples diluted in different solutions were tested for FII and FV.

Chromatography was performed using 5ml columns pre-packed with Eshmuno Q at pH 8.0 (C63R 2). 1IU/ml heparin was added to CDP loading material, filtered through PES 0.2 μm Supor filter, and pH was adjusted to 7.9. 75ml CDP (batch #X33Y 140) was loaded into the column and 86ml UB (which included a 2CV rinse) was collected. Bound material was eluted with 1m nacl+20mm sodium citrate buffer, and the volume was then divided into 7 samples:

a) Untreated (pH 8.0)

B) Untreated, adjust to pH 7.2

C)PuW

D)PuW+1mM CaCl ₂

E) 20mM citrate+1 mM CaCl ₂

F) 20mM citrate+5 mM CaCl ₂

G) 50mM glycine+1 mM CaCl ₂

All samples were incubated at 3 different temperatures for 24 hours: the additional samples (T-0) were tested at-65℃and 2℃to 8℃and at room temperature on the same day.

Results

The starting material exhibited FII at 1.14IU/ml and FV at 0.80 IU/ml.

The untreated FII sample should contain the same composition as the buffer in which it is eluted, i.e., 20mM citrate+5 mM CaCl ₂ . When the pH is maintained at pH 8.0 (A), the sample exhibits very similar values (values between 0.90IU/ml and 0.97 IU/ml) when incubated for 24 hours at different temperatures compared to T-0 (0.96 IU/ml). The pH adjustment to 7.2 (B) showed no significant improvement, had a lower value at T-0 (0.83 IU/ml), and was found to be very low<Has values of 0.92IU/ml, 0.82IU/ml and 1.01IU/ml respectively after 24 hours at-65 ℃, 2 ℃ to 8 ℃ and room temperature. FII appeared to be stable after 24 hours at all conditions and incubation temperatures when the treated samples were analyzed, with no evidence of any benefit by dilution with PuW (C and D), citrate (E and F) or glycine (G). All values after 24 hours of incubation ranged from 0.80IU/ml to 0.95IU/ml, indicating recovery rates between 0.70% and 0.83% (FIG. 7A).

Untreated FV samples at pH 8.0 exhibited 0.38IU/ml at T-0 and slightly lower values of 0.33IU/ml after 24 hours at < 65 ℃ (related to freeze-thawing), while substantial degradation was observed after 24 hours at 2℃to 8℃and room temperature. Similarly, untreated samples adjusted to pH 7.2 exhibited 0.44IU/ml and 0.40IU/ml at T-0 and 24 hours at < 65 ℃, respectively, which is slightly better than pH 8.0. Complete degradation was observed only at 2℃to 8℃and 0.3IU/ml at room temperature. The treated samples showed different patterns: all dilutions appeared to have increased stability at T-0 when compared to untreated samples (A and B), with associated values of 0.70IU/ml (C), 0.78IU/ml (D), 0.53IU/ml (E), 0.73IU/ml (F) and 0.85IU/ml (G), indicating recovery relative to the loaded value (0.80 IU/ml) >88%, G is the best result (50 mM glycine, 1mM calcium).

Considering that these values are obtained immediately after collection, it appears that rapid degradation occurs at the moment of collection unless the eluate is immediately diluted with one of these solutions. When the stability after 24 hours at < 65℃is analyzed, the freeze-thaw effect appears to reduce the activity by exactly 0.2U/ml in all cases compared to T-0: 0.70IU/ml to 0.50IU/ml (C), 0.78IU/ml to 0.60IU/ml (D), 0.53IU/ml to 0.32IU/ml (E), 0.73IU/ml to 0.52IU/ml (F) and 0.85IU/ml to 0.65IU/ml (G), which in turn are higher results, indicating a recovery of 81% relative to loading. Dilution of the sample with 20mM citrate+1 mM calcium (E) appears to result in almost complete degradation at 2℃to 8℃and room temperature (0.08 IU/ml and 0.10IU/ml, respectively).

