FX activation method and its use in the preparation of FXa composition

Abstract

The present invention relates to the preparation of high-purity coagulation factor Xa (FXa or activated coagulation factor X) and an activation and purification method for obtaining the high-purity and highly activated FXa, which does not require the addition of a protein activator during the preparation process.

Description

Technical Field

[0001] The present invention relates to the preparation of high-purity coagulation factor Xa (FXa or activated coagulation factor X) and an activation and purification method for obtaining the high-purity and high-activation degree FXa, which does not require the addition of a protein activator during the production process. Background Art

[0002] Coagulation factor Xa (FXa) is a two-chain glycoprotein generated by the activation of its vitamin K-dependent zymogen coagulation factor X (FX) during the whole coagulation process. In vivo, FXa can be produced by the external pathway (i.e., by activation of factor VIIa) or by the intrinsic pathway (i.e., by activation of activated coagulation factors IX (FIXa) and VIII (FVIIIa)). The molecule undergoes enzymatic cleavage of the Arg15-Ile16 bond, releasing a 52 amino acid activation peptide. In the presence of its cofactors, coagulation factor Va (FVa), calcium (Ca) ions, and negatively charged phospholipid membranes, FXa binds to them to form a prothrombin (FII) activation complex (Toso et al. 2008).

[0003] In addition to these natural activation pathways, there are several in vitro methods that utilize trypsin (Bajaj et al., 1973) or non-human activators, wherein the non-human activators are derived from the venom of the viper (Fujikawa et al., 1974) or fungi (Rüchel et al., 1983), bacteria (Imamura et al., 1997) or plants (Richter et al., 2002). WO 2008 / 145989A1 teaches two techniques that activate FX to FXa by using venom, in particular the venom of the viper as an FX activator in combination with calcium ions. The venom of the viper contains the substances RVV-V and RVV-X, which directly activate factor V and FX. RVV-X is a Zn2+-dependent serine protease. In the first technique, FX is incubated in a solution containing 25mM calcium chloride and 0.01U / ml (RVV-X). In contrast, in the second technique, RVV-X is immobilized in an activated The resin is then removed and FXa is retained in the supernatant. As a result, the addition of snake-derived activators requires an effective method to remove potential leachables (venom) from FXa preparations, especially if intended for human use. Other separation techniques are needed to minimize concerns about the safety and tolerability of the finished product.

[0004] WO 2016 / 025601A1 describes a method for FX activation that does not rely on venom activators but instead utilizes an AEX resin. The bound FX is auto-activated, where it is exposed to a solution containing calcium ions for several hours before being eluted from the resin. The yield of FXa eluted from the AEX resin is approximately 5% of the co-eluted FX, which is a drawback of the production process.

[0005] Therefore, an activation method that can achieve highly reproducible conversions using synthetically prepared pharmaceutical-grade quality activators would be a major breakthrough in this field.

[0006] FXa is produced physiologically near the end of the coagulation cascade. It behaves as a protease necessary for the conversion of prothrombin (FII) to thrombin (FIIa), and is therefore also necessary for the conversion of fibrinogen to fibrin, which functions in wound closure and hemostasis: FXa is located downstream of FVIII in the cascade reaction, so FXa can in principle also drive coagulation in the absence of FVIII or when FVIII is inhibited, and can therefore be considered to have "factor eight bypassing activity".

[0007] Based on its factor VIII bypass activity, FXa can be used to treat patients with hemophilia A. Hemophilia A is an X-linked inherited disease that results in severe bleeding due to a deficiency or dysfunction of factor VIII. Progressive development of neutralizing alloantibodies has been reported in patients with hemophilia (Kempton 2009). Such patients with high-titer inhibitors cannot be effectively treated with factor VIII (FVIII) replacement because the administered FVIII is neutralized by these inhibitors. Patients experience bleeding episodes that can be treated with activated prothrombin complex concentrate (aPCC) (Kempton 2009). aPCC consists of a complex mixture of coagulation factors (partially activated) and other plasma proteins. Another treatment approach is the use of recombinantly produced activated coagulation factor (rFVIIa). rFVIIa is able to directly activate FX on the platelet surface (Kempton 2009). By administering FVIII, usually in combination with immunomodulatory substances and / or by-passing products, immune tolerance induction (ITI) can be achieved in 70% of patients receiving conventional and long-term treatment (Kempton 2009).

[0008] Because the amplification pathway is impaired in patients with hemophilia A and hemophilia B, blood clotting time is prolonged and blood loss is dramatically increased. Due to FXa's central role in coagulation, FXa may temporarily restore normal coagulation function. In addition, hemophiliacs who suffer from the neutralization of supplemented coagulation FVIII by neutralizing antibodies are particularly susceptible to FXa treatment. Giles et al. (1988) found that a combination of phosphatidylcholine, phosphatidylserine, and FXa has the potential to bypass FVIII, but the dose administered is critical to the net hemostatic outcome.

[0009] Given the recent common use of novel oral anticoagulants (NOACs) or direct oral anticoagulants (DOACs), another potential use of FXa is the reversal of these compounds. An example of such a compound is rivaroxaban. Rivaroxaban is a direct, reversible inhibitor of FXa used for anticoagulation therapy in patients with nonvalvular atrial fibrillation and venous thromboembolism. Patients receiving DOACs such as rivaroxaban must undergo systemic DOAC reversal in the setting of surgery (Lindhoff-Last 2017). Summary of the invention

[0010] The inventors surprisingly found that FX derived from plasma can be converted to FXa in vitro when the concentration of extrinsic and intrinsic activators (i.e., FVIIa, FIXa, and FVIIIa) in the composition is below the detection limit, without the need to add a protein activator, such as RVV-X. The risk of bringing such a protein activator into the finished product is thus eliminated. Activation is achieved by contacting the composition containing FX with synthetic phosphatidylserine (PS) and calcium.

[0011] According to a first aspect, the present invention provides a method for activating FX to FXa, wherein the method is performed in vitro and comprises the following steps:

[0012] a. Providing a composition containing FX; and

[0013] b. contacting the FX with PS, particularly synthetic PS (sPS), and calcium to form an activated composition; and

[0014] c. Incubating the activation composition to allow conversion of FX to FXa.

[0015] The activation method according to the first aspect may in particular be part of a method for preparing a FXa composition. Thus, according to a second aspect, the present invention provides a method for preparing a FXa composition, the method comprising the method for activating FX to FXa according to the first aspect, and further comprising at least one purification step.

[0016] Furthermore, the inventors have found that by using a heparin affinity column, in particular using The resin can specifically remove the remaining FX from the composition after activating FX to FXa without reducing the amount of FXa. Therefore, according to a third aspect, the present invention relates to a method for removing FX from a solution containing FX and FXa, the method comprising the following steps:

[0017] a. Under conditions suitable for binding FXa, particularly at a pH in the range of 5.8 to 9.0, a solution containing FX and FXa is mixed with a heparin affinity resin, particularly Resin contact;

[0018] b. separating the heparin affinity resin from the solution;

[0019] c. optionally, washing the heparin affinity resin; and

[0020] d. Optionally, eluting FXa from the heparin affinity resin under conditions suitable for elution.

[0021] According to a fourth aspect, the present invention relates to Use of a resin for removing FXa or FX from a composition containing FXa and FX.

[0022] Finally, according to a fifth aspect, the present invention relates to a FXa composition prepared according to the method of the second aspect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 depicts SDS-Page (Coomassie blue staining) and Western blot assay (probed with a polyclonal antibody against FXa; Abeam #111171) under non-reducing conditions.

[0024] Lanes 1+10: Molecular weight markers

[0025] Lane 2: Commercial FX products

[0026] Lane 3: Commercial FXa standard derived from plasma (Coachrom) - a mixture of α-FXa and β-FXa.

[0027] Lane 4: Loading with Heparin Material on the column

[0028] Lane 5: Passing through Heparin Column effluent

[0029] Lanes 6-8: Heparin Dilution series of eluent 1 (0.25 M NaCl)

[0030] Lane 9: Heparin Eluent 2 (El.2; 1M NaCl)

[0031] An immunoreactive band for coagulation factor FXa was detected in lanes 4 to 9 at approximately 50 kDa (compared to the α-FXa / β-FXa standard in lane 3). Lanes 6-8 of eluate 1 show pure single band β-FXa. While the load and pass-through fractions (lanes 4 and 5) contain various other immunoreactive FX / Fxa species. These signals may represent FX proenzyme and activation fragments as well as truncated FX / FXa. In addition, eluate 2 (lane 9) contains FXa present as a smaller immunoreactive species, most likely a truncated form of FXa.

[0032] Figure 2 Depicted is a comparison of FXa purified by the method described in this patent with a commercial activated prothrombin complex concentrate (aPCC), which is made from FX derived from plasma and activated and purified with Viperina viper venom (RVV). The graph shows the reduction [%] of aPTT in factor VIII inhibitor plasma as a function of FXa concentration. FXa purified by the method described in this patent shows very similar dose-dependent properties in this aPTT test system compared to the commercial FXa.

[0033] Figure 3 Depicted is a comparison of FXa purified by the method described in this patent with a commercially available preparation made from FX derived from plasma, activated with RVV and purified. The graph shows TGA mediation in Factor VIII inhibitor plasma as a function of FXa concentration. FXa purified by the method described in this patent shows very similar dose-dependent properties in this TGA test system compared to commercial FXa.

[0034] Figure 4 The results of the thrombogenic adverse reaction safety test of FXa prepared by the method described in this patent are described, compared with the commercially available and licensed activated prothrombin complex concentrate (aPCC) product. In contrast to the dose-dependent increase in thrombogenic potential in the aPCC product, pure FXa does not cause any thrombogenic problems.

[0035] Detailed description

[0036] Activation method

[0037] A first aspect of the present invention is a method for activating FX to FXa, wherein the method is performed in vitro and comprises the following steps:

[0038] a. Providing a composition containing FX; and

[0039] b. contacting the FX with (PS) and calcium to form an activated composition; and

[0040] c. Incubating the activation composition to allow conversion of FX to FXa.

[0041] The method of activating FX to FXa is also referred to as "activation method". The method is specifically an in vitro method. A composition containing FX is also referred to as a FX composition. As used herein, "contact" refers to introducing components into the same composition to allow the components to contact each other. In order to contact FX with PS and calcium and form an activated composition, any known method of mixing components can be used. In addition, the components FX, synthetic phosphatidylserine and calcium can be added in any possible order. For example, PS and calcium can be mixed into a composition, and the composition can be added to a composition containing FX to form an activated composition. Alternatively, both PS and calcium can be prepared into a solution. In this regard, in one embodiment, calcium is first added to a composition containing FX, and then PS. According to an alternative embodiment, PS is first added to a composition containing FX, and then calcium. According to another alternative embodiment, all components of the activated composition except the FX composition are premixed, and the FX composition is added to the mixture.

[0042] As used herein, the term "comprising" includes, in addition to its literal meaning, the expressions "consisting essentially of" and "consisting of." Therefore, the expression "comprising" refers not only to embodiments in which the subject matter "comprising" the specifically listed elements does not include other elements, but also to embodiments in which the subject matter "comprising" the specifically listed elements can and / or does include other elements. Likewise, the expression "having" should be understood like the expression "comprising," and also includes the expressions "consisting essentially of" and "consisting of."

[0043] According to one embodiment, the composition containing FX is an intermediate in the manufacture of an intermediate, in particular an intermediate in the manufacture of a blood protein product. The FX composition is preferably an intermediate in the manufacture of a FX or FXa product. The manufacture of coagulation factors is based on a purification process starting from a plasma or recombinant product source. Therefore, the FX composition is preferably a purified intermediate. According to one embodiment, FX is derived from plasma. According to one embodiment, the FX composition is a plasma fractionation product. The composition containing FX can be derived from a side fraction of plasma, which can be used as a starting material for an activation process. This side fraction of plasma fractionation can come from a method as described in Hoffer et al. (1995) or Roemisch and Pock (2012). The virus-inactivated side fraction is particularly beneficial for pathogen safety, and solvent / detergent (S / D) treatment can be proposed as an example of virus inactivation.

[0044] As mentioned above, the method does not need to add protease or other proteins to the activation composition. Therefore, according to one embodiment of the activation method, no protein activator is added. In particular, before and during the incubation of the activation composition, no protein activator is added in the composition. More preferably, no protein activator is added after the incubation. As used herein, "protein activator" is any peptide, particularly a polypeptide that can activate FX. The protein activator is preferably a FX-specific serine protease, such as FVIIa, FVIIIa and FIXa, or a serine protease derived from snake venom, such as RVV-X.

[0045] Usually, the initial concentration of FX in the activated composition is not limited to a specific range. In the activation method, since FX can be converted into FXa, its concentration changes over time. Therefore, the FX concentration when each component is mixed is called "initial concentration". In order to effectively convert, the initial concentration of FX is preferably in the range of 2 to 80IU / ml. The initial concentration of FX in the activated composition can be 2IU / ml, 3IU / ml, 4IU / ml, 5IU / ml, 6IU / ml, 7IU / ml, 8IU / ml, 9IU / ml, 10IU / ml, 12IU / ml, 15IU / ml, 20IU / ml, 25IU / ml, 30IU / ml, 1U / ml or 40IU / ml, 45IU / ml, 50IU / ml, 55IU / ml or 60IU / ml. Preferably, the initial concentration of FX in the activated composition is in the range of 2 to 40IU / ml. More preferably, the starting concentration of FX in the activating composition is in the range of 5 to 25 IU / ml.

[0046] Phosphatidylserine (abbreviated as Ptd-L-Ser or PS) is a phospholipid and is one of the components of cell membranes. Synthetic PS (sPS) is PS synthesized in vitro using chemical synthesis or biotechnology. The PS used is preferably synthetic PS. According to one embodiment, the concentration of PS in the activated composition is in the range of 0.1 to 100 mg / ml. According to another embodiment, the concentration of PS in the activated composition is in the range of 0.1 to 50 mg / ml. The PS concentration can be 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.8 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 7 mg / ml, 10 mg / ml, 12 mg / ml, 15 mg / ml, 17 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml or 40 mg / ml or 45 mg / ml. In addition, the concentration of PS in the activated composition can be in the range of 0.2 to 10 mg / ml. Preferably, the concentration of PS in the activated composition is in the range of 0.2 to 1 mg / ml.

[0047] According to one embodiment, the calcium used in the activation process is in ionic form, in particular Ca 2+ According to one embodiment, calcium is added to FX, preferably in the form of a salt selected from calcium phosphate, calcium chloride or calcium citrate.

[0048] According to one embodiment, the concentration of calcium ions in the activated composition is in the range of 1 to 100mM. The concentration can be 1mM, 2mM, 3mM, 4mM, 5mM, 7mM, 10mM, 12mM, 15mM, 17mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM or 80mM. According to one embodiment, the concentration of calcium ions in the activated composition is in the range of 2.5 to 50mM. According to one embodiment, the concentration of calcium ions in the activated composition is in the range of 5 to 40mM. According to a preferred embodiment, the concentration of calcium ions in the activated composition is in the range of 10 to 35mM.

[0049] According to one embodiment of the method for activating FX to FXa, the pH of the activated composition is in the range of 5.0 to 9.0. The pH may be 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0. According to one embodiment, the pH in the activated composition is in the range of 6.0 to 8.0. Preferably, the pH is in the range of 7.3 to 7.7.

[0050] According to one embodiment of the method for activating FX to FXa, during the incubation, the temperature of the activation composition is in the range of 15 to 40° C. The temperature may be 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 15° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C. or 40° C. Preferably, the incubation temperature is in the range of 15 to 35° C. More preferably, the incubation temperature is about room temperature.

[0051] In the activation process, FX is converted into FXa. Thus, the composition of the activated composition changes over time until equilibrium is reached or the process ends. With respect to FX and FXa, the terms "conversion" and "activation" are used interchangeably. According to one embodiment, the activation process ends after a defined time, i.e., an "incubation time" or "activation time". According to one embodiment, the activation process can in particular be ended by adding EDTA.

[0052] According to another embodiment of the first aspect, the incubation time is in the range of 1 to 48 hours. The incubation time can be 1 hour, 2 hours, 3, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours or 46 hours. Preferably, the incubation time is in the range of 4 to 20 hours.

[0053] As shown in the examples using the method according to the first aspect, the yield from FX to FXa is at least 10%. As used herein, the yield from FX to FXa or "FX conversion" shows the percentage of FX molecules converted to FXa, i.e., the percentage of the number of FXa molecules at the end of the activation method relative to the number of FXa molecules at the beginning of the activation method. Depending on the conditions, up to 100% conversion can be achieved using the activation method. The conversion can be at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%. According to one embodiment of the first aspect, their FX conversion is in the range of 20% to 100%.