At the other extreme, C and G proved to be the most stable conditions, presenting very similar results: 0.42IU/ml and 0.43IU/ml at 2℃to 8℃and 0.47IU/ml and 0.50IU/ml at room temperature. This suggests that when the sample is diluted and no specific buffer such as glycine is used, the decrease in citrate concentration may stabilize FV, probably because citrate combines with free calcium in solution into a chelate, which is important for stabilization. Other possibilities are that high concentrations of salts in the elution buffer (1M NaCl) interfere with FV activity. Dilution of NaCl to 250nM reduces this interference. Furthermore, the addition of 1mM calcium (in final concentration) appears to increase stability, as evidenced by the significantly lower levels obtained in C, where no calcium was added to PuW (0.23 IU/ml and 0.18IU/ml at 2 ℃ to 8 ℃ and room temperature, respectively). It was observed that even when diluted with citrate solution, the addition of higher concentration of calcium (5 mM in the final solution) appears to compensate for the negative effects of citrate, probably because it biases the equilibrium citrate-calcium towards the latter. Finally, in general, incubation of the samples at room temperature appears to be slightly more advantageous than 2 ℃ to 8 ℃ for reasons that are not clear; this phenomenon can be explained by the possible partial activation of some procoagulants or degradation of some inhibitors (fig. 7B).

EXAMPLE 6 pH with calcium 7.2 dilution with buffer 1/4

The purpose of this experiment was to verify that when used a final concentration of 1mM CaCl was included ₂ FII and FV stability results obtained when diluting the eluted fraction from the AEX column by 1/4 with pH 7.2 buffer. Two chromatography runs were performed at pH 8.0 using a 5ml column prefilled with Eshmuno Q(C63R 3 and C63R 4). 1IU/ml heparin was added to CDP loading material, filtered through PES 0.2 μm Supor filter, and pH was adjusted to 8.0. 75ml CDP (batch #X33Y140) was loaded into the column and 80ml to 87ml UB (including 2CV washes) was collected. Bound material was eluted with 3CV 1M NaCl+20mM sodium citrate and FII and FV were immediately tested as follows:

a) Untreated with

B) 1/4 of the dilution was performed with buffer pH 7.2. Final concentration 1mM CaCl ₂ And 1IU/ml heparin.

C) 1/4 of the dilution was performed with buffer pH 7.2. Final concentration 1mM CaCl ₂ 1IU/ml heparin and 50mM glycine.

Results

Based on fig. 8, FII showed similar values in both the untreated and eluted fractions diluted with calcium-containing buffer, all ranging between 0.78IU/ml and 0.88IU/ml, with no observable trend for each experiment, 1.14IU/ml and 1.16IU/ml, respectively, being loaded. On the other hand, it is evident that the loading in the collection dilution buffer has a positive effect on the stability of FV. Experiment C63R3 started with loading of 0.80IU/ml and only 0.26IU/ml was observed in the untreated elution fraction, whereas 0.46IU/ml (B) and 0.52IU/ml (C) were present when diluted 1/4 with calcium. In addition, experiment C63R4 showed a similar trend with starting material of 0.68IU/ml and eluent of 0.26IU/ml (A) when untreated, whereas 0.46IU/ml was obtained when diluted 1/4 in calcium buffer.

EXAMPLE 7 calcium concentration Range

The purpose of this experiment was to determine the extent to which calcium acts as a stabilizer for coagulation factors V and IX in the eluted fraction without causing activation. An appropriate amount of CDP (batch # W35Y 440) containing 1IU/ml heparin was filtered through a PES 0.2 μm Supor filter and the pH was adjusted to 7.2. 20ml was loaded onto a 1ml Eshmuno Q column, in which 21ml UB was collected and bound protein eluted with 3ml (3 CV) buffer containing 1M NaCl and 50mM glycine (pH 7.2). The eluted sample was divided into 6 aliquots, one of which was kept untreated (A), 5 of which was treated with a sample containing different concentrations of CaCl ₂ 1/4 diluted with 50mM glycine solution pH 7.2 to obtain the final concentration: b) 0.1mM, C) 0.5mM, D) 1.0mM, E) 2.0mM and F) 5.0mM. FV and FIX in all samples were determined immediately after treatment (T ₀ ) And at<FV and FIX were determined in all samples after 24 hours incubation at-65 ℃ (freeze-thaw), 2 ℃ to 8 ℃ and Room Temperature (RT).