[0054] According to one embodiment, the composition that contains FX also contains FII.As shown in the examples, when there is FII in the activated composition, it is converted into FIIa.This shows that the condition determined according to first aspect is suitable for utilizing FXa that FII (prothrombin) is converted into FIIa (thrombin).According to one embodiment, the initial concentration of FII in the activated composition is in the scope of 0.01 to 80IU / ml. The FII initial concentration in the activated composition can be 0.01 IU / ml, 0.05 IU / ml, 0.1 IU / ml, 0.5 IU / ml, 1 IU / ml, 2 IU / ml, 3 IU / ml, 4 IU / ml, 5 IU / ml, 7 IU / ml, 8 IU / ml, 10 IU / ml, 12 IU / ml, 15 IU / ml, 17 IU / ml, 20 IU / ml, 25 IU / ml, 30 IU / ml, 35 IU / ml, 40 IU / ml, 45 IU / ml, 50 IU / ml, 55 IU / ml, 60 IU / ml, 65 IU / ml, 70 IU / ml or 75 IU / ml. Preferably, the initial concentration of FII in the activated composition is in the scope of 0.01 to 40 IU / ml. More preferably, the initial concentration of FII in the activated composition is in the scope of 2 to 25 IU / ml. According to another embodiment, the initial concentration of FII is in the scope of 0.01 to 2 IU / ml. According to another embodiment, the initial concentration of FII is in the scope of 0.1 to 0.8 IU / ml.

[0055] According to one embodiment of the first aspect, in the composition containing FX, the concentration of the intrinsic activator and the extrinsic activator of FX is below the detection limit. In particular, the concentration of FVII / FVIIa, FVIII / FVIIIa and FIX / FIXa in the composition containing FX is below the detection limit as determined by a chromogenic assay.

[0056] The composition containing FX can be an initial protein mixture, which comprises FX and FII of 1-80IU / ml (particularly 1-60IU / ml) and 0.03-80IU / ml (particularly 0.03-60IU / ml) in a biologically acceptable buffer in the range of pH 6-8, and its total protein content is higher than 0 to 60mg / ml. Total protein can also be in the range of 1 to 50mg / ml. Total protein can further be in the range of 2 to 25mg / ml. Such an initial protein mixture can be obtained from affinity chromatography, for example, heparin affinity chromatography. Those skilled in the art are familiar with biologically acceptable buffers, and buffers based on citrate, phosphate, TRIS or imidazoles can be proposed as examples. An example of an acceptable buffer is a buffer containing 20mM TRIS and 150mM NaCl at pH 7.5 ± 0.2. The initial protein mixture with above-mentioned total protein, FX and FII content can be used as the starting material in the activation method.

[0057] According to another embodiment of the first aspect of the invention, the initial protein mixture originates from plasma fractionation or biotechnological production of FX obtained by recombinant or transgenic expression and immunoaffinity chromatography.

[0058] The plasma fraction used to generate the initial protein mixture is preferably cryo-poor plasma.

[0059] The initial protein mixture may contain about 40 IU / ml of FX and 0.01-80 IU / ml of FII in a buffer consisting of 20 mM TRIS and 150 mM NaCl at pH=7.5, and its total protein content is 1-60 mg / ml.

[0060] The initial protein mixture can be obtained by subjecting a blood fraction to DEAE-Sepharose or heparin affinity chromatography and buffer exchange.

[0061] Cold supernatant plasma can be stabilized with heparin and adsorbed onto anion exchange resin QAE-Sephadex. After washing, vitamin K dependent factors are eluted with a buffer of higher ionic strength. For example, the ionic strength can be 1600-2200mosmol / kg. The vitamin K dependent factors in the eluate are stabilized by adding 3IU heparin / ml. The stabilized eluate can be further subjected to S / D treatment by incubation with 0.3% TnBP / 1% polysorbate 80. If so, the S / D reagent can be removed by adsorption on DEAE-Sepharose and washing with a buffer of 260-310mosmol / kg. The protein can then be eluted with a buffer of 700-780mosmol / kg. The eluate can then be subjected to buffer exchange, for example, with a 10kDa membrane, by ultrafiltration / diafiltration. The new buffer can have about 330-400mosmol / kg. After buffer exchange, it can be concentrated to 30IU IX factor / ml.

[0062] Alternatively, cold supernatant plasma can be adsorbed on anion exchange resin (such as DEAE-Sephadex A50), and then the resin is washed. By eluting with a high ionic strength buffer of 2M sodium chloride and 15mM sodium citrate at about neutral pH, eluent can be obtained. The eluent can be concentrated to 380-420mosmol / kg. Anion exchange purification can be carried out on DEAE resin such as DEAE-SepharoseFF to (concentrated) eluent. After washing, the DEAE eluent can be obtained by utilizing a buffer of 360mM sodium chloride, 5mM sodium citrate and 5mM disodium hydrogen phosphate. Virus inactivation can be carried out to the DEAE eluent by incubation of 0.3% TnBP / 1% polysorbate 80. Afterwards, affinity chromatography can be carried out on Heparin-Sepharose CL 6B to the eluent processed by S / D. After washing with a buffer composed of 20 mM sodium citrate at pH 7.0-8.0, elution is performed with a buffer composed of 250 mM sodium chloride and 20 mM sodium citrate at the same pH, thereby obtaining an eluate.

[0063] To obtain the initial protein mixture, each purification step was followed by buffer exchange by ultrafiltration / diafiltration using a membrane with a cut-off of 10 kDa into a buffer consisting of 20 mM TRIS and 150 mM NaCl at pH = 7.5.

[0064] When starting from recombinant expression products, immunorecombinant affinity chromatography can be used in particular to obtain the initial protein mixture. In particular, antibodies specific for FX and / or FXa, e.g. Calcium-dependent monoclonal antibody; Catalog No. AX100083, can be immobilized on an activated chromatographic resin, such as The column containing such immunoaffinity resin can be pretreated with an equilibration buffer having a pH of 7.0-8.0 and a conductivity of 1-50 mS / cm and containing 5-100 mM TRIS, 1-250 mM NaCl, 10-25 mM Ca ions and 0.005-1% of a nonionic surfactant (such as polysorbate), and an acceptable equilibration buffer can contain 10-40 mM TRIS, 100-150 mM NaCl, 5-20 mM Ca ions and 0.01-0.2% polysorbate, having a pH of 7.4±0.2 and a conductivity of about 10 to 25 mS / cm. Before the initial protein mixture is loaded onto the immunoaffinity column, a buffer exchange with the equilibration buffer can be performed. After extensive washing with equilibration buffer, the flow-through / wash fraction containing unbound impurities is discarded and the bound FX is eluted from the column with an elution buffer having a pH of 6.0-8.0 and containing 5-100 mM TRIS, 1-250 mM NaCl, 1-200 mM EDTA and 0.005-1% non-ionic surfactant. The preferred elution buffer contains 10-40 mM TRIS, 100-150 mM NaCl, 20-60 mM EDTA and 0.01-0.2% polysorbate and has a pH of 7.4±0.2%. The initial protein mixture thus obtained is ultrafiltered / diafiltered with a buffer solution of pH 6.0-8.0 and composed of 5-100 mM TRIS, 50-500 mM NaCl and 0.005-1% nonionic surfactant, so that the FX content is 5-80 IU / ml, particularly 30-50 IU / ml, an acceptable buffer solution may comprise 10-40 mM TRIS, 130-170 mM NaCl and 0.01-0.2% polysorbate and its pH is 7.5 ± 0.2, and the membrane has a cut-off value of 10 kDa. The initial protein mixture may be activated using an activation method selected from Examples 1 to 4 and 9 without further purification thereafter. Therefore, the initial protein mixture obtained may also be used as a starting material in the activation method.

[0065] The risk of carrying these compounds into the finished product is thus eliminated. The initial protein mixture can be activated under various conditions in the presence of a phosphatidylserine dispersion (PS dispersion), in particular a synthetic phosphatidylserine dispersion. The PS dispersion can be prepared with a concentration of 0.1-100 mg / mL of synthetic phosphatidylserine in a biologically acceptable buffer containing 5-100 mM buffering compound (e.g. maleate, imidazole, carbonate or TRIS) and 5-500 mM NaCl and having a pH range of 6.0-8.0. A buffer with a pH of 7.5±0.2 and containing 10-40 mM TRIS and 130-170 mM NaCl is particularly suitable and is used as a reaction buffer. PS can be prepared by high shear mixing (e.g. using a 20-60°C buffer) at 20-60°C. The phospholipid vesicles are dispersed in a buffer solution for about 1-240 minutes in a dispersing machine to obtain dispersed particles with a majority of diameters less than <25 μm, especially less than <10 μm, and then cooled to room temperature. Other techniques well known to technicians in the field of phospholipid vesicle preparation are, for example, lipid film hydration, detergent removal, reverse phase evaporation, and organic solvent injection, and can also be used for preparation. The final dispersion can be subjected to subsequent steps to further reduce the particle size, such as ultrasonic treatment or extrusion.

[0066] In one embodiment of the first aspect, the initial protein mixture can be mixed with a PS dispersion and a Ca ion solution to obtain a mixture comprising 1-25 mg / ml total protein, 1-30 IU / ml FX, 0.003-0.3 U / ml FII, 0.1-2 mg / ml PS and 2.5-50 mM Ca ions, and the pH value can be adjusted to a range of 5.0-9.0, in particular the pH value is adjusted to 7.4 to 7.6. The mixture can be incubated at 15-40° C. for 1-48 hours, in particular 4-16 hours, even more particularly at 15-35° C. for 4-10 hours, with slow stirring, and then a buffer is added, the buffer also containing a chelating agent such as EDTA or any other chelating agent that can effectively prevent Ca ions from further activating FX to FXa. For example, 50 mM EDTA is sufficient to effectively prevent 25 mM Ca ions from further activating FX to FXa. After incubation and addition of EDTA, the dispersed PS is solubilized by adding 0.01-10% of a surfactant such as Triton X-100, sodium deoxycholate or CHAPS to obtain a clear or almost clear solution that can be filtered through a 0.2-1.0 μm filter (particularly a 0.45 μm filter) to recover the FXa-containing intermediate. FXa analysis is performed at this stage of the method.

[0067] The mixture thus prepared has, as defined in the assay description, a FXa-specific activity of 1-30 IU / ml, a thrombin level <50 IU / ml and a FXa yield of about 25-100%, in particular 30-75%, based on the FX content activated with PS and Ca ions.

[0068] In one embodiment of the first aspect, an initial protein mixture comprising 1-50 mg / ml total protein, 1-60 IU / ml FX and 0.03-60 U / ml FII is diluted with a reaction buffer. The amount of the reaction buffer is about 90% of the amount of the initial protein mixture, and a reaction buffer containing about 75 mM Ca ions is additionally mixed. The amount of the reaction buffer containing Ca ions is the same as the amount of the undiluted initial protein mixture. A PS dispersion (about 10% of the undiluted initial protein mixture) is carefully added to the above mixture in a continuous operation (steering) manner, and the pH can be adjusted to a range of 5.0-9.0, in particular to 7.4-7.6. The obtained mixture containing PS can be incubated at 15-40° C. (in particular at 15-35° C.) for 1-48 hours (in particular 4-20 hours) with gentle stirring, and then additional reaction buffer containing a chelating agent is added. The steps of stopping the activation of FX to FXa, dissolving the PS dispersion and filtering to recover the intermediate containing FXa may be performed as described in the first embodiment.

[0069] The mixture thus prepared has a FXa-specific activity of 1-60 IU / ml, less than 10,000 IU / ml of FIIa and a total protein content of about 9.9 mg / ml, and a FXa yield of about 30-75% based on the FX content activated with PS and Ca ions.

[0070] Preparation method

[0071] According to a second aspect, the present invention provides a method for producing a FXa composition, the method comprising the method for activating FX to FXa according to the first aspect, and further comprising at least one purification step.

[0072] According to one embodiment of the second aspect, the at least one purification step is selected from immobilized metal affinity chromatography (IMAC), anion exchange chromatography (AEX) and affinity chromatography (AF). AF can specifically be immunoaffinity chromatography or heparin affinity chromatography.

[0073] The preparation method according to the second aspect may include various purification steps. Suitable methods for protein purification steps are, for example, IMAC, AEX, cation exchange chromatography (CEX), AF, particularly immunoaffinity chromatography or heparin affinity chromatography, size exclusion chromatography (SEC). The method may be used in any combination or in any order. Between purification steps, steps such as buffer exchange, such as diafiltration or ultrafiltration / diafiltration (UDF) may be included. The activation treatment may be carried out before the purification step, between two purification steps, or after the purification step (i.e., for the purified product).

[0074] According to one embodiment, the production process starts with an activation method according to the first aspect, followed by AEX. In one embodiment of the second aspect, a column filled with an anion exchange resin is pretreated with a biologically acceptable equilibrium buffer having a pH in the range of 5.8-8.0, the resin can be selected from a weak or strong anion exchange resin, examples of which are worth mentioning Such biologically acceptable buffers are well known to those skilled in the art, examples of which can be mentioned are buffers based on citrate, TRIS or imidazole. The equilibrium buffer may contain 1-100 mM imidazole, with a pH range of 6.1-7.0 and a conductivity of 1-20 mS / cm. The intermediate containing FXa may be diluted or buffer exchanged with the equilibrium buffer until the conductivity is less than 20 mS / cm. The diluted FXa solution may be loaded onto a pretreated anion exchange column to bind FXa while unbound proteins and impurities (such as thrombin and PS) are in the flow through fraction and subsequent wash fraction. The chromatographic column may be thoroughly cleaned with a buffer having a reduced pH range of 5.8-8.0 or an increased conductivity or a combination of the two. Such a wash buffer may contain 1-100 mM imidazole and 0.1-0.3 M NaCl and a pH of 5.8-7.0 until the OD280 reading of the monitored UV flow cell returns to the baseline level. FXa can be eluted from the anion exchange resin using a biologically acceptable elution buffer having a pH of 5.8-9.0. Bound proteins can be eluted by lowering the pH and / or increasing the conductivity. Thus, an elution buffer having a pH of 5.8-9.0 and comprising 1-100 mM TRIS and at least 0.3 M NaCl (particularly 0.3-1.5 M NaCl) can be used.

[0075] Subsequently, the anion exchange eluate may be diluted to a diluted eluate having a pH range of 5.8-9.0 (preferably 7.4±0.2) and containing 1-30 mM (preferably 1-3 mM) imidazole and 0.4-4 M (preferably 0.5-1 M) NaCl.

[0076] Then, AEX can be followed by IMAC. In one embodiment of the second aspect, the diluted eluate from the anion exchange column is loaded onto an immobilized metal affinity chromatography (IMAC) resin with copper ions. Under these conditions, FXa can be found in the IMAC flow-through fraction and can be obtained therefrom with a purity of 20-45 U / mg protein, while impurities such as α2 macroglobulin, inter-α trypsin inhibitor, protein C or vitronectin, etc., are bound to the resin.

[0077] Optionally, IMAC can be followed by heparin affinity chromatography, in particular using In one embodiment of the second aspect, the buffer of the IMAC flow-through fraction is replaced to obtain FXa dissolved in 1-100 mM TRIS buffer at a pH of 5.8-9.0, in particular 6.8-8.0. The FXa solution obtained is subjected to heparin affinity chromatography on a resin which has been balanced with a buffer having a pH range of 5.8-9.0 (particularly 6.8-8.0) and containing 1-100 mM TRIS and 0.0005-1% nonionic surfactant (such as polysorbate). Under these conditions, FXa is bound to the resin, while proenzyme FX and impurities (such as F1.2, which is an activated fragment of FII) are not bound and pass through the chromatographic column. After washing with the equilibrium buffer and the monitored OD280 signal returns to baseline, FXa can be eluted from the resin by increasing conductivity. This elution buffer can comprise 1-100 mM TRIS, 0.0005-1% nonionic surfactant (such as polysorbate) and 0.01-0.30 M NaCl, and its pH range is 5.8-9.0, particularly 6.8-8.0. FXa eluted from the resin has a purity of 80-217 U / ml, particularly 150-210 U / ml.

[0078] In an alternative embodiment of the second aspect, after AEX, immunoaffinity chromatography is performed using FX / FXa specific immunoaffinity resin. In this regard, the column (described previously) equipped with FX / FXa specific immunoaffinity resin is pretreated using an equilibrium buffer having a pH of 7.0-8.0 and a conductivity of 1-250 mS / cm and containing 5-100 mM TRIS, 1-250 mM NaCl, 10-25 mM Ca ions and 0.005-1% nonionic surfactant. An acceptable equilibrium buffer may comprise 10-40 mM TRIS, 100-150 mM NaCl, 5-20 mM Ca ions and 0.01-0.2% polysorbate, having a pH of 7.4 ± 0.2 and a conductivity of about 10 to 20 mS / cm. Prior to loading onto the immunoaffinity column, the eluent of the anion exchange chromatography is buffer exchanged to exchange for the equilibrium buffer. After extensive washing with the equilibration buffer, the flow-through / wash fraction containing unbound impurities is discarded and the bound FXa is eluted from the column with an elution buffer having a pH of 7.4±0.2 and a conductivity of 1-250 mS / cm and containing 5-100 mM TRIS, 1-250 mM NaCl, 5-40 mM EDTA and 0.005-1% polysorbate. In particular, such an elution buffer may comprise 10-40 mM TRIS, 100-150 mM NaCl, 10-40 mM EDTA and 0.05% polysorbate and a pH of 7.4±0.2 and a conductivity of 19±2 mS / cm. The purity of the FXa eluted from the FX / FXa specific immunoaffinity resin is over 95%.

[0079] Optionally, immunoaffinity chromatography can be followed by heparin affinity chromatography, in particular using In one embodiment of the second aspect, FXa is eluted from the immunoaffinity resin and purified by heparin affinity chromatography using the above Further purification occurs, wherein, if desired, FX is separated from FXa.