Results

As observed in fig. 9A, FV activity was significantly reduced from 0.82IU/ml in the loaded sample to 0.14IU/ml and 0.15IU/ml in the untreated sample after 24 hours incubation at 2 ℃ to 8 ℃ and room temperature, respectively. Whereas 24 hours freeze-thaw also affected FV activity, but to a lesser extent (0.55 IU/ml). At T ₀ Samples containing between 0.1mM and 2.0mM calcium exhibited low variability with less FV reduction (0.68 IU/ml to 0.73 IU/ml) when tested, and at the same time<Incubation at-65℃did not impair activity.

As shown in FIG. 9B, FIX activity in the untreated sample was also affected after 24 hours of incubation, changing from 0.96IU/ml to 0.83IU/ml, 0.72IU/ml and 0.74IU/ml in the loaded sample at < -65 ℃, 2 ℃ to 8 ℃ and room temperature, respectively. Under all conditions, the presence of between 0.1mM and 2.0mM calcium stabilized the activity of FIX, while the addition of 5mM triggered activation of the cascade, as described above, which was demonstrated by the immediate clotting (FV and FIX deficiency clotting assays) following the addition of 25mM calcium.

As shown in fig. 9C, incubation at 2 ℃ to 8 ℃ and RT improved FV stability with increasing calcium concentration, with maximum at 2mM (0.66 IU/ml) at 2 ℃ to 8 ℃, while addition of 5mM triggered activation of the cascade.

Based on the results described herein, FII was shown to be stable over a wide range of conditions. However, during the purification of FV from plasma using AEX, FV is highly unstable, undergoes immediate degradation from the moment it elutes from the column, and may even degrade during operation (higher flow rates show higher FV recovery). It has been proposed that one effective way to stabilize coagulation factors during manufacture of the formulation is to use a pH 7.2 buffer containing 1mM calcium.

Furthermore, it is important that when storedCitrate is kept low during any purification step to avoid chelation of free calcium in solution, which is critical for FV stabilization. The buffer may also contain glycine as a good choice for parenteral drug production methods, as well as heparin to keep procoagulants inactive. When the ion exchange column is used in the binding mode, it is recommended that the eluted fraction is collected directly into a dilution buffer previously adjusted to pH 7.2. Thus, FII and FV can be in<Stable at 23 ℃ for at least 24 hours. Freeze-thaw is preferably avoided under any conditions. Finally, caCl ranging between 0.1mM and 2.0mM in the absence of chelating agents such as citrate ₂ Suitable for stabilization, best results were obtained at 2.0 mM. Exceeding this value may cause activation of the coagulation cascade.

EXAMPLE 8 lyophilization of formulations

In order to improve the stability of the final product for long-term storage, a lyophilization process is performed. For this, 320ml CDP (batch #W33Y140) at pH 7.0 was loaded at 200cm/h into a 16ml Eshmuno Q column. The bound material was eluted with 56ml buffer (pH 7.2) containing 1M NaCl and 20mM glycine and the purified protein was concentrated 6.3-fold (loading/elution ratio). The eluate was divided into 3 aliquots: a) Calcium was not added, B) 0.1mM CaCl was added ₂ And C) adding 2mM CaCl ₂ . The sample was centrifuged in a 10KDa PES Ultra Spin column (Thermo) between 6000g and 7000g at 4 ℃ for 5 hours to concentrate the protein content 11-fold. Samples were diluted to between 1/3.8 and 1/4.3 with 20mM glycine pH 7.2 while compensating for calcium concentration to maintain the original values of A, B and C, resulting in osmotic pressures of A, B and C of 282mOsm, 233mOsm and 295mOsm, respectively. Using the method described in Table 8 and FIG. 10, three samples were each taken with 1ml of sample in a 5ml vial and lyophilized using a Christ freeze dryer model Epsilon 2-8D.