[0080] In one embodiment of the second aspect of the invention, the column (described previously) containing the FX / FXa-specific immunoaffinity resin is pretreated with an equilibration buffer having a pH of 7.0-8.0 and a conductivity of 1-250 mS / cm and containing 5-100 mM TRIS, 1-250 mM NaCl, 10-25 mM Ca ions and 0.005-1% nonionic surfactant. An acceptable equilibration buffer may contain 10-40 mM TRIS, 100-150 mM NaCl, 5-20 mM Ca ions and 0.01-0.2% polysorbate and has a pH of 7.4 ± 0.2 and a conductivity of about 10 to 25 mS / cm. Prior to loading onto the immunoaffinity column, the initial protein mixture is buffer exchanged to the equilibration buffer. After extensive washing with the equilibration buffer, the flow-through / wash fraction containing unbound impurities is discarded and the bound FX is eluted from the column with an elution buffer having a pH of 7.4±0.2 and a conductivity of 1-50 mS / cm and containing 5-100 mM TRIS, 1-250 mM NaCl, 5-40 mM EDTA and 0.005-1% polysorbate. In particular, such an elution buffer may comprise 10-40 mM TRIS, 100-150 mM NaCl, 10-40 mM EDTA and 0.05% polysorbate and a pH of 7.4±0.2 and a conductivity of 19±2 mS / cm. After buffer exchange, the FX eluted from the immunoaffinity column may be activated by one of the activation methods described previously, thereby obtaining a mixture of FX and FXa, the ratio of which depends on the activation yield.

[0081] In one embodiment of the second aspect, FX is eluted from the immunoaffinity resin and activated using the method according to the first aspect and Further purification.

[0082] How to remove FX

[0083] According to a third aspect, the present invention relates to a method for removing FX from a solution containing FX and FXa, comprising the following steps:

[0084] a. Under conditions suitable for binding FXa, particularly at a pH in the range of 5.8 to 9.0, a solution containing FX and FXa is mixed with a heparin affinity resin (particularly Resin) in contact;

[0085] b. separating the heparin affinity resin from the solution;

[0086] c. optionally, washing the heparin affinity resin; and

[0087] d. Optionally, eluting the FXa from the heparin affinity resin under conditions suitable for elution.

[0088] This method can be used in combination with the activation method of the first aspect. However, the method can be used for any composition comprising FXa and FX. The method can also be used to remove FXa from a solution containing FX and FXa. Therefore, according to an alternative embodiment of the third aspect, the present invention relates to a method for removing FX from a solution containing FX and FXa, the method comprising the following steps:

[0089] a. Under conditions suitable for binding FXa, particularly at a pH in the range of 5.8 to 9.0, a solution containing FX and FXa is mixed with a heparin affinity resin (particularly Resin) in contact;

[0090] b. separating the heparin affinity resin from the solution;

[0091] c. Collect the solution containing FX;

[0092] d. Optionally, washing residual amounts of FX from the heparin affinity resin; and

[0093] e. Optionally, using the FX-containing solution obtained in steps c) and (optionally) d) for further processing.

[0094] As used herein, "removal" or "removal" refers to reducing the concentration of the component to be removed in the composition. Removal is not limited to but specifically includes the possibility of completely eliminating the component from the composition.

[0095] The pH of the contacting step is in the range of 5.8 to 9.0. The pH can be 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0. Preferably, the pH is in the range of 6.8 to 8.0.

[0096] The composition for contacting heparin with FX / FXa preferably also comprises 0.0005-1% of a nonionic surfactant. The percentage of the nonionic surfactant may be, for example, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5% or 1%. Preferably, the nonionic surfactant may be, for example, polysorbate.

[0097] The composition preferably comprises 1-100 mM TRIS. TRIS can be present, for example, at the following concentrations: 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7 mM, 10 mM, 12 mM, 15 mM, 17 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM. Preferably, the nonionic surfactant can be, for example, polysorbate.

[0098] Under these conditions, FXa binds to the resin, while the proenzyme FX and impurities (such as F1.2, which is an activated fragment of FII) pass through the column unbound.

[0099] In order to elute FXa from the column, a higher salt concentration may be used. According to one embodiment, the elution buffer comprises a salt at a concentration of 0.01 to 0.30 M. The salt is preferably NaCl. The elution buffer may comprise NaCl at a concentration of, for example, 0.01 M, 0.02 M, 0.03 M, 0.05 M, 0.07 M, 0.1 M, 0.12 M, 0.15 M, 0.17 M, 0.2 M, 0.22 M, 0.25 M, 0.27 M or 0.3 M.

[0100] For example, the composition containing FXa and FX is dissolved in 1-100 mM TRIS buffer at a pH of 5.8-9.0, especially 6.8-8.0. The obtained FXa solution is subjected to heparin affinity chromatography on a resin which has been equilibrated with a buffer having a pH range of 5.8-9.0, particularly 6.8-8.0 and containing 1-100 mM TRIS and 0.0005-1% of a nonionic surfactant (such as polysorbate). Under these conditions, FXa binds to the resin, while proenzyme FX and impurities (such as F1.2, which is an activated fragment of FII) pass through the chromatographic column without being bound. After washing with the equilibration buffer and the monitored OD280 signal returning to the baseline, FXa can be eluted from the resin by increasing the conductivity. This elution buffer can contain 1-100 mM TRIS, 0.0005-1% of a nonionic surfactant (such as polysorbate) and 0.01-0.30 M NaCl, and has a pH range of 5.8-9.0, particularly 6.8-8.0. FXa eluted from the resin has a purity of 80-217 U / ml, particularly 150-210 U / ml.

[0101] The method according to the third aspect can also be used to remove FXa from a FX composition. According to a fourth aspect, the present invention relates to Use of the resin for removing FXa or FX from a composition containing FXa and FX. For the use according to the fourth aspect, the same conditions as defined for the third aspect apply.

[0102] According to one embodiment of the second aspect, the preparation method comprises the heparin affinity chromatography method defined according to the third aspect and the activation process according to the first aspect.

[0103] Composition and use in medicine

[0104] According to a fifth aspect, the present invention provides a FXa composition prepared according to the method of the second aspect.

[0105] According to one embodiment, in the FXa composition of the fifth aspect, the ratio of FXa to FX is greater than 10:1. Preferably, the ratio of FXa to FX is greater than 20:1. More preferably, the ratio of FXa to FX is greater than 50:1. According to an embodiment of the fifth aspect, the composition further comprises FIIa. Preferably, the ratio of FIIa to FXa is in the range of 1:30 to 1:5000.

[0106] The pathogen safety of the formulation containing FXa is achieved by incorporating a pathogen removal or inactivation step at an appropriate point in the production process, such as solvent / detergent treatment, nanofiltration, or other means known to those skilled in the art, or a combination thereof. The FXa intermediate obtained by one of the methods described in the third aspect of the present invention is formulated by ultrafiltration / diafiltration (UDF) using a formulation buffer based on 5-100 mM of a biologically and pharmaceutically acceptable buffer substance such as histidine, citrate, citrate-phosphate, phosphate, succinate or others, 10-300 mM NaCl or other salts, and finally 0.001-0.5% w / v nonionic surfactants such as polysorbates or poloxamers, or polymers such as albumin, Or polyethylene glycol is used as a stabilizer.

[0107] Finally, one or more excipients and stabilizers selected from sugars, sugar alcohols, polyols, amino acids, polymers or other biocompatible compounds, such as sucrose, trehalose, glucose, lactose, maltitol, mannitol, glycine, L-arginine or PVP (polyvinylpyrrolidone, e.g. 12PF or 17PF) to complete this formula. According to one embodiment, the FXa composition of the fifth aspect further comprises a stabilizer selected from sugars, polyols, amino acids, polymers or polyvinylpyrrolidone (PVP).

[0108] The pH range of the FXa composition is preferably in the range of 6.0-7.5. The formulated FXa composition is stored frozen, or can be lyophilized to a final FXa concentration of 0.05-10 IU / ml. Table 1 lists an example of such a formulation.

[0109] In particular, the FXa composition of the fifth aspect can be formulated and finally lyophilized for use in or on a carrier material, which can be used for local treatment of bleeding diseases or for local induction of coagulation. Therefore, according to one embodiment of the fifth aspect, the FXa composition is a lyophilized composition. According to one embodiment, the FXa composition is stable for at least 48 hours after reconstitution. Alternatively, the FXa composition is stored frozen.

[0110] According to an embodiment of the fifth aspect, the FXa composition is used to treat a bleeding disorder. The bleeding disorder can be hemophilia A, hemophilia B, von Willebrand disease, congenital hemophilia A with inhibitors, or acquired hemophilia A with inhibitory autoantibodies to FVIII, congenital hemophilia B with inhibitors, or acquired hemophilia B with inhibitory autoantibodies to FIX, traumatic blood loss, FVII deficiency, FV deficiency, FX deficiency, FXI deficiency, FXIII deficiency, fibrinogen deficiency, prothrombin deficiency, dilutional coagulopathy, thrombocytopenia, high-risk surgical blood loss, intracerebral hemorrhage, von Willebrand disease with inhibitors to von Willebrand factor, or a combination thereof.

[0111] According to one embodiment of the fifth aspect, the bleeding disorder is caused by the administration of a novel oral anticoagulant (NOAC) or a direct oral anticoagulant (DOAC).

[0112] The present invention enables the production of FXa with an economical and pathogen-free process to manage bleeding events caused by NOAC- / DOAC during surgery or injury in patients receiving oral anticoagulants. Due to the role of FXa in the treatment of coagulation of congenital and acquired bleeding defects, the bleeding caused by NOAC / DOAC belonging to the direct thrombin inhibitor class can also be managed with a preparation comprising a mixture of FXa and FIIa (which can also be obtained by the method of the present invention).

[0113] It is generally preferred to use biologically acceptable buffers, such as those based on citrate, phosphate, TRIS, imidazole and more.

[0114] In order to confirm the stability and activity of the reconstituted FXa formulation of the present invention, the lyophilized composition was reconstituted to its original volume and subjected to analytical tests, in particular, the determination of FXa specific activity and thrombin generation (TGA) in FVIII-inhibitor plasma. The FXa specific activity test showed that more than 75% of the initial activity was retained after lyophilization, and the TGA test showed that more than 85% of the initial activity was retained. The results are listed in Table 2.

[0115] The selected formulations 1, 2 and 6 were reconstituted and stored in liquid form at room temperature, particularly 20-25°C and 2-8°C for 48 hours to determine the residual activity after liquid storage. After 48 hours of liquid storage at room temperature, the FXa-specific activity test showed that more than 90% of the activity of the reconstituted formulation before liquid storage was retained, and the TGA test showed that more than 85% of the activity was retained. The results are summarized in Table 3.

[0116] According to one embodiment, the use comprises administering the FXa composition by a parenteral administration method such as intravenous injection or infusion. The composition obtained by the method of the present invention can be administered intravenously, but can also be administered topically and topically.

[0117] In the patient using direct oral anticoagulants for treatment, immobilized and topically applied FXa, that is, non-intravenously applied FXa, or the mixture containing FXa and FIIa can make the recovery of normal coagulation function confined to the treatment site.Because FXa and FIIa are the target molecules of direct oral anticoagulants, the immobilized FIIa and / or FXa of topically applied will remove excessive inhibitor and recover the local effect of internal FXa and FIIa.In addition, immobilized FIIa can cause blood coagulation and form primary blood clot at injured part.The compositions containing FXa can be fixed on or in the sponge, patch, film, bandage or other commonly used carrier materials (such as powder or particle) through coating or soaking, with topically applied.

[0118] According to one embodiment of the fifth aspect, the use comprises topical application of the composition via a coated or soaked sponge, patch, film, bandage or other commonly used carrier material, such as a powder or granules. For topical application, a hemostatic tool such as a film, patch, bandage or sponge soaked with the composition of the invention or deposited thereon is suitable for local hemostasis. The hemostatic tool is also suitable for local hemostasis in patients undergoing anticoagulant therapy with DOAC, particularly if thrombin is additionally deposited on or in the tool.

[0119] The compositions obtained by the method of the present invention have a lower thrombogenic potential as demonstrated by aPTT assay, thrombin generation assay (TGA) and related animal studies for determining the results of the Wessler test (Wessler et al. 1959) and as shown in Table 4.

[0120] Analysis (FX / FXa and FII / FIIa)

[0121] The product and intermediate obtained are analyzed and tested, i.e. FX ELISA, TGA (thrombin generation assay), Fxa specific activity assay and FIIa specific colorimetric assay. Although the chromogenic substrate for measuring FXa and FIIa is all very specific to each protease, it is not 100%, particularly under high protease concentration, the cracking of other potential targets of a small part can occur. In order to determine the specific activity of FXa / FIIa, the highly specific inhibitor of FXa (i.e. rivaroxaban) and the highly specific inhibitor of FIIa (i.e. dabigatran) are used to determine the inhibitory amount of corresponding protease, which represents protease specific activity. Calculate activation efficacy, and be expressed as the percentage of the FXa produced from the total FX added to the reaction.

[0122] TGA – Thrombin Generation Assay

[0123] Pre-diluted samples (dilution buffer: 20 mM TRIS, 130 mM NaCl, 0.5% human serum albumin (HSA); pH 7.4) were mixed with FVIII inhibitor plasma (Technoclone; Item No. 5159008) in a ratio of 1 pre-diluted sample + 4 inhibitor plasma and immediately measured. The Technothrombin TGA kit (Technoclone; Technothrombin RC high kit; Item No. 5006010) was used according to the manufacturer's instructions for measurement. The parameter read out was the peak thrombin concentration (PTC) in nM.

[0124] FXa-specific activity assay (measurement of FXa in the presence of cross-reacting proteins)

[0125] FXa was measured in the presence of cross-reacting proteins using a FXa-specific activity assay. Samples and standards containing FXa were diluted with dilution buffer (50 mM TRIS, 0.2% BSA (bovine serum albumin); pH 8.4). The chromogenic substrate CS11 (32) was measured in the diluted samples over 5 min with and without the addition of 10 mM rivaroxaban on a temperature-controlled microplate reader (405 nm). The assay is calibrated with FXa concentrate (purified human factor Xa from Coachrom; EZ007A). The FXa-specific activity contained in each sample is calculated by subtracting the uninhibited activity measured in the sample treated with rivaroxaban from the total activity measured in the sample not treated with rivaroxaban. If thrombin is present at a high concentration, for example 1000 U / ml, the substrate specific for FXa is also cleaved and a false high FXa activity is simulated.

[0126] FIIa-specific activity assay (FIIa measurement in the presence of cross-reacting proteins)

[0127] Use FIIa specific activity assay to measure FIIa in the presence of quite high concentration of protein, which may also cause thrombin chromogenic substrate to crack, although the rate is relatively low. Use dilution buffer (50mM TRIS, 100mM NaCl, 1% PEG 6000; pH 8.3) dilute the sample and standard containing thrombin. Add and do not add 10 μM dabigatran (incubated 30 minutes at 37 ℃), on the microplate reader (405nm) of temperature control, measure the chromogenic substrate CS01 (38) in the diluted sample in 5 minutes. The conversion rate of 1:1.0 ...

[0128] Total Protein

[0129] According to the manufacturer's instructions, use The total protein was measured by Bradford analysis.

[0130] Measurement of FII and FX

[0131] FII and FX in the unactivated mixture were measured using the Instrumentation Laboratories (IL) assay according to the ACL instrument manufacturer's instructions.

[0132] Measurement of FX:Ag

[0133] According to the manufacturer's instructions, use (#RK033A) The assay measures FX in the activation mixture.

[0134] SDS PAGE:

[0135] According to the manufacturer's Gel system (Thermo Scientific; ) and SDS PAGE was performed using 4-20% acrylamide gradient gel under non-reducing conditions.

[0136] Western Blotting:

[0137] Under non-reducing conditions according to the manufacturer's SDS PAGE (4-20% acrylamide gradient gel) and electrotransfer were performed according to the instructions of the gel system and XCell II Item and XC Thermo Scientific. Super Block manufactured by Thermo Scientific was used. The membrane was blocked with ready-to-use blocking buffer. Thereafter, the membrane was probed with polyclonal anti-FX / FXa antibody (anti-FXa polyclonal antibody; Abcam #111171) diluted in blocking buffer. As secondary antibody, an appropriate antibody with HRP label was used, also diluted in blocking buffer. The probed protein was detected by using ECL substrate (Thermo Scientific), and a time series of images was recorded.

[0138] Example

[0139] Starting material from plasma purification

[0140] The initial protein mixture contained approximately 40 IU / ml FX and 0.01-80 IU / ml FII in a buffer consisting of 20 mM TRIS and 150 mM NaCl at pH=7.5, and its total protein content was 1-60 mg / ml.

[0141] The initial protein mixture is obtained from DEAE agarose or heparin affinity chromatography side fractions from coagulation factor purification and buffer exchange using ultrafiltration / diafiltration.

[0142] Specifically, make the cold supernatant plasma through heparin stabilization adsorbed on the anion exchange resin QAE-Sephadex, then wash this resin.Then increase to the buffer elution vitamin K dependent factor of 1600-2200mosmol / kg with ionic strength, and stabilize it by adding 3IU heparin / ml.S / D treatment is carried out to the stabilization eluent by 0.3%TnBP / 1% polysorbate 80 incubation, and by adsorption on DEAE-Sepharose and washing with 260-310mosmol / kg buffer to remove S / D reagent.Elute protein with the buffer of 700-780mosmol / kg.Finally, use ultrafiltration / diafiltration to carry out buffer exchange and be concentrated to 30IU Factor IX / ml on 10kDa membrane utilizing the buffer of about 330-400mosmol / kg, obtain initial protein mixture.