TABLE 8 freeze drying process

Figure 10 shows a freeze drying process. When the sample is inserted into a vial prior to lyophilization, the concentration of coagulation factor in the product is about 17 times that of the starting material, considering no degradation. After 60 days the samples were reconstituted with 1ml PuW.

Results

The compact obtained from freeze-dried samples exhibited a normal visual appearance, meaning that no collapse was observed. The lyophilized samples were reconstituted with 1ml PuW for no more than 10 minutes. It is suggested that, at least under the above conditions, the calcium-stable formulation obtained from the AEX column can be concentrated and lyophilized for later reconstitution and use.

Claims

1. A formulation comprising concentrated factor V, at least one vitamin K-dependent coagulation protein and at least one stabilizer, wherein the concentration of the at least one stabilizer is such that the factor V and/or the at least one vitamin K-dependent coagulation protein is in zymogen form.

2. The formulation of claim 1, wherein the factor V and the at least one vitamin K-dependent coagulation protein are present in the formulation in zymogen form for at least 4 hours at a temperature of up to about 30 ℃.

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

4. The formulation of any one of claims 1 to 3, wherein the concentration of factor V is at least 20% of at least one vitamin K dependent coagulation protein.

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

6. The sealant formulation according to 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 the divalent cation is at a concentration of up to about 5mM.

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

10. The formulation of claim 9, wherein the anticoagulant protein is AT least one of protein C, protein S, or antithrombin III (AT III).

11. The formulation of any one of claims 1 to 10, having a pH 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 the excipient is at least one of mannitol, maltose, sucrose, glycerol, or glycine.

14. The formulation according to any one of claims 1 to 13, which is derived from cryoprecipitated plasma (CDP).

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

16. The formulation according to any one of claims 1 to 15, which is suitable for systemic administration.

17. A formulation comprising concentrated factor V, at least one vitamin K-dependent clotting protein, and free calcium ion at a concentration of less than about 5 mM.

18. The formulation of claim 17, comprising zymogens of factor II, factor VII, factor IX and factor X.

19. A method for treating, inhibiting, preventing or delaying bleeding or any disorder associated therewith in a subject in need thereof, the method comprising the steps of: administering to the subject an effective amount of a formulation comprising concentrated factor V, at least one vitamin K-dependent clotting protein, and at least one stabilizer.

20. A method according to claim 19, wherein the formulation is as defined in any one of claims 1 to 18.

21. The method of claim 19 or 20, wherein the bleeding disorder is a genetic or acquired bleeding disorder.

22. The method of any one of claims 19-21, wherein the bleeding disorder is factor V deficiency.

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

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

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

26. A formulation comprising concentrated factor V, at least one vitamin K-dependent coagulation protein and at least one stabilizer for use in treating, inhibiting, preventing or delaying bleeding or any disorder associated therewith in a subject in need thereof.

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

28. The formulation for use according to claim 26 or 27, wherein the bleeding disorder is a genetic or acquired bleeding disorder.

29. The formulation for use according to any one of claims 26 to 28, wherein the bleeding disorder is factor V deficiency.

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

31. A kit, the 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 up to about 5mM, and (b) instructions for using the formulation to treat, inhibit, prevent, or delay bleeding or any disorder associated therewith in a subject in need thereof.

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

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

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

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

36. The kit of any one of claims 35, wherein the applicator is a syringe.

37. The kit of any one of claims 31-36, wherein the bleeding disorder is a genetic or acquired bleeding disorder.

38. The kit of any one of claims 31-37, wherein the bleeding disorder is factor V deficiency.

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

40. A process for preparing a formulation comprising concentrated factor V and at least one vitamin K-dependent coagulation 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 coagulation protein obtained in step (i).

41. The method of claim 40, wherein the blood, plasma, or any product/fraction is cryoprecipitated plasma (CDP).

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

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

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

45. The method of claim 44, wherein the collection solution comprises divalent cations at a concentration of up to about 5.0 mM.

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

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