[0143] Alternatively, cold supernatant plasma is adsorbed to DEAE-Sephadex A50, then the resin is washed. Eluent is obtained by eluting with the high ionic strength buffer of 2M sodium chloride and 15mM sodium citrate of about neutral pH, concentrated to 380-420mosmol / kg, and DEAE-Sepharose FF is used to carry out anion exchange purification to the concentrated eluent. After washing, the DEAE-eluent is obtained with the buffer of 360mM sodium chloride, 5mM sodium citrate and 5mM disodium hydrogen phosphate, and the DEAE-eluent is inactivated by virus incubation of 0.3%TnBP / 1% polysorbate 80. Afterwards, the eluent processed by S / D is carried out to affinity chromatography on heparin-Sepharose CL 6B. After washing with a buffer composed of 20 mM sodium citrate at pH 7.0-8.0, elution was performed with a buffer composed of 250 mM sodium chloride and 20 mM sodium citrate having the same pH value to obtain an eluate.

[0144] To obtain the initial protein mixture, after each purification process, a buffer exchange was performed using ultrafiltration / diafiltration using a membrane with a cut-off of 10 kDa to a buffer consisting of 20 mM TRIS and 150 mM NaCl at pH = 7.5.

[0145] Starting material for FX manufacturing from plasma purification or biotechnology using immunoaffinity chromatography

[0146] Antibodies specific for FX and FXa are immobilized on activated chromatographic resins, such as The column containing the immunoaffinity resin was pretreated with an equilibration buffer having a pH of 7.4 and a conductivity of 16.8 mS / cm and containing 20 mM TRIS, 130 mM NaCl, 10 mM Ca ions and 0.05% polysorbate 80. Before loading on the immunoaffinity column, the initial protein mixture was subjected to a buffer exchange to the equilibration buffer. After thorough washing with the equilibration buffer, the flow-through / wash fraction containing unbound impurities was discarded and the bound FX was eluted from the column with an elution buffer having a pH of 7.4 and a conductivity of 19.2 mS / cm and containing 20 mM TRIS, 130 mM NaCl, 40 mM EDTA and 0.05% polysorbate 80. The FX content of the initial protein mixture thus obtained was brought to about 40 IU / ml by ultrafiltration / diafiltration with a buffer consisting of 20 mM TRIS and 150 mM NaCl at pH = 7.5 and a membrane with a cut-off value of 10 kDa. The initial protein mixture can be activated using one of the activation methods selected from Examples 1 to 4 and 9 without further purification.

[0147] Example 1 (activation)

[0148] 40 mg of synthetic phosphatidylserine (SPS) was mixed with 10 ml of reaction buffer (20 mM TRIS and 150 mM NaCl at pH 7.5) and heated at 50°C. The mixture was dispersed in a disperser for 30 minutes and cooled to room temperature to obtain an SPS dispersion.

[0149] The 55ml initial protein mixture comprising 2mg / ml total protein, about 40IU / ml FX and about 0.5U / ml FII was mixed with 27.5ml SPS dispersion, 27.5ml reaction buffer and 55ml reaction buffer containing 75mM calcium ion. The mixture was incubated at room temperature for 6 hours with gentle stirring at pH 7.5, and then 55ml reaction buffer with 200mM EDTA was added. Dispersed PS was dissolved by adding 1.5mM Triton X-100, and the clear solution obtained was filtered through a 0.45μm PES membrane filter to reclaim the intermediate containing FXa.

[0150] The mixture thus prepared, as defined in the assay instructions, had a FXa specific activity of about 9.6 IU / ml, a thrombin level below 50 IU / ml and a FXa yield of about 69% based on the FX content activated with PS and calcium ions.

[0151] Example 2 (activation)

[0152] 100 ml of the initial protein mixture containing 40 mg / ml total protein, about 60 IU / ml FX and about 60 U / ml FII was mixed with 90 ml of reaction buffer and 100 ml of reaction buffer containing 75 mM Ca ions. 10 ml of PS dispersion was added dropwise in a continuous operation. The mixture was incubated at 22° C. for 16 hours with gentle stirring at a pH of 7.0, and then 100 ml of reaction buffer with 200 mM EDTA was added. The dispersed PS was dissolved by adding 1.5 mM Triton X-100, and the clear solution obtained was filtered through a 0.45 μm PES membrane filter to recover the intermediate containing FXa.

[0153] The mixture thus prepared had a FXa-specific activity of 8.2 IU / ml, a FIIa-specific activity of 619 IU / ml, and a total protein content of 9.9 mg / ml. The yield of FXa was 33% based on the FX content activated with PS and Ca ions.

[0154] Example 3 (activation)

[0155] The initial protein mixture was diluted with reaction buffer, mixed with PS dispersion and Ca ions to obtain a solution containing 40 IU / ml FX, 0.33 mg / ml PS and 25 mM Ca ions. The mixture was incubated at 15° C. for 10 hours while gently stirring, and then the reaction was terminated with 50 mM EDTA. The dispersed PS was dissolved by adding 1.5 mM Triton X-100, and the obtained clear solution was filtered through a 0.45 μm PES membrane filter to recover the FXa-containing intermediate.

[0156] The mixture thus prepared had a FXa-specific activity of 4.1 IU / ml, a FIIa-specific activity of 3.1 IU / ml, a total protein content of 0.4 mg / ml, and a FXa yield of 41% based on the FX content activated with PS and calcium ions.

[0157] Example 4 (Activation)

[0158] A portion of the reaction mixture before incubation in Example 3 was taken out and used for incubation for 10 hours at 33° C. The results of this activation treatment were: 2.8 IU / mL FXa, 16.4 IU / mL FIIa, 0.6 mg / mL protein content, and a FX / FXa conversion yield of 27% based on the FX content activated with PS and Ca ions.

[0159] Example 5 (Collection of FXa and Removal of FIIa—AEX)

[0160] The strong ion exchange resin was loaded with an equilibration buffer having a pH of 6.3 and a conductivity of 1.0 mS / cm and containing 10 mM imidazole. The column was pretreated with FXa. The intermediate containing FXa was diluted with the equilibrium buffer until the conductivity was less than 15.0 mS / cm. The diluted FXa solution was loaded onto the pretreated anion exchange column to bind FXa and leave unbound proteins and impurities (such as thrombin and PS) in the flow-through fraction and subsequent wash fractions. The chromatographic column was thoroughly cleaned with a wash buffer having a pH of 6.3 and a conductivity of 21.4 mS / cm and containing 10 mM imidazole and 0.2 M NaCl until the OD280 reading of the monitored UV flow cell returned to the baseline level. FXa was eluted from the anion exchange resin with an elution buffer having a pH of 7.4 and a conductivity of 89.3 mS / cm and containing 20 mM TRIS and 1 M NaCl. The anion exchange eluate obtained had a FXa purity of 8 U per mg of protein.

[0161] Example 6 (Removal of copper binding proteins - IMAC)

[0162] The anion exchange eluate was diluted with a buffer containing 20 mM TRIS and 4 mM imidazole at pH 7.4 to reduce the NaCl content from 1 M to 0.5 M and adjust the imidazole concentration to 2 mM. The diluted eluate was loaded onto an immobilized metal affinity chromatography (IMAC) resin with copper ions, and the purity of FXa present in the IMAC flow-through fraction was 43 U / mg protein.

[0163] Example 7 (Separation of FXa from FX by heparin affinity chromatography)

[0164] The IMAC flow-through fraction was buffer exchanged to obtain FXa dissolved in 20 mM TRIS buffer at pH 7.4 and conductivity 1.6 mS / cm and The obtained FXa solution was subjected to heparin affinity chromatography on a resin which had been equilibrated with a buffer having a pH value of 7.4 and a conductivity of 1.6 mS / cm and containing 20 mM TRIS and 0.005% polysorbate 80. Under these conditions, FXa was bound to the resin, while the zymogen FX and the impurity F1.2 were not bound and passed through the column. After washing with the equilibration buffer and the monitored OD280 signal returned to the baseline, FXa was eluted from the resin with an elution buffer having a pH value of 7.4 and a conductivity of 26.1 mS / ml and containing 20 mM TRIS, 0.005% polysorbate 80 and 0.25 M NaCl. The purity of FXa eluted from the resin was 199 U / ml.

[0165] Example 8 (Immunoaffinity chromatography-activation)

[0166] A column containing an FX / FXa-specific immunoaffinity resin with covalently bound anti-factors X and Xa was pretreated with an equilibration buffer having a pH of 7.4 and a conductivity of 16.8 mS / cm and containing 20 mM TRIS, 130 mM NaCl, 10 mM Ca ions and 0.05% polysorbate 80. Calcium-dependent monoclonal antibody (Catalog No.: AX100083). Before loading on the immunoaffinity column, the initial protein mixture not obtained from the immunoaffinity chromatography was subjected to buffer exchange to the equilibration buffer. After thorough washing with the equilibration buffer, the flow-through / wash fraction containing unbound impurities was discarded and bound FX and FXa were eluted from the column with an elution buffer having a pH of 7.4 and a conductivity of 19.2 mS / cm and containing 20 mM TRIS, 130 mM NaCl, 40 mM EDTA and 0.05% polysorbate 80.

[0167] Example 9 (preparation)

[0168] The FXa intermediate was formulated by ultrafiltration / diafiltration (UDF) using a basic formulation buffer containing 10 mM histidine, 100 mM NaCl and 0.005% w / v Polysorbate, supplemented with an excipient, as shown in Table 1, pH 7.2. Prior to lyophilization, the formulated FXa composition was made to have a final FXa concentration of 0.3 IU / ml and a protein content of less than 0.125 mg / ml.

[0169] Example 10 (lyophilization)

[0170] Several purified FXa preparations were formulated for lyophilization and packaged in vials using the buffers shown in Table 1. The FXa preparations in vials were subjected to a lyophilization cycle to ensure that the residual water content was less than 1.5% as determined by Karl Fischer (1935).

[0171] result

[0172]

[0173]

[0174] Table 1

[0175]

[0176] Table 2

[0177]

[0178] Table 3

[0179]

[0180] Table 4: Wessle Thrombosis Safety Test Results

[0181] References

[0182] Toso et al. The conformational switch from the factor X zymogen to protease state mediates exosite expression and prothrombinase assembly;The Journal of Biological Chemistry, 2008;283(27):18627–18635. - DOI:10.1074 / jbc.M802205200.

[0183] Bajaj, S.P. Mann, K.G. Simultanenous Purification of Bovine Prothrombin and Factor X.;J.Biol.Chem., 1973;248(22):7729 - 7741.

[0184] Fujikawa, K., Coan, M.H., Legaz, M.E. & Davie, E.W. The mechanism of activation of bovine factor X(Stuart factor) by intrinsic and extrinsic pathways;Biochemistry, 1974;13:5290–5299.

[0185] Rüchel, R. On the renin - like activity of Candida proteinases and activation of blood coagulation in vitro;Zentralblatt für Bakteriologie, Mikrobiologie und Hygiene 1.Abt.Originale A., 1983;255:368–379.

[0186] Imamura,T.,Potempa,J.,Tanase,S.&Travis,J.Activation of bloodcoagulation factor X by arginine-specific cysteine proteinase(Gingiparin-Rs)from Porphyromonas gingivalis.;Journal of Biological Chemistry,1997;272:16062–16067.

[0187] Richter,G.et al.Activation and inactivation of human factor X byproteases derived from Ficus carica.;British Journal of Haematology,2002;19:1042-1051.

[0188] Kempton and White.How we treat a hemophilia A patient with a factorVIII inhibitor;Blood,2009;113(1):11–17.-DOI:10.1182 / blood-2008-06-160432.

[0189] Giles,A.R.et al.A combination of factor Xa and phosphatidylcholine-phosphatidylserinevesicles bypasses factor VIII in vivo;British Journal ofHaematology,1988;69:491-497.Lindhoff-Last,Edelgard.Direct oral anticoagulants(DOAC)-Management of emergency situations;Hamostaseologie,2017;37(04):257-266.-DOI:10.5482 / HAMO-16-11-0043.

[0190] Hoffer, L., Schwinn, H., Biesert, L., Josic, D. Improved virus safety and purity of a chromatographically produced factor IX concentrate by nanofiltration.;Journal of chromatography.B,Biomedical applications,1995;669(2):187–196.

[0191] Roemisch J. and Pock, K. In: Production of Plasma Proteins for Therapeutic Use, J. Bertolini, N. Goss, and J. Curling (eds.), John Wiley & Sons Inc., Hoboken, New Jersey;2012;65–79.

[0192] Wessler et al.Biologic assay of a thrombosis-inducing activity inhuman serum;J.Appl.Physiol.,1959;14(6):943-946.

[0193] K. Fischer. New method for the mass-analytical determination of the water content of liquids and solids Angewandte Chemie, 1935; 48:394–396.-DOI:10.1002 / anie.19350482605.

Claims

1. A method for activating coagulation factor X (FX) to activated coagulation factor X (FXa), wherein the method is performed in vitro and comprises the following steps: a. Providing a composition containing FX; and b. contacting the composition containing FX with phosphatidylserine PS and calcium to form an activated composition; and C. incubating the activated composition to convert FX into FXa, wherein no protein activator is added to the composition before, during, and after the incubation; wherein the concentration of phosphatidylserine in the activation composition is in the range of 0.05 to 100 mM, and the phosphatidylserine is synthetic phosphatidylserine; and Wherein, the calcium is in ionic form, and the concentration of calcium ions in the activation composition is in the range of 1 to 100 mM.

2. The method for activating FX to FXa according to claim 1, wherein: The FX is plasma-derived FX.

3. The method for activating FX to FXa according to claim 1 or 2, wherein: The yield of conversion of FX to FXa is at least 10%.

4. The method for activating FX to FXa according to claim 3, wherein: The yields of this conversion ranged from 20% to 100%.

5. The method for activating FX to FXa according to any one of claims 1, 2 and 4, wherein The starting concentration of FX in the activating composition ranges from 2 to 80 IU / ml.

6. The method for activating FX to FXa according to claim 5, wherein: The starting concentration of FX in the activating composition ranged from 2 to 40 IU / ml.

7. The method for activating FX to FXa according to any one of claims 1, 2, 4 and 6, wherein The concentration of phosphatidylserine in the activating composition ranges from 0.1 to 50 mM.

8. The method for activating FX to FXa according to claim 7, wherein: The concentration of phosphatidylserine in the activating composition is in the range of 0.2 to 10 mM.

9. The method for activating FX to FXa according to claim 8, wherein: The concentration of phosphatidylserine in the activating composition is in the range of 0.2 to 1 mM.

10. The method for activating FX to FXa according to any one of claims 1, 2, 4, 6, 8 and 9, wherein The concentration of calcium ions in the activating composition is in the range of 2.5 to 50 mM.

11. The method for activating FX to FXa according to claim 10, wherein: The concentration of calcium ions in the activating composition is in the range of 5 to 40 mM.

12. The method for activating FX to FXa according to claim 11, wherein: The concentration of calcium ions in the activating composition is in the range of 10 to 35 mM.

13. The method for activating FX to FXa according to any one of claims 1, 2, 4, 6, 8, 9, 11 and 12, wherein The FX-containing composition further comprises FII, and wherein the concentration of FII in the activated composition is in the range of 0.01 to 30 IU / ml.

14. The method for activating FX to FXa according to claim 13, wherein: The concentration of FII in the activation composition ranges from 0.01 to 1 IU / ml.

15. The method for activating FX to FXa according to any one of claims 1, 2, 4, 6, 8, 9, 11, 12 and 14, wherein The pH of the activating composition is in the range of 5.0 to 9.

0.

16. The method for activating FX to FXa according to claim 15, wherein: The pH of the activating composition is in the range of 7.4 to 7.

6.

17. The method for activating FX to FXa according to any one of claims 1, 2, 4, 6, 8, 9, 11, 12, 14 and 16, wherein The temperature of the activation composition during incubation was in the range of 15-40°C.

18. The method for activating FX to FXa according to claim 17, wherein: The temperature of the activation composition during incubation was in the range of 15-35°C.

19. The method for activating FX to FXa according to any one of claims 1, 2, 4, 6, 8, 9, 11, 12, 14, 16 and 18, wherein The incubation time ranges from 1 to 48 hours.

20. The method for activating FX to FXa according to claim 19, wherein: The incubation time ranges from 4 to 20 hours.

21. A method for preparing a FXa composition comprising the method for activating FX to FXa according to any one of claims 1 to 20, and further comprising at least one purification step.

22. The method according to claim 21, wherein: The at least one purification step is selected from the group consisting of immobilized metal affinity chromatography IMAC, anion exchange chromatography AEX and affinity chromatography AF.

23. The method according to claim 21 or 22, wherein: The activation method is followed by AEX.

24. The method according to claim 23, wherein: This was followed by heparin affinity chromatography.

25. The method according to claim 24, wherein: Heparin affinity chromatography using Heparin- Resin.

26. The method of claim 23, wherein: The AEX is followed by IMAC or immunoaffinity chromatography using an FX / FXa-specific immunoaffinity resin.

27. The method according to claim 26, wherein: This was followed by heparin affinity chromatography.

28. The method according to claim 27, wherein: Heparin affinity chromatography using Heparin- Resin.

29. The method according to any one of claims 21-22 and 24-28, further comprising at least one virus removal step and one virus inactivation step to improve pathogen safety of the finished product.

